Book - Sex and internal secretions (1961) 16: Difference between revisions

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M. X. Zarrow, Ph.D. Professor Of Zoology, Purdue University, Lafayette, Indiana  
M. X. Zarrow, Ph.D. Professor Of Zoology, Purdue University, Lafayette, Indiana  


I. Introduction 958
II. Length of Gestation 958
III. Normal Reproductive Potential... 959
IV. Environment 962
A. Crowding 962
B. Body Temperature and Hypoxia. . . 962
V. Maternal Hormone Levels during Gestation 963
A. Estrogens 964
B. Gestagens 964
C. Sources of Gestagens 972
D. Relaxin 973
E. Sources of Relaxin 976
F. Adrenal Cortex 976
L Hydrocortisone 976
2. Aldosterone 979
G. Thyroid Gland 979
H. Growth Hormone 980
I. Prolactin 981
J. Placental Gonadotrophins 982
1. Human chorionic gonadotrophin (HCG) 983
2. Equine gonadotrophin (PMS). . . 985
VI. Pregnancy Tests 986
VII. Water and Electrolyte Balance.. 988
VIII. Plasma Proteins 993
IX. Renal Function 994
X. Enzymes 995
A. Histaminase 995
B. Carbonic Anhydrase 996
XI. Factors in the Maintenance of Gestation 997
A. Thyroid Gland 997
B. Adrenal Cortex 1000
C. Pancreas 1003
D. Ovarv: Progesterone, Estradiol, and Relaxin 1003
E. Pituitarv Gland 1005
F. Placenta 1006
G. Pelvic Adaptation 1008
H. Dilation of the Uterine Cervix .... 1011
XII. Uterine Myometrial Activity 1013
XIII. Parturition 1015
A. Progesterone 1015
B. Oxvtocin 1015
C. Relaxin 1016
D. Labor 1017
^ Aided by grants from the Purdue Research Foundation.
XIV. Conclusion.
XV. References


__TOC__


==I. Introduction==
==I. Introduction==


Reproduction in the animal kingdom is  
Reproduction in the animal kingdom is  
Line 2,727: Line 2,635:
termination of pregnancy in 24 hours. Castration later in pregnancy resulted in abortion approximately 2 days after the operation. In 1928, Corner showed that an extract  
termination of pregnancy in 24 hours. Castration later in pregnancy resulted in abortion approximately 2 days after the operation. In 1928, Corner showed that an extract  
of the corpus luteum could induce a progestational endometrium in the castrated rabbit. This was soon followed by the demonstration that this extract could induce  
of the corpus luteum could induce a progestational endometrium in the castrated rabbit. This was soon followed by the demonstration that this extract could induce  
implantation of the fertilized egg in the  
implantation of the fertilized egg in the rabbit and maintain pregnancy in the castrated animal (Allen and Corner, 1929;  
 
 
 
rabbit and maintain pregnancy in the castrated animal (Allen and Corner, 1929;  
1930). Purification of the extract of the  
1930). Purification of the extract of the  
corpus luteum led to the chemical identification of the active substance by Butenandt,  
corpus luteum led to the chemical identification of the active substance by Butenandt,  
Line 2,738: Line 2,642:
and Slotta (1935) agreed to the name pi^ogesterone for this hormone of the corpus  
and Slotta (1935) agreed to the name pi^ogesterone for this hormone of the corpus  
luteum.  
luteum.  


These events were soon followed by tlu'  
These events were soon followed by tlu'  
Line 2,743: Line 2,648:
the urine as tlie glucuronide of pregnanediol and i)regnanolone, metabolites of progesterone. Studies of urinary products of  
the urine as tlie glucuronide of pregnanediol and i)regnanolone, metabolites of progesterone. Studies of urinary products of  
progesterone were immediately undertaken  
progesterone were immediately undertaken  
and a marked increase in urinary pregnancdiol was observed in the human female  
and a marked increase in urinary pregnancdiol was observed in the human female throughout pregnancy, especially in the  
 
 
 
966
 
 
 
SPERM, OVA, AND PREGNANCY
 
 
 
throughout pregnancy, especially in the  
second half (Fig. 16.3).  
second half (Fig. 16.3).  


Line 2,772: Line 2,665:
established.  
established.  


The concentration of gestagen in the
blood of pregnant sheep (Neher and Zarrow, 1954) , women (Forbes, 1951 ; Schultz,
1953; Fujii, Hoshino, Aoki and Yao, 1956),
rabbits (Zarrow and Neher, 1955), and
mice (Forbes and Hooker, 1957) has been
determined by the Hooker-Forbes test and




Fig. 16.3. Urinary excretion of estrogens and
pregnanediol throughout gestation in the human
being. (From E. Venning, Macy Foundation, Conferences on Gestation, 3, 1957.)


80
70


o


2  
The concentration of gestagen in the
blood of pregnant sheep (Neher and Zarrow, 1954) , women (Forbes, 1951 ; Schultz,
1953; Fujii, Hoshino, Aoki and Yao, 1956),
rabbits (Zarrow and Neher, 1955), and
mice (Forbes and Hooker, 1957) has been
determined by the Hooker-Forbes test and expressed as /^g. equivalents of progesterone. The data obtained from pregnant
women by the different investigators are
in marked disagreement. Whereas both
Forbes (1951) and Schultz (1953) failed
to observe any significant rise in blood gestation of pregnant women throughout gestation, Fujii, Hoshino, Aoki and Yao
(1956) obtained a conspicuous rise during
this period. The data reported by Forbes
(1951) indicate an extremely low level for
protein-bound progesterone (0.5 /^g. per
ml. plasma or less) and a maximum of 2  
/xg. per ml. free progesterone (Fig. 16.4). The
concentration of the hormone in the blood
showed a series of irregular peaks throughout gestation and varied from less than
0.3 /xg. to 2 /xg. per ml. plasma. In general,
these results were confirmed by Schultz
(1953) who assayed the blood from 46
women at 6 to 17 weeks of pregnancy.
Again the results failed to reveal any consistent change with the length of pregnancy. Both investigators (Forbes, 1951;
Schultz, 1953) were led to question the
importance of progesterone during gestation in the primate. Fujii, Hoshino, Aoki
and Yao (1956), on the other hand, reported a significant increase in the level of
circulating progesterone throughout gestation. Again these investigators used the
Hooker-Forbes assay but indicated that
the plasma was not treated in any way
except for dilution before the assay. The
results obtained by this latter group revealed a rise from a level of 6 /*g. progesterone per ml. plasma during the luteal
phase of the cycle to a high of 25 /i,g. during:
the last trimester of pregnancy (Fig. 16.5).
The concentration showed a steady increase
from the 4th to the 24th week of pregnancy,,
and a plateau from the 24th week until
term. A sharp drop occurred within 12 to
24 hours after parturition with zero values
noted by 72 hours postpartum. Analysis of
the urine for pregnanediol showed a rather
good correlation between the two curves although the plasma levels rose sooner than
the urinary pregnanediol.


16
The curve for the concentration of progesterone in the pregnant mouse is
markedly different from those reported for
other species (Forbes and Hooker, 1957).
Again the Hooker-Forbes assay was used








Fig. 16.4. Free and bound ge.stagen in the plasma of the pregnant human female. (From




Line 2,799: Line 2,732:




 
Fig. 16.5. Concentration of gestagen in the blood plasma and pregnanediol in the
 
uterine of the pregnant human female. Gestagen levels were determined by the HookerForbes test. (From K. Fujii, K. Hoshino, I. Aoki and J. Yao, Bull. Tokyo Med. & Dent.
60
Univ., 3, 225, 1956.)
 
 
■12
 






as with the other species and the values
expressed as activity equivalent to progesterone. The values for the bound action
were consistently low and, in general, less
than 1 fjig. per ml. plasma (Fig. 16.6). The
concentration of the free hormone showed
marked variations on the first day or so of
pregnancy. Actually a variation from 1
fig. per ml. plasma to 8 fig. per ml. plasma
was seen on day 0. This type of fluctuation
has also been seen in the rabbit and is without explanation at the present time. However, such marked variations disappeared
by the 4th day of pregnancy and the results
became much more consistent. The average curve for the concentration of gestagen in
the blood of the pregnant mouse showed
two peaks, one the 7th to the 9th day and a
second the 15th day. The concentration
increased from 2 fxg. per ml. plasma the 4th
day of gestation to an average of approximately 8 fig. the 7th day. This level was
maintained until day 9 and fell thereafter
with a second peak occurring on day 15 and
an immediate drop on day 16. Thereafter
the levels remained low throughout the remainder of pregnancy.


Although it may be assumed that the
initial peak in the concentration of the
gestagen is due to an increased activity








Fig. 16.6. Concentration of free and bound gestagen in the plasma of the pregnant
mouse. Gestagen levels were determined by the Hooker-Forbes test. (From T. R. Forbes
and C. W. Hooker, Endocrinology, 61, 281, 1957.)


//I




50
40


e
on the part of the corpora lutea, an explanation of the second peak and the drop
between the two peaks offers more difficulty. The latter may reflect a diminished
luteal activity. This could be assumed on
the grounds that the corpus luteum is the
only source of gestagen during this period
of gestation and that the luteal cells show
cytologic signs of regressive changes, although the drop in serum progestogen antidates the cytologic changes by several
days. An explanation for the second peak
would probably involve increased secretory
activity !)y the placenta. Progestational
activity has l)een found in i:)lacental extracts and progesterone has been isolated
from the placentae of human beings and
mares (Salhanick, Noall, Zarrow^ and
Samuels, 1952; Pearlman and Cerceo,
1952; Zander, 1954; Short, 1956). Thus, the
drop in serum gestagen seen on day 10
could be due to loss in the activity of the
corpora lutea and the second rise as a contribution from the placentas. It is of interest
that the low levels on days 10 to 13 and between day 16 to term appear to have no
counterpart in other species. The physiologic
significance of this is still unknown and will
require further work on additional species and on the mouse before an explanation is forthcoming. It is of interest that the
concentration of gestagen in the blood dur




ing the first 12 days of pregnancy corresponds with the intensity of the response to
progesterone exhibited by the endometrium
during the same period (Atkinson and
Hooker, 1945). This w^ould suggest that
the serum gestagen levels reflect the physiologic state of the animal.


Serum gestagen levels in the rabbit reveal a curve of increasing concentration
throughout pregnancy (Zarrow and Neher,
1955). Initial values of 0.3 to 1 yug. per
ml. serum were noted at the time of mating,
with a sharp rise beginning on the 4th day
of gestation. The concentration rose to a
level of 6 to 8 /xg. per ml. by the 12th day
and thereafter showed only a slight rise to
a maximal concentration of 8 to 10 /xg. per
ml. serum at parturition (Fig. 16.7). No
drop in serum hormone level was observable at parturition or 1 hour later. The
first significant drop occurred at 6 to 12
hours postpartum when the gestagen level
had decreased 50 per cent. It is of interest
that the serum progestagen levels did not
fall until after the conceptus had been expelled.


castrated the 12th,
of gestation aborted
following removal of
the ovaries (Zarrow and Neher, 1955). In
all instances the serum gestagen levels fell
before the abortion. Figure 16.8 shows the






Pregnant rabbits
19th, or 24th day
within 1 to 3 days


/




>
GESTATION


.'estriol


/


969


30




± 5
3
NORMAL PREGNANCY
RABBIT 25 o
RABBIT 26 •
RABBIT 31 e
- RABBIT 27 x
RABBIT 30 ^
Fig. 16.7. Concentration of gestagen in the blood of the normal pregnant rabbit as determined by the Hooker-Forbes test. (From M. X. Zarrow and G. M. Neher, Endocrinology,
56, 1, 1955.)


PREGNANEDIOL




r








/




20 ■
10 ■




4


Fig. 16.8. The effect of castration on serum progestogen levels and maintenance of gestathe rabbit. Gestagen levels were determined by the Hooker-Forbes test. (From M. X. and G. M. Xeher. Endocrinology, 56, 1, 1955.)






/  
changes in serum gestagen levels before
and after castration of a pregnant rabbit.
The concentration increased from a level of
0.3 ing. per ml. at day to 10 /xg. per ml. on
day 24 when the rabbit was castrated. A
60 per cent drop in serum gestagen level is
seen 12 hours after castration with a fur




/
ther drop at the 36th hour, when the animal
aborted.


Studies on the concentration of serum
gestagen in the pregnant ewe (Neher and
Zarrow, 1954) permit a comparison with
the results obtained in the rabbit. Such a
comparison is extremely valuable in view


ESTRONE -t-ESTRADIOL






Fig. 16.3. Urinary excretion of estrogens and
pregnanediol throughout gestation in the human
being. (From E. Venning, Macy Foundation, Conferences on Gestation, 3, 1957.)


Fig. 16.9. Concentration of gestagen in the blood of the pregnant ewe. Gestation levels
were determined bv the Hooker-Forbes test. (From G. M. Neher and M. X. Zarrow, J.
Endocrinol., 11,323,1954.)




expressed as /^g. equivalents of progesterone. The data obtained from pregnant
women by the different investigators are
in marked disagreement. Whereas both
Forbes (1951) and Schultz (1953) failed
to observe any significant rise in blood gestation of pregnant women throughout gestation, Fujii, Hoshino, Aoki and Yao
(1956) obtained a conspicuous rise during
this period. The data reported by Forbes
(1951) indicate an extremely low level for
protein-bound progesterone (0.5 /^g. per
ml. plasma or less) and a maximum of 2
/xg. per ml. free progesterone (Fig. 16.4). The
concentration of the hormone in the blood
showed a series of irregular peaks throughout gestation and varied from less than
0.3 /xg. to 2 /xg. per ml. plasma. In general,
these results were confirmed by Schultz
(1953) who assayed the blood from 46
women at 6 to 17 weeks of pregnancy.
Again the results failed to reveal any consistent change with the length of pregnancy. Both investigators (Forbes, 1951;
Schultz, 1953) were led to question the
importance of progesterone during gestation in the primate. Fujii, Hoshino, Aoki
and Yao (1956), on the other hand, reported a significant increase in the level of
circulating progesterone throughout gestation. Again these investigators used the
Hooker-Forbes assay but indicated that
the plasma was not treated in any way
except for dilution before the assay. The
results obtained by this latter group revealed a rise from a level of 6 /*g. progesterone per ml. plasma during the luteal
phase of the cycle to a high of 25 /i,g. during:
the last trimester of pregnancy (Fig. 16.5).
The concentration showed a steady increase
from the 4th to the 24th week of pregnancy,,
and a plateau from the 24th week until
term. A sharp drop occurred within 12 to
24 hours after parturition with zero values
noted by 72 hours postpartum. Analysis of
the urine for pregnanediol showed a rather
good correlation between the two curves although the plasma levels rose sooner than
the urinary pregnanediol.


The curve for the concentration of progesterone in the pregnant mouse is
of the fact that castration of the rabbit
markedly different from those reported for
invariably leads to abortion whereas castration of the pregnant ewe does not do so
other species (Forbes and Hooker, 1957).
if the ovaries are removed during the second half of pregnancy. Again the progesterone determinations were carried out on
Again the Hooker-Forbes assay was used
untreated serum and the samples assayed
by the Hooker-Forbes technique using progesterone as a standard. An initial rise in
the serum gestagen level occurred soon
after mating and seemed to level off at a
concentration of 6 ixg. per ml. approximately the 50th day of gestation (Fig.
16.9). Thereafter, the concentration remained unchanged for approximately 50
days, when a second rise to a level of 8 to
12 fj.g. occurred. These levels remained unchanged until at least 30 minutes after parturition was complete.


Castration at various times after the
66th day of pregnancy failed to influence
the concentration of circulating gestagen
or interfere with the pregnancy. The data
in Figure 16.10 show a normal concentration of 8 to 10 fxg. gestagen from the 114th
day of gestation to parturition although the
animal was ovariectomized the 114th day.
Pregnancy was normal in all castrated ewes and the expected drop in scrum gestagen was observed following parturition.


It can now be stated that the human
being, the monkey, the ewe, the rabbit, the
mouse, and probably the guinea pig (Herrick, 1928; Ford, Webster and Young,
1951) have met the problem of a second
source of progesterone supply with varying
degrees of success. In the ewe, placental replacement of the ovary as a source of progesterone can be considered as complete
by approximately the 66th day of pregnancy. Castration at this time will neither
interfere with the pregnancy nor with the
concentration of the hormone in the blood.
In the monkey, castration as early as the
25th day of gestation (Hartman, 1941)
does not interfere with pregnancy and in
the human being castration as early as the
41st day after the last menstrual period
may not interfere with pregnancy (Melinkoff, 1950; Tulsky and Koff, 1957). One
may conclude, therefore, that the placenta
can adequately take on the role of the
ovary in this regard. On the other hand,
aspects of the situation in the human female are still puzzling, especially the blood
gestagen values; but despite this ambiguity


GESTATION






96/
Fig. 16.10. The effect of castration on gestagen levels in the pregnant ewe. Gestagen
le\els were obtained by the Hooker-Forbes test. Note that castration failed to interfere
with the pregnancy or the level of gestagen in the blood. (From G. M. Neher and M. X.
Zarrow, J. Endocrinol., 11, 323, 1954.)






it might be concluded that here also the
placenta has successfully replaced the
ovary. In the rabbit, on the other hand,
castration at any time during pregnancy
vvill cause a decrease in the level of the
circulating hormone and terminate the
pregnancy. Hence, in this species, the placenta has failed to replace completely the
ovary. The mouse is another instance in
which castration leads to abortion so that
one can assume a failure on the part of the
placenta to replace the endocrine activity of
the ovary. In this case, however, the second
peak of circulating gestagen has been ascribed to the placenta and this presents the
possibility of a partial replacement of the
ovary by the placenta but a replacement
that is not adequate since pregnancy is
terminated by ovariectomy.


S 2  
As indicated above, a marked discrepancy exists between the bioassays and the
chemical determinations of gestagens in
the blood and other tissues. The chemical
determinations of progesterone invariably
give results that are far lower than those
obtained by bioassay methods. Edgar and
Ronaldson (1958) found a maximal concentration of approximately 2 /xg. progesterone per ml. ovarian venous blood during






.t=^t=^t^
gestation in the ewe. This concentration
was no higher than that seen in the ewe
during a normal estrous cycle. The maximal level reached during the estrous cycle
was maintained when pregnancy supervened and remained fairly constant until
the last month of pregnancy. Thereafter
the concentration fell and no progesterone
was detectable at 15 days prepartum (Fig.
16.11). Inasmuch as no progesterone was
found in the peripheral blood of the ewe,
this poses again the following question:
What was being measured in the peripheral
blood by the bioassay procedure? In addition, a second question is posed by the
earlier discussion on the need of the ovary
in the maintenance of pregnancy as to the
relative contributions of the ovary and the
placenta to the concentration of this hormone in the body.  


That the biologic methods are measuring
more than progesterone is obvious from the
many reports emphasizing the high levels
obtained by bioassay and the low levels
obtained by chemical techniques. In addition to the above data. Short (1957, 1958a,
1958b) reported the presence of progesterone in the peripheral blood of the pregnant




■J^ri.


Fig. 16.11. The con«?ntiation of progesterone in the ovarian venous blood of the pregnant ewe. Progesterone was determined by chemical methods. (From D. G. Edgar and
J. W. Ronaldson, J. Endocrinol., 16, 378, 1958.)






19 21 23 25 27
0.0098 ixg. per ml. plasma. It is of interest
 
that the level remained constant from the
H8 Weeks since start of L.M.P.  
32nd to about the 256th day of pregnancy
 
and then decreased several days before
 
parturition. In the human being values of
 
0.17 to 0.44 fig. per ml. during the final trimester of pregnancy have recently been
^^
reported (Oertel, Weiss and Eik-Nes ( 1959 ) .
 
Numerous investigators have suggested
 
that the discrepancy between the chemical
 
and biologic assays is due to the presence
29 31 33 35 37 39 41
of unknown gestagens in the blood. This
 
has been validated in part by the discovery
Caesarian I2!I5 A.M.  
of 2 metabolites in the blood of the pregnant human female (Zander, Forbes, Neher
Fig. 16.4. Free and bound ge.stagen in the plasma of the pregnant human female. (From
and Desaulles, 1957). They have been identified as 20a-hydroxypregn-4-en-3-one and
20^-hydroxypregn-4-en-3-one and have
been shown to be active in both the Clauberg and Hooker-Forbes tests (Zander,
Forbes, von IMiinstermann and Neher
1958 ) . The 20/?-epimer was twice as active
as progesterone in the Hooker-Forbes test
and the 20a-epimer one-fifth as active. It is likely that more unidentified gestagens
occur in the blood and other tissues.




===C. Sources of Gestagens===


T. R. Forbes, Endocrinology, 49, 218, 1951.)
The second question asked above concerning the role of the placenta versus the
ovary as a source of progesterone probably
cannot be answered in a simple manner.  
Wide differences exist between species (1)
in the need of the ovary for maintenance
of pregnancy, (2) in the concentration of
the hormone in peripheral blood, (3) in the
activity of the placenta in secreting progesterone, and (4) in the presence of extraovarian and extraplacental sources of the
hormone.  


The presence of progesterone in the placenta of the human being has been confirmed (Salhanick, Noall, Zarrow and Samuels, 1952; Pearlman and Cerceo, 1952)
and a high output of progesterone demonstrated. Zander and von ]\Iiinstermann
(1956) and Pearlman (1957) independently reported the production of approximately
250 mg. progesterone into the peripheral
circulation every 24 hours. This and other
evidence tends to prove that the placenta
is the major source of progesterone in the
human species. However, with respect to
other species, progesterone has been found
only in the placenta of the mare (Short,
1957) although in amounts much less than
in the human being. Placentas of the cow,
ewe, sow, or bitch were all negative. Although the placenta of the mare contains
progesterone and castration does not lead
to abortion after day 200 of gestation, no
progesterone was found in the peripheral
blood or uterine vein blood. The ewe offers
an even more intriguing problem inasmuch
as (1) a discrepancy exists between the
biologic and chemical values for progesterone in the peripheral blood, (2) the placentas contain no progesterone, and (3) no
{progesterone is found in the uterine vein
blood (Edgar, 1953). This has led to the
conclusion that the maintenance of pregnancy in the ewe may be dependent on an
extra-ovarian, extraplacental source of progesterone.


If such a conclusion is correct, and it
must be added that the evidence is still
tenuous, then the adrenal cortex must be
considered as a possible source. Beall and
Reichstein isolated a small amount of progesterone from the adrenal cortex in 1938
and Heehter, Zaffaroni, Jacobson, Levy,
Jeanloz, Schenker and Pincus (1951) demonstrated from perfusion experiments that
progesterone is an important intermediate
metabolite in the synthesis of the adrenal
corticoids. In addition, it has long been
known that desoxycorticosterone possesses
progesterone-like activity (Courrier, 1940)
which is due to a conversion of the desoxycorticosterone molecule to a gestagen. This
has been shown by experiments in vivo in
the monkey (Zarrow, Hisaw and Bryans,
1950), rat, and rabbit (Lazo-Wasem and
Zarrow, 1955), and by an incubation experiment with rat tissue (Lazo-Wasem and
Zarrow, 1955). In addition, Zarrow and
Lazo-Wasem reported the release of a gestagen from the adrenal cortex of the rat
and rabbit following treatment with
ACTH. The substance was obtained from
the peripheral blood and measured by the Hooker-Forbes test, but it was not identified chemically. This was followed by the
finding that pregnanediol is present in the
urine of ovariectomized women, but not
ovariectomized, adrenalectomized women
(Klopper, Strong and Cook, 1957), and by
the finding that progesterone is present in
the adrenal venous blood of the cow, sow,
and ewe (Balfour, Comline and Short,
1957). In all instances the concentration
of progesterone in the adrenal venous blood
was 10 to 100 times greater than the concentration in the arterial blood. Thus the
total evidence that the adrenal cortex can
secrete progesterone is more than adequate.
The question remains as to whether the
adrenal cortex contributes to the progesterone pool of the body during pregnancy
and whether a species difference exists here.


■?6
===D. Relaxin===
 
 
nfl^
 
 
iTrHi
 
 
-f-i ^
 
 
Z2
18
 
 
W"


/•' o °
The initial discovery by Hisaw (1926,
o'^i o
1929) of the presence of an active substance
in the blood and ovaries responsible for
relaxation of the pubic symphysis of the
guinea pig has led in recent years to a consideration of this substance as a hormone
of pregnancy (Hisaw and Zarrow, 1951).
Some doubt as to the existence of relaxin
was raised in the 1930's by investigators
who were able to show that pubic relaxation in the guinea pig could be obtained
with estrogen alone or estrogen and progesterone (de Fremery, Kober and Tausk,
1931; Courrier, 1931; Tapfer and Haslhofer, 1935; Dessau, 1935; Haterius and
Fugo, 1939). This matter was resolved by
the demonstration that pubic relaxation in
the guinea pig following treatment with the
steroids or relaxin differed in ( 1 ) time required for relaxation to occur, (2) histologic changes in the pubic ligament, and (3)
treatment with estrogen and progesterone
which induced the formation of relaxin
(Zarrow, 1948; Talmage, 1947a, 1947b).
Subsequent discoveries of additional biologic activities possessed by relaxin and further purification of the hormone has led to
the conclusion that relaxin is an active substance in the body, and that it plays a significant role during parturition. The hormone has been found in the blood or other
tissues of the dog, cat, rabbit, sheep, cow,
rat, and man. The specific action of this hormone varies with the species involved.
Still unsolved is the question as to whether
the water-soluble extract obtained from the
ovary and referred to as relaxin is a single
substance or a group of active substances
(Friedcn and Hisaw, 1933; Sher and Martin, 1956).


The concentration of relaxin in the blood
increases as pregnancy progresses until a
plateau is reached. This has been demonstrated in the rabbit (Marder and Money,
1944), guinea pig (Zarrow, 1947), cow
(Wada and Yuhara, 1955), and human
being (Zarrow, Holmstrom and Salhanick,
1955). Relaxin has also been found to increase in the ovary of the sow (Hisaw and
Zarrow, 1949). In general, the shape of the
curve for the concentration of relaxin in
the blood as a function of the length of
pregnancy has been more or less the same
for all species studied. Figure 16.12 indicates that the concentration of relaxin in
the blood of the pregnant rabbit rises from
a level of 0.2 guinea pig unit (G.P.U.) per
ml. for the first trimester of pregnancy, i.e..
until day 12, to a level of 10 G.P.U. per ml.






^wA^
on day 24. This concentration was then
maintained until parturition. After delivery
of the young, the concentration of the hormone decreased 80 per cent in 6 hours. On
the 3rd day postpartum no hormone could
be detected.


As indicated above, the concentration of
relaxin in the blood of the pregnant cow
and human being showed approximately
the same type of curve. In the cow the concentration rose gradually from a level of 1
G.P.U. per ml. to a maximum of approximately 4 G.P.U. at 6 months (Fig. 16.13).
Thereafter the level remained unchanged
until parturition, wdien the level dropped at
a rate comparable to that seen in the rabbit. The curve for the concentration of relaxin in the blood serum of the pregnant
woman followed the general pattern described above (Fig. 16.14). The concentration rose from a level of 0.2 G.P.U. per ml.
the 6th week of i:)regnancy to a maximum
of 2 G.P.U. the 36th week. Thereafter the
level remained unchanged until delivery.
Again the postpartum fall was precipitous
and the hormone was not detectable at 24


-14


I'M




o




GO




%
Fig. 16.12. Concentration of relaxin in the blood of the rabbit during pregnancy. Parturition (P) occurred 32 days after mating. Guinea pig units (G.P.U.) of relaxin are plotted
against days pregnant. (From S. N. Marder and W. L. Money, Endocrinology, 34, 115,
1944.)




2 °o


'- o o o


Fig. 16.13. Concentration of I'elaxin in the blood of the cow during pregnancy. Partiuition is indicated by P. (From H. Wada and M. Yuhara, Jap. J. Zootech. Sc, 26, 12, 1955.)


% °








1 c
Fig. 16.14. The concentration of relaxin in the blood serum of normal pregnant women.
(From M. X. Zarrow, E. G. Holmstrom and H. A. Salhanick, Endocrinology. 15, 22. 1955.)






L' 16 ^0 Z4 28 32 36 40 1
hours postpartum. Studies in the guinea pig
revealed a marked rise in relaxin on day
21 of gestation to a maximal concentration
of 0.5 G.P.U. per ml. serum on day 28  
(Zarrow, 1948). Thereafter the level remained unchanged for approximately 4
weeks. Contrary to the results obtained in
the rabbit, cow, and human being a drop in
the concentration of the hormone in the
pregnant guinea pig was noted before parturition. The concentration of relaxin fell to 0.33 G.P.U. per ml. on the 63rd day of
gestation and then dropped to nondetectable levels within 48 hours postpartum.


Although no studies have been carried
out on the blood levels of relaxin in the sow
as a function of the length of pregnancy,
analysis of the ovary for relaxin has revealed a situation comparable to that reported for the blood in other species. The
concentration rose from 5 G.P.U. per gm.
ovarian tissue during the luteal phase of the cycle to approximately 10,000 G.P.U.
per gm. fresh ovarian tissue by the time a
fetal length of 5 inches had been reached
(Hisaw and Zarrow, 1949).




90H
===E. Sources of Relaxin===
 
70
 
50|
 
30
 
3 4 5
 
 
 
Weeks of Pregnancy


The ovaries, placentas, and uteri are
possible sources of relaxin in different species. It seems from the extremely high concentration in the ovary of the sow during
pregnancy that this organ is the major site
of relaxin synthesis at this time. However,
studies on other species indicate that both
the placenta and uterus may be involved.


Treatment of castrated, ovariectomized
rabbits with estradiol and progesterone
stimulated the appearance of relaxin in the
blood of the rabbit as indicated by the
ability of the blood to induce relaxation of
the pubic symphysis of estrogen-primed
guinea pigs (Hisaw, Zarrow, Money, Talmage and Abramovitz, 1944). Similar experiments on castrated, hysterectomized
rabbits failed to reveal the presence of the
hormone in the blood of the treated animals. Treatment with estradiol alone also
failed to stimulate the release of relaxin.
It is obvious then that, if the bioassay is
specific for relaxin, the uterus is a definite
source of this hormone. Comparable results
were also obtained in the guinea pig (Zarrow, 1948). Treatment with estradiol and
progesterone caused pubic relaxation and
the presence of relaxin in the l)lood after
approximately 3 days of treatment with
progesterone. In the absence of the uterus
relaxin was not demonstrable in the blood.


Days after Delivery
The concentration of relaxin in the blood
of the rabbit castrated the 14th day of i^regnancy and maintained with progesterone
remained unaffected by removal of the
ovaries, provided the pregnancy was maintained (Zarrow and Rosenberg, 1953). Figure 16.15 shows a typical curve for the
relaxin content of the blood of such an
animal. The concentration of the hormone
rose between days 12 and 24 to a maximal
concentration of 10 G.P.U. per ml. and was
maintained till the time of normal parturition. It is of interest that in those instances
in which the placentas were not maintained
in good condition, the concentration of the
hormone fell. Analysis of the reproductive tract revealed concentrations of 5 G.P.U.
per gm. fresh ovarian tissue during pseudopregnancy and approximately 25 G.P.U.
during the last trimester of gestation. The
uterus contained 50 G.P.U. per gm. fresh
tissue during pseudopregnancy and an
equal concentration the first 24 days of
pregnancy. The 26th day of pregnancy the
concentration fell to 15 G.P.U. per gm.
The highest concentration was in the placenta which contained from 200 to 350
G.P.U. per gm. Some evidence indicated
that after treatment with estradiol minimal
amounts of relaxin, i.e., 5 G.P.U. per gm.,
were present in the vaginal tissue (Table
16.5).


===F. Adrenal Cortex===


====1. Hydrocortisone====


Fig. 16.5. Concentration of gestagen in the blood plasma and pregnanediol in the  
Initial studies on the possible role of the
uterine of the pregnant human female. Gestagen levels were determined by the HookerForbes test. (From K. Fujii, K. Hoshino, I. Aoki and J. Yao, Bull. Tokyo Med. & Dent.  
adrenal cortex in gestation involved the
Univ., 3, 225, 1956.)  
determination of the two urinary metabolites of the gland, i.e., the 17-ketosteroids
and the corticoids. Inasmuch as the 17-ketosteroids are believed to be associated with
the androgenic activity of the adrenal cortex,
bioassays for adrenogenic activity in the  
urine were carried out. Dingemanse, Borchart and Laqueur (1937) found no increase
in urinary androgen by the 6th to the 8th
month of pregnancy whereas Hain (1939)
reported that pregnant women secreted even
less androgen than nonjircgnant women.  
Pincus and Pearlman (1943) found no
change in the urinary 17-ketosteroids of
the pregnant and nonpregnant woman although Dobriner (1943), by the use of
chromatograi)hic separation, showed a
marked decrease in androsterone. Venning
(1946) found no change in the urinary
ketosteroids as measured by the antimony
trichloride reagent described by Pincus
(1943), but the ketosteroids measured by
the Zimmerman reagent (dinitrobenzene)
showed a significant rise in the latter part
of pregnancy. The discrepancy between the
two determinations can be explained by the
fact that other ketonic substances besides
17-ketosteroids give a color in the Zimmerman reaction. These are the 20-ketosteroids
and to a limited extent the 3-ketosteroids.  
V^enning (1946) believes most of this in






as with the other species and the values
expressed as activity equivalent to progesterone. The values for the bound action
were consistently low and, in general, less
than 1 fjig. per ml. plasma (Fig. 16.6). The
concentration of the free hormone showed
marked variations on the first day or so of
pregnancy. Actually a variation from 1
fig. per ml. plasma to 8 fig. per ml. plasma
was seen on day 0. This type of fluctuation
has also been seen in the rabbit and is without explanation at the present time. However, such marked variations disappeared
by the 4th day of pregnancy and the results
became much more consistent. The average


Fig. 16.15. Concentration of relaxin in the blood of a pregnant rabbit castrated the 14th
day of gestation and maintained with 4 nig. progesterone daily until the 32nd day. Postmortem examination revealed 8 placentas and 2 dead fetuses. (From M. X. Zarrow and B.
Rosenberg, Endocrinology, 53, 593, 1953.)




curve for the concentration of gestagen in
the blood of the pregnant mouse showed
two peaks, one the 7th to the 9th day and a
second the 15th day. The concentration
increased from 2 fxg. per ml. plasma the 4th
day of gestation to an average of approximately 8 fig. the 7th day. This level was
maintained until day 9 and fell thereafter
with a second peak occurring on day 15 and
an immediate drop on day 16. Thereafter
the levels remained low throughout the remainder of pregnancy.


Although it may be assumed that the
TABLE 16.5
initial peak in the concentration of the
gestagen is due to an increased activity


Relaxin content of the blood serum and tissue of the reproductive tract of the rabbit


(From M. X. Zarrow and B. Rosenberg, Endocrinology, 53, 593, 1953.)


968






SPERM, OVA, AND PREGNANCY


No. of Rabbits




E 6
Relaxin Concentration in G.P.U.


M


o
Treatment


a- 5


u
Per ml. serum
u


UJ


a. 3
Per gm fresh tissue
2
I








lii i fV^» 15 « ? i f
Ovary




Uterus


T


12 3 4 5 6
Placenta
whole


Day* afl«r finding vaginal plug


Fig. 16.6. Concentration of free and bound gestagen in the plasma of the pregnant
Placenta
mouse. Gestagen levels were determined by the Hooker-Forbes test. (From T. R. Forbes
fetal
and C. W. Hooker, Endocrinology, 61, 281, 1957.)




Placenta
maternal


15


Pseudopregnant


Chorionic gonadotrophin . . .


17


3


4
3
2
2


Ttrm
2


1




on the part of the corpora lutea, an explanation of the second peak and the drop
0.2-0.3
between the two peaks offers more difficulty. The latter may reflect a diminished
0.2
luteal activity. This could be assumed on
1.0
the grounds that the corpus luteum is the
10.0
only source of gestagen during this period
10.0
of gestation and that the luteal cells show
10.0
cytologic signs of regressive changes, although the drop in serum progestogen antidates the cytologic changes by several
10.0
days. An explanation for the second peak
would probably involve increased secretory
activity !)y the placenta. Progestational
activity has l)een found in i:)lacental extracts and progesterone has been isolated
from the placentae of human beings and
mares (Salhanick, Noall, Zarrow^ and
Samuels, 1952; Pearlman and Cerceo,
1952; Zander, 1954; Short, 1956). Thus, the
drop in serum gestagen seen on day 10  
could be due to loss in the activity of the
corpora lutea and the second rise as a contribution from the placentas. It is of interest
that the low levels on days 10 to 13 and between day 16 to term appear to have no
counterpart in other species. The physiologic
significance of this is still unknown and will
require further work on additional species and on the mouse before an explanation is forthcoming. It is of interest that the
concentration of gestagen in the blood dur




ing the first 12 days of pregnancy corresponds with the intensity of the response to
5
progesterone exhibited by the endometrium
5
during the same period (Atkinson and
30
Hooker, 1945). This w^ould suggest that
25
the serum gestagen levels reflect the physiologic state of the animal.
20
25
25


Serum gestagen levels in the rabbit reveal a curve of increasing concentration
throughout pregnancy (Zarrow and Neher,
1955). Initial values of 0.3 to 1 yug. per
ml. serum were noted at the time of mating,
with a sharp rise beginning on the 4th day
of gestation. The concentration rose to a
level of 6 to 8 /xg. per ml. by the 12th day
and thereafter showed only a slight rise to
a maximal concentration of 8 to 10 /xg. per
ml. serum at parturition (Fig. 16.7). No
drop in serum hormone level was observable at parturition or 1 hour later. The
first significant drop occurred at 6 to 12
hours postpartum when the gestagen level
had decreased 50 per cent. It is of interest
that the serum progestagen levels did not
fall until after the conceptus had been expelled.


castrated the 12th,
50
of gestation aborted
50
following removal of
50
the ovaries (Zarrow and Neher, 1955). In
50
all instances the serum gestagen levels fell
30
before the abortion. Figure 16.8 shows the
15




75
50
50


Pregnant rabbits
75
19th, or 24th day
within 1 to 3 days
 




GESTATION
10
20
25






969


Pregnant 24 davs




± 5
250




3
Pregnant 25 days


Pregnant 26 days


NORMAL PREGNANCY
RABBIT 25 o
RABBIT 26 •
RABBIT 31 e
- RABBIT 27 x
RABBIT 30 ^


350
200




Pregnant 28 days




Line 3,232: Line 3,453:




crease in ketosteroid excretion during pregnancy is the result of increased output of
the stereoisomers of pregnanolone:


1
Measurement of urinary glucocorticoids
by the glycogen deposition test showed an
initial increase in the first trimester of
pregnancy in the human being. After the
initial rise, the urinary excretion level returned to normal with a second increase the
140th to 160th day of pregnancy. Values
of 200 to 300 /xg. equivalent of 17,hydroxy11-dehydrocorticosterone per 24 hours of
urine were obtained at days 200 to 240. In
most instances the urinary outj^ut fell several weeks before parturition.
' Analysis of the blood levels for 17ahydroxycorticosterone in the jiregnant wo


]


man confirmed the results obtained with
the urine (Gemzell, 1953; Seeman, Varangot, Guiguet and Cedard, 1955). Gemzell
( 1953) reported a rise from approximately
5 /xg. per 100 ml. plasma to an average of
approximately 22 fxg. per cent (Fig. 16.16).
A further rise to 36 /xg. per cent was noted
at the time of labor. This has been confirmed by McKay, Assali and Henley
(1957) who found an average rise of approximately 40 /xg. per cent during labor
lasting more than 6 hours. Although McKay, Assali and Henley reported values
still well above normal on the 4th to 6th




Line 3,242: Line 3,483:




 
Fig. 16.16. Correlation between the concentration of 17-hydroxycorticosteroids in the
 
blood of pregnant women and the duration of pregnancy (in weeks). Conception at zero
i:^ A •«
time. (From C. A. Gemzell, J. Clin. Endocrinol.,13, 898, 1953.)
 
 
fc
 




Line 3,254: Line 3,491:




The mechanism whereby labor induces
a marked stimulation of the adrenal cortex
is still obscure. It is possible that labor is
a stressful state and the stress induced by
both the pain and the muscular work act to
stimulate the increased release of ACTH
resulting in increased adrenocortical activity. Some confirmation of this may be
obtained from the fact that significant in(■i'eas(> in }ilasma 17a-hydroxycorticoids is
noted only if the labor lasts more than 6
hours.


Analysis of the rise in plasma levels of
hydrocortisone during pregnancy has suggested that the phenomenon is not simply
the result of an increased rate of secretion
from the adrenal cortex, but rather the result of an increased retention and an alteration in the metabolism of the hormone
< Cohen, Stiefel, Redely and Laidlaw, 1958).


====2. Aldosterone====


The isolation for aldosterone by Simi)son,
Tait, Wettstein, Neher, von Euw, Schindler
and Reichstein (1954) and its identification
as the hormone regulating fluid and mineral
metal)olism stimulated marked interest in
the role of this hormone. Among the items
of interest was its significance in pregnancy
and in the toxemia of pregnancy. Early
studies by Chart, Shipley and Gordon
( 1951 1 revealed the presence of a sodium
retention factor in the urine that increased
from a normal pregnancy value of 36 to 106
fxg. equivalent of desoxycorticosterone acetate (DOCA) per 24 hours to a maximum of
1008 |U.g. equivalent in pregnancy toxemia.
These results were confirmed by Venning,
Simpson and Singer (1954) and by Gordon,
Chart, Hagedorn and Shipley (1954). In
addition a slight increase in the sodium retaining factor was observed in gravid women as compared to nongravid women.


The discovery that the greater part of
the aldosterone in urine is present in the
conjugated fraction led to a repetition of
the above work using both acid hydrolysis
and incubation with /3-glucuronidase (Venning and Dyrenfurth, 1956; Venning, Primrose, Caligaris and Dyrenfurth, 1957). The
results show little change in the excretion
of free aldosterone throughout pregnancy,
but the glucuronidase and acid-liydrolyzed






fractions increased markedly (Fig. 16.17).
The urinary excretion values increased
from a prepregnancy normal of 1 to 6 /xg.
aldosterone (average for women was 3.8 ±
14 fig. per 24 hours; Venning, Dyrenfurth
and Giroud, 1956) to approximately 25 /xg.
per 24 hours. The first significant rise occurred about the third month of gestation
and an increased concentration was obtained until after parturition, when there
was a rapid fall to the nonpregnant values.




===G. Thyroid Gland===


Clinical data have long indicated a possible involvement of the thyroid gland in
gestation (Salter, 1940). In regions where
the iodine supply is low this is demonstrated by an enlargement of the thyroid
during pregnancy. The formation of a
goiter has been interpreted as evidence for
an increased need for iodine during gestation. Scheringer (1930) and Bokelmann and
Scheringer ( 1930) reported a rise in the
iodine content of the blood of pregnant
women during the first trimester of pregnancy with a peak at the seventh month.
The increased concentration is maintained
until shortly after parurition. In the goat,
however, Leitch (1927) reported no change
in serum iodine during gestation until just
before parturition. Analysis of umbilical
vein blood revealed values that were normal, i.e., lower than in the mother (Leipert,
1934). Increased thyroid secretion (Scheringer, 1931 ) and increased urinary excretion
of iodine have been reported in pregnant
women (Nakamura, 1932; 1933). However,
Salter (1940) concluded in his review that
no reliable evidence of increased thyroid
hormone levels in the blood during jiregnancy is available.


Peters, Man and Heinemann (1948) reported a range of 4 to 8 fig. per cent of
serum-precipitable iodine in the normal,
nonpregnant woman with a rise to 8.3 fig.
per cent (range 6 to 11.2 fig. per cent) in the
pregnant woman (Fig. 16.18). It is of interest that the elevation in the proteinbound iodine (PBI) does not follow the
course of changes in the basal metabolic
rate. Whereas the former is already high by
the second month of pregnancy the basal
metabolic rate rises gradually after approximately the 4th month of pregnancy (Rowe
and Boyd, 1932; Javert, 1940). No other
sym})toms of hyperthyroidism are seen in
pregnancy which leads to the question of
the significance of the rise in protein-bound
iodine. A somewhat comparable paradox
exists in the guinea pig in which a rise in
the rate of oxygen consumption during
pregnancy is not accompanied by an increase in heart rate (Hoar and Young,
1957).


Recently, AVerner (1958) rcj^orted a decrease in the I^-^^ up-take after treatment
with triiodothyronine in both the normal
and pregnant woman. From this and other
data he ruled out any abnormal pituitarythyroid relationship or marked secretion of
thyroid-stimulating hormone (TSH) by the
placenta and concluded that the increased
PBI in pregnancy is due to an increased
binding capacity of the serum protein.


• A
Feldman ( 1958) failed to find any increase
in the level of serum-hutanol-extracted iodine throughout pregnancy in the rat. Actually the values were consistently lower
than in the controls and similarly the total
amount of PBI in the thyroid of the pregnant rat was consistently lower. He did find
an increase in the rate of excretion of V-''\
a diminished up-take of I^^^ by the thyroid,
and a decreased half-life for thyroxine in the
pregnant rats. It is obvious that these results
are quite dissimilar from those obtained in
the pregnant women. One can only conclude
at this time that pregnancy has an effect on
iodine metabolism and a species difference
exists.


===H. Growth Hormone===


•xoe
Although it has been possible to demonstrate the presence of growth-promoting substance (STH) in the blood plasma, there are
few data bearing on the identity of the substance and few ciuantitative measurements.
Westman and Jacobsohn ( 1944) first showed
the ]irescnce of a growth-]5romoting sub




• 00






 
Fig. 16.17. Urinary excretion of aldosterone throughout pregnancy in the human being.
 
O, free fraction only; •, free and acid-hydrolyzed fraction; O, free, enzyme and acidhA'drolyzed. (From E. H. Venning and I. Dyrenfurth. J. Clin. Endocrinol,, 16, 426, 1956.)
0»
 
 
 
 
X ex






stance in the blood by cross transfusion between a normal and hypophysectomized rat
united in parabiosis. Gemzell, Heijkenskjold
and Strom (1955), using the technique of
adding exogenous growth hormone to the
sample of blood, failed to find any growthjiromoting substance in 23-ml. equivalents
of blood. However, retroplacental blood from
human beings gave a positive response at a
level of 7- to 15-ml. equivalents of plasma
without the addition of exogenous STH. Increase in the width of the proximal tibial
epiphysis of the rat was used as an end
l)oint. A comparable concentration of 650
fxg. eciuivalent of the standard STH per 100
ml. plasma was also found in the blood from
the umbilical cord.


Contopoulos and Simpson (1956, 1957)
measured the STH of the plasma in the
pregnant rat, using the tibial cartilage, tail
length, and body weight increase. No significant increase in plasma STH was noted
on the 5th day of pregnancy, however, a significant rise was observed by the 9th day.
An estimated 3-fold increase in plasma STH
during pregnancy was reported from calculations on both the tibial cartilage and the
tail length tests. No changes were reported
in the STH activity of the pituitary gland
throughout pregnancy. Recently, the persistence of greater than normal amounts of
growth-promoting activity was reported in
the plasma of pregnant rats after hypophysectomy. Since the fetal pituitary probably
does not contribute to the STH pool of the
mother, at least in early pregnancy, it is
likely that the placenta may be a source of
the hormone.


===I. Prolactin===


Few data are available on the concentration of prolactin during gestation. This has
been due, in part, to the minute amounts of
the hormone present in the urine and blood
and to the inadequacy of the available assays. Although Hoffmann ( 1936 ) failed to
find any prolactin in the urine of women
before parturition, Coppedge and Segaloff
(1951) and Fujii and Schimizu (1958) reported measurable amounts of prolactin in
the urine of pregnant women. Coppedge and
Segaloff reported a gradual rise in the excretion of prolactin throughout pregnancy and
a gradual decline following parturition even
though lactation was maintained. The number of observations, however, was limited
and the authors point out that the results
were ecjuivocal. Fujii and Shimizu observed
an initial drop in the prolactin output during the first month of pregnancy followed by
a rise to approximately 32 P.C.U. (one pigeon crop sac unit is equivalent to 0.3 I.U.)
per 24 hours during the second trimester of
pregnancy in women. (Fig. 16.19). This was
followed by a drop to approximately 10
P.C.U. per^24 hours between the 30th and
38th wrecks of pregnancy and a marked rise
to 64 P.C.U. per 24 hours during the lactation period.




•


FiG. 16.18. The level of protein-bound iodine in the pregnant woman. (From J. P.
Peters, E. B. Man and M. Heinemann, in The Normal and Pathologic Physiology oj
Pregnancy, The Williams & Wilkins Co., 1948.)








Fig. 16.19. Urinary excretion of prolactin throughout gestation in the human being.
One pigeon crop unit (P.C.U.) is equivalent to 0.3 I.U. (From K. Fujii and A. Shimizu,
Bull. Tokyo Med. & Dental Univ., 5, 33, 1958.)






===J. Placental Gonadotrophins===


Placental gonadotrophins have been found
in the monkey, chimpanzee, human being,
mare, and rat (Hisaw and Astwood, 1942).
The physiologic activities of these placental
hormones differ among the three groups of
niannnals and appear to represent divergent
evolutionary steps in the adoption of pituitary function by the placenta. The physiologic properties of the placental gonadotrophins differ not only among themselves
but also from the pituitary gonadotrophins.
The gonadotrophin from the rat placenta
(luteotrophin) has been shown to be leuto




trophic with the ability to maintain a functional corpus luteum in the hypophysectomized rat (Astwood and Greep, 1938). The
hormone has no effect on follicular growth
or ovulation. Its function appears to be that
of maintaining the secretory activity of the
corjius luteum in the rat from the 10th day
of pregnancy to term.


The placental hormones of the human being (HCG) and the mare (PMS) have been
studied in much greater detail. These two
hormones differ markedly in both chemical
and physiologic properties. The presence of
HCG in the urine and the absence of P]\IS in
the urine would alone indicate a marked difference in the size of the two molecules.
Physiologically, PMS is highly active in producing follicular growth and some luteinization, whereas HCG has no effect on follicular growth but will induce ovulation and a
delay in the onset of menstruation. This
would indicate a luteotrophic action. Although chorionic gonadotrophin has been reported in the macaque (Hamlett, 1937) between the 18th and 25th day of pregnancy,
and in the chimpanzee from the 25th to the
130th day of gestation (Zuckerman, 1935;
Schultz and Snyder, 1935), little work has
been done on the characterization and identification of these substances except in man
and horse.








©/iO
Fig. 16.20. The relative time of appearance of placental gonadotropliins in the pregnant
mare and the woman. (From E. T. Engle, in Sex and Internal Secretions, 2nd ed., The
Williams & Wilkins Company, Baltimore, 1939.)




It is of some interest to note that the appearance of the placental gonadotrophins in
the blood and urine of horse and man occurs
at approximately the same relative time in
pregnancy (Fig. 16.20). The role played by
these hormones in gestation is still not clear,
but it is significant that their appearance
corresponds with the time of implantation
of the blastocyst and their disappearance
roughly with the time when ovariectomy no
longer interferes with the maintenance of the
pregnancy.


===K. Human Chorionic Gonndotrophin (HCG)===


The discovery of the presence of a gonadotrophic hormone in human pregnancy urine
by Aschheim and Zondek (1927j was soon
followed by a description of its biologic activity and quantitative determinations of
its concentration in the urine throughout
pregnancy (Ascheim and Zondek, 1928).
Recently a number of investigators have determined the titer of chorionic gonadotrophin in the serum of pregnant women. These
curves agree very well with the values obtained from the urine. Figure 16.21 is a typical curve for the concentration of chorionic
gonadotrophin in the blood of pregnant
women (Haskins and Sherman, 1952). A
peak value of 120 I.U. per ml. of serum was
obtained on the 62nd day after the last
menses and a rapid decline was noted to a
low of approximately 10 I.U. per ml. of serum on day 154. A subsequent rise to 20
I.U. was noted by day 200 and this was
maintained until the end of pregnancy.
These results are in excellent argeement with
those reported by Wilson, Albert and Randall (1949) using the ovarian hyperemia
test in the immature rat. These authors obtained a peak concentration of approximately 70 I.U. per ml. of serum on the 55th
day after the last menses. A gradual decrease
occurred thereafter to a low of approximately 20 I.U. per ml. of serum which remained unchanged from day 100 to parturition although the data indicate a slight
rise towards the end of pregnancy.








1






 
Fig. 16.21. Concentration of human chorionic gonadotiophin in the blood of the normal
»XA
pregnant woman. The hormone levels were determineti b>' the male frog test. (From A. L.
 
Haskins and A. I. Sherman. J. CUn. Endocrinol., 12, 385, 1952.)
 
 
 
 
 
 
 
• •
• •






The significance of the excretion pattern and concentration of the hormone in the
serum is still a matter of conjecture. Browne,
Henry and Venning (1938) suggested that
the peak level of chorionic gonadotrophin in
the blood reflects an increased production
and a physiologic need in order to maintain
a functional corpus luteum during early
pregnancy. Recent evidence has tended to
confirm this opinion in that HCG has been
found to be active in the maintenance of the
secretory activity of the corjius luteum in
the primate (Hisaw, 1944; Brown and Bradbury, 1947; Bryans, 1951 j. In addition, histologic studies reveal a direct proportion between the number of Langhans' cells and the
amount of hormone excreted (Stewart, Sano
and Montgomery, 1948; Wislocki, Dempsey
and Fawcett, 1948) .


The possibility that the kidney plays a role in the changes in the concentration of
HCG was investigated by Gastineau, Albert
and Randall (1948) . The renal clearance was
relatively constant throughout all stages of
pregnancy although the urine and serum
concentrations of the hormone varied as
much as 20- fold. In addition, the mean, renal
clearance found during pregnancy was not
markedly different from that found in cases
of hydatiform mole and testicular chorioma.
Inasmuch as the renal elimination of the
hormone remained constant, it was obvious
that two possible explanations existed : these
were (1) changes in the secretion rate, and
(2) changes in extrarenal disposal of the
hormone. Studies on the latter were contradictory. Whereas Friedman and Weinstein (1937) and Bradbury and Brown
(1949) reported an excretion of 20 per cent and higher of HCG following the injection
of HCG, Johnson, Albert and Wilson (1950)
found an excretion of 5.8 per cent in pregnant women during the immediate postpartum period. Zondek and Sulman (1945)
reported a 5 to 10 i)er cent elimination of
HCG in the urine of animals. Thus Bradbury and Brown felt that there is relatively
little destruction or utilization of the hormone in the body; Wilson, Albert and
Randall ( 1949) believed that 94 per cent of
the circulating hormone is affected by extrarenal factors and that the fluctuating character of hormonal level in serum or urine
depends entirely on changes in rate of hormone production.


24
An analysis of the distribution of chorionic
gonadotrophin in the mother and fetus led
Bruner (1951) to conclude that the ratio of
maternal blood to urinary gonadotrophin is
not constant although the ratio of gonadotrophin in the chorion to maternal blood is
constant. Consequently, she concluded that
the concentration of gonadotrophin in the
urine does not depend entirely on the rate of
production of the hormone and that the method of gonadotrophin elimination
changes during pregnancy. She also pointed
out that a significant amount of chorionic
gonadotrophin is found in the fetus and that
this is due to the fact that, although the
chorion releases the hormone into the maternal blood, secondarily some of it passes the
placental barrier and enters the fetal system across the wall of the chorionic vesicle.


====2. Equine Goyiadotrophin (PAIS)====


The presence of a gonadotrophin in the
blood of the pregnant mare was first described by Cole and Hart in 1930. The hormone appears in the blood about the 40th
day of pregnancy and increases rapidly to a
concentration of 50 to 100 rat units (R.U.)
per ml. by the 60th day of pregnancy (Cole
and Saunders, 1935). This concentration is
maintained for approximately 40 to 65 days.
By day 170 it has fallen to a nondetectable
level (Fig. 16.22).


28
Catchpole and Lyons (1934) suggested
that the placenta is the source of the gonadotrophin and indicated that the chorionic
epithelium is the probable source. Cole and






4 8 12 16 20


TIME IN DAYS
Fig. 16.7. Concentration of gestagen in the blood of the normal pregnant rabbit as determined by the Hooker-Forbes test. (From M. X. Zarrow and G. M. Neher, Endocrinology,
56, 1, 1955.)


Fk;. 16.22. Tlie concentration of i)rognant mare's serum in the blood of the mare throug
out pregnancy. (From H. H. Cole and F. J. Saunders, Endocrinology. 19, 199, 1935.)






Goss (1943), on the other hand, concluded
that the endometrial cups are the source of
the hormone. Recent evidence tends to confirm the endometrial cups as the source of
the hormone (Clegg, Boda and Cole, 1954).
The endometrial cups form in the endometrium opposite the chorion in the area
where the allantoic blood vessels fan out.
The cups develop precisely at the time when
the hormone is first obtained in the serum
of the pregnant mares and desquamation of
the enclometrial cups is complete at the time
of the disappearance of the hormone from
the maternal blood. Analyses of the cups for
gonadotrophin content reveal a correlation
between the concentration of the hormone in
the maternal blood and the concentration in
the endometrial cups. Finally, histochemical
stains for glycoprotein indicate the presence
of this substance only in the epithelial cells
lining the uterine lumen and the uterine
glands in the cup area ( for complete discussion of the subject see the chapter by Wislocki and Padykulal.




==VI. Pregnancy Tests==


INTERRUPTED PREGNANCY
The discovery of gonadotrophic activity
in the urine of pregnant women by Aschheim and Zondek in 1927 led to introduction
of the first valid test for pregnancy (Aschheim and Zondek, 1928). These investigators
used the innnature mouse and reported the
presence of corpora hemorrhagica as indicative of the presence of a gonadotrophin in
the urine and a positive reaction for pregnancy. The Aschheim-Zondek test for pregnancy was the first successful test of its kind
and has been used both as a qualitative and
quantitative test. In the latter instance, a
serial dilution of the urine is made in order
to obtain the minimal effective dose.


It is not too surprising that many tests for pregnancy have been described. In general, all of the successful tests involve the
detection of chorionic gonadotrophin in the
urine, and to some extent in the blood. The
changes that have appeared in the development of new pregnancy tests have been
those concerned with the use of different
species of animals, the rapidity with which
the test could be completed, and convenience to the laboratory. Thus the Friedman
test (Friedman, 1929; Friedman and Lapham, 1931 ) followed soon after the Aschheim-Zondek test and in turn was succeeded by several newer tests.


Ap])roximately five reliable tests are now
available (Table 16.6). All are concerned
with the detection of HCG and have an accuracy of 98 to 100 per cent. The AschheimZondek suffers from a time requirement of
96 hours and was largely supplanted by
the Friedman test that used the isolated
rabbit and required only 48 hours. Within
recent years several new tests have been
reported using the frog, toad, and immature rat. Frank and Berman (1941) first
noted the occurrence of hyperemia in the
ovary of the immature rat, following the
injection of HCG. Albert (1949) reported
excellent results with the use of this test
in 1000 cases. Comparison of the rat hyperemia test with the Friedman test was
on the whole very good and revealed the
same order of accuracy for both tests. The
Friedman test, however, will detect about
5 I.U. of HCG which would mean a concentration of 500 I.U. of HCG per 24-hour output of urine ( assuming a 24-hour urine output of 1500 ml.). Positive results in the rat
test require a 24-hour output of 1000 I.U.,
indicating that the ovarian hyperemia test
in the rat is about one-half as sensitive as
the Friedman test. Nevertheless, the rat




Line 3,368: Line 3,949:




TABLE 16.6
Pregnancy tests with an accuracy




oj 98 to 100 per cent




Animal


RABBIT 31


Sex




Observed End Point




Time


(CASTRATED)


 
Reference
10




Immature mouse
Isolated rabbit
Xenopus laevis
Bufo arenarum
Immature rat




F
F
F
M
F




Corpora hemorrhagica
Corpora hemorrhagica
Extrusion of ova
Extrusion of sperm
Hyperema of ovary




hr.


96


48
8-12
2-4


4




9
Aschheim and Zondek, 1928
Friedman and Lapham, 1931
Shapiro and Zwarenstein, 1934a
Galli-Mainini, 1947
Frank and Berman, 1941








test requires only 4 hours and a larger number of animals can be utilized, thus decreasing the error due to use of inadequate numbers of animals. Comparison of the rat
hyperemia and the Friedman tests revealed
that the former is slightly more accurate
but a little less sensitive (Albert, 1949).


Within two years after the publication of
the Friedman test for pregnancy, Shapiro
and Zwarenstein (1934a, 1934b, 1935) and
Bellerby (1934) reported the use of the
African toad {Xenopus laevis, D) in the
diagnosis of pregnancy. Again the test was
based on the ability of HCG to induce the
extrusion of ova by the frog following the
injection of the urine into the dorsal lymph
sac. Extrusion of the ova occurred in 6 to
15 hours and the test was shown to compare
favorably with both the Aschheim-Zondek
and Friedman tests, although it did not give
tiie graded response seen with the A-Z test
(Crew, 1939). Weisman and Coates (1944)
found an accuracy of 98.9 per cent with the
Xenopus test over a 5-year period during
which 1000 clinical cases were examined.


Galli-Mainini (1947) first reported the
use of the male batrachian in the diagnosis
of pregnancy and Robbins, Parker and
Bianco (1947) simultaneously reported the
release of sperm by Xenopus following
treatments with gonadotrophins. Galli-Mainini (1948» pointed out that this reaction
is not restricted to a single toad, but would
]irobably be found in many frogs and toads.
He added that care should be used to employ animals with a continuous spermatogenesis. This was immediately confirmed by
reports from different countries using various species of frogs and toads endogenous
to the areas. Immediate use of Rana pipiens
was reported in the United States and this
species became very popular in that country
( Wiltberger and :\Iiller, 1948) .


The advantages of the sperm-release test
are the time requirements, simplicity, end
point, and opportunity to use many animals.
On the other hand, the reaction is all or
none and shows no gradation in degree of
reaction. In general, the urine is injected
into the dorsal lymph sac and the cloaca
aspirated for sperm 1 to 3 hours later. Although this is the most recent of the pregnancy tests, many reports have appeared and some evaluation as to accuracy may be
attempted. Galli-IVIainini (1948) reported
an accuracy of 98 to 100 per cent in a summary of more than 3000 tests and 100 per
cent accuracy for negative results in more
than 2000 controls. Robbins (1951 ) reported
an accuracy of 89.5 per cent in the first trimester of pregnancy. Pollak (1950) indicated that as many as 20 per cent of the
negative tests obtained in the summer were
false. This suggested the existence of a
refractory state at this season. Bromberg,
Brzezinski, Rozin and Sulman (1951) reported on a comparison of several tests
including 700 cases. An accuracy of 85 per
cent was obtained with the male frog test,
99 per cent with the rat hyperemia test, 98.5
per cent with the Friedman test and 98 per
cent with the Aschheim-Zondek test. The
authors indicate that the 15 per cent failures
to get a positive reaction in the frog could
be due in part to the poor sensitivity of the
animal which could only be overcome by
concentrating and detoxifying the urine.
Comparison of the minimal amounts of
HCG to elicit a positive reaction are Vs
I.U. for the rat hyperemia test, 1 I.U. for
the Aschheim-Zondek and Hyla tests, 2
I.U. for the Rana and Bufo tests, and 5
I.U. for the Friedman test. Reinhart, Caplan
and Shinowara (1951) reported an accuracy
of 99 per cent with 840 urine specimens;
only 3 false negatives were noted in 346
specimens from known pregnant women
and no false positives noted in 125 nonpregnant women. The authors attribute the
high degree of accuracy to standardization
of the procedure by which extraneous factors were eliminated. These include ( 1 ) the
use of 2 or more 30- to 40-gm. frogs for
each test, (2) elimination of all animals suffering from red leg and other diseases, (3)
adequate time for sperm release, (4) concentration of the urine, (5) maintenance
of frogs at 15 to 22°C., and (6) during the
summer the injection of an increased volume
of urine and an increase to 4 hours in the
period for sperm release. The maintenance
of frogs in a hibernating state by keeping
them in a refrigerator at 38°F. has been rei:)orted to insure a high degree of sensitivity
regardless of the season (Allison, 1954).
Although it is obvious that more data are needed, the present results are very promising for the "frog-sperm" test and if the
seasonal effect can be eliminated, this test
will be the equal of the other four.
VII. Water and Electrolyte Balance
The changes in the various components of
the blood during pregnancy have been described in a number of species (Tables 16.7
and 16.8). It is generally agreed that a marked increase in the blood and plasma
volume and a decrease in the relative
amounts of erythrocytes and hemoglobin
occur during the last trimester. Inasmuch
as the increase in the plasma volume in man
exceeds the concurrent increase in the total
cell volume, the resultant hemodilution produces an anemia which has been described
as the "physiologic'' anemia of jM-egnancy.






TABLE l(i.7
The average percentage of change in the constituents of the blood and in the extracellular fluid volume during


 
normal pregnancy in man
8
 
 
 
 
 
 
 
 






Extracellular
space




7


Adams, 1954.






Bibb. 1941


Caton, Robv, Reid and Gibson, 2nd, "l949


Caton, Roby, Reid, Caswell.
Maletskos, Fluharty and
Gibson, 1951






Chesley, 1943


Dieckmann and Wegner,


1934a, b, c, d


6
Ferguson, 1950






Freis and Kenny, 1948


Friedman, Goodfriend, Berlin


and Goldstein, 1951


Ganguli, 1954






Gemzell, Robbe and Sjostrand, 1954






Hamilton and Higgins, 1949. .


Jarosova and Daum, 1951 ...


Lambiotte-Escoffier, Moore
and Tavlor, Jr.. 1953
Lund, 1951
McLennan and Corey, 1950..


McLennan and Thouin, 1948. .


5
Miller, Keith and Rownetree,
1915


Mukherjee and Mukherjee,
1953


Roscoe and Donaldson, 1946.


Thompson, Hersheimer, Gibson and Evans, Jr., 1938 . . .


Tysoe and Lowenstein, 1950 .


White, 1950






59






25






4
15




V


Blood
volume






49
None






45




|o


23


3




30






59






32






Plasma
volume


2




>
22






49






55






(♦(ABORTION)
25


1


1 j


1
32






Total
hemoglobin




12 16


TIME IN DAYS
20






20
59






Fig.
20
tion in
Zarrow






16.8. The effect of castration on serum progestogen levels and maintenance of gestathe rabbit. Gestagen levels were determined by the Hooker-Forbes test. (From M. X.
Hematocrit
and G. M. Xeher. Endocrinology, 56, 1, 1955.)






changes in serum gestagen levels before
10  
and after castration of a pregnant rabbit.
The concentration increased from a level of
0.3 ing. per ml. at day to 10 /xg. per ml. on
day 24 when the rabbit was castrated. A
60 per cent drop in serum gestagen level is
seen 12 hours after castration with a fur




ther drop at the 36th hour, when the animal
aborted.


Studies on the concentration of serum
15
gestagen in the pregnant ewe (Neher and
 
Zarrow, 1954) permit a comparison with
 
the results obtained in the rabbit. Such a
comparison is extremely valuable in view


10




970


12




SPERM, OVA, AND PREGNANCY


16




12


11
RBC


10
count


_ 9


DO


I ^
14


^ 5


^ A


3
21


2




22


1 ^




Gram %
hemoglobin


Normal pregnancy
Sheep SI °


Sheep S3 •


Sheep S 2 «
25


Sheep S2A x


Additional sheep A


15


25% in
50% of
patients


I
A


!


I
10


I


-A ho
I


I
Time of Determinat
I
in Pregnancy


I


I
I


I
6th week antepartum
I
3rd trimester
I


I
3rd trimester






JL_L
60 days antepar


tum
3rd trimester


3rd trimester


20




3rd trimester


40
At term


Throughout pregnancy


8th week antepartum


60 80
3 months antepartum


Time (days)
9th monlli


10th lunar month
3rd trimester
10th lunar month
At term


3rd trimester


100
3rd trimester
3rd trimester


9th lunar month


At term


120
3rd trimester






140
GESTATION






Fig. 16.9. Concentration of gestagen in the blood of the pregnant ewe. Gestation levels
989
were determined bv the Hooker-Forbes test. (From G. M. Neher and M. X. Zarrow, J.
Endocrinol., 11,323,1954.)






of the fact that castration of the rabbit
TABLE 16.8
invariably leads to abortion whereas castration of the pregnant ewe does not do so
 
if the ovaries are removed during the second half of pregnancy. Again the progesterone determinations were carried out on
 
untreated serum and the samples assayed
by the Hooker-Forbes technique using progesterone as a standard. An initial rise in
the serum gestagen level occurred soon
after mating and seemed to level off at a
concentration of 6 ixg. per ml. approximately the 50th day of gestation (Fig.
16.9). Thereafter, the concentration remained unchanged for approximately 50
days, when a second rise to a level of 8 to
12 fj.g. occurred. These levels remained unchanged until at least 30 minutes after parturition was complete.


Castration at various times after the
The  
66th day of pregnancy failed to influence
the concentration of circulating gestagen
or interfere with the pregnancy. The data
in Figure 16.10 show a normal concentration of 8 to 10 fxg. gestagen from the 114th
day of gestation to parturition although the
animal was ovariectomized the 114th day.
Pregnancy was normal in all castrated




average percentage of cl


ewes and the expected drop in scrum gestagen was observed following parturition.


It can now be stated that the human
aiu/e in  
being, the monkey, the ewe, the rabbit, the
mouse, and probably the guinea pig (Herrick, 1928; Ford, Webster and Young,
1951) have met the problem of a second
source of progesterone supply with varying
degrees of success. In the ewe, placental replacement of the ovary as a source of progesterone can be considered as complete
by approximately the 66th day of pregnancy. Castration at this time will neither
interfere with the pregnancy nor with the
concentration of the hormone in the blood.
In the monkey, castration as early as the
25th day of gestation (Hartman, 1941)
does not interfere with pregnancy and in
the human being castration as early as the
41st day after the last menstrual period
may not interfere with pregnancy (Melinkoff, 1950; Tulsky and Koff, 1957). One
may conclude, therefore, that the placenta
can adequately take on the role of the
ovary in this regard. On the other hand,
aspects of the situation in the human female are still puzzling, especially the blood
gestagen values; but despite this ambiguity




the constituents


GESTATION


of the blood and




971
in the extracellular fluid






10
9


J'
volume during norn


w> 7
E 6


BO -*
al pregnancy in


o


3
variouf
2




laboratory and domestic animals


— p o






Ovariectomy during pregnancy
Increase




Decrease


_ ]


— k-(ovariectonny)




Animals


1




114






125




Total
hemoglobin




Time (days)






Fig. 16.10. The effect of castration on gestagen levels in the pregnant ewe. Gestagen
le\els were obtained by the Hooker-Forbes test. Note that castration failed to interfere
with the pregnancy or the level of gestagen in the blood. (From G. M. Neher and M. X.
Zarrow, J. Endocrinol., 11, 323, 1954.)






it might be concluded that here also the
Author
placenta has successfully replaced the
ovary. In the rabbit, on the other hand,
castration at any time during pregnancy
vvill cause a decrease in the level of the
circulating hormone and terminate the
pregnancy. Hence, in this species, the placenta has failed to replace completely the
ovary. The mouse is another instance in
which castration leads to abortion so that
one can assume a failure on the part of the
placenta to replace the endocrine activity of
the ovary. In this case, however, the second
peak of circulating gestagen has been ascribed to the placenta and this presents the
possibility of a partial replacement of the
ovary by the placenta but a replacement
that is not adequate since pregnancy is
terminated by ovariectomy.


As indicated above, a marked discrepancy exists between the bioassays and the
chemical determinations of gestagens in
the blood and other tissues. The chemical
determinations of progesterone invariably
give results that are far lower than those
obtained by bioassay methods. Edgar and
Ronaldson (1958) found a maximal concentration of approximately 2 /xg. progesterone per ml. ovarian venous blood during






gestation in the ewe. This concentration
Blood
was no higher than that seen in the ewe
during a normal estrous cycle. The maximal level reached during the estrous cycle
was maintained when pregnancy supervened and remained fairly constant until
the last month of pregnancy. Thereafter
the concentration fell and no progesterone
was detectable at 15 days prepartum (Fig.
16.11). Inasmuch as no progesterone was
found in the peripheral blood of the ewe,
this poses again the following question:
What was being measured in the peripheral
blood by the bioassay procedure? In addition, a second question is posed by the
earlier discussion on the need of the ovary
in the maintenance of pregnancy as to the
relative contributions of the ovary and the
placenta to the concentration of this hormone in the body.


That the biologic methods are measuring
more than progesterone is obvious from the
many reports emphasizing the high levels
obtained by bioassay and the low levels
obtained by chemical techniques. In addition to the above data. Short (1957, 1958a,
1958b) reported the presence of progesterone in the peripheral blood of the pregnant
cow but onlv in the order of 0.0074 to


Pasma




972
Cell




Hema


SPERM, OVA, AND PREGNANCY
RBC




Gram %




Line 3,853: Line 4,494:




volume




volume




volume




tocrit




count


No. of observations


hemoglobin




Line 3,870: Line 4,516:




%




%




%




%




%


5 5


%


8




6




9


Rat


11




7




7




7




6




8


40


7


20-40


7


Beard and Mvers, 1933


5




14


41


11




7




4


20


7 7


10
29




18
30
25


I 1 1
• Ewes under


7
31
33


■— 1 — r


2 years
Bond, 1958
Newcomer, 1947
van Donk, Feldman and
Steenbock, 1939




■ 1
Rabbit




-T—
50








T


— T"




— r


— r~
17




T
20




"T"
10-20
17




1
Salvesen, 1919


Zarrow and Zarrow, 1953


1




1


6




12


1 1




Line 3,989: Line 4,635:




12




13




Horger and Zarrow, 1957




Sheep




25




22




10




Line 4,009: Line 4,662:




Initial decrease, normal at term




Barcroft, Kenned}- and Mason, 1939




Cow




Slight




Slight




Line 4,022: Line 4,680:




» Ewes over 2




years




Line 4,034: Line 4,690:




Reynolds, 1953






Comparable changes were observed in the
blood constituents and plasma volumes of
the rat and rabbit during the latter third
of gestation. Although there is no increase
in the total cell volume, the resultant "physiologic" anemia of pregnancy in the rabbit
follows the same general pattern as that
rei)orted in the human being (Horger and
Zarrow, 1957).


A significant decrease in the erythrocyte
number, hemoglobin concentration, and hematocrit, and an increase in the blood volume have been noted in the rat during
pregnancy (Table 16.8). However, the increase in blood volume is correlated with
an increase in body weight and the ratio
of blood volume to body weight remains unchanged (Bond, 1948). Calculation of the
total number of erythrocytes and grams of
hemoglobin actually showed an increase in
these constituents during gestation, indicating that the anemia of pregnancy in the
rat is due to a hemodilution in which
the blood volume increases proportionately
faster than the number of erythrocytes.


Comparable results were also reported in
the rabbit (Zarrow and Zarrow, 1953). A
marked drop in the relative number of
circulating erythrocytes and percentage of
hemoglobin is seen invariably towards the
end of gestation (Fig. 16.23). A marked fall in hematocrit occurs concomitantly with
the fall in the two blood constituents along
with an increase in the reticulocytes. The
time of onset of the increase in reticulocytes
is variable and seems to occur during the
second trimester of gestation. Their number
returns to normal before parturition in spite
of the increasing severity of the anemia. A
second rise in the reticulocytes is seen during the first week postpartum. All the other
constituents return to normal values during
the first or second week postpartum.








Fig. 16.23. Changes in the relative number of
circulating erythrocytes, reticulocytes, percentage of hemoglobin, hematocrit, and nonprotein
nitrogen of the blood of the rabbit during pregnancy and after parturition. (From M. X. Zarrow
and I. G. Zarrow, Endocrinology, 52, 424, 1953.)




Line 4,049: Line 4,745:




Disagreement exists as to whether there
is a change in the volume of the extracellular
fluid compartment during pregnancy in the
human being. Whereas certain investigators
have reported rather marked increases in
the extracellular space (Chesley and Chesley, 1941; Chesley, 1943; Freis and Kenny,
1948; Caton, Roby, Reid and Gibson, 1949;
Friedman, Goodfriend, Berlin and Goldstein, 1951; Jarosova and Damn, 1951),
others have reported that the changes in
this fluid compartment are proportional to
changes in the body weight (LambiotteEscofiier, Moore and Taylor, 1953; Seitchik
and Alper, 1954). The results obtained in
the rabbit support the findings of the latter
authors as no disproportionate increase in
the thiocyanate space was observed during
pregnancy in the rabbit. The slight increase
that occurred during the last trimester of gestation was in good agreement with the
fluid accumulation by the developing fetus.
Similarly the increase in blood volume in
the rat is correlated with increase in body
weight.




Line 4,054: Line 4,771:




Fig. 16.24. Changes in blood plasma and total
erythrocyte volume in the ovariectomized rabbit
treated with 1 mg. estradiol daily. (From L. M.
Horger and M. X. Zarrow, Am. J. Physiol., 189,
407, 1957.)








1'
Fig. 16.25. Changes in the blood plasma and
 
total erythrocyte volume during pregnancy.
 
(From L. M. Horger and M. X. Zarrow, Am. J.
 
Physiol., 189, 407, 1957.)
 
 
 
 
 
••
 
 
 
 






Thus the anemia of pregnancy as observed in the rabbit and rat is very similar
to that reported for man. It can be characterized as a normochromic and normocytic
anemia. Although a decrease in the relative
concentrations of hemoglobin and erythrocytes occurs, the total amounts of these
components of the blood remain unchanged.
Consequently, the anemia of pregnancy is
due to a hemodilution.


The anemia induced by treatment with
estradiol is similar to the anemia of pregnancy in many respects. Witten and Bradbury (1951) treated 16 women with 5 mg.
estrone or 0.4 mg. estradiol dipropionate
and noted an erythrocyte drop of 14.8 per
cent, a hemoglobin drop of 8.5 per cent, a
hematocrit drop of 15 per cent, and a blood
volume increase. Treatment of the castrated
rabbit with 1 mg. estradiol daily caused a
20 per cent decrease in both erythrocyte
count and hemoglobin with no significant
changes in total hemoglobin or number of
erythrocytes. Estradiol also caused an increase in plasma and blood volume (Fig.
16.24) which was comparable to that seen
during pregnancy (Fig. 16.25), but no significant change in cell voluiiic was obtained.
The estradiol-induced anemia is both normochromic and normocytic and is caused by
a htMnodilution. However, in addition to the
ciianges in the blood and plasma volumes,
estradiol induces a significant increase in
the thiocyanate space. Furthermore, only
the massive dosage of 1.0 mg. estradiol per
day elicits an anemia comjKirable to that
observed in pregnancy. This dosage level
is probably toxic since there is a decrease
in the body weight of most rabbits which received this treatment. Thus, in spite of
the similarities of these anemias, it is
likely that estrogen is not the sole etiologic
agent in the anemia of pregnancy.


Progesterone alone at dosages of 4 mg.
daily has little effect on the plasma volume
or the thiocyanate space. This steroid does
exert a significant influence on the action
of estradiol on the blood and plasma volume, but it is to be noted that rather large
dosages of estradiol were still needed to induce a significant hypervolemia and that
the effect depends on the ratio of the concentration of the two hormones. The hypervolemia induced by the treatment with 4
mg. progesterone in combination with 0.1
mg. estradiol was greater than that caused
by the estradiol alone, whereas the treatment with 4 mg. progesterone in combination with 1.0 mg. estradiol resulted in an
inhibition of the estrogenic activity (Fig.
16.261. Thus progesterone may play a dual
role in the water metabolism of the gravid
female. In the presence of low titers of
estrogen, progesterone augments its action
which may be a means of insuring an adequate fluid retention to provide for the
fluid requirements of the fetus. However,
if the titers of the estrogens and possibly of
other steroids affecting salt and water metabolism became excessively high, the progesterone may provide a protective measure
by inhibiting the activity of these substances. This concept is in accord with reports describing the diuretic action of progesterone in the iiypophysectomized rat
(Selye and Bassett, 1940) and the inhibition of the salt- and water-retaining action
of DOCA and cortisone by progesterone
(Landau, Bergenstal, Lugibihl and Kascht,
1955).






Fig. 16.26. Changes in the blood volume of
the ovariectomized rabbit treated with 0.1 mg.
and 1.0 mg. estradiol daily and with a combination
of the two estrogen treatments and 4 mg. progesterone. (From L. M. Horger and M. X. Zarrow,
Am. J. Pliysiol., 189, 407, 1957.)


It is also of interest that no anemia was
observed in animals treated with various
combinations of estrogen and progesterone
(Horger and Zarrow, 1957). Progesterone
elicits an increase in the cell volume which
api^roximates that of the plasma volume.
Because no erythrocyte counts were made in
this study, it is not possible to state whether
this increase in the cell volume is caused
by a macrocytosis or an increase in the number of erythrocytes. Vollmer and Gordon
( 1941 ) reported that progesterone caused an
increase in the erythrocyte count of the
rat but that the action was inconsistent.
Hence it is possible that the increase in
the cell volume is due to an enhancement of
hematopoiesis by the progesterone. This
possibility is not inconsistent with the
absence of an increase in the reticulocyte
count in response to these treatments since
a reticulocytosis usually occurs only after
an intense stimulation of the hematopoietic
tissue such as by hemorrhage.


In view of the previous discussion, it is
improbable that the anemia of pregnancy
is due entirely to the interaction of estrogen
and progesterone. These hormones appear to play an important role in the salt and
water metabolism of the gravid female.
Furthermore, it is noted that the cow
exhibits a hypervolemia but no anemia
during pregnancy (Reynolds, 1953) and
that a similar condition is produced in the
rabbit by the treatment with various combinations of these steroids. Hence the interaction of estrogen and progesterone may be
responsible for this species difference.


Inasmuch as no antidiuretic hormone
(ADH) could be detected in any of the
plasma samples, it is apparent that the
plasma titers of ADH did not rise above
10 fiV. per ml. during the experimental
period. However, in view of the increased
ability of the blood to inactivate ADH during pregnancy (McCartney, Vallach and
Pottinger, 1952; Croxatto, Vera and Barnafi, 1953), there may be an increased
rate of turnover of ADH during gestation.
Consequently the data obtained in this
study neither substantiate nor eliminate
ADH as an etiologic agent in the anemia of
pregnancy.


A number of investigators have attributed
the hypervolemia of pregnancy to structural
changes in the circulatory system. Burwell
(1938) observed a marked similarity between the circulatory changes observed in
pregnancy and those observed in a patient
with an arteriovenous fistula. He noted that
in both conditions there is an increase in
the blood volume, cardiac output, pulse
rate, pulse pressure, and an increased venous
pressure near the opening of the fistula. He
concluded that the changes in the circulation of the pregnant woman are caused by
an arteriovenous leak through the placenta
and the obstruction of the venous return
by the enlarged uterus. Bickers (1942) correlated the intensity of the edema of the
right or left leg with the location of the
placenta in the uterus. The edema was observed to be consistently greater on the same
side as the location of the placenta whereas
the edema of the legs was equal when implantation occurred on the anterior or posterior wall of the uterus. However, when
the uterus was lifted off the interior vena
cava, there was no precipitous drop in the
venous pressure in the femoral vein. Thus
this study supports the arteriovenous shunt
theory of Burwell.






One objection to this theory is that it i


does not account for the decrease in the i


blood volume during the 10th lunar month j


of pregnancy. However, it has been reported i


that during the latter part of pregnancy
there is an increase in the resistance to the
flow of blood through the placenta. This increase is due to the increased number of
villi and to the anastomizing of the villi in
the placenta. Since an increase in the peripheral resistance to blood flow results in
a hemoconcentration, this would account for
the decrease in the blood volume during
the last lunar month of gestation (Kline,
1951; McGaughey, 1952).


Other objections to the arteriovenous
shunt theory were reported by Kellar ( 1950)
who found that blood flow through the
placenta is sluggish rather than rapid as
in an arteriovenous shunt. He also observed
that the uterine venous blood is not exceedingly rich in oxygen as is the venous
return of an arteriovenous aneuryism and
lie concluded that, although the uterus is
an area of decreased resistance to blood
flow, the effect is not entirely due to the
placenta. He suggested that thyroxine may
be partially responsible for the expansion of
the blood volume since mild thyrotoxicosis
is common in pregnancy. This concept is
supported by the observation that there
is a tendency for vasodilation in the upper
extremities during the latter months of gestation (Burt, 1950). Furthermore, the basal
metabolic rate increases during this period
(Sandiford and Wheeler, 1924; Rowe and
Boyd, 1932). It is to be noted, however,
that the changes in the blood flow in the
extremities are closely correlated with the
cardiovascular changes occurring during
pregnancy but not with the changes in the
hormonal levels in the blood (Herbert,
Banner and Wakim, 1954 ) .


Since there is no disproportionate increase in the thiocyanate space of the rabbit
during pregnancy, the increase in the blood
volume can best be explained on the basis
of cardiovascular changes during the latter
part of gestation. However, in view of the
previous discussion, it is extremely improbable that this hypervolemia is induced
by any one factor. Rather, it is more probable that the condition is produced by a
multiplicity of factors. On the basis of the previous reports, it is evident that the
placenta, due to its similarity to an arteriovenous aneuryism, is partially responsible
for the hypervolemia. The marked increase
in the uterine size and vascularity during
pregnancy (Barcroft and Rothschild, 1932)
will also account for a considerable amount
of the increase in the blood volume. In addition, the tendency for dilation of the
peripheral blood vessels may account for
another portion of the increase in the blood
volume. Thus, in general, the hypervolemia
of pregnancy can be attributed primarily
to structural changes in the circulatory system.


It cannot be denied, however, that the
cardiovascular system is influenced by the
changes in the endocrine balance during
gestation. It is well known that the placenta
elaborates large amounts of sex steroids
and corticoids. It has also been suggested
that there is an increased production of
thyroxine and ADH at this time. In addition, water-soluble extracts of the pregnant sow's ovaries have been shown to
cause water retention and anemia in the
rabbit (Zarrow and Zarrow, 1953). The
resultant hormonal balance becomes somewhat precarious as the additional secretions
of the glands tend to build up the blood
titers of the sex steroids and other substances which influence water metabolism.
It is possible that when a proper balance of
these factors is maintained, the pregnancy
is normal and the various requirements of
the fetus are provided without disrupting
the distribution of the body fluids outside
of the vascular system. However, if the
balance is not maintained, the animal tends
to accumulate fluid, and edema and other
pathologic complications result.




==VIII. Plasma Proteins==


 
It has long been known that the plasma
Ji
proteins play a significant role in the fluid
 
balance of the organism and as such are
 
also involved in water balance during pregnancy (]\Iack, 1955). In addition, the
 
plasma proteins are of importance in many
other functions, such as heat and energy
source and replacement of tissue in which
function they act as a protein source whenever needed and form the metabolic pool.
These proteins are synthesized in general in the liver and reticuloendothelial system
and may be classified as albumins or globulins although many different entities of
these two classifications are known to
exist.




The maintenance and stabilization of
blood volume and the equilibrium of fluid
exchange between the extravascular and
intravascular compartments is a function
of the albumin fraction of special significance in i)regnancy, in addition to its other
functions of acting as carrier for other
substances and sui^jilying of nutrients. The
regulation of blood volume by albumin
depends on its osmotic action and is of
much greater significance than the globulins. Approximately 4.6 gm. albumin and
3.17 gm. globulin per 100 ml. of plasma
are found in the normal, nonpregnant
woman. At least four types of glol)ulins are
present in the plasma among which are
found the lipoproteins, prothrombin, fibrinogen, antibodies, and several hormones.




Although both the plasma proteins and
albumin drop during pregnancy, this does
not necessarily indicate a drop in the total
available albumin protein. An increase in
the plasma volume compartment of 25 per
cent as seen in pregnancy could easily
result in an increase in the total amount
of circulating protein. However, as the
total circulating blood volume increases in
pregnancy, the albumin fraction and yglobulin seem to be diluted whereas the
other globulins become more concentrated.
Nevertheless, the globulins cannot compensate for the albumin loss and the total
protein decreases. Mack (1955) has listed
several possible explanations for the above
paradox : ( 1 ) the small albumin molecule
may diffuse more freely into tissues and
across placental membrane, and (2) albumin synthesis cannot keep pace with utilization.




Innumerable studies on the plasma i^-oteins of women during pregnancy have I'evealed markedly consistent changes in the
albumin-globulin ratio of the plasma. The
concentration of total protein and albumin
decreases while the total globulin increases.
The trend is apparent by the first trimester
and continues throughout gestation. A return to the nonpregnant pattern is seen
shortly after parturition. The total protein dropped 13 per cent and the albumin 26
per cent. The various globulin fractions
showed a rise except for the y-globulin
(Mack, 1955). As a result of these changes,
the albumin-globulin ratio declines
throughout pregnancy and shows the well
known reversal (Fig. 16.27) and recovery
to normal by 6 weeks postpartum.


X








Fig. 16.27. Progressive decreu.se in the albuminglobulin ratio of the plasma during pregnancy in
women. (From H. C. Mack, The Plasma Proteins
in Pregnancy, Charles C Thomas, Springfield, 111..
1955.)








Fig. 16.28. Changes in the glomerular filtration
rate throughout pregnancy in the woman. (From
W. J. Dignam, P. Titus and N. S. Assali, Proc.
Soc. Exper. Biol. & Med., 97, 512, 1958.)






Although it is obvious that the albumin
fraction is important in maintaining the blood fluid compartment, the changed
albumin-globulin ratio cannot solely account for the retention of water and edema
present in pregnancy. Although it has been
argued that the hypoalbuminemia through
diminished colloid osmotic pressure is the
cause of water retention in the tissues,
the occurrence of the postpartum diuresis at
the time when the albumin is lowest would
tend to indicate some other mechanism
(Dieckmann and Wegner, 1934a-d). Additional mechanisms, such as changes in the
hormone level, especially the sex steroids
and adrenal corticoids, may be responsible.


IX. Renal Function


Studies on renal function during pregnancy have resulted in contradictory reports. The earlier investigations failed to
show any effect of pregnancy on renal function (Chesley and Chesley, 1941; Welsh,
Wellen and Taylor, 1942; Dill, Isenhour,
Cadden and Schaffer, 1942) , whereas recent
studies indicate a marked change in renal
function during gestation (Bucht, 1951;
Dignam, Titus and Assali, 1958). Part of
the explanation for the divergent results
could be the type of patient studied, the
periods when studied, and the types of
controls. Dignam, Titus and Assali studied
both the renal plasma flow and glomerular
filtration rate in various patients throughout gestation and immediately following
l^arturition. Care was taken to select individuals without any history of cardiovascular or renal disease. Both the renal
plasma flow and the glomerular filtration
rate (Fig. 16.28) were increased throughout gestation. The initial rise was extremely marked during the 1st and 2nd
trimesters of pregnancy. A slight rise was
noted during the 3rd trimester and a return to normal by 6 to 8 weeks postpartum.


Recently, de Alvarez (1958) reported a
50 to 60 per cent rise in the glomerular
filtration rate and a 60 per cent rise in the
renal plasma flow during the 1st trimester
of pregnancy in the human being. This is
in agreement with the findings of Dignam,
Titus and Assali (1958). However, de Alvarez reported, in addition, a progressive
decline in both the glomerular filtration
rate and renal plasma flow during the 2nd
and 3rd trimesters. The filtration factor (glomerular filtration rate divided by the
renal plasma flow) remained low in the first
2 trimesters and increased in the last trimester. This is evidence for an increase in
tubular resorption of water and electrolyte.
It can only be concluded, therefore, that
kidney function is altered during pregnancy, especially the 1st trimester. Results
from investigations involving the 2nd and
3rd trimesters are contradictory. De Alvarez concludes that the changes in renal
hemodynamics during pregnancy are mediated by the endocrine system because the
alterations in renal function seem to be
related to the sodium and water retention.
If the changes are progressive throughout
gestation, it would be possible to correlate
the phenomenon with a number of hormones that increase during pregnancy. On
the other hand, if the phenomenon is transient, i.e., only during the 1st trimester, then
the phenomenon can only be correlated with
HCG.


==X. Enzymes==


===A. Histamixase===


The presence of histaminase or diamine
oxidase in tissues of the body has been
known for some time. As yet the enzyme
lias not been crystallized but is believed to
he a flavoprotein (Swedin, 1943). The enzyme is not specific for histamine because it
inactivates other diamines such as cadaverine and putrescine. Histaminase determinations, in general, are based on incubation of
the test material with histamine dihydrochloride for a fixed period of time and the
bioassay of the residual histamine carried
out on an isolated strip of guinea pig intestine.


Histaminase has been found in the
l)lasma of men and women with an increase
(luring ])regnancy from a value of between
0.003 and 0.008 /^g. per ml. per hr. to a
value of between 3.5 and 10 at parturition
(Ahlmark, 1944, 1947). This has been confirmed by Swanberg (1950), who determined the histaminolytic activity in pei-iplK>ral blood throughout pregnancy (Fig.
16.29). A marked rise is observed from
the 10th to the 20th week of pregnancy,
and thereafter the concentration plateaus
until after parturition.






Fig. 16.29. Tlie histaminase activity of the
peripheral blood of the human female during
pregnancy (•) and at parturition (®). (From
H. Swanberg, Acta scandinav., Suppl. 79, 23,
1950.)


X
Both the maternal placenta and the
 
decidual tissue have been identified as
 
major sites for formation of the enzyme.
•
Danforth and Gorham (1937) reported the
 
presence of histaminase in the placenta
 
of a series of patients at term. This was
 
confirmed by Swanberg (1950) who, in
 
addition, separated the placenta by a series
i 3
of slices parallel to the surface of the organ
 
and reported that the layer adjacent to
 
the uterine wall, consisting of practically
 
only the thin decidual membrane, contained
 
a mean value of 614 /xg. per gm. per hr. of
 
histaminase as compared to 38 for the fetal
 
portion of the placenta. Confirmation of the
 
concept that the maternal placenta is the
 
main source of histaminolytic activity can
X
be obtained from the finding of histaminase
 
in decidual tissue of nonpregnant females
 
and in the maternal placentas of animals.
,  
In cases in which maternal and fetal
 
placentas can be separated easily, the
 
maternal placenta contained from 14- to
 
100-fold the activity seen in the fetal
 
placenta. Comparison of the histaminolytic
 
activity in the decidual tissue of the sterile
 
horn and the control pregnant horn of the
X
uterus of a rabbit revealed 319 fig. per gm.
per hr. and 222 fxg. per gm. per hr., respectively. Treatment with progesterone or induction of jiseudopregnancy caused a
marked rise in the histaminase of the
endometrium to upwards of 1000 fig. per
gm. per hr. Nonetheless, histaminase was not observed in the blood plasma of the
progesterone treated rabbits whereas progesterone treatment of two nonpregnant
women caused a marked rise iti plasma
histaminase.


The physiologic significance of histaminase is still unknown. A consideration of
this problem must take into account not
only the action of the enzyme and changes
in its concentration under different physiologic conditions, but also the species problem. In regard to the latter point, the data
are extremely inadequate. Only two species
have been studied in any detail and these
are the human being and the ral)bit. One
can conclude from the available data that
histaminase is produced by the maternal
placenta, decidua, and uterine endometrium.
It increases with pregnancy in these tissues
and its concentration may be correlated
with the progestational hormone. It increases in the blood of tlie human being,
I'at, and guinea pig during pregnancy but
nut in the cat or rabbit (Swanberg, 1950;
Carlsten, 1950). The obvious hypothesis
that histaminase })rotects the uterus from
the stimulating action of histamine has not
been confirmed. But it is somewhat jiai'adoxical to note that urinary histamine also
increases during pregnancy. Kahlson,
Rosengren and Westling (1958) reported a
daily 24-hour excretion of 18 to 43 /xg.
of histamine during the first 2 weeks of
pregnancy in the human being. A marked
increase was noted on the 15th day with a
peak of 123 to 835 /xg. per 24 hr. at the
peak of excretion which occurred 1 to 2
days before parturition. As yet no role
can be attributed to this substance. It is
of interest that the increased histaminase
present during pregnancy can serve the role
of protecting the uterus from the musclecontracting action of this substance. Because the amount of urinary histamine excreted is correlated with the number of
young and no changes are apparent in the
concentration of histamine in the tissues
during pregnancy, it would seem that the
excessive formation of histamine during the
last trimester of pregnancy takes place in
the uterus and its contents and the basic
action of histaminase is protective.


It was shown recently that the excessive
formation of histamine during the last trimester of pregnancy in the rat is due to
an increase in the rate of histidine decarboxylase activity (Kahlson, Rosengren,
Westling and White, 1958). Inasmuch as
removal of the fetuses without other interference with the pregnancy abolishes the
increased urinary histamine, it can l)e concluded that the site of formation is in the
fetus. This histamine could escape into the
maternal circulation and eventually be
eliminated via the kidneys.


Roberts ( 1954) reported that aminoguanidine leads to a general disturbance of
pregnancy in the rat; large doses tended to
jiroduce death of the mother and smaller
doses tended to kill all or part of the litters
and some of the mothers. Again one could
conclude a protective action on the part of
histaminase dui'ing tlie latter i)art of i^regnancy.


===B. Carbonic Anhydrase===


Carbonic anhydrase was discovered by
Aleldrum and Roughton in 1933 and soon
shown to catalyze the following reaction,
H,CO, z:± CO. + HoO. The enzyme was
found to occur in many tissues and was
generally located within the cell especially
in cells possessing a secretory function. The
discovery by Lutwak-Mann and Laser
(1954) that carbonic anhydrase is present
in tlu' uterine mucosa led to a thorougli
study of the changes in the concentration
of the enzyme and the factors controlling
its presence (Lutwak-Mann, 1955; Lutwak-Mann and Adams, 1957). The enzyme
has been found to be present in the reproductive tract of a wide variety of
mammals. In general, the uterine endometrium, placenta, and Fallopian tubes are
the main loci of activity although there
are marked differences among different
species. Carbonic anhydrase activity was
found consistently in all the animals studied
such as the rat, hamster, guinea pig, rabbit,
pig, and ewe. No activity was noted in the
uterine mucosa of the nonpregnant animal
except the ewe and the rabbit. In several
species, such as the cow, human being,
and pig, carbonic anhydrase was also found
in the Fallopian tube.


A marked rise in carbonic anhydrase of
the endometrium of the rabbit was noted
during the first trimester of pregnancy






(Fig. 16.30). The value rose from a prepregnancy level of 20 enzyme units (E.U.)
per gm. of fresh tissue to a maximum of 100
E.U. per gm. at approximately the 8th day
of pregnancy. This level was maintained
until the 12th day and then declined to
approximately the prepregnancy level by
about the 20th day. Examination of the
placentas at this time revealed marked activity, 68 E.U. per gm. of maternal placenta
and 25 E.U. per gm. of fetal placenta. The
curve for the concentration of carbonic
anhydrase in the uterine mucosa during
pscudopregnancy is essentially the same
as that seen during pregnancy, although
some minor differences exist.


It is obvious from the above data and
from the evidence involving the increased
concentration of carbonic anhydrase in the
uterine mucosa following treatment with
progesterone, that the enzyme is probably
under the control of the luteoid hormone.
Indeed, an excellent correlation has been
shown between the degree of {progestational
proliferation in the uterus and the concentration of carbonic anhydrase. In the ewe,
however, the carbonic anhydrase of the
uterus is independent of the ovary. A possible explanation for this discrepancy between the two species has been offered on
the basis of differences in the blood level
of progesterone. However, no explanation is
forthcoming for the failure to maintain
the carbonic anhydrase level throughout
pregnancy in the rabbit, even though the
circulating progesterone remains liigh.


 
The significance of this enzyme in the
*
physiology of reproduction is still unknown.
 
From the data on the rabbit, it miglit be
 
inferred that the carbonic anhydrase contributes to the maintenance of bicarbonate
5
in the blastocyst fluid. The universal presence of the enzyme in placental tissue could
 
also lead to the assumption that carbonic
anhydrase is involved in fetal metabolism.
Lutwak-Mann (1955) indicates that the
enzyme might be involved in the transmission of calcium across the placenta.
^^'hether carbonic anhydrase is essential for
fetal (Icvelopment and successful pregnancy
is still unanswered. Treatment with carbonic anhydrase inhibitors (Diamox)
failed to affect adversely the pregnancy or
fetuses in pregnant rats even though no enzyme acti\'ity was present either in the
matei'nal blood or placenta.








Fig. 16.30. Carhonic anhydrase activity in the
uteiu.s of the rabbit during pregnancy, i),seudopregnancy, large doses of gonadotrophin, and
pregnant mare's serum (PMS). Pregnancy,
• •; i^seudopregnancy, O O; gonadotrophin, D D: PMS, x" X. (From C. Lut
\val<-Mann. .1. Kndocrinol.. 13, 26. 1955.)








==XI. Factors in the Maintenance of Gestation==


.  
===A. Thyroid Gland===


Several recent reviews have pointed out
that the extract role of the thyroid gland in
reproductive physiology is still in need of
elucidation (Peterson, Webster, Rayner and
Young, 1952; Reineke and Soliman, 1953).
Numerous investigations over the past half
century have definitely indicated that the
thyroid gland is involved in reproduction
but the site and manner of action are still
not well known. In addition, contradictory
reports indicate that each species and even
each strain may have to be studied independently (Alaqsood, 1952). Some evidence foi- the involvement of the thyroid
gland in gestation has already been considered. The increase in FBI at the onset of
jiregnancy and the incidence of miscarriage
in the human female when the FBI fails to
rise tend to involve the thyroid hormone in
the maintenance of pregnancy. Habitual
abortion in women is usually associated
with t'ithcr hypo- or hyperthyroidism
(Litzenberg, 1926). Litzenberg and Carey
(1929 » I'eported that in 70 married women
with low basal metabolic rates appi'oximately 45 per cent had one or more abortions or stillbirthsrtf one eliminates the
sterile woman from the group, the figure for
women showing abortion or stillborn rises
to approximately 35 per cent. However, the
results are still controversial both with
regard to data obtained within a single
species and from different species.




Hypothyroidism in the rat induced by
the prolonged administration of thiouracil
resulted in a resorption of the fetus in 100
per cent of the cases (Jones, Delfs and
Foote, 1946). Rogers (1947) reported a reduction in litter size following sulfaguanidine and Krohn and White (1950) reported
a reduction in litter size following thyroidectomy in the rat. Thyroidectomy early in
pregnancy caused a resorption of the
fetuses and if performed at a later stage
in pregnancy resulted in the birth of stillborn young (Chu, 1945). Following the induction of pregnancy in thyroidectomized
rabbits, either a resorption of the young or
abortion or prolongation of gestation was
noted and the newborn young were usually
dead. Chu concluded that the thyroid hormone was concerned with the vitality and
growth of the embryos during gestation. In
the pig the average duration of pregnancy
was 114 days for normal gilts and 124.5
days for thiouracil-treated animals. In addition, the controls farrowed an average of
8.67 pigs per litter compared with 3.25 per
litter for the thiouracil-treated .sows ( Lucas,
Brunstad and Fowler, 1958 ) . The difference
was significant in both instances. Bruce and








 
Fig. 16.31. Tlie effect of tliyioid deficiency on
«
litter size. O, 422 litters from tliyroid-defieient
 
mice; •, 423 litters from normal control mothers.
 
(From H. M. Bruce and H. A. Sloviter, J. Endocrinol., 15, 72. 1957.)
>
 
 






Sloviter (1957) pointed out that part of
the conflicting reports on the role of the
thyroid in gestation might be due to the
different methods used in producing a
thyroid-deficient state. Surgical removal of
the gland generally results in the loss of
the parathyroids which may be also important in the maintenance of gestation
(Krichesky, 1939), although adequate information is lacking. The use of antithyroidal substances offers more serious objections because these drugs not only pass
through the placenta but they are nonspecific and interfere with other glands such
as the adrenal cortex (Zarrow and Money,
1949; McCarthy, Corley and Zarrow, 1958),
with nutrition, and with the general status
of the animal. Consequently, Bruce and
Sloviter preferred to establish a thyroidectomized state in mice by the use of radioactive iodine after establishing the dose
necessary to induce total destruction of the
thyroid without damage to the parathyroid
or ganiete.s.


X
Although ( lorbman ( 1950 ) rei)orted a
 
complete loss of reproductive activity in the
•
mouse following treatment with P'*\ Bruce
 
and Sloviter (1957) reported no effect on
 
the ability of the mouse to conceive or bear
young. This discrepancy could be due in
part to the strain differences in the sensitivity of the ovary to the I^^^. Bruce and
Sloviter (1957), however, noted a decrease
in the average litter size of thyroid-deficient
mice (Fig. 16.31 ). The data indicate a maximum of 6 young per litter in thyroid-deficient mice versus 10 young per litter for
the normal mice. It is apparent that the
entire curve for the litter size of thyroiddeficient mice is shifted toward a smaller
size. This has also been observed in the rat
following thyroidectomy (Nelson and Tobin, 1937). The thyroid-deficient mice also
showed a prolongation of gestation as reported in rats, guinea pigs, and sows. Of
the thyroid-deficient rats, 46 per cent
showed a gestation period of more than 19
days whereas only 15 per cent of the normal
controls showed a gestation period of more
than 19 days whereas only 15 per cent of
the normal controls showed a gestation period of more than 19 days (Table 16.9).
Analysis of the data based on grouping
according to litter size showed clearly an effect of litter size on length of gestation.
The smaller litter size gave a higher inci(l(>nce of prolonged gestation.


Studies on oxygen consumption in the
guinea i)ig revealed a slight but significant
rise of 8 per cent at the end of gestation
(Hoar and Young 1957). The increase in
oxygen consumption is consistent but slight
for the first 60 days of pregnancy after
which the significant increase occurs (Fig.
16.32). The rise continued until 5 days
postpartum and then fell rapidly. In a second set of experiments oxygen consumption
was measured in control, thyroidectomized,
and thyroxine-injected, pregnant guinea
pigs. Measurements were taken at the
time of mating and at parturition. In all
three instances, an increase in the oxygen
consumption was noted at parturition as
compared with the values at the time of


X
TABLE 16.9


Ejfcct of thyroid-deficiency and litter size on length


,
of gestation in mice


(From H. M. Bruce and H. A. Sloviter, J.
Endocrinol., 15, 72, 1957.)




Line 4,222: Line 5,530:




Thyroid-deficient




Control




No. of


Young In


Litter




1.  
No. of
pregnancies




> 19 days




No. of
pregnancies




> 19 days




1^
No.




Per
cent




X
No.




Per


cent




1-5


6-9
10-14


Totals




36
40
20


96




24


13


7


44


•


67
33
35


46




16


38


28


82




7
3
2


12




X •
44


7
8


■X
15




:/


 
mating (Fig. 16.33). Again the control
r>
guinea pigs showed a 7.9 per cent gain in
oxygen consumption by the end of pregnancy, but both the thyroidectomized pregnant guinea pigs and the thyroxinc-treated
guinea pigs also showed an increase in oxygen consumption of 11.9 and 16.2 per cent,
respectively. The increase in oxygen consumption was not paralleled by increases in
heart rate; actually the heart rate decreased
in several instances. In addition, neither
the weight of the thyroid gland nor the
histology of the gland was changed during
pregnancy. It is obvious then that an explanation for the rise in oxygen consumption during pregnancy may not involve the
thyroid gland. On the basis of changes in
its appearance. Hoar and Young (1957)
suggested the possibility that the adrenal
cortex is involved and that the increased
oxygen consumption is due to an increased
release of adrenal corticoids. More evidence
is needed before this suggestion can be fully
accepted.




^
Further work from the same laboratory
has led to the concept that one locus of
action of thyroxine during pregnancy is at
parturition (Hoar, Goy and Young, 1957).
These investigators used an inbred strain
of guinea pigs that is characteristically hypothyroid and a genetically heterogeneous
stock in which the level of thyroid activity
is presumed to be higher. It had been previously shown that pregnancy wastage was
high in the hypothyroid guinea pigs. Treatment with thyroxine reduced the percentage
of stillborn from 40 to 13.6 in the hypothy




X




5


X


Fig. 16.32. Oxvgen consumption in the guinea pig during gestation. (From K. M. Hoar
and W. C. Young, Am. J. Physiol., 190, 425, 1957.)


,^




'"^^




^J__
Fig. 16.33. Oxygen con.suinption in the pregnant guinea pig treated with thyroxine or
thvroiclpctomized before mating. (From R. M. Hoar and W. C. Young, Am. J. PhysioL,
190, 425, 1957.)




X


X
roid guinea pig!>, i.e., to a level !?een in the
untreated heterogeneous group. Treatment
of the heterogeneous group with thyroxine
not only failed to reduce the percentage
of stillborn but actually increased the abortion rate particularly in the 2nd and 3rd
trimesters. The most consistent result, however, was a decrease in length of gestation
following treatment with thyroxine, and an
increase following thyroidectomy. From
these experiments it was concluded that
the thyroid hormone facilitates parturition
and need be present only late in gestation
to exert its action.


It is apparent that in some species the
thyroid hormone is involved directly in
pregnancy. In the absence of the hormone,
certain species tend to resorb or to abort;
or if pregnancy is maintained gestation
tends to be lengthened. This is probably
due to an interference with the mechanism
of parturition. In certain species such as
the guinea pig only a parturitional problem
has been demonstrated; in others an entire
galaxy of symptoms may be present. Reduction in the size, number, and viability
of the young give added emphasis to an essential role for thyroxine in the phenomenon of gestation.


J


===B. Adrenal Cortex===


•
Removal of the adrenal cortex without
 
further treatment invariably leads to disturbances in rejM'oductive i)hysiology and
X
the termination of pregnancy. Although the
early results were controversial in that
some investigators reported that adrenalectomy failed to affect gestation in the rat
(Lewis, 1923; Ingle and Fisher, 1938),
others reported that adrenalectomy led to
abortion (Wyman, 1928; Dessau, 1937) or
to some other disturbance of gestation (McKcown and Spurrel, 1940). Davis and
Plotz (1954) adrenalectomized two groups
of pregnant rats on the 4th to 6th and the
14th to 16th day of pregnancy. Abortion
occurred in all 12 rats adrenalectomized
during the first half of pregnancy whereas
only 1 of the 12 adrenalectomized during
the second half of pregnancy aborted. However, even in those adrenalectomized during
the second half of gestation, an effect on
jiregnancy was observed. A significantly
higher incidence of stillborn and sickly
young (14.4 per cent) and a marked decrease in the weight of the fetuses were
noted (Table 16.10).
 
Early results indicated that extracts of
the adrenal cortex could readily replace
the absent adrenal gland and maintain successful pregnancies. Within recent years it
has been demonstrated that many steroids
such as cortisone and 9a-chlorohydrocortisone at 10 /xg. per day (Llaurado, 1955)
permit fecundation and successful maintenance of pregnancy. Successful maintenance




X


GESTATION


.




X
1001






TABLE Ki.lO


Effects of adrenalectomy on the character of the litter,


and on fetal body weight and adrenal weight


(From M. E. Davis and E. J. Plotz,


Endocrinology, 54, 384, 1954.)






/T^




SwV
Pregnant
Controls




Adrenalectomy
2nd Half of
Pregnancy




Percentage
versus
Pregnant
Controls




^*^
No. of litters ....




«w--*
21




"•x
11








Dead and




Line 4,363: Line 5,792:




XJL,




<*Ay
"sickly" young.
Vigorous voung . . .




5


182




13


78




M 1
<0.01




-p
Fetal hodv weight




'^ •




X




•




.  
(gm.)




••
6.13


X


5.22


XXX


<0.01








(=b0.10)«




(±0.30)"


X




V>


Fetal adrenal


X
X




-f


XX
X
X
X




T


X


weight (mg.). . . .


s^


0.49()


X


0.554


•


>0.3








- 1
(±0.017)"




(±0.041)«




\ —




Fetal l)()dv weight/




— i_




— 1—




1. .




_L_
fetal adrenal




.1 .




-.1




-i—




J—
weight X 1000. . .




i_
12.35




.,.,I.,„
9.42




1
<0.01




1




•
(0.34±)''


1 .


(±0.31)"


••xx^xxxxxx


1 1






1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Calculation of standard error of the mean:
Weeks of pregnancy


Fig. 16.11. The con«?ntiation of progesterone in the ovarian venous blood of the pregnant ewe. Progesterone was determined by chemical methods. (From D. G. Edgar and
J. W. Ronaldson, J. Endocrinol., 16, 378, 1958.)




 
S.E.  
0.0098 ixg. per ml. plasma. It is of interest
that the level remained constant from the
32nd to about the 256th day of pregnancy
and then decreased several days before
parturition. In the human being values of
0.17 to 0.44 fig. per ml. during the final trimester of pregnancy have recently been
reported (Oertel, Weiss and Eik-Nes ( 1959 ) .
Numerous investigators have suggested
that the discrepancy between the chemical
and biologic assays is due to the presence
of unknown gestagens in the blood. This
has been validated in part by the discovery
of 2 metabolites in the blood of the pregnant human female (Zander, Forbes, Neher
and Desaulles, 1957). They have been identified as 20a-hydroxypregn-4-en-3-one and
20^-hydroxypregn-4-en-3-one and have
been shown to be active in both the Clauberg and Hooker-Forbes tests (Zander,
Forbes, von IMiinstermann and Neher
1958 ) . The 20/?-epimer was twice as active
as progesterone in the Hooker-Forbes test
and the 20a-epimer one-fifth as active. It






is likely that more unidentified gestagens
/ Ed-^
occur in the blood and other tissues.


]/ nin




C. SOURCES OF GESTAGENS
1)


The second question asked above concerning the role of the placenta versus the
ovary as a source of progesterone probably
cannot be answered in a simple manner.
Wide differences exist between species (1)
in the need of the ovary for maintenance
of pregnancy, (2) in the concentration of
the hormone in peripheral blood, (3) in the
activity of the placenta in secreting progesterone, and (4) in the presence of extraovarian and extraplacental sources of the
hormone.
The presence of progesterone in the placenta of the human being has been confirmed (Salhanick, Noall, Zarrow and Samuels, 1952; Pearlman and Cerceo, 1952)
and a high output of progesterone demonstrated. Zander and von ]\Iiinstermann
(1956) and Pearlman (1957) independently




of a pregnancy has also been reported in
an adrenalectomized human female maintained on hydrocortisone 9a-fliiorohydrocortisone (Laidlaw, Cohen and Gornal,
1958). In this instance measurements of
urine excretion of aldosterone revealed an
increase to 4.4 fxg. per 24 hours during the
last trimester of pregnancy and a postpartum value of 0.5 fig. Inasmuch as the value
is only 1/10 of that seen in a normal pregnancy the authors concluded that the adrenal cortex of the mother is the major
source of aldosterone during pregnancy and
that a high output is not a major prerequisite for a normal pregnancy.
Treatment with either 0.9 per cent saline
drinking water or with cortisone increased
the number of successful pregnancies following adrenalectomy during the first half
of gestation. Pregnancy was normal in 8
of 11 adrenalectomized rats (Davis and
Plotz, 1954). Treatment with 2 mg. of cortisone acetate resulted in successful pregnancies in 13 of 14 rats adrenalectomized
on the 4th to 6th day of gestation and 12 of
12 rats adrenalectomized on the 14th to 16th
day of gestation. However, complete maintenance was not obtained. The body weight
of the mothers and the weight of the fetuses
were significantly lower than in the controls, and the number of stillborn and sickly
young was increased.
A comparison of the pregnancy-maintenance activity in a number of adrenal corticoids indicated that a combination of a
glucocorticoid and mineralocorticoid provides the best protection in the adrenalectomized rat (Cupps, 1955). Nulliparous rats
were adrenalectomized, placed on treatment, and mated. Under these conditions
the adrenalectomized controls and the rats
treated with desoxycorticosterone acetate
failed to become pregnant inasmuch as no
implantation sites were obtained (Table
TABLE 16.11
Effect of adrenal steroids on reproduction in
adrenalectomized female rats
(From P. T. Cupps, Endocrinology, 57, 1, 1955.)


GESTATION




Daily Treatment


973




Control


reported the production of approximately
Cortisone acetate
250 mg. progesterone into the peripheral
^i mg  
circulation every 24 hours. This and other
evidence tends to prove that the placenta
is the major source of progesterone in the
human species. However, with respect to
other species, progesterone has been found
only in the placenta of the mare (Short,
1957) although in amounts much less than
in the human being. Placentas of the cow,
ewe, sow, or bitch were all negative. Although the placenta of the mare contains
progesterone and castration does not lead
to abortion after day 200 of gestation, no
progesterone was found in the peripheral
blood or uterine vein blood. The ewe offers
an even more intriguing problem inasmuch
as (1) a discrepancy exists between the
biologic and chemical values for progesterone in the peripheral blood, (2) the placentas contain no progesterone, and (3) no
{progesterone is found in the uterine vein
blood (Edgar, 1953). This has led to the
conclusion that the maintenance of pregnancy in the ewe may be dependent on an
extra-ovarian, extraplacental source of progesterone.


If such a conclusion is correct, and it
Cortisone acetate
must be added that the evidence is still
^i mg
tenuous, then the adrenal cortex must be
considered as a possible source. Beall and
Reichstein isolated a small amount of progesterone from the adrenal cortex in 1938
and Heehter, Zaffaroni, Jacobson, Levy,
Jeanloz, Schenker and Pincus (1951) demonstrated from perfusion experiments that
progesterone is an important intermediate
metabolite in the synthesis of the adrenal
corticoids. In addition, it has long been
known that desoxycorticosterone possesses
progesterone-like activity (Courrier, 1940)
which is due to a conversion of the desoxycorticosterone molecule to a gestagen. This
has been shown by experiments in vivo in
the monkey (Zarrow, Hisaw and Bryans,
1950), rat, and rabbit (Lazo-Wasem and
Zarrow, 1955), and by an incubation experiment with rat tissue (Lazo-Wasem and
Zarrow, 1955). In addition, Zarrow and
Lazo-Wasem reported the release of a gestagen from the adrenal cortex of the rat
and rabbit following treatment with
ACTH. The substance was obtained from
the peripheral blood and measured by the


Cortisone acetate
1'^ mg


Cortisone acetate
2,4 mg


Hooker-Forbes test, but it was not identified chemically. This was followed by the
Hydrocortisone
finding that pregnanediol is present in the
acetate Vi mg.. . ,
urine of ovariectomized women, but not
ovariectomized, adrenalectomized women
(Klopper, Strong and Cook, 1957), and by
the finding that progesterone is present in
the adrenal venous blood of the cow, sow,
and ewe (Balfour, Comline and Short,
1957). In all instances the concentration
of progesterone in the adrenal venous blood
was 10 to 100 times greater than the concentration in the arterial blood. Thus the
total evidence that the adrenal cortex can
secrete progesterone is more than adequate.
The question remains as to whether the
adrenal cortex contributes to the progesterone pool of the body during pregnancy
and whether a species difference exists here.  


D. RELAXIN
Cortisone acetate
Vi mg. plus


The initial discovery by Hisaw (1926,
Desoxycorticosterone acetate ^
1929) of the presence of an active substance
mg
in the blood and ovaries responsible for
relaxation of the pubic symphysis of the
guinea pig has led in recent years to a consideration of this substance as a hormone
of pregnancy (Hisaw and Zarrow, 1951).
Some doubt as to the existence of relaxin
was raised in the 1930's by investigators
who were able to show that pubic relaxation in the guinea pig could be obtained
with estrogen alone or estrogen and progesterone (de Fremery, Kober and Tausk,
1931; Courrier, 1931; Tapfer and Haslhofer, 1935; Dessau, 1935; Haterius and
Fugo, 1939). This matter was resolved by
the demonstration that pubic relaxation in
the guinea pig following treatment with the
steroids or relaxin differed in ( 1 ) time required for relaxation to occur, (2) histologic changes in the pubic ligament, and (3)
treatment with estrogen and progesterone
which induced the formation of relaxin
(Zarrow, 1948; Talmage, 1947a, 1947b).
Subsequent discoveries of additional biologic activities possessed by relaxin and further purification of the hormone has led to
the conclusion that relaxin is an active substance in the body, and that it plays a significant role during parturition. The hormone has been found in the blood or other
tissues of the dog, cat, rabbit, sheep, cow,
rat, and man. The specific action of this


Desoxycorticosterone acetate ^^
mg


Desoxycorticosterone acetate ^
mg


^74
Desoxycorl icosterone acflate 1 mg ,


Adrenalectomized
control




SPERM, OVA, AND PREGNANCY


No. of
Rats




hormone varies with the species involved.  
No.  
Still unsolved is the question as to whether
Born
the water-soluble extract obtained from the
Alive
ovary and referred to as relaxin is a single
substance or a group of active substances
(Friedcn and Hisaw, 1933; Sher and Martin, 1956).


The concentration of relaxin in the blood
(average)  
increases as pregnancy progresses until a
plateau is reached. This has been demonstrated in the rabbit (Marder and Money,
1944), guinea pig (Zarrow, 1947), cow
(Wada and Yuhara, 1955), and human
being (Zarrow, Holmstrom and Salhanick,
1955). Relaxin has also been found to increase in the ovary of the sow (Hisaw and
Zarrow, 1949). In general, the shape of the
curve for the concentration of relaxin in
the blood as a function of the length of
pregnancy has been more or less the same
for all species studied. Figure 16.12 indicates that the concentration of relaxin in
the blood of the pregnant rabbit rises from
a level of 0.2 guinea pig unit (G.P.U.) per
ml. for the first trimester of pregnancy, i.e..
until day 12, to a level of 10 G.P.U. per ml.




Implantation
Sites
(average)


on day 24. This concentration was then
maintained until parturition. After delivery
of the young, the concentration of the hormone decreased 80 per cent in 6 hours. On
the 3rd day postpartum no hormone could
be detected.


As indicated above, the concentration of
7
relaxin in the blood of the pregnant cow
and human being showed approximately
the same type of curve. In the cow the concentration rose gradually from a level of 1
G.P.U. per ml. to a maximum of approximately 4 G.P.U. at 6 months (Fig. 16.13).
Thereafter the level remained unchanged
until parturition, wdien the level dropped at
a rate comparable to that seen in the rabbit. The curve for the concentration of relaxin in the blood serum of the pregnant
woman followed the general pattern described above (Fig. 16.14). The concentration rose from a level of 0.2 G.P.U. per ml.
the 6th week of i:)regnancy to a maximum
of 2 G.P.U. the 36th week. Thereafter the
level remained unchanged until delivery.
Again the postpartum fall was precipitous
and the hormone was not detectable at 24




8.2


lf\


11.0




6




3.5'>




5.6^


9


6




3.6*


yO ^0 ^


6.2'^




5


8


3.5


.


8.6"


/


5




5.8


2


10.0




7


r


5.0




8.5"


3


5




9.0




9.6




4


oc 7




/




Line 4,768: Line 6,100:




5


lij '




Line 4,777: Line 6,109:




4


tn




Line 4,786: Line 6,118:




5


U.6




/




Line 4,796: Line 6,127:




Weight
Change
during
Pregnancy
(average)"


o




gm.
46.4


-30. S''


-1.5«
17.2^
12.8^
30.7






44.6


o5




/
"Weight change of mother from day of breeding to day after parturition.
" Significant at 0.05 level.
'= Significant at 0.01 level.




o


1002






O
SPERM, OVA, AND PREGNANCY






16.11). Treatment with 2.5 mg. cortisone
acetate per day was partially effective in
restoring reproductive capacity. Injections
of 1.25 mg. hydrocortisone acetate per day
gave results comparable with those obtained when cortisone was given, although
the ratio of young born alive to implantation sites indicated that hydrocortisone
acetate was more effective. It was definitely
more effective than cortisone acetate in
maintaining the body weight of the mother.
However, reproduction was completely restored to normal in the adrenalectomized
rat following treatment with desoxycorticosterone acetate and cortisone acetate.


Interference with gestation in the normal
animal has been reported by several investigators following treatment with ACTH
or adrenal corticoids (Courrier and Colonge, 1951; Robson and Sharaf, 1952;
Velardo, 1957). This is taken to indicate
that there is a finely balanced requirement
for adrenocortical hormones during gestation ; and that suboptimal or supra-optimal
amounts of the hormone interfere with
pregnancy. Courrier and Colonge found
that cortisone administered to intact rabbits in the second half of pregnancy interfered with gestation. Robson and Sharaf
treated both pregnant rabbits and mice
with ACTH and reported a marked effect
on gestation. Abortion or resorption occurred in 8 of 9 mice and in 8 of 11 rabbits.
Contamination by posterior pituitary hormones or gonadotrophins can be excluded.
A subsequent experiment with cortisone
also caused marked interference with pregnancy in the rabbit when 20 mg. were given ;
10 mg. were without effect. Administration
of cortisone to castrated or hypophysectomized pregnant rabbits maintained with
progesterone also caused damage to the
pregnancy. Since the hormone was not acting by way of the ovary or pituitary gland,
the authors felt that cortisone was acting
directly on the uterus and the uterine contents.


In the rat, however, ]Meunier, Duluc and
Mayer (1955) observed an effect on pregnancy only when cortisone acetate was injected at the time of mating. Rats injected
with 10 to 25 mg. cortisone acetate daily for 5 to 6 days beginning on day 12 or day
14 of gestation had a normal pregnancy.




Velardo (1957) reinvestigated the problem in the rat and reported a marked reduction in litter size and an increase in the
number of stillborn following ACTH treatment. Although quantitative differences
appeared, a significant decrease in litter
size w^as observed only when the hormone
was given (1) before mating, (2) immediately after mating, or (3) between the 11th
and 15th day after mating. However, the
greatest effect was noted when the ACTH
was administered immediately after mating.
Surprisingly enough, litter size was markedly reduced only if adrenalectomy was
performed on day 7 of gestation. Adrenalectomy on day 8 to 14 of gestation had no
effect on live litter size. However, a total
of 6, 9, and 13 stillbirths were obtained
following adrenalectomy on days 8, 9, and
11. It is interesting that the number of stillbirths decreased from 21 following adrenalectomy on day 7 to none following adrenalectomy on day 14. It is apparent that
the adverse effects of adrenalectomy on
gestation decrease as pregnancy progresses.
It is also apparent from these and other
experiments that the action of ACTH is
mediated by the adrenal cortex. From these
results and others described above, it seems
likely that the adrenal corticoids may be
acting on the uterus.


ccA
Mayer and Duluc (1955) found that
adrenalectomy of the I'at on the 14th to
the 16th day of pregnancy led to variable
results. In 17 pregnant adrenalectomized
rats, gestation was terminated in 8, but
no interference was observed in 9. The rats
that failed to maintain pregnancy died
witiiin 2 to 3 days. Again it would appear
that delicate hormonal balances are involved. In a further investigation of this
problem Aschkenasy-Lelu and Aschkenasy
( 1957) reported that a diet adequate in salt
and proteins would prevent interference
with pregnancy in rats adrenalectomized
before mating. On a low protein diet, pregnancy could be maintained only in the intact rat (80 per cent) and then only if
daily injections of progesterone were given.
These authors believe that the role of the adrenal corticoids in pregnancy is concerned with stimulation of appetite and
mobilization and degradation of proteins
to amino acids. The latter action would
permit the replacement of body protein in
the absence of a normal jirotcin intake.




===C. Pancreas===


The impact of diabetes mellitus on the
course of pregnancy has been of interest
to the clinician for many years. In a recent
review of the subject, Reis, DeCosta and
Allweiss (1952) came to the conclusion that
"the carefully controlled diabetic aborts
no more frequently than the nondiabetic."
On the other hand, it has been well known
for many years that uncontrolled diabetes
and pregnancy are basically incompatible
(Eastman, 1946).


Studies in the rat have given controversial results with regard to the influence
of insulin on pregnancy. Davis, Fugo and
Lawrence (1947) reported that in the alloxan diabetic rat pregnancy was normal for
the first 12 days. Thereafter death of the
fetuses occurred followed by resorption.
Sinden and Longwell (1949) and Levi and
Weinberg (1949) reported no detrimental
effect from diabetes on the course of i^regnancy. The latter group obtained 12 pregnancies from 25 rats made permanently diabetic with alloxan. Eleven of the 12 rats
went to term and delivered normal fetuses
and 1 died during pregnancy. Recently,
Wells, Kim, Runge and Lazarow (1957)
reported a 14 per cent loss in fetal weight,
an increase in gestation length from a normal of 538 to 563 hours, and an increase in
fetal or neonatal mortality in the pregnant
rat made diabetic by pancreatectomy or
treatment with alloxan.


In general, the clinical data indicate that
uncontrolled diabetes has a detrimental effect on pregnancy, but that the abortion
rate in the controlled diabetics approaches
that seen in the ''normal" population. Since
the crux of the matter seems to hinge on the
severity of the diabetes, one might conclude
that the effect of insulin is an indirect one
by virtue of its action in maintaining a good
metabolic state. The conflicting reports
from animal experimentation may be due to the differences resulting from uncontrolled environmental and dietary factors.




===D. Ovary: Progesterone, Estradiol, and Relaxin===


Marshall and Jolly (1905) were probably
the first to point out that ovariectomy during pregnancy leads to abortion or resorption of the fetuses in the rat. Subsequently,
a number of investigators repeated these
experiments and confirmed the findings in
all species tested thus far, provided ovariectomy is performed before implantation. Removal of the ovaries after gestation is well
under way, however, does not disturb the
course of pregnancy in all species. The human being, monkey, horse, ewe, and cow are
examples of species not dependent on the
ovary for the maintenance of pregnancy
once it has been well established. Species
such as the rabbit and the rat require the
presence of the ovary throughout pregnancy.


lU
The importance of progesterone for i)regnancy was established by Allen and Corner
 
(1929) who first showed that an extract
 
of the corpus luteum will maintain pregnancy in the castrated rabbit. Identification
 
of the active substance in the extract as
 
progesterone led to the use of the hormone
 
in many other species. Allen (1937) reported
 
that crystalline progesterone was inferior
 
to the crude luteal extract in the maintenance of pregnancy in the castrated rabbit.
 
From these and other data, such as the enhancing action of estrogen on the progesterone-induced progestational reaction, he
Q.  
inferred that a combination of estrogen and
 
progesterone should be superior to progesterone alone in the maintenance of pregnancy. However, he pointed out with proper
 
caution that the dosages would have to be
 
carefully regulated because estrogen could
 
also antagonize progesterone. Although Robson (1936) failed to enhance the action of
 
progesterone with estrone in the pregnant
 
hypophysectomized rabbit, Pincus and
 
Werthessen (1938) obtained enhancement
 
with both the androgens and estrogen.
3
Whereas the early work indicated that a
 
pregnancy maintenance dose of progesterone varied from 0.5 to 2 mg. (Allen and
 
Corner, 1930), later experimentation indicated that the dosage varied with the stage
/
of pregnancy. An adequate dose of approximately 1 mg. progesterone in the early
 
stages of pregnancy needs to be increased
 
to 5 mg. in the later stages (Allen and
 
Heckel, 1939; ComTier and Kehl, 1938a, b).
These investigators also revealed that an
optimal effect could be obtained by using a
progesterone-estrogen combination in the
ratio of 750 to 1. Chang (1951) transferred
ova to nonovulated intact rabbits and noted
that massive doses in the order of 25 mg.
macrocrystalline progesterone injected for
three times were required to obtain a 50 per
cent maintenance of pregnancy. He also reported that under the conditions of his experiment an initially high dose was needed
for the passage of the ova, implantation,
and early maintenance. Since then, further
experimentation, especially on other species, has revealed a significant role by estrogen in enhancing the pregnancy-maintaining action of progesterone.
 
A vast literature exists for the human
being on the prevention of threatened abortion by progesterone which is beyond the
scope of this review. Variation from negative results to excellent maintenance is reported. It is obvious that a great deal of
variability exists here and, to some extent,
this is explained by a need for more objective criteria in evaluating threatened abortion and the therapy (Guterman and Tulsky, 1949). It is obvious that if the
 
TABLE 16.12
Maintenance of pregnancy in the rat castrated on


the 12th day of gestation


(From J. Yochim and M. X. Zarrow, Fed. Proc,


iA
18, 174, 1959.)




Line 4,870: Line 6,365:




Progester






t




Line 4,881: Line 6,376:




§2




o/


one


<




>




1




G i e ,








L


No.




12 15 18 21 24


DAYS AFTER MATINO


Estradiol
Daily




27
Implantation
Site




No. of
Fetuses


30


No. of


Fetuses


35
Alive




Preg


Fig. 16.12. Concentration of relaxin in the blood of the rabbit during pregnancy. Parturition (P) occurred 32 days after mating. Guinea pig units (G.P.U.) of relaxin are plotted
Rats
against days pregnant. (From S. N. Marder and W. L. Money, Endocrinology, 34, 115,
1944.)




Daily
dose


GESTATION


No.
daily




975.
nancy
Index








6 7 8 9
mg.


• — Pregnancy (in months)






2 6 10 15
Mg.


After parturition (in days) — *






Fig. 16.13. Concentration of I'elaxin in the blood of the cow during pregnancy. Partiuition is indicated by P. (From H. Wada and M. Yuhara, Jap. J. Zootech. Sc, 26, 12, 1955.)








6 12 18 24 30 36


LENGTH OF PREGNANCY - WEZKS


4




HOURS POSTPARTURITON






Fig. 16.14. The concentration of relaxin in the blood serum of normal pregnant women.
(From M. X. Zarrow, E. G. Holmstrom and H. A. Salhanick, Endocrinology. 15, 22. 1955.)






hours postpartum. Studies in the guinea pig
40
revealed a marked rise in relaxin on day
 
21 of gestation to a maximal concentration
 
of 0.5 G.P.U. per ml. serum on day 28
37
(Zarrow, 1948). Thereafter the level remained unchanged for approximately 4
weeks. Contrary to the results obtained in
the rabbit, cow, and human being a drop in
the concentration of the hormone in the
pregnant guinea pig was noted before parturition. The concentration of relaxin fell




37


to 0.33 G.P.U. per ml. on the 63rd day of
gestation and then dropped to nondetectable levels within 48 hours postpartum.


Although no studies have been carried
0.925
out on the blood levels of relaxin in the sow
as a function of the length of pregnancy,
analysis of the ovary for relaxin has revealed a situation comparable to that reported for the blood in other species. The
concentration rose from 5 G.P.U. per gm.
ovarian tissue during the luteal phase of




4


976


2




SPERM, OVA, AND PREGNANCY
1






the cycle to approximately 10,000 G.P.U.
per gm. fresh ovarian tissue by the time a
fetal length of 5 inches had been reached
(Hisaw and Zarrow, 1949).


E. SOURCES OF RELAXIN
47


The ovaries, placentas, and uteri are
possible sources of relaxin in different species. It seems from the extremely high concentration in the ovary of the sow during
pregnancy that this organ is the major site
of relaxin synthesis at this time. However,
studies on other species indicate that both
the placenta and uterus may be involved.


Treatment of castrated, ovariectomized
32
rabbits with estradiol and progesterone
stimulated the appearance of relaxin in the
blood of the rabbit as indicated by the
ability of the blood to induce relaxation of
the pubic symphysis of estrogen-primed
guinea pigs (Hisaw, Zarrow, Money, Talmage and Abramovitz, 1944). Similar experiments on castrated, hysterectomized
rabbits failed to reveal the presence of the
hormone in the blood of the treated animals. Treatment with estradiol alone also
failed to stimulate the release of relaxin.
It is obvious then that, if the bioassay is
specific for relaxin, the uterus is a definite
source of this hormone. Comparable results
were also obtained in the guinea pig (Zarrow, 1948). Treatment with estradiol and
progesterone caused pubic relaxation and
the presence of relaxin in the l)lood after
approximately 3 days of treatment with
progesterone. In the absence of the uterus
relaxin was not demonstrable in the blood.


The concentration of relaxin in the blood
of the rabbit castrated the 14th day of i^regnancy and maintained with progesterone
remained unaffected by removal of the
ovaries, provided the pregnancy was maintained (Zarrow and Rosenberg, 1953). Figure 16.15 shows a typical curve for the
relaxin content of the blood of such an
animal. The concentration of the hormone
rose between days 12 and 24 to a maximal
concentration of 10 G.P.U. per ml. and was
maintained till the time of normal parturition. It is of interest that in those instances
in which the placentas were not maintained
in good condition, the concentration of the
hormone fell. Analysis of the reproductive


27




tract revealed concentrations of 5 G.P.U.
0.574
per gm. fresh ovarian tissue during pseudopregnancy and approximately 25 G.P.U.
during the last trimester of gestation. The
uterus contained 50 G.P.U. per gm. fresh
tissue during pseudopregnancy and an
equal concentration the first 24 days of
pregnancy. The 26th day of pregnancy the
concentration fell to 15 G.P.U. per gm.
The highest concentration was in the placenta which contained from 200 to 350
G.P.U. per gm. Some evidence indicated
that after treatment with estradiol minimal
amounts of relaxin, i.e., 5 G.P.U. per gm.,
were present in the vaginal tissue (Table
16.5).  


F. ADRENAL CORTEX


1. Hydrocortisone
7


Initial studies on the possible role of the
adrenal cortex in gestation involved the
determination of the two urinary metabolites of the gland, i.e., the 17-ketosteroids
and the corticoids. Inasmuch as the 17-ketosteroids are believed to be associated with
the androgenic activity of the adrenal cortex,
bioassays for adrenogenic activity in the
urine were carried out. Dingemanse, Borchart and Laqueur (1937) found no increase
in urinary androgen by the 6th to the 8th
month of pregnancy whereas Hain (1939)
reported that pregnant women secreted even
less androgen than nonjircgnant women.
Pincus and Pearlman (1943) found no
change in the urinary 17-ketosteroids of
the pregnant and nonpregnant woman although Dobriner (1943), by the use of
chromatograi)hic separation, showed a
marked decrease in androsterone. Venning
(1946) found no change in the urinary
ketosteroids as measured by the antimony
trichloride reagent described by Pincus
(1943), but the ketosteroids measured by
the Zimmerman reagent (dinitrobenzene)
showed a significant rise in the latter part
of pregnancy. The discrepancy between the
two determinations can be explained by the
fact that other ketonic substances besides
17-ketosteroids give a color in the Zimmerman reaction. These are the 20-ketosteroids
and to a limited extent the 3-ketosteroids.
V^enning (1946) believes most of this in


1


GESTATION


2




977




76


RABBIT N0.80


12




2


■e-e


0.026


15
AFTE R


9




21
1.5




2


3:^






J3
99




61


MATING


49




9 12
0.495


DAYS


Fig. 16.15. Concentration of relaxin in the blood of a pregnant rabbit castrated the 14th
7
day of gestation and maintained with 4 nig. progesterone daily until the 32nd day. Postmortem examination revealed 8 placentas and 2 dead fetuses. (From M. X. Zarrow and B.
Rosenberg, Endocrinology, 53, 593, 1953.)




2


TABLE 16.5


Relaxin content of the blood serum and tissue of the reproductive tract of the rabbit
2


(From M. X. Zarrow and B. Rosenberg, Endocrinology, 53, 593, 1953.)






85




No. of Rabbits
65




Relaxin Concentration in G.P.U.
63




Treatment
0.741




Per ml. serum
6




Per gm fresh tissue
1




2




Ovary
0.1




Uterus
69




Placenta
50
whole




Placenta
48
fetal




Placenta
0.696
maternal




Pseudopregnant
5


Chorionic gonadotrophin . . .


1.5


3


4
3
2
2  
2  


2


1  
0.1  
 


51


0.2-0.3
0.2
1.0
10.0
10.0
10.0
10.0


48


5
5
30
25
20
25
25


46


50
50
50
50
30
15


0.900


75
50
50


75
5




10
2
20
25




2




Pregnant 24 davs
0.1




250
60




Pregnant 25 days
55


Pregnant 26 days


54


350
200


0.900


Pregnant 28 days




threatened abortion were the result of some
disturbance other than progesterone, that
progesterone therapy might be without success. Indirect evidence for the need for
progesterone to maintain a successful pregnancy in the human being and for the lack
of need for the corpus luteum once pregnancy is established has been presented by
Tulsky and Koff (1957). Corpora lutea
were removed from day 35 to day 77 of
pregnancy in 14 women. Two of the women
exhibited spontaneous abortion and a
marked drop in pregnanediol excretion. The
remaining 12 maintained a normal pregnancy and pregnanediol excretion. The data
can be interpreted to indicate a need for
progesterone during pregnancy and that
this need can be met by a nonovarian
source, i.e., the i)lacenta.




In both the rat and mouse, successful
maintenance of pregnancy after castration
has been obtained with progesterone or a
combination of progesterone and estrogen.
However, partial maintenance following
castration can be obtained in the rat under
special circumstances. Haterius (1936) removed all the fetuses except one and left all
placentas intact. Under these conditions the
remaining fetus was carried beyond term.
Alexander, Fraser and Lee (1955) found
that castration of the rat on the 9th day
resulted in 100 per cent abortion, whereas
60 per cent of the fetuses were retained until
term if castration was on the 17th day.
Dosage of progesterone as high as 5 to 10
mg. daily following castration the 9th day
gave only partial maintenance. It is possible
that better results would have followed
multiple daily injections. Yochim and Zarrow (1959) castrated rats on day 12 of gestation and obtained a pregnancy index (no.
of fetuses alive at day 20 h- no. of implantation sites at day 12) of 0.741 when 2 mg.
progesterone were gi^'en in two divided
daily doses and 0.495 when 1.5 mg. progesterone was given (Table 16.12). However,
the addition of 0.1 /^.g. estradiol daily
markedly enhanced the action of the progesterone so that a pregnancy index of 0.9, i.e.,
equivalent to the normal controls, was obtained with 1.5 mg. progesterone.


•978
Finally, Hall (1957) has indicated that
relaxin synergizes with estradiol and progesterone in the maintenance of pregnancy in the castrated mouse. One nig. progesterone per day maintained pregnancy in 83
per cent of the mice castrated on day 14 of
gestation, but 0.5 mg. maintained pregnancy in only 30 per cent of the animals.
The addition of 1.5 ^g. estradiol per day
was without effect. On the other hand, the
addition of relaxin to the estradiol and 0.5
mg. progesterone gave pregnancy maintenance in over 80 per cent of the mice as
compared with 30 per cent when progesterone alone was given.


Smithberg and Runner (1956) induced
ovulation and mating in prepubertal mice
(age 30 to 35 days) and obtained 100 per
cent implantation with 0.5 to 1 mg. progesterone daily and approximately 90 per cent
successful pregnancies when 2 mg. progesterone were given. A comparison of the
amount of progesterone required for maintenance of pregnancy in the normal and
castrated prepubertal mouse is given in Figure 16.34. In an interesting application of
the information available on the induction
of ovulation and maintenance of pregnancy,
Smithberg and Runner (1957) were able to
obtain successful pregnancies in genetically
sterile, obese mice.


Haterius (1936) observed that distortion
of the fetus occurred following ovariectomy
in the rat. This has been confirmed by
Zeiner (1943) in the rat and by Courrier
and Colonge (1950) in the rat and rabbit.
It was noted that castration greatly compressed the fetuses and eventually caused
death. Courrier and Colonge (1950) in very
elegant experiments showed that removal of
the rabbit fetus into the peritoneal cavity
prevented the distortion and death which
ordinarily followed castration. Frazer
(1955) obtained similar results in the rat
and concluded that fetal death after castration of the mother follows a rise in intrauterine pressure which is associated with an
increased tone of the circular uterine muscle fibers. Consequently the increased survival of the extra-uterine fetuses following
ovariectomy in the mother is the result of
the removal of this pressure by the circular
muscle of the uterus.


SPERM, OVA, AND PREGNANCY




Fig. 16.34. Daily dose of progesterone required
to maintain pregnancy in the normal and castrated prepubertal mouse. (From M. Smithberg
and M. N. Runner. J. Exper. Zool., 133, 441,
1956.)


crease in ketosteroid excretion during pregnancy is the result of increased output of  
Many investigators have demonstrated
the stereoisomers of pregnanolone:
that gestation can be prolonged by inhibiting parturition. Both the injection of large
doses of progesterone or the formation of a new set of functional corpora lutea during
pregnancy will prevent parturition. The injection of an ovulating dose of HCG on the
25th day of pregnancy in the rabbit delayed
parturition for 15 days after the injection,
i.e., until the 40th day of gestation (Snyder,
1934). The fetuses survived in utero for
only 3 days and grew to greater than normal size during this period. The placentas
persisted until day 41 of gestation. Comparable results were obtained following
daily injections of progesterone into pregnant rabbits (Zarrow, 1947a). Haterius
(1936) obtained prolongation of pregnancy  
in the castrated rat by removing all the
fetuses except one, leaving all placentas
intact. Recently a comparable experiment
was performed in tlie rabbit with intact
ovaries (Hafez, Zarrow and Pincus, 1959).
In 2 of 10 rabbits, live fetuses were obtained
l)y cesarean section on day 36. However, in
8 of the 10, delivery was delayed beyond
day 36, although some degree of fetal resorption was present in all instances. Prolongation of pregnancy in the rat was obtained by the injection of prolactin (Meites
and Shelesnyak, 1957), but only if the ovaries were present.


Measurement of urinary glucocorticoids
by the glycogen deposition test showed an
initial increase in the first trimester of
pregnancy in the human being. After the
initial rise, the urinary excretion level returned to normal with a second increase the
140th to 160th day of pregnancy. Values
of 200 to 300 /xg. equivalent of 17,hydroxy11-dehydrocorticosterone per 24 hours of
urine were obtained at days 200 to 240. In
most instances the urinary outj^ut fell several weeks before parturition.
' Analysis of the blood levels for 17ahydroxycorticosterone in the jiregnant wo


===E. Pituitary Gland===


man confirmed the results obtained with
In general, hypophysectomy before midpregnancy leads to resorption. This is especially true of the rat and mouse. On the other hand, hypophysectomy at midpregnancy or later does not interfere in the
the urine (Gemzell, 1953; Seeman, Varangot, Guiguet and Cedard, 1955). Gemzell
maintenance of gestation in these species
( 1953) reported a rise from approximately
(Pencharz and Long, 1933; Selye, Collip
5 /xg. per 100 ml. plasma to an average of
and Thompson, 1933a, b; Pencharz and  
approximately 22 fxg. per cent (Fig. 16.16).  
Lyons, 1934 ) . In the dog, ferret, and rabbit,
A further rise to 36 /xg. per cent was noted
hypophysectomy leads to abortion (Aschner, 1912; McPhail, 1935a; White, 1932),  
at the time of labor. This has been confirmed by McKay, Assali and Henley
whereas the results in the cat seem contradictory (Allan and Wiles, 1932; McPhail,
(1957) who found an average rise of approximately 40 /xg. per cent during labor
1935b) .  
lasting more than 6 hours. Although McKay, Assali and Henley reported values
still well above normal on the 4th to 6th
day postpartum, Gemzell (1953) reported
a drop to 1.99 /xg. per cent on the 6th day
postpartum.  


Hypophysectomy of the rhesus monkey
does not always interfere with pregnancy.
Smith (1954) obtained normal pregnancies
in 10 of 20 hypophysectomized rhesus monkeys. The remaining animals aborted. Although more data are needed, it seems that
the pituitary gland can be removed very
early in gestation without disturbing the
pregnancy. Whereas hypophysectomy before midterm invariably leads to abortion
or resorption in the rat or mouse, 1 of the
4 monkeys hypophysectomized between the
29th and 34th day of gestation carried its
young to term. Inasmuch as Hartman and
Corner (1947) showed that the placenta secretes sufficient progesterone by the 25th
day of gestation to maintain pregnancy, it
is apparent that the placenta in the monkey
is able to maintain its endocrine secretory
activity independent of the pituitary and
at a sufficiently high level to replace the
ovary.


Little, Smith, Jessiman, Selenkow, van't
Hoff, Eglin and Moore (1958) reported a
successful pregnancy in the 37-year-old
woman hypophysectomized the 25th week
of pregnancy. The mother w^as maintained
on thyroid, cortisone, and pitressin tannate
replacement therapy. The excretion of chorionic gonadotropin and pregnandiol was
not markedly different from that seen in
normal gestation. Estrogen excretion was
slightly reduced and the 17-hydroxy corticosteroids dropped to zero when cortisone
therapy was discontinued. It would seem
that this phase of adrenocortical activity
was reduced and that ACTH or corticoidlike substances from the placenta were inadequate. No interference in aldosterone
output was observed.


E
Hypophysectomy on the 10th day of
 
gestation in mice terminated the pregnancy in only 3 of 19 animals (Gardner and Allen,
o
1942). Sixteen mice carried their litters to
o
term although 7 of the 16 had a difficult
 
and prolonged parturition. Body weight
curves were normal and the corpora lutea
appeared unaffected by the loss of the pituitary gland, indicating either the independence of the corpus luteum or the presence
of a placental luteotrophin. Marked involution of the adrenal cortex was noted in all
instances.


Simultaneous measurements of the concentration of cholesterol in the adrenal
gland and ACTH in the pituitary of the rat
revealed a drop in adrenal cholesterol and
pituitary ACTH on the 15th day of gestation (Poulton and Reece, 1957). This was
followed by a marked increase of both substances on the 21st day of pregnancy and a
sharp drop at parturition. The authors concluded that a gradual increase occurs in
the secretory activity of the adrenal cortex
which reaches a peak on the 15th day of
pregnancy in the rat. Thereafter the activity decreased until parturition when a
marked increase was observed. The initial
decrease in pituitary ACTH potency followed by an increase after day 15 is interpreted as an initial increase in ACTH release followed by a decreased release. The
decrease in pituitary ACTH potency at
parturition is compatible with the marked
increase in adrenocortical activity at this
time if the decreased pituitary ACTH activity is interpreted as indicative of ACTH
release.


20
Maintenance of pregnancy in rats hyl')ophysectomized early in pregnancy was
obtained with prolactin by Cutuly (1942),
although Lyons, Simpson and Evans (1943)
reported negative results with a purified
prolactin. However, a partial maintenance
of pregnancy was obtained with purified
prolactin and estrone.




Portus
===F. Placenta===
©36t2,7


(n=J7)  
The placenta is not only involved in the
synthesis of hormones during pregnancy but
also in the transfer of substances between
mother and fetus. It is obvious that the
transfer of substances is limited and the
l^lacenta does offer a barrier. This problem
bears not onlv on the matter of fetal nutrition, but also on the fetal environment
and as such is important in the sexual development of the fetus (see chapter by
Burns) .




The presence of estriol in the urine of
newborn male infants has led to the conclusion that estrogens can pass through the
placenta because of their low molecular
weight (Diczfalusy, Tillinger and Westman,
1957). Studies on the transfer of estrogens
across the placental barrier in the guinea
pig with C^'^-labeled estradiol revealed an
extremely rapid disappearance of radioactivity from the maternal blood following
intravenous injection of the hormone into
the mother, and the appearance of large
amounts of water-so.luble radioactivity in
the fetal plasma (Dancis, Money, Condon
and Levitz, 1958). However, no estradiol
was found in the fetal plasma. Replacement
of fetal circulation with a perfusion system
indicated that estradiol did not j^ass the
placenta although estriol was readily transferred in both directions. These authors reported that the placenta was relatively impermeable to the water-soluble estrogens
found in the urine, wliich are essentially
glucuronides.


The discovery in 1927 of large amounts
of estrogens and gonadotrophins in the
blood and urine of pregnant w^omen led to
the cjuestion as to whether the placenta is
a gland of internal secretion. This can be
answered with an uneciuivocal yes. Nevertheless, several questions are still unanswered: (1) the number of hormones produced by the placenta, (2) the quantities,
and (3) the secretory activity of the placenta in different species.


Data on the presence of gonadotrophins
in the placenta have already been discussed.
At least three different types of gonadotrophins have been extracted from the placentas of the human being, mare, and rat.
These have been defined physiologically and
appear to be different in the three species.
Cole and his co-workers have identified the
endometrial cups as the source of PJVIS in
the mare, whereas the elegant experiments
of Stewart, Sano and Montgomery (1948)
indicate that HCG in the human being is
secreted by the Langhans cells. These investigators grew human placental cells in tissue culture and obtained ^ gonadotrophin
in the culture. They also noted a direct correlation between the growth of the Langhans cells and the production of gonadotrophic hormone (see also the discussion of
this subject in the chapter by Wislocki and
Padykula).


The initial discovery of a progressive rise
in the secretion of adrenal corticoids in
pregnancy (Venning, 1946) has been confirmed by numerous investigators. Gemzell
( 1953) attributed the steady rise to a stimulation of the adrenal glands by excessive
amounts of estrogen present during pregnancy and to hyperactivity of the fetal
adrenals. The hypertrophy of the fetal adrenal cortex in the rat following adrenalectomy of the pregnant mother was first reported by Ingle and Fisher in 1938 and
confirmed by Walaas and Walaas (1944),
and Knobil and Briggs (1955). However,
the 17-ketosteroid and corticoid level of
fetal urine is very low (Day, 1948; Jailer
and Knowlton, 1950) as are the 17-hydroxycorticosteroids in the blood of the newborn
infant (Klein, Fortunato and Papados,
1953). ACTH-like activity has been found
in extracts of the placenta (Jailer and
Knowlton, 1950; Tarantino, 1951; Opsahl
and Long, 1951) and corticoid activity has
been found in the placenta of horses and
human beings, as demonstrated by the glycogen deposition and growth-survival test
in adrenalectomized rats (Johnson and
Haines, 1952). Berliner, Jones and Salhanick (1956) isolated 17a-hydroxy corticoids from the human placenta.


Pincus (1956) reported that ACTH can
stimulate steroidocorticogenesis in the perfused placenta. Using the ascorbic acid depletion test, Assali and Hamermesz (1954)
assayed the blood in the intervillous space
and the chorionic villous tissue for ACTH.
Good activity was observed in the blood
from the intervillous spaces and in the tissue of the chorionic villi. Corticotrophic activity was also obtained by Lundin and
Holmdahl (1957) from placentas obtained
at full term, but the activity was small compared with that obtained from the pituitary
gland.


•
The possible role of the fetal pituitary
 
was investigated by Knobil and Briggs (1955) who noted that hypophysectomy of
 
the mother prevented the fetal adrenal
1
weight increase observed following adrenalectomy of the pregnant mother. However,
complete atrophy of the adrenal gland was
not observed in the pregnant mother if the
conceptus was present. It was concluded
that ACTH can cross the placental barrier
and that the fetus or placenta or both produce a sufficient amount of ACTH, to influence the maternal adrenal gland in the
absence of the maternal hypophysis. It is
still questionable, however, whether these
sources, i.e., placenta and fetal pituitary,
are of sufficient magnitude to account for
the increased release of adrenal corticoids.
Hofmann, Knobil and Caton (1954) showed
that the ability of the hypophysectomized
nonpregnant rat to secrete a water load is
not greater than that of the hypophysectomized pregnant rat. Hence the contribution
of the fetal pituitary or j^lacenta to the
corticoid pool is not of sufficient magnitude
to influence water balance.




As with the gonadotrophins, the increased
amounts of estrogen ancl pregnanediol during pregnancy were thought to be derived
from the placenta. In 1933, Selye, Collip
and Thompson presented evidence to indicate that the placentas of rats jiroduce both
estrogen and gestagen. Many physiologic
data have been accumulated to prove this
point, but completely convincing evidence
was obtained only when these hormones
were identified in placental extracts and in
fluid perfused through the placenta. Diczfalusy and Lindkvist (1956) identified estradiol in the placenta and the presence of
progesterone was described by Salhanick,
Noall, Zarrow and Samuels (1952) and by
Pearlman and Cerceo (1952).


Perfusion experiments on human placentas have revealed that this organ secretes a
number of steroids (Pincus, 1956). These
include progesterone, desoxycorticosterone
Cortisol, and a number of unidentified steroids. Addition of ACTH to the perfusate
had no effect on the concentration of Cortisol, but it did increase the concentration of
the reduced corticosteroids, namely, the
tetrahydro derivatives of cortisone and Cortisol. This was interpreted as a stimulation
of the placenta by ACTH resulting in an increased release of the corticoid as demonstrated by the increase in the degradation
products.


•
The identification of the placenta as a
 
source of both sex steroids and certain gonadotrophins clarifies the manner by which
 
jiregnancy can be maintained in certain
•
species in the absence of the pituitary
, '
gland or ovary (see sections above on ovary
 
and pituitary gland). Newton and Beck
 
(1939) and others showed the hypophysectomy of the pregnant mouse does not precipitate abortion. Studies of the ovary reveal that, if the placentas are retained, the
^
corpora lutea remain normal but removal
 
of the placentas causes immediate degeneration of the corpora lutea (Deanesly and
 
Newton, 1940). A comparable situation appears to exist in the rabbit and rat ; it is assumed, therefore, that the placenta takes
y^
over control of the corpus luteum in pregnancy in those species that require the
 
ovary for successful gestation. In other species, such as man, sheep, cattle, and guinea
 
pig, it seems that the placenta can supplant
the ovary after pregnancy has progressed
to a certain stage.




===G. Pelvic Adaptation===


The discovery that pelvic changes are
under hormonal control in certain species
was the result of extensive studies on pelvic
adaptations associated with parturition (see
reviews by Allen, Hisaw and Gardner, 1939;
Hisaw and Zarrow, 1951). It has been argued that, in general, a narrow pelvis is
present in mammals living in burrows. This
would have the advantage of permitting an
animal to turn within narrow confines, but
a narrow pelvis would also interfere with
the delivery of the young at parturition. As
Hisaw pointed out in his extensive studies,
this problem has been met by special adaptations on the part of different species. This
has varied from a resorption of the cartilaginous pubic arch in the male and female
mole iScalopiis aquaticus machrinus, Raf.)
which is independent of the endocrine system (Hisaw and Zilley, 1927) to elongation
of the pubic ligament which is directly under hormonal control (Hisaw and Zarrow,
1951).


The symphysis pubis of the pocket gopher, Geomys bursarius (Shaw), behaves as
a female secondary sexual character so that a sex dimorphism exists in this species. The
pubic cartilages ossify in both sexes and
unite to form a complete pelvis with a rigid
symphysis pubis. At this stage, the pelvis is
too small for the passage of the young, but
with the first estrus in the female, the pubic
bones are gradually resorbed, leaving the
pelvis open ventrally. The pelvis in the male
remains intact (Hisaw, 1925). Treatment
with estrogen alone can readily bring about
the resorption of the pubic bones.


A third type of adaptive mechanism has
been described in great detail in the guinea
pig and led to the discovery of the hormone,
relaxin. A sex dimorphism of the pelvis exists in the guinea pig, as in the pocket gopher, but in addition parturition is further
facilitated by marked relaxation of the
pubic ligaments and of the sacroiliac joint.
Thus far extensive pelvic relaxation has
been described in the guinea pig (Hisaw,
1926, 1929 », mouse (Gardner, 1936; Newton
and Lits, 1938; Hall and Newton, 1946a),
women (see review by Hisaw and Zarrow,
1951), and rhesus monkey (Straus, 1932;
Hartman and Straus, 1939). No relaxation
of the pubic symphysis has been reported in
the ewe but a relaxation of the sacroiliac
joint and an elongation of the sacrosciatic
ligament was noted the 2nd to 3rd month of gestation. These changes increased as pregnancy progressed (Bassett and Phillips,
1955). Treatment with stilbestrol alone
caused a marked loosening of the sacroiliac
joint and the sacrosciatic ligament. The addition of relaxin to the treatment was without effect (Bassett and Phillips, 1954).


The role of relaxin in the relaxation of
the pubic symphysis has been studied most
extensively in the guinea pig and mouse.
The work before 1950 was reviewed by Hisaw and Zarrow in 1951. The controversies
(de Fremery, Kober and Tausk, 1931 ; Haterius and Fugo, 1939) as to whether such
a hormone exists need not be discussed here,
in detail, except to point out that the evidence supporting this opinion is more than
adeciuate. Zarrow ( 1946, 1948) showed that
pubic relaxation could be induced by estradiol alone, by a combination of estradiol and progesterone, or by relaxin in
an estrogen primed animal (Table 16.13).
The difference in the time required to induce relaxation, i.e., 23 days for estrogen
alone, 13 days for estrogen and progesterone,
and 6 hours for relaxin, and data indicating
that progesterone caused the presence of
relaxin in the blood of guinea pig only if a
uterus was present led to the concept that
pubic relaxation may be produced independ




•
TABLE 16.13


Relaxation of the symphysis pubis and relaxin content of blood, urine, and uteri of castrated and castrated,


•
hysterectomized guinea pigs after treatment with moderate doses of estradiol and progesterone


(From M. X. Zarrow, Endocrinology, 42, 129, 1948.)






•




Treatment, Daily




Average Relaxation Time




10
Relaxin Content




No. of Guinea Pigs




Estradiol




Progesterone




Total


^


After progesterone
treatment


^


Blood serum


•


Urine




Uterus


•




•


MS


•
mg.








days




•
days




G.P.U./ml.




G.P.U./ml.




•
G.P.U./gm.




Castrated
9«




10




1 from day




11




13.5


(13-14)


^/^


3.5


•


0.5




0.3




10




10




10




2 from day




11




13.0




3




0.5




0.5




10




10




10






,






(12-14)


^
23.7
(16.31)








^
Negative at
4 ml.




•
Negative at
5 ml.




Negative




Castrated, hys




•




terectomized
11




10




1 from day




11




#
23.7
(17-30)




13.7




Negative at
4 ml.




Negative at
8 ml.








10




10




Line 5,512: Line 7,259:




•
25.6
(18-32)




.




•
Negative at
4 ml.




Negative at
4 ml.








•


One guinea pig not included in the table refiuired 22 days of treatment for pubic relaxation.






ently by estradiol (prolonged treatment) or
relaxin (single injection). It is also possible
to conclude that the action of progesterone
is indirect and due to the formation of relaxin in the uterus (Zarrow, 1948; Hisaw,
Zarrow, Money, Talmage and Abramovitz,
1944) . In the mouse, however, progesterone
inhibits the action of relaxin on the pubic
symphysis (Hall, 1949).


Further evidence that two hormones are
involved in pubic relaxation was provided
by histologic examination of the pubic ligament. Symphyseal relaxation following estrogen appeared to be due to a resorption of
bone and a proliferation of loose fibrous
connective tissue with an increase in mucoid
alkaline phosphatase and water content
(Talmage, 1947a, 1947b, 1950; Heringa and
van der Meer, 1948). Relaxin produced a
breakdown and splitting of the collagenous
fibers into thin threads and a similar change
was noted with progesterone (Talmage,
1947a, 1950).


Histochemical and biocliemical studies of
the pubic symphysis have recently been reviewed (Frieden and Hisaw, 1933) and tend
to show that relaxin produces specific
changes. These include loss of metachromasia (Heringa and van der Meer, 1948) ,
accumulation of Evans blue m vivo, and increased solubility of the glycoproteins in
the McManus-Hotchkiss reaction, all of
wiiich indicate that a depolymerization of
the ground substance and basement membrane glycoproteins had occurred (Perl and
Catchpole, 1950) . Frieden and Hisaw (1951)
found an increase in water content of the
symphyseal tissue, but failed to find a decrease in the water-soluble hexose and hexoseamine following a single injection of relaxin. On the basis of a depolymerization
of ground substance, a decrease should have
occurred. However, repeated injections of
relaxin led to a decrease in the insoluble
hexoses and hexoseamines. In addition, consistent decreases in collagen content and
trypsin-resistant protein content were noted.
No hyaluronidase was found, but ^-glucuronidase was increased during relaxation.
Gersh and Catchpole (1949) reported the
presence of a collagenase from histochemical studies, but no confirmation has been
forthcoming. Relaxin also has a protein






anabolic effect which occurs in the absence
of pubic relaxation (Frieden, 1956). This
action was demonstrated by the increased
up-take of labeled glycine by the connective
tissue proteins of the pubic symphysis. Recent experiments indicate that relaxin not
only acts in conjunction with the female
sex steroids but can also act alone (Brennan and Zarrow, 1959). However, it is apparent that the available data are still inadequate for a clear understanding of the
mechanism of action of relaxin.


•
Relaxation of the pubic symphysis of the
 
mouse has been studied in great detail by
 
Hall. In a series of reports she showed that
•
pubic relaxation occurs in the mouse during pregnancy and following treatment with
 
estradiol and relaxin (Hall and Newton,
 
1946a, b). This was later confirmed by Kliman, Salhanick and Zarrow (1953). Contrary to the results reported following work
 
on the guinea pig, progesterone not only
 
failed to influence the effect of estrone on
 
the pubic symphysis of the mouse, but progesterone also inhibited the action of relaxin. It was suggested that this inhibition
is the result of an antagonism by progesterone on the action of relaxin and that a true
species difference exists (Hall, 1949, 1955).
Histologic studies revealed that changes in
the pubic symphysis during pregnancy and
after treatment with relaxin and estradiol
are similar (Hall, 1947) . These changes consist of proliferation of articular hyaline cartilage, resorption of the medial ends of the
pubes, lengthening of the pubic ligament by
formation of new cartilage, and reversion of
the cartilage to collagenous connective tissue. Hall (1956) suggested that estradiol
causes a depolymerization of the mucopolysaccharides through enzymatic action resulting in a matrix sufficiently pliable to
respond to the tensions set up by relaxin.
Evidence presented in support of this concept was the loss of metachromasia and the
increase in water. In addition, a two-step
effect was seen with relaxin: (1) complete
degradation of the matrix, and (2) the appearance of a gap in the cranial part of the
cartilage produced by stretching of the symphyseal cleft. Some data in support of the
latter part of this concept were presented by
van der Meer (1954) who showed that inhihition of pelvic muscle tension inhibited
relaxation in the guinea pig. In a similar
type of experiment Crelin (1954) tied together the innominate bones of a mouse before pregnancy and obtained some dorsoventral displacement of the pubic symphysis
but normal relaxation was inhibited.




===H. Dilation of the Uterine Cervix===


Dilation or softening of the uterine cervix
in the pregnant woman at the time of labor
has been known for a long time. This reaction has been used to determine whether
delivery can be anticipated. Within recent
years this phenomenon has been described
in a number of animals and some analysis
of the hormonal control of the reaction has
been attempted.


Relaxation of the uterine cervix of the
rat during pregnancy was first reported by
de Vaal in 1946 and confirmed by Uyldert
and de Vaal in 1947. Relaxation was measured by the insertion of a gauging pin into
a cervix that had been removed and the
diameter determined at the point where resistance is first felt. The measurements revealed a marked rise from approximately
3.5 mm. on the 17th day of pregnancy to 10
mm. at parturition. Recently, both Harkness and Harkness (1956) and Yochim and
ZaiTow (1959) have taken in vitro measurements of the relaxation of the uterine cervix
of the rat and observed marked relaxation
during the latter part of gestation and at
parturition. Yochim and Zarrow (1959) removed the cervix, suspended it from a rod
and measured the stretch due to weights
added at fixed intervals until the cervix
broke. The amount of relaxation of the
cervix was determined by the amount of
stretch obtained with a weight of 50 gm.
Under these conditions, the curve for relaxation of the cervix showed two sloi^es as
pregnancy progressed (Fig. 16.35). The initial slope between day 12 and day 20 showed
a rise of approximately 4 mm., with an extremely abrupt rise of 14 mm. on day 21.
By 24 hours after parturition the degree of
dilation had fallen to 3 mm. It is of interest
that the curve for the tensile strength of the
cervix (expressed in grams force necessary
to tear 1 mg. cervical tissue in a rat weighing 100 gm.) was the opposite to that seen for cervical dilation. The tensile strength
fell from approximately 50 gm. force to a
low of 3 gm. at parturition and then rose
during the postpartum period. The drop in
tensile strength preceded the changes in the
dilation of the cervix and was essentially
completed 5 to 6 days before parturition or
when the abrupt increase in dilatability of
the cervix occurred.


^'^ *
Similar changes have been described in
the dilatability of the cervix of the mouse
(Steinetz, Beach and Kroc, 1957) with increased dilatability progressed beyond the
15th day (Fig. 16.36). The diameter of the
cervix increased from a])proximately 2 mm.
to about 5 mm. at delivery. It is apparent
that the rate of the reaction, i.e., dilation, is
much more rapid in the rat, although it is
possible that the method of measurement is
responsible for the differences.


The induction of cervical dilation by relaxin was reported by Graham and Dracy
(1953) in the cow, and by Zarrow, Sikes
and Neher (1954) in the sow and the heifer.
Treatment with stilbestrol followed by relaxin caused a dilation of the uterine cervix
of the gilt from % or % inch to 1 inch
(Zarrow, Neher, Sikes, Brennan and Bullard, 1956). Measurements were made by
the passage of aluminum rods, and, although the technique is not too exact, the
differences are significant. Stilbestrol given
alone or in combination with progesterone
had no effect on the cervical dilation. On
the other hand. Smith and Nalbandov
(1958) have recently reported that estrogen
treatment constricted the uterine cervix of
the sow and that relaxin was without effect.
A cue with respect to the mechanism of action of relaxin is given by the similarity of
the action of relaxin on the pubic symphyseal ligament and the uterine cervix. In
both instances, an increase in water content
and a marked dei)olymerizatioii occurs.






'
Fig. 16.35. Dilation and ten.sile strength of the uterine cervLx of the rat during estrus,
pregnancy, and 2 days postpartum. The dihition of the cervix in mm. of stretch per 50 gm.
of added weight. The tensile strength is expressed in grams force necessary to tear 1 mg.
cervical tissue in a rat weighing 100 gm. E = estrus; P = parturition. (From J. Yochim and
M. X. Zarrow, Fed. Proc, 18, 174, 1959.)




, :
•
•


 
Cullen and Harkness ( 1958) observed relaxation of the uterine cervix of the rat with
•
estradiol alone, or with estradiol and progesterone, or with estradiol and relaxin, but
maximal dilation was obtained only with
a combination of estradiol, progesterone,
and relaxin. In general Kroc, Steinetz and
Beach (1959b) obtained comparable results
in the rat. Estrogen alone caused some in
crease in dilatability when 5 fxg. estradiol
cyclopentylpropionate were given, and a decrease when 50 /Ag. were given. Progesterone
had no consistent effect either alone or in
estrogen-primed animals. Relaxin alone
caused some softening of the cervix, but
gave a maximal effect only when given with
progesterone in estrogen-primed animals.
Normal cervical dilation was also obtained
in pregnant rats castrated the 15th day of
gestation and maintained with progesterone,
estradiol, and relaxin (Kroc, Steinetz and
Beach, 1959; Yochim and Zarrow, 1959).
Data on dilation of the uterine cervix of the mouse are rather sparse; nevertheless, softening of the cervix with relaxin has been
reported (Kroc, Steinetz and Beach, 1959).
It is not the purpose of this review to
evaluate the data on cervical softening in
the human female. The nature of the action
of relaxin in the human female is controversial. Nevertheless, softening of the cervix
following treatment with relaxin has been
reported (Eichner, Waltner, Goodman and
Post, 1956; Stone, Sedlis and Zuckerman,
1958) although McGaughey, Corey and
Thornton (1958) reported no effect on the
cervix following relaxin.








Fig. 16.36. Increased length of the pubic ligament, inciea.sed cervical dilatability, and increased responsiveness to oxytocin with the length of pregnanc.y in the mouse. L = lactating; NL = not lactating. (From B. G. Steinetz, V. L. Beach and R. L. Kroc, Endocrinology, 61, 271, 1957.)






==XII. Uterine Myometrial Activity==


The classical and well known description
of uterine muscular activity has been more
than adequately reviewed by Reynolds
(1949). Since then Csapo and his colleagues
have reported a series of elegant experiments involving the action of estrogen and
progesterone on the uterine myometrium
and have evolved the concept of "i)rogesterone block" in the control of uterine activity
(1956a, 1956b). It has been shown that the
ovarian steroid hormones regulate myometrial activity and that the uterine contractions are dependent on the relative
amounts of the two hormones. Contractility
is dependent basically on the concentration
of the high energy phosphates which are
maintained by estrogen w^iich in turn is
involved in the synthesis of these substances
(Csapo, 1950; Menkes and Csapo, 1952).
Discovery of the staircase phenomenon in
the uterine myometrium similar to that exhibited by cardiac muscle led to a marked
difference between the action of estrogen
and progesterone (Csapo and Corner, 1952 ) .






With decreasing freciuency of electrical
stimidation in an isometric arrangement,
tension decreased if the uterus was dominated by estrogen and increased if it was
dominated by progesterone. Uteri from castrated rabbits were insensitive to the frequency of electrical stimulation. Thus estrogen induced a "positive staircase" response
and progesterone a "negative staircase" response, although in the latter instance some
estrogen is also present. These staircase responses have been used successfully as a
measure of hormone domination and have
been shown to be a function of the Na+ and
K+ gradients across the myometrial cell
membrane.


Uterine motility during estrus, the diestrum, and pregnancy has been described
by many investigators in great detail (for
a review see Reynolds, 1949). The diestrous
uterus shows extremely slow, feeble, uncoordinated movements. The contractions may
arise in any part of the uterus and extend
in any direction. At estrus, the uterine contractions become rhythmic and sweep over






Fig. 16.37. Change from a positive to a negative staircase as the hormone dominance of
the myometrium moves from the estrus to the progestational state after mating. X and
O indicate the two strains of rabbits used. (From B. M. Schofield, J. Physiol., 138, 1,
1957.)


•




the uterine horn in a wave starting at the
tubal end. Both amplitude and rate are increased. During pregnancy the uterus becomes relatively quiescent. In general this
pattern of myometrial activity has been reproduced with both hormones, estradiol and
progesterone.


Recently Schofield 11957), using the
Csapo technique, has studied, in vivo, myometrial activity in the rabbit. In a series of
experiments she was able to show in several
strains of rabbits that, when mating occurs
during estrus, the uterine myometrium is
dominated by estrogen. Within 20 to 28






STAIRCASE
Negative


Transient
Positive


6 days after




o




o
o




o
o




8x


OX




o


,




•




Line 5,617: Line 7,592:




ox




X
X
X




Line 5,632: Line 7,611:




partus


o
o
o




ox


X




.






26 27 28 29 30 31
Day of pregnancy






•
32




•


 
Fig. 16.38. Change from negative through
.  
transient to positive staircase as the hormone
 
dominance reverses at the end of pregnane}', indicating estrogen dominance. X and O indicate the
 
two strains of rabbits used. (From B. M. Schofield,
•
J. Physiol. 138, 1, 1957.)






hours after mating, the positive staircase
effect passes through a transient effect to a
negative effect indicating the development
of progesterone dominance (Fig. 16.37j.
This condition remained in effect throughout pregnancy until 24 hours before parturition when a reversion to estrogen domination was indicated by the positive staircase
response ( Fig. 16.38) . Thus the progesteronedominated uterus is maintained throughout
pregnancy and the uterus is nonreactive to
oxytocin. Csapo (1956a) and others have
shown that labor cannot be induced by
oxytocin in the rabbit before day 30 of gestation, but 24 hours later, on removal of the
progesterone block, 96 per cent of the rabbits delivered following treatment with oxytocin. He believes that the specific action of
progesterone involves a blocking of the excitation-contraction coupling which is a
consequence of the disturbed ionic balance
in the myometrial cell. Thus a block is set
up to the propagation of the contraction
wave which can be removed only by a decrease in the level of progesterone.


The role of the water-soluble extract, relaxin in myometrial activity, is still uncertain. That an inhibition of estrogen-induced uterine contractions is obtained in
certain species, such as the rat, mouse, and
guinea pig, with relaxin preparations is un(luestionable. However, we still have not
answered the questions as to w'hether this
hormone plays a role in uterine contractions
under normal physiologic conditions and
whether the uterine contraction-inhibiting substance is relaxin or a contaminant of the
relaxin extract.


Krantz, Bryant and Carr (1950) reported
than an aqueous extract of the corpus luteum would produce an inhibition or decrease of uterine activity in the guinea pig
and rabbit previously primed with estrone.
This has been amply confirmed with both
in vivo and in vitro preparations involving
spontaneous contractions measured isometrically in the guinea pig (Felton, Frieden
and Bryant, 1953; Wada and Yuhara, 1956;
JMiller, Kisley and Murray, 1957) , rat (Sawyer, Frieden and Martin, 1953; Wada and
Yuhara, 1956; Bloom, Paul and Wiqvist,
1958), and mouse (Kroc, Steinetz and
Beach, 1959). However, Miller, Kisley and
Murray (1957) failed to show any action of
relaxin on uterine motility in the rabbit
and the human being in vitro. Thus, the information on the rabbit is contradictory and
a similar situation exists with regard to the
human female for whom both positive and
negative results have been reported following treatment with relaxin for threatened
abortion (McGaughey, Corey and Thornton, 1958; Stone, Sedlis and Zuckerman,
1958; Eichner, Herman, Kritzer, Platock
and Rubinstein, 1959). In briefly summarizing the action of relaxin on the uterine
myometrium it should be pointed out that
( 1 ) relaxin inhibits uterine motility in an
estrogen-primed animal, (2) the action may
be species-limited, and (3) relaxin treatment docs not interfere with the action of
pitocin.




==XIII. Parturition==


===A. Progesterone===


A number of theories have been suggested
to explain the hormonal control of parturition. The most popular is that parturition
is due to a decrease in the level of progesterone which allows oxytocin to exert its effect on the uterus. Evidence has already
been presented indicating that pregnancy
can be maintained in the castrated rabbit
by an extract of corpora lutea, or progesterone, and even prolonged in rats (Nelson,
Pfiffner and Haterius, 1930; Miklos, 1930),
mice (Mandelstamm and Tschaikowsky,
1931), and rabbits (Zarrow, 1947a). Snyder (1934) and Koff and Davis (1937) prolonged gestation in rabbits by inducing the
formation of new corpora lutea during the
last trimester of pregnancy.


Knaus (1930) originally noted a marked
antagonism between posterior pituitary extract and the corpus luteum hormone and
Koff and Davis (1937) reported that in prolonged gestation induced by progesterone,
posterior pituitary extract was ineffective
until two days after the last injection.
Csapo (1956a) performed a series of elegant
experiments and concluded that progesterone blocks the uterine contractions, and
that premature labor could not be induced
with oxytocin before the 30th day of gestation in the rabbit except for a very small
percentage of animals. This has been confirmed by Fuchs and Fuchs (1958).


Zarrow and Neher (1955) found the serum gestagen levels in the pregnant rabbit
fell only after parturition was under way.
Hence the problem arose as to how parturition could begin while a high blood concentration of gestagen was present. A partial
answer was obtained in experiments by
Csapo (1956b) and Schofield (1957) who
showed that the progesterone-dominated
uterus of the pregnant rabbit becomes estrogen-dominated and responsive to oxytocin 24 hours before parturition. Hence the
concentration of progesterone in the serum
is meaningless by itself and it could be
theorized that the significant point is the
ratio of estrogen to progesterone. Csapo
(1956a), however, offered an alternative solution. He observed a local effect of placental progesterone on the myometrium so
that the myometrium closest to the placenta
is under a greater progesterone-dominance
than that portion of the myometrium lying
more distant. Hence the local level of progesterone would be the significant factor in
the onset of parturition and not the systemic
level.




===B. Oxytocin===


It is now generally believed that parturition is the result of the action of the posterior pituitary hormone on the myometrium of the uterus sensitized by estrogen.
The development of this hypothesis followed
from the well known fact that oxytocin produces uterine contractions and induces labor
and delivery of the young. It is apparent,
however, that a mass of contradictory data
exist and the hypothesis is still in need of
better evidence before it can be fully accepted (for review of early literature see
Reynolds, 1949) .


Some of the evidence supporting the
above hypothesis is the fact of the presence,
to a limited degree, of a deficiency syndrome in parturition following removal of
the posterior pituitary gland. The data,
however, are still equivocal. Labor is apparently prolonged in the monkey (Smith,
1946) and guinea pig (Dey, Fisher and
Ranson, 1941 ) after total hypophysectomy.
Nevertheless, parturition will occur normally after removal of the pituitary gland
in the rabbit (Robson, 1936), cat (Allen
and Wiles, 1932), mouse (Gardner and Allen, 1942), and rat (Smith, 1932). Even
where there is some indication of interference with labor, delivery occurs. However,
the lack of consistent results and species
differences may be due to the recent finding
that the posterior pituitary hormones are
synthesized in the hypothalamus and that
removal of the posterior pituitary is only
effective under limited conditions because
the source of the hormone is still present.
These experiments have also been criticized on the ground that the anterior pituitary was also removed and hence interference with many other hormones occurred.


Additional evidence in favor of a role
for the neurohypophysis in the delivery of
the young is the increase in uterine motility
following stimuli that bring about release
of the posterior pituitary hormones, and the
lack of an effect on the uterus when release
of the hormone is blocked.


Positive evidence for the release of oxytocin at the time of parturition is still lacking as are measurements of the concentration in the blood. Fitzpatrick (1957) takes
the view that oxytocin is liberated as an essential part of normal parturition and cites
the following evidence. (1) A superficial
similarity exists between spontaneous labor
and that induced by oxytocin. Harris (1955)
also stresses the similarity in the uterine response to oxytocin and to electrical stimulation of the supraoptic hypophyseal nucleus.
(2) Mechanical dilation of the uterus or cervix evokes an increase in uterine contractions presumably by way of a nervous
reflex release of oxytocin (Ferguson, 1941).
(3) Oxytocin is decreased in the posterior
pituitary gland of the rat and the dog after
labor (Dicker and Tyler, 1953).


Evidence from the attempts to measure
the concentration of oxytocin in body fluids
at the time of parturition is inadequate. The
early reports of higher concentrations in
the urine (Cockrill, Miller and Kurzrok,
1934) and blood (Bell and Morris, 1934;
Bell and Robson, 1935) during parturition
are questioned because of the inadequate
methods of extraction and lack of specificity
in the assay. Recently, Hawker and Robertson (1957, 1958) reinvestigated the problem
and concluded that two oxytocic substances
are present in the blood and hypothalamus
of cats, cows, and rats and blood of women.
However, they found that the concentration
of oxytocin in the blood fell during labor
from a high during pregnancy. It is apparent that this presents a paradoxical situation in view of the fact that the concentration of oxytocin is low at the time of
parturition; a time when the hormone is
supposedly exerting its greatest effect. The
situation is further complicated by the presence of two oxytocic factors and the presence of an oxytocinase in the blood and
l)lacenta (von Fekete, 1930; Page, 1946;
Woodbury, Ahlquist, Abreu, Torpin and
Watson, 1946; Hawker, 1956). Although
more work is required on this problem and
esi)ecially with regard to the specificity and
concentration of the oxytocinase, there is
some indication of a fall in enzyme level
before parturition. Tyler (1955) reported a
decrease in the blood level of the enzyme
towards the end of pregnancy and Sawyer
(1954) reported a decrease in oxytocinase
activity in rat tissues at the end of pregnancy.


===C. Relaxin===


(3,1,99 tost
Recently, the discovery of the action of
 
relaxin on the pubic symphysis, uterine
 
cervix, and uterine motility has raised the
0.  
question of the role of this hormone in parturition. Certainly in the species that normally show pubic relaxation, relaxin would
 
appear to play a significant role. However,
 
this phenomenon is a special adaptation and the question of cervical dilatability becomes
 
more important because it seems to occur
 
in all species examined thus far. It would
 
seem that relaxin can induce cervical dilatability in conjunction with the sex steroids and that cervical dilation is a necessary
 
event in parturition, but whether relaxin
 
controls this event under physiologic conditions is still unknown and direct evidence
 
is unavailable. It is also apparent in some
•
species that relaxin can inhibit uterine contractions w'ithout interfering with the action
 
of oxytocin. Kroc, Steinetz and Beach
 
(1959) reported that relaxin actually restored responsiveness to oxytocin in mice
 
treated with progesterone. Again the question is raised as to whether this is merely
 
a good experiment or a part of the normal
 
physiologic events.
 
 
 
*
 
 
 
 
 
 
 
 






In a general way the events leading to
labor may be summarized as follows. As
pregnancy approaches term, the uterus becomes subject to increasing pressure from
within, due to a differential change in the
growth rates of the fetus and the uterus
(Woodbury, Hamilton and Torpin, 1938).
Concurrently, a reversal from progesterone
to estrogen domination occurs, which also
contributes to an increase in uterine tonus.
As intra-uterine tension increases, spontaneous contractions acquire a greater efficiency and forcefulness. Because the radius
of curvature in the human uterus is greater
at the fundus than at the cervix, and because the myometrium is thicker at the upper pole (by a factor of 2) the contractile
force is stronger at the fundus than at the
cervical end. This contractile gradient i^roduces a thrust toward the cervix.


Utilization of a type of strain gauge, the
tokodynamometer, has afforded information on the rate and strength of contraction
of the various parts of the parturient uterus
simultaneously (Reynolds, Heard, Bruns
and Hellman, 1948). These measurements
have indicated that, during the first stage of
labor, the fundus exerts strong contractions
of rather long duration. The corpus exhibits
less intense contractions, usually of shorter
duration, which frequently diminish in force
as labor advances. The lower uterine segment is almost inactive throughout the first stage of parturition. According to Reynolds
(1949), both the fundus and the midportion
contract at the same time, but the fundus
remains contracted for a longer period of
time than the corpus beneath, thus building
up a force downward. If cervical dilation
has not occurred, the three areas of the
uterus will continue to contract. As cervical
dilation begins, the contractions in the midportion of the uterus decrease in intensity
and the contractions in the lower segment
disappear. Cervical dilation has been observed only when there is a preponderance
of rhythmic activity of the fundus over the
rest of the uterus.


When amniotic fluid is lost after the rupture of the membranes, the absolute tension
within the wall of the uterus is reduced so
that the ratio of force between fundus and
cervix is increased. Thus rupture of the
membranes decreases the tension in the cervix more than the fundus and the net effect
is an increased force from the fundus. This
change tends to precipitate the parturition
more rapidly.


As pregnancy nears term, both increased
tonus of the myometrium and rapid growth
of the fetus cause a rise in intra-uterine
pressure. This rise results in a decrease of
effective arterial blood pressure in the placenta. During this period also, thrombosis
is observed in many of the venous sinuses
of the placenta and many of the blood vessels become more or less obstructed by giant
cells. During parturition, the systemic blood
pressure of the mother rises with each contraction, but, due to the increased intrauterine pressure produced by the contractions, the effective maternal arterial blood
pressure in the placenta decreases to zero.
Thus maternal circulation is cut off from
the fetus.


Measurements of intra-uterine pressure
at term show that the human uterus contracts with a pressure wave which varies
from 25 to 95 mm. Hg (Woodbury, Hamilton and Torpin, 1938). The uterine wall is
subjected to an average tension of 500 gm.
per cm.- and, during delivery of the head,
may, with the aid of abdominal musculature, develop as much as 15 kg. force.


In animals giving birth to multiple young
(rat and mouse) evacuation of the horn proceeds in an orderlv fashion beginning at the cervical end. As evacuation of the lowest
implantation site starts, changes occur in
the periods of contractions of segments of
uterine artery near its entrance into the
uterine wall (Knisely, 1934; Keiffer, 1919).
Gradually the constriction phase becomes
proportionately longer than the dilation
phase until the arterial lumen is obliterated.
The myometrium in the area of the constricting segments becofes more active and,
after long intense local contractions of the
uterine muscle, the fetuses and the placentas separate and are discharged through the
dilated cervix. After evacuation, a relaxation of the contracted segment of uterus
occurs and the process is repeated at the
next implantation site.


Recently, Cross (1958) re-examined the
problem of labor in the rabbit. He concluded
that (1) oxytocin in physiologic amounts
can induce labor that is comparable to the
events normally seen, (2) oxytocin is released during a normal labor, and (3) oxytocin can induce delivery without supplementary mechanisms. He noted that
straining movements involving reflex abdominal contractions initiated by distention
of the vagina and cervix aided in expulsion
of the fetus. It is also possible that this
might cause reflexly an increased secretion
of oxytocin. Other reflex mechanisms have
been suggested, but evidence is inadequate.
Cross cites a report by Kurdinowski published in 1904 in which the entire process of
labor and delivery in an isolated full-term
rabbit uterus perfused with Locke's solution
is described. In these experiments orderly
delivery of the viable fetuses was affected
by the contractile efforts of the uterus and
vagina in absence of any hormonal or nervous stimuli.




==XIV. Conclusion==


(n-IO)
Although we have garnered much information, no major conclusions can be drawn
at this time concerning gestation in the
mammal. This is probably true because of
the vastness of the subject and the lack of
sufficient data, especially that of a comparative nature. It is probably fitting to
close this chapter with the final statement
written by Newton in the second edition of
Sex and Internal Secretion, "It seems rather that the investigation of endocrine relationships during pregnancy is still in the
exploratory stage and that the time is not
ripe for systematization."


It is true that many data have been accumulated in the last two decades since the
publication of the second edition of this
book. It is also probably true that some systematization can now be started. But above
all we need more data on different species
in order to systematize fully the role of the
various hormones and glands in pregnancy
and to evaluate the metabolic and other
changes that occur at this time.


==XV. References==


Adams, J. Q. 1954. Cardiovascular physiology in
normal pregnancy: studies with the dye dilution technique. Am. J. Obst. & Gynec, 67,
741.


Ahl.mark, a. 1944. Studies on the histaminolytic
jiower of i)lasma with special reference to
pregnancy. Acta physiol. scandinav., 9, suppl.
28.


Ahlmark, a. 1947. The histaminolytic power of
]ilasma with special reference to pregnancy.
In 17th International Physiological Congress, p.
127. Oxford.


■n
AiTKKX, E. H., AND Preedy, J. R. K. 1957. The
determination of plasma estiogen levels in late
pregnancy. Ciba Foundation Colloquia Endocrinol., 11, 331.


Albp:rt, a. 1949. Evaluation of the hypermia test
for ])regnancy as a routine clinical laboratory
procedvu'e : comparison of results with those of
1000 consecutive Friedman tests. Proc. Staff
Meet. Mayo Clin., 24, 259.


^
Alexander, D. A., Fr.«er, J. F. D., and Lee, J.
1955. The effect of steroids on the maintenance of pregnancy in the spayed rat. J. Physiol., 130, 148.


Allan, H., and Wile-s, P. 1932. The role of the
pituitary gland in pregnancy and parturition,
hypophysectomy in the cat. J. Physiol., 75, 23.


^^
Allen, E., Hisaw, F. L., .\nd Gardner. W. U. 1939.
The endocrine function of the ovaries. In Sex
and Internal Secretions, 1st ed., E. Allen, Ed.
Baltimore : The Wilhams & Wilkins Company.


Allen, W. M. 1937. Some effects of estrin and
progestin in the rabbit. Cold Spring Harbor
Symposia Quant. Biol., 5, 104.


T^
Allen, W. M., Butenandt, A., Corner, G. W., and
Slotta, K. H. 1935. Nomenclature of corpus
luteum hormone. Science, 82, 153.


Allen, W. M., and Corner, G. \V. 1929. Physiology of corpus luteum. III. Normal growth
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^^
Allen, W. M. and Corner, G. W. 1930. Physiology of corpus luteuni. VII. Maintenance of
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Allen, W. M., .and Heckel. G. P. 1939. Maintenance of pregnancy by progesterone in rabbits castrated on the 11th dav. Am. J. Phvsiol.,
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Aschke.\asy-Lelu. p.. and Aschkenasy, a. 1957.
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n"
A.SDELL, S. A. 1946. Patterns oj Mammalian Reproduction. Ithaca, N. Y.: Comstock Publishing Company, Inc.


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^
A.STWooD, E. B., -AND Creep, R. O. 1938. A corpus-stimulating substance in the rat placenta.
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Atkinson, W. B., and Hooker, C. W. 1945. Day
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^
Balfour, W. E., Comline. R. S., and Short, R. V.
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Barcroft, J., Kennedy, A., and Mason, M. F.
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^
Barcroft, J., and Rothschild, P. 1932. The volume of blood in the uterus during pregnancy.
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Bassett, E. G., and Phillips, D. S. M. 1954.
Pelvic relaxation in sheep. Nature, London,
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^■^^
B.as.sett, E. G., and Phillips, D. S. M. 1955,
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Be.all, D., and Reich.stein. T. 1938. Isolation of
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w^
Be.ard, H. H., and Myers, V. C. 1933. Studies in
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Bell, G. H., and Morris, S. 1934. The oxytocic
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Bell. G. H., .and Robson, J. M. 1935. O.xytocic
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Bellerby, C. W. 1934. A rapid test for the diagnosis of pregnancy. Nature, London, 133, 194.


Berliner, D. L., Jones, J. E., and S.alhanick, H. A.
1956. The isolation of adrenal-like steroids
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223, 1043.


Bibb. J. D. 1941. Protein and hemoglobin in
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. 1 .,,.,.. .  
Bickers, W. 1942. The placenta: a modified
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Bloom, G., Paul, K. G., and Wiqvist. N. 1958.
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Bokelmann, O., and Scheringer, W. 1930. Beitrag zur Kenntnis der Schilddriisenfunktion
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to
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20
Brennan, D. M., AND Z.ARROw, M. X. 1959. Water
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Time, weeks
Bromberg, Y. M., Brzezinski, A., Rozix, S., and
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Browne, J. S. L., Henry, J. S., and Venning, K. H.
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JO
Bruce, H. M., and East. J. 1956. Number and
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AO
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Fig. 16.16. Correlation between the concentration of 17-hydroxycorticosteroids in the
BUTENANDT, A., WeSTPH.\L, U., AND COBLER, H.  
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GESTATION
Castellani. a., and Chalmers, A. S. 1919. Monital of Tropical Medicine, 3rd ed. New York:
William Wood & Company.


Catchpole, H. R.. and Lyons. W. R. 1934. The
gonad-stimulating hormone of pregnant mares.
Am. J. Anat., 55, 167.


Caton, W. L., Roby, C. C, Reid, D. E., and Gibson,
J. G., 2nd. 1949. Plasma yolume and extravascular fluid \olume during pregnancy and
the puerperium. Am. J. Obst. & Gynec, 57,
471.


979
Caton, W. L., Rosy, C. C, Reid, D. E., Caswell,
 
C. J., Meletskos, C. J., Fluharty, R. G., and
Gibson, J. G. 1951. The circulating red cell
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Gynec, 61, 1207.


Chang, M. C. 1951. Maintenance of pregnancy
in intact rabbits in the absence of corpora
lutea. Endocrinology, 48, 17.


The mechanism whereby labor induces
Chart, J. J., Shipley, E. G., and Gordon, E. S.
a marked stimulation of the adrenal cortex
1951. Evidence for a sodium retaining factor
is still obscure. It is possible that labor is
in toxemia of pregnancy. Proc. Soc. Exper.
a stressful state and the stress induced by
Biol. & Med., 78, 244.  
both the pain and the muscular work act to
stimulate the increased release of ACTH
resulting in increased adrenocortical activity. Some confirmation of this may be
obtained from the fact that significant in(■i'eas(> in }ilasma 17a-hydroxycorticoids is
noted only if the labor lasts more than 6
hours.  


Analysis of the rise in plasma levels of
Chesley, L. C. 1943. Study of extracellular
hydrocortisone during pregnancy has suggested that the phenomenon is not simply
water changes in pregnancy. Surg. Gvnec. AObst., 76, 589.  
the result of an increased rate of secretion
from the adrenal cortex, but rather the result of an increased retention and an alteration in the metabolism of the hormone
< Cohen, Stiefel, Redely and Laidlaw, 1958).  


2. Aldosterone
Chesley. L. C, AND Chesley, E. R. 1941. Extracellular water in late pregnancy and its relation to the development of toxemia. Am. J.
Obst. ife Gynec, 42, 976.  


The isolation for aldosterone by Simi)son,  
Chitty, H. 1957. The estrous cycle and gestation period in the lactating field vole, Microtiis
Tait, Wettstein, Neher, von Euw, Schindler
agreslui. J. Endocrinol., 15, 279.  
and Reichstein (1954) and its identification
as the hormone regulating fluid and mineral
metal)olism stimulated marked interest in
the role of this hormone. Among the items
of interest was its significance in pregnancy
and in the toxemia of pregnancy. Early
studies by Chart, Shipley and Gordon
( 1951 1 revealed the presence of a sodium
retention factor in the urine that increased
from a normal pregnancy value of 36 to 106
fxg. equivalent of desoxycorticosterone acetate (DOCA) per 24 hours to a maximum of
1008 |U.g. equivalent in pregnancy toxemia.  
These results were confirmed by Venning,  
Simpson and Singer (1954) and by Gordon,
Chart, Hagedorn and Shipley (1954). In
addition a slight increase in the sodium retaining factor was observed in gravid women as compared to nongravid women.  


The discovery that the greater part of
Christian, J. J., .and Lemunyan, C. D. 1958.  
the aldosterone in urine is present in the
Adverse efTects of crowding on lactation and
conjugated fraction led to a repetition of
reproduction of mice and two generations of  
the above work using both acid hydrolysis
their progeny. Endocrinology, 63, 517.
and incubation with /3-glucuronidase (Venning and Dyrenfurth, 1956; Venning, Primrose, Caligaris and Dyrenfurth, 1957). The
results show little change in the excretion
of free aldosterone throughout pregnancy,  
but the glucuronidase and acid-liydrolyzed


Chu, J. P. 1945. The influence of the thyroid
on pregnancy and parturition in the rabbit.
J. Endocrinol., 4, 109.


Clegg, M. T., Boda, J. M., AND Cole, H. H: 1954.
The endometrial cups and allantochorionic
pouches in the mare with emphasis on the
source of equine gonadotrophin. Endocrinology, 54, 448.


fractions increased markedly (Fig. 16.17).  
Cockrill. J. R., Miller, E. G., and Kurzrok, R.  
The urinary excretion values increased
1934. Presence of oxytocic substance in urine during labor. Proc. Soc. Exper. Biol. & Med.,
from a prepregnancy normal of 1 to 6 /xg.  
31,527.  
aldosterone (average for women was 3.8 ±
 
14 fig. per 24 hours; Venning, Dyrenfurth
Cohen, M., Stiefel, M.. Reddy, W. J., and LaidLAW, J. C. 1958. The secretion and disposition of Cortisol during pregnancy. J. Clin. Endocrinol., 18, 1076.  
and Giroud, 1956) to approximately 25 /xg.  
per 24 hours. The first significant rise occurred about the third month of gestation
and an increased concentration was obtained until after parturition, when there
was a rapid fall to the nonpregnant values.  


G. THYROID GLAND
Cole, H. H., and Goss, H. 1943. The source of
eciuine gonadotrophin. In Essays in Biology in
Honor of Herbert H. Evans, p. 107. San Francisco: University of California Press.  


Clinical data have long indicated a possible involvement of the thyroid gland in
Cole, H. H., and Hart, G. H. 1930. The potency
gestation (Salter, 1940). In regions where
of blood serum of mares in progessive stages
the iodine supply is low this is demonstrated by an enlargement of the thyroid
of i)regnancy in effecting the sexual maturity
during pregnancy. The formation of a
of the immature rat. Am. J. Physiol.. 93, 57.  
goiter has been interpreted as evidence for
an increased need for iodine during gestation. Scheringer (1930) and Bokelmann and
Scheringer ( 1930) reported a rise in the
iodine content of the blood of pregnant
women during the first trimester of pregnancy with a peak at the seventh month.
The increased concentration is maintained
until shortly after parurition. In the goat,
however, Leitch (1927) reported no change
in serum iodine during gestation until just
before parturition. Analysis of umbilical
vein blood revealed values that were normal, i.e., lower than in the mother (Leipert,
1934). Increased thyroid secretion (Scheringer, 1931 ) and increased urinary excretion
of iodine have been reported in pregnant
women (Nakamura, 1932; 1933). However,  
Salter (1940) concluded in his review that
no reliable evidence of increased thyroid
hormone levels in the blood during jiregnancy is available.  


Peters, Man and Heinemann (1948) reported a range of 4 to 8 fig. per cent of
Cole, H. H., and Saunders, F. J. 1935. The concentration of gonad-stimulating hormone in  
serum-precipitable iodine in the normal,  
blood serum and of estrin in the urine throughout pregnancy in the mare. Endocrinology,
nonpregnant woman with a rise to 8.3 fig.  
19, 199.  
per cent (range 6 to 11.2 fig. per cent) in the
pregnant woman (Fig. 16.18). It is of interest that the elevation in the proteinbound iodine (PBI) does not follow the
course of changes in the basal metabolic
rate. Whereas the former is already high by
the second month of pregnancy the basal
metabolic rate rises gradually after approx


CONTOPOULOS. A. N., AND SiMPSON, M. E. 1956.


980
Increased FSH and ICSH content in the pituitary of pregnant rats. Anat. Rec, 124, 276.


CoNTOPOULOS, A. N., AND SiMPSON, M. E. 1957.


Increased growth promoting substance in the
plasma of pregnant rats. Endocrinology. 61,
765.


SPERM, OVA, AND PREGNANCY
CONTOPOI'LOS, A. N., AND Sl.MPSON. M. E. 1959.


Growth-promoting activity of pregnant rat
plasma after hypophysectomy and after thyroidectomy. Endocrinology, 64, 1023.


Coppedge. R. L., and Segaloff. A. 1951. Urinary
prolactin excretion in man. J. Clin. Endocrinol., 11,465.


imately the 4th month of pregnancy (Rowe
Corner, G. W. 1929. Pliysiology of the corpus
and Boyd, 1932; Javert, 1940). No other
luteum. II. Production of a special uterine
sym})toms of hyperthyroidism are seen in
reaction (progestational proliferation) by extracts of corpus luteum. Am. J. Physiol., 88,  
pregnancy which leads to the question of
326.  
the significance of the rise in protein-bound
iodine. A somewhat comparable paradox
exists in the guinea pig in which a rise in
the rate of oxygen consumption during
pregnancy is not accompanied by an increase in heart rate (Hoar and Young,  
1957).  


Recently, AVerner (1958) rcj^orted a decrease in the I^-^^ up-take after treatment
CouRRiER. R. 1931. Reclierches sur le Mechanisme de la Crise Genital du Nouveau-Ne. In
with triiodothyronine in both the normal
Proceedings Second Inlernational Congress for
and pregnant woman. From this and other
Sex Research, p. 355. Edinburgh: Oliver &
data he ruled out any abnormal pituitarythyroid relationship or marked secretion of
Boyd.  
thyroid-stimulating hormone (TSH) by the
placenta and concluded that the increased
PBI in pregnancy is due to an increased
binding capacity of the serum protein.  


Feldman ( 1958) failed to find any increase
CouRRiER, R. 1940. La desoxycorticosterone est
in the level of serum-hutanol-extracted io
Capable de Maintenir la Grossesse on de Provoquer I'avortement. Presse med., 48, 658.


CouRRiER. R., and Colonge, R. a. 1950. Nouvelles remarques concernant I'effet de I'ovariectomie sur la gestation. Compt. rend. Siances,
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dine throughout pregnancy in the rat. Actually the values were consistently lower
CouRRiER. R., AND CoLONGE, R. A. 1951. Cortisoue
than in the controls and similarly the total
et gestation chez la lapine. Compt. rend. Acad.  
amount of PBI in the thyroid of the pregnant rat was consistently lower. He did find
Sc, 232, 1164.  
an increase in the rate of excretion of V-''\
a diminished up-take of I^^^ by the thyroid,  
and a decreased half-life for thyroxine in the
pregnant rats. It is obvious that these results
are quite dissimilar from those obtained in
the pregnant women. One can only conclude
at this time that pregnancy has an effect on
iodine metabolism and a species difference
exists.  


H. GROWTH HORMONE
CouRRiER, R.. AND Kehl, R. 1938a. Donnees
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biol., 127, 529.  


Although it has been possible to demonstrate the presence of growth-promoting substance (STH) in the blood plasma, there are
CouRRiER, R., AND Kehl, R. 1938b. Sur le besoin
few data bearing on the identity of the substance and few ciuantitative measurements.  
hormonal quantitatif chez la lapine gestante
Westman and Jacobsohn ( 1944) first showed
castree. Compt. rend. Soc. biol., 128, 188.
the ]irescnce of a growth-]5romoting sub


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■z.  
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'•
Cs.\po, A., AND Corner. G. 1952. The antagonistic
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2
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z
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®^  
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IT
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• • •
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•
qO
• •
•
MONTHS 2
DAYS
3 4 5 6 7 8 9
Fig. 16.17. Urinary excretion of aldosterone throughout pregnancy in the human being.
O, free fraction only; •, free and acid-hydrolyzed fraction; O, free, enzyme and acidhA'drolyzed. (From E. H. Venning and I. Dyrenfurth. J. Clin. Endocrinol,, 16, 426, 1956.)
GESTATION
981
stance in the blood by cross transfusion between a normal and hypophysectomized rat
united in parabiosis. Gemzell, Heijkenskjold
and Strom (1955), using the technique of
adding exogenous growth hormone to the
sample of blood, failed to find any growthjiromoting substance in 23-ml. equivalents
of blood. However, retroplacental blood from
human beings gave a positive response at a
level of 7- to 15-ml. equivalents of plasma
without the addition of exogenous STH. Increase in the width of the proximal tibial
epiphysis of the rat was used as an end
l)oint. A comparable concentration of 650
fxg. eciuivalent of the standard STH per 100
ml. plasma was also found in the blood from
the umbilical cord.
Contopoulos and Simpson (1956, 1957)
measured the STH of the plasma in the
pregnant rat, using the tibial cartilage, tail
length, and body weight increase. No significant increase in plasma STH was noted
on the 5th day of pregnancy, however, a significant rise was observed by the 9th day.
An estimated 3-fold increase in plasma STH
during pregnancy was reported from calcu
lations on both the tibial cartilage and the
tail length tests. No changes were reported
in the STH activity of the pituitary gland
throughout pregnancy. Recently, the persistence of greater than normal amounts of
growth-promoting activity was reported in
the plasma of pregnant rats after hypophysectomy. Since the fetal pituitary probably
does not contribute to the STH pool of the
mother, at least in early pregnancy, it is
likely that the placenta may be a source of
the hormone.
I. PROLACTIN
Few data are available on the concentration of prolactin during gestation. This has
been due, in part, to the minute amounts of
the hormone present in the urine and blood
and to the inadequacy of the available assays. Although Hoffmann ( 1936 ) failed to
find any prolactin in the urine of women
before parturition, Coppedge and Segaloff
(1951) and Fujii and Schimizu (1958) reported measurable amounts of prolactin in
the urine of pregnant women. Coppedge and
Segaloff reported a gradual rise in the excre
FiG. 16.18. The level of protein-bound iodine in the pregnant woman. (From J. P.
Peters, E. B. Man and M. Heinemann, in The Normal and Pathologic Physiology oj
Pregnancy, The Williams & Wilkins Co., 1948.)
982
SPERM, OVA, AND PREGNANCY
28 30 32 3i 36 38 aO 1 23 45 6 78 2 4 6 8 10 12 14 28
Weeks of pregnancy Days post partum Weeks of lactation
Fig. 16.19. Urinary excretion of prolactin throughout gestation in the human being.
One pigeon crop unit (P.C.U.) is equivalent to 0.3 I.U. (From K. Fujii and A. Shimizu,
Bull. Tokyo Med. & Dental Univ., 5, 33, 1958.)
tion of prolactin throughout pregnancy and
a gradual decline following parturition even
though lactation was maintained. The number of observations, however, was limited
and the authors point out that the results
were ecjuivocal. Fujii and Shimizu observed
an initial drop in the prolactin output during the first month of pregnancy followed by
a rise to approximately 32 P.C.U. (one pigeon crop sac unit is equivalent to 0.3 I.U.)
per 24 hours during the second trimester of
pregnancy in women. (Fig. 16.19). This was
followed by a drop to approximately 10
P.C.U. per^24 hours between the 30th and
38th wrecks of pregnancy and a marked rise
to 64 P.C.U. per 24 hours during the lactation period.
J. PL.\CENTAL GONADOTROPHINS
Placental gonadotrophins have been found
in the monkey, chimpanzee, human being,
mare, and rat (Hisaw and Astwood, 1942).
The physiologic activities of these placental
hormones differ among the three groups of
niannnals and appear to represent divergent
evolutionary steps in the adoption of pituitary function by the placenta. The physiologic properties of the placental gonadotrophins differ not only among themselves
but also from the pituitary gonadotrophins.
The gonadotrophin from the rat placenta
(luteotrophin) has been shown to be leuto
trophic with the ability to maintain a functional corpus luteum in the hypophysectomized rat (Astwood and Greep, 1938). The
hormone has no effect on follicular growth
or ovulation. Its function appears to be that
of maintaining the secretory activity of the
corjius luteum in the rat from the 10th day
of pregnancy to term.
The placental hormones of the human being (HCG) and the mare (PMS) have been
studied in much greater detail. These two
hormones differ markedly in both chemical
and physiologic properties. The presence of
HCG in the urine and the absence of P]\IS in
the urine would alone indicate a marked difference in the size of the two molecules.
Physiologically, PMS is highly active in producing follicular growth and some luteinization, whereas HCG has no effect on follicular growth but will induce ovulation and a
delay in the onset of menstruation. This
would indicate a luteotrophic action. Although chorionic gonadotrophin has been reported in the macaque (Hamlett, 1937) between the 18th and 25th day of pregnancy,
and in the chimpanzee from the 25th to the
130th day of gestation (Zuckerman, 1935;
Schultz and Snyder, 1935), little work has
been done on the characterization and identification of these substances except in man
and horse.
It is of some interest to note that the ap
GESTATION
983
MAX CONJCENTRATION
CONTROP HORMONE
lOOr n ,'-— X
WOMAN
MARE
10 20 30 40 50
7. OF DURATION OF PREGNANCY
60
70
80
90
100
Fig. 16.20. The relative time of appearance of placental gonadotropliins in the pregnant
mare and the woman. (From E. T. Engle, in Sex and Internal Secretions, 2nd ed., The
Williams & Wilkins Company, Baltimore, 1939.)
pearance of the placental gonadotrophins in
the blood and urine of horse and man occurs
at approximately the same relative time in
pregnancy (Fig. 16.20). The role played by
these hormones in gestation is still not clear,
but it is significant that their appearance
corresponds with the time of implantation
of the blastocyst and their disappearance
roughly with the time when ovariectomy no
longer interferes with the maintenance of the
pregnancy.
/. Human Chorionic Gonndotrophin (HCG)
The discovery of the presence of a gonadotrophic hormone in human pregnancy urine
by Aschheim and Zondek (1927j was soon
followed by a description of its biologic activity and quantitative determinations of
its concentration in the urine throughout
pregnancy (Ascheim and Zondek, 1928).
Recently a number of investigators have determined the titer of chorionic gonadotrophin in the serum of pregnant women. These
curves agree very well with the values ob
tained from the urine. Figure 16.21 is a typical curve for the concentration of chorionic
gonadotrophin in the blood of pregnant
women (Haskins and Sherman, 1952). A
peak value of 120 I.U. per ml. of serum was
obtained on the 62nd day after the last
menses and a rapid decline was noted to a
low of approximately 10 I.U. per ml. of serum on day 154. A subsequent rise to 20
I.U. was noted by day 200 and this was
maintained until the end of pregnancy.
These results are in excellent argeement with
those reported by Wilson, Albert and Randall (1949) using the ovarian hyperemia
test in the immature rat. These authors obtained a peak concentration of approximately 70 I.U. per ml. of serum on the 55th
day after the last menses. A gradual decrease
occurred thereafter to a low of approximately 20 I.U. per ml. of serum which remained unchanged from day 100 to parturition although the data indicate a slight
rise towards the end of pregnancy.
The significance of the excretion pattern
984
SPERM, OVA, AND PREGNANCY
o
o
o
ii
o
X
u o
U. CQ
o
5 ♦
ui 2 ■
MAXIMUM TITER
/\
^^\
/N,
20 60 100 140 180 ZZO 260
DURATION OF PREGNANCY DAYS AFTER THE LAST MENSES
Fig. 16.21. Concentration of human chorionic gonadotiophin in the blood of the normal
pregnant woman. The hormone levels were determineti b>' the male frog test. (From A. L.
Haskins and A. I. Sherman. J. CUn. Endocrinol., 12, 385, 1952.)
and concentration of the hormone in the
serum is still a matter of conjecture. Browne,
Henry and Venning (1938) suggested that
the peak level of chorionic gonadotrophin in
the blood reflects an increased production
and a physiologic need in order to maintain
a functional corpus luteum during early
pregnancy. Recent evidence has tended to
confirm this opinion in that HCG has been
found to be active in the maintenance of the
secretory activity of the corjius luteum in
the primate (Hisaw, 1944; Brown and Bradbury, 1947; Bryans, 1951 j. In addition, histologic studies reveal a direct proportion between the number of Langhans' cells and the
amount of hormone excreted (Stewart, Sano
and Montgomery, 1948; Wislocki, Dempsey
and Fawcett, 1948) .
The possibility that the kidney plays a
role in the changes in the concentration of
HCG was investigated by Gastineau, Albert
and Randall (1948) . The renal clearance was
relatively constant throughout all stages of
pregnancy although the urine and serum
concentrations of the hormone varied as
much as 20- fold. In addition, the mean, renal
clearance found during pregnancy was not
markedly different from that found in cases
of hydatiform mole and testicular chorioma.
Inasmuch as the renal elimination of the
hormone remained constant, it was obvious
that two possible explanations existed : these
were (1) changes in the secretion rate, and
(2) changes in extrarenal disposal of the
hormone. Studies on the latter were contradictory. Whereas Friedman and Weinstein (1937) and Bradbury and Brown
(1949) reported an excretion of 20 per cent
GESTATION
985
and higher of HCG following the injection
of HCG, Johnson, Albert and Wilson (1950)
found an excretion of 5.8 per cent in pregnant women during the immediate postpartum period. Zondek and Sulman (1945)
reported a 5 to 10 i)er cent elimination of
HCG in the urine of animals. Thus Bradbury and Brown felt that there is relatively
little destruction or utilization of the hormone in the body; Wilson, Albert and
Randall ( 1949) believed that 94 per cent of
the circulating hormone is affected by extrarenal factors and that the fluctuating character of hormonal level in serum or urine
depends entirely on changes in rate of hormone production.
An analysis of the distribution of chorionic
gonadotrophin in the mother and fetus led
Bruner (1951) to conclude that the ratio of
maternal blood to urinary gonadotrophin is
not constant although the ratio of gonadotrophin in the chorion to maternal blood is
constant. Consequently, she concluded that
the concentration of gonadotrophin in the
urine does not depend entirely on the rate of
production of the hormone and that the
method of gonadotrophin elimination
changes during pregnancy. She also pointed
out that a significant amount of chorionic
gonadotrophin is found in the fetus and that
this is due to the fact that, although the
chorion releases the hormone into the maternal blood, secondarily some of it passes the
placental barrier and enters the fetal system across the wall of the chorionic vesicle.
2. Equine Goyiadotrophin (PAIS)
The presence of a gonadotrophin in the
blood of the pregnant mare was first described by Cole and Hart in 1930. The hormone appears in the blood about the 40th
day of pregnancy and increases rapidly to a
concentration of 50 to 100 rat units (R.U.)
per ml. by the 60th day of pregnancy (Cole
and Saunders, 1935). This concentration is
maintained for approximately 40 to 65 days.
By day 170 it has fallen to a nondetectable
level (Fig. 16.22).
Catchpole and Lyons (1934) suggested
that the placenta is the source of the gonadotrophin and indicated that the chorionic
epithelium is the probable source. Cole and
leae/7(f
• Co/7ce/7irct/o/? of oes^r/y?.
— /re/7<y of ///tf co/7cs/7^rot/o/f of oasin.
/PO /ss
//s ^oo ^^s ^so ^7s joo ses
^^ayj prfyrjo/?^
Fk;. 16.22. Tlie concentration of i)rognant mare's serum in the blood of the mare throug
out pregnancy. (From H. H. Cole and F. J. Saunders, Endocrinology. 19, 199, 1935.)
986
SPERM, OVA, AND PREGNANCY
Goss (1943), on the other hand, concluded
that the endometrial cups are the source of
the hormone. Recent evidence tends to confirm the endometrial cups as the source of
the hormone (Clegg, Boda and Cole, 1954).
The endometrial cups form in the endometrium opposite the chorion in the area
where the allantoic blood vessels fan out.
The cups develop precisely at the time when
the hormone is first obtained in the serum
of the pregnant mares and desquamation of
the enclometrial cups is complete at the time
of the disappearance of the hormone from
the maternal blood. Analyses of the cups for
gonadotrophin content reveal a correlation
between the concentration of the hormone in
the maternal blood and the concentration in
the endometrial cups. Finally, histochemical
stains for glycoprotein indicate the presence
of this substance only in the epithelial cells
lining the uterine lumen and the uterine
glands in the cup area ( for complete discussion of the subject see the chapter by Wislocki and Padykulal.
VI. Pregnancy Tests
The discovery of gonadotrophic activity
in the urine of pregnant women by Aschheim and Zondek in 1927 led to introduction
of the first valid test for pregnancy (Aschheim and Zondek, 1928). These investigators
used the innnature mouse and reported the
presence of corpora hemorrhagica as indicative of the presence of a gonadotrophin in
the urine and a positive reaction for pregnancy. The Aschheim-Zondek test for pregnancy was the first successful test of its kind
and has been used both as a qualitative and
quantitative test. In the latter instance, a
serial dilution of the urine is made in order
to obtain the minimal effective dose.
It is not too surprising that many tests
for pregnancy have been described. In general, all of the successful tests involve the
detection of chorionic gonadotrophin in the
urine, and to some extent in the blood. The
changes that have appeared in the development of new pregnancy tests have been
those concerned with the use of different
species of animals, the rapidity with which
the test could be completed, and convenience to the laboratory. Thus the Friedman
test (Friedman, 1929; Friedman and Lapham, 1931 ) followed soon after the Aschheim-Zondek test and in turn was succeeded by several newer tests.
Ap])roximately five reliable tests are now
available (Table 16.6). All are concerned
with the detection of HCG and have an accuracy of 98 to 100 per cent. The AschheimZondek suffers from a time requirement of
96 hours and was largely supplanted by
the Friedman test that used the isolated
rabbit and required only 48 hours. Within
recent years several new tests have been
reported using the frog, toad, and immature rat. Frank and Berman (1941) first
noted the occurrence of hyperemia in the
ovary of the immature rat, following the
injection of HCG. Albert (1949) reported
excellent results with the use of this test
in 1000 cases. Comparison of the rat hyperemia test with the Friedman test was
on the whole very good and revealed the
same order of accuracy for both tests. The
Friedman test, however, will detect about
5 I.U. of HCG which would mean a concentration of 500 I.U. of HCG per 24-hour output of urine ( assuming a 24-hour urine output of 1500 ml.). Positive results in the rat
test require a 24-hour output of 1000 I.U.,
indicating that the ovarian hyperemia test
in the rat is about one-half as sensitive as
the Friedman test. Nevertheless, the rat
TABLE 16.6
Pregnancy tests with an accuracy
oj 98 to 100 per cent
Animal
Sex
Observed End Point
Time
Reference
Immature mouse
Isolated rabbit
Xenopus laevis
Bufo arenarum
Immature rat
F
F
F
M
F
Corpora hemorrhagica
Corpora hemorrhagica
Extrusion of ova
Extrusion of sperm
Hyperema of ovary
hr.
96
48
8-12
2-4
4
Aschheim and Zondek, 1928
Friedman and Lapham, 1931
Shapiro and Zwarenstein, 1934a
Galli-Mainini, 1947
Frank and Berman, 1941
GESTATION
987
test requires only 4 hours and a larger number of animals can be utilized, thus decreasing the error due to use of inadequate numbers of animals. Comparison of the rat
hyperemia and the Friedman tests revealed
that the former is slightly more accurate
but a little less sensitive (Albert, 1949).
Within two years after the publication of
the Friedman test for pregnancy, Shapiro
and Zwarenstein (1934a, 1934b, 1935) and
Bellerby (1934) reported the use of the
African toad {Xenopus laevis, D) in the
diagnosis of pregnancy. Again the test was
based on the ability of HCG to induce the
extrusion of ova by the frog following the
injection of the urine into the dorsal lymph
sac. Extrusion of the ova occurred in 6 to
15 hours and the test was shown to compare
favorably with both the Aschheim-Zondek
and Friedman tests, although it did not give
tiie graded response seen with the A-Z test
(Crew, 1939). Weisman and Coates (1944)
found an accuracy of 98.9 per cent with the
Xenopus test over a 5-year period during
which 1000 clinical cases were examined.
Galli-Mainini (1947) first reported the
use of the male batrachian in the diagnosis
of pregnancy and Robbins, Parker and
Bianco (1947) simultaneously reported the
release of sperm by Xenopus following
treatments with gonadotrophins. Galli-Mainini (1948» pointed out that this reaction
is not restricted to a single toad, but would
]irobably be found in many frogs and toads.
He added that care should be used to employ animals with a continuous spermatogenesis. This was immediately confirmed by
reports from different countries using various species of frogs and toads endogenous
to the areas. Immediate use of Rana pipiens
was reported in the United States and this
species became very popular in that country
( Wiltberger and :\Iiller, 1948) .
The advantages of the sperm-release test
are the time requirements, simplicity, end
point, and opportunity to use many animals.
On the other hand, the reaction is all or
none and shows no gradation in degree of
reaction. In general, the urine is injected
into the dorsal lymph sac and the cloaca
aspirated for sperm 1 to 3 hours later. Although this is the most recent of the pregnancy tests, many reports have appeared
and some evaluation as to accuracy may be
attempted. Galli-IVIainini (1948) reported
an accuracy of 98 to 100 per cent in a summary of more than 3000 tests and 100 per
cent accuracy for negative results in more
than 2000 controls. Robbins (1951 ) reported
an accuracy of 89.5 per cent in the first trimester of pregnancy. Pollak (1950) indicated that as many as 20 per cent of the
negative tests obtained in the summer were
false. This suggested the existence of a
refractory state at this season. Bromberg,
Brzezinski, Rozin and Sulman (1951) reported on a comparison of several tests
including 700 cases. An accuracy of 85 per
cent was obtained with the male frog test,
99 per cent with the rat hyperemia test, 98.5
per cent with the Friedman test and 98 per
cent with the Aschheim-Zondek test. The
authors indicate that the 15 per cent failures
to get a positive reaction in the frog could
be due in part to the poor sensitivity of the
animal which could only be overcome by
concentrating and detoxifying the urine.
Comparison of the minimal amounts of
HCG to elicit a positive reaction are Vs
I.U. for the rat hyperemia test, 1 I.U. for
the Aschheim-Zondek and Hyla tests, 2
I.U. for the Rana and Bufo tests, and 5
I.U. for the Friedman test. Reinhart, Caplan
and Shinowara (1951) reported an accuracy
of 99 per cent with 840 urine specimens;
only 3 false negatives were noted in 346
specimens from known pregnant women
and no false positives noted in 125 nonpregnant women. The authors attribute the
high degree of accuracy to standardization
of the procedure by which extraneous factors were eliminated. These include ( 1 ) the
use of 2 or more 30- to 40-gm. frogs for
each test, (2) elimination of all animals suffering from red leg and other diseases, (3)
adequate time for sperm release, (4) concentration of the urine, (5) maintenance
of frogs at 15 to 22°C., and (6) during the
summer the injection of an increased volume
of urine and an increase to 4 hours in the
period for sperm release. The maintenance
of frogs in a hibernating state by keeping
them in a refrigerator at 38°F. has been rei:)orted to insure a high degree of sensitivity
regardless of the season (Allison, 1954).
Although it is obvious that more data are
988
SPERM, OVA, AND PREGNANCY
needed, the present results are very promising for the "frog-sperm" test and if the
seasonal effect can be eliminated, this test
will be the equal of the other four.
VII. Water and Electrolyte Balance
The changes in the various components of
the blood during pregnancy have been described in a number of species (Tables 16.7
and 16.8). It is generally agreed that a
marked increase in the blood and plasma
volume and a decrease in the relative
amounts of erythrocytes and hemoglobin
occur during the last trimester. Inasmuch
as the increase in the plasma volume in man
exceeds the concurrent increase in the total
cell volume, the resultant hemodilution produces an anemia which has been described
as the "physiologic'' anemia of jM-egnancy.
TABLE l(i.7
The average percentage of change in the constituents of the blood and in the extracellular fluid volume during
normal pregnancy in man
Extracellular
space
Adams, 1954.
Bibb. 1941
Caton, Robv, Reid and Gibson, 2nd, "l949
Caton, Roby, Reid, Caswell.
Maletskos, Fluharty and
Gibson, 1951
Chesley, 1943
Dieckmann and Wegner,
1934a, b, c, d
Ferguson, 1950
Freis and Kenny, 1948
Friedman, Goodfriend, Berlin
and Goldstein, 1951
Ganguli, 1954
Gemzell, Robbe and Sjostrand, 1954
Hamilton and Higgins, 1949. .
Jarosova and Daum, 1951 ...
Lambiotte-Escoffier, Moore
and Tavlor, Jr.. 1953
Lund, 1951
McLennan and Corey, 1950..
McLennan and Thouin, 1948. .
Miller, Keith and Rownetree,
1915
Mukherjee and Mukherjee,
1953
Roscoe and Donaldson, 1946.
Thompson, Hersheimer, Gibson and Evans, Jr., 1938 . . .
Tysoe and Lowenstein, 1950 .
White, 1950
59
25
15
Blood
volume
49
None
45
23
30
59
32
Plasma
volume
22
49
55
25
32
Total
hemoglobin
20
59
20
Hematocrit
10
15
10
12
16
RBC
count
14
21
22
Gram %
hemoglobin
25
15
25% in
50% of
patients
10
Time of Determinat
in Pregnancy
6th week antepartum
3rd trimester
3rd trimester
60 days antepar
tum
3rd trimester
3rd trimester
3rd trimester
At term
Throughout pregnancy
8th week antepartum
3 months antepartum
9th monlli
10th lunar month
3rd trimester
10th lunar month
At term
3rd trimester
3rd trimester
3rd trimester
9th lunar month
At term
3rd trimester
GESTATION
989
TABLE 16.8
The
average percentage of cl
aiu/e in
the constituents
of the blood and
in the extracellular fluid
volume during norn
al pregnancy in
variouf
laboratory and domestic animals
Increase
Decrease
Animals
Total
hemoglobin
Author
Blood
Pasma
Cell
Hema
RBC
Gram %
volume
volume
volume
tocrit
count
hemoglobin
%
%
%
%
%
%
Rat
40
20-40
Beard and Mvers, 1933
41
20
10
29
18
30
25
7
31
33
Bond, 1958
Newcomer, 1947
van Donk, Feldman and
Steenbock, 1939
Rabbit
50
17
20
10-20
17
Salvesen, 1919
Zarrow and Zarrow, 1953
6
12
12
13
Horger and Zarrow, 1957
Sheep
25
22
10
Initial decrease, normal at term
Barcroft, Kenned}- and Mason, 1939
Cow
Slight
Slight
Reynolds, 1953
Comparable changes were observed in the
blood constituents and plasma volumes of
the rat and rabbit during the latter third
of gestation. Although there is no increase
in the total cell volume, the resultant "physiologic" anemia of pregnancy in the rabbit
follows the same general pattern as that
rei)orted in the human being (Horger and
Zarrow, 1957).
A significant decrease in the erythrocyte
number, hemoglobin concentration, and hematocrit, and an increase in the blood volume have been noted in the rat during
pregnancy (Table 16.8). However, the increase in blood volume is correlated with
an increase in body weight and the ratio
of blood volume to body weight remains unchanged (Bond, 1948). Calculation of the
total number of erythrocytes and grams of
hemoglobin actually showed an increase in
these constituents during gestation, indicating that the anemia of pregnancy in the
rat is due to a hemodilution in which
the blood volume increases proportionately
faster than the number of erythrocytes.
Comparable results were also reported in
the rabbit (Zarrow and Zarrow, 1953). A
marked drop in the relative number of
circulating erythrocytes and percentage of
hemoglobin is seen invariably towards the
end of gestation (Fig. 16.23). A marked
Fig. 16.23. Changes in the relative number of
circulating erythrocytes, reticulocytes, percentage of hemoglobin, hematocrit, and nonprotein
nitrogen of the blood of the rabbit during pregnancy and after parturition. (From M. X. Zarrow
and I. G. Zarrow, Endocrinology, 52, 424, 1953.)
^90
SPERM, OVA, AND PREGNANCY
fall in hematocrit occurs concomitantly with
the fall in the two blood constituents along
with an increase in the reticulocytes. The
time of onset of the increase in reticulocytes
is variable and seems to occur during the
second trimester of gestation. Their number
returns to normal before parturition in spite
of the increasing severity of the anemia. A
second rise in the reticulocytes is seen during the first week postpartum. All the other
constituents return to normal values during
the first or second week postpartum.
Disagreement exists as to whether there
is a change in the volume of the extracellular
fluid compartment during pregnancy in the
human being. Whereas certain investigators
have reported rather marked increases in
the extracellular space (Chesley and Chesley, 1941; Chesley, 1943; Freis and Kenny,
1948; Caton, Roby, Reid and Gibson, 1949;
Friedman, Goodfriend, Berlin and Goldstein, 1951; Jarosova and Damn, 1951),
others have reported that the changes in
this fluid compartment are proportional to
changes in the body weight (LambiotteEscofiier, Moore and Taylor, 1953; Seitchik
and Alper, 1954). The results obtained in
the rabbit support the findings of the latter
authors as no disproportionate increase in
the thiocyanate space was observed during
pregnancy in the rabbit. The slight increase
that occurred during the last trimester of
ESTHAOIOL TREATMENT
DAYS OF TREATMENT
Fig. 16.24. Changes in blood plasma and total
erythrocyte volume in the ovariectomized rabbit
treated with 1 mg. estradiol daily. (From L. M.
Horger and M. X. Zarrow, Am. J. Physiol., 189,
407, 1957.)
DAYS ANTE PARTUM
Fig. 16.25. Changes in the blood plasma and
total erythrocyte volume during pregnancy.
(From L. M. Horger and M. X. Zarrow, Am. J.
Physiol., 189, 407, 1957.)
gestation was in good agreement with the
fluid accumulation by the developing fetus.
Similarly the increase in blood volume in
the rat is correlated with increase in body
weight.
Thus the anemia of pregnancy as observed in the rabbit and rat is very similar
to that reported for man. It can be characterized as a normochromic and normocytic
anemia. Although a decrease in the relative
concentrations of hemoglobin and erythrocytes occurs, the total amounts of these
components of the blood remain unchanged.
Consequently, the anemia of pregnancy is
due to a hemodilution.
The anemia induced by treatment with
estradiol is similar to the anemia of pregnancy in many respects. Witten and Bradbury (1951) treated 16 women with 5 mg.
estrone or 0.4 mg. estradiol dipropionate
and noted an erythrocyte drop of 14.8 per
cent, a hemoglobin drop of 8.5 per cent, a
hematocrit drop of 15 per cent, and a blood
volume increase. Treatment of the castrated
rabbit with 1 mg. estradiol daily caused a
20 per cent decrease in both erythrocyte
count and hemoglobin with no significant
changes in total hemoglobin or number of
erythrocytes. Estradiol also caused an increase in plasma and blood volume (Fig.
16.24) which was comparable to that seen
during pregnancy (Fig. 16.25), but no sig
GESTATION
991
nificant change in cell voluiiic was obtained.
The estradiol-induced anemia is both normochromic and normocytic and is caused by
a htMnodilution. However, in addition to the
ciianges in the blood and plasma volumes,
estradiol induces a significant increase in
the thiocyanate space. Furthermore, only
the massive dosage of 1.0 mg. estradiol per
day elicits an anemia comjKirable to that
observed in pregnancy. This dosage level
is probably toxic since there is a decrease
in the body weight of most rabbits which received this treatment. Thus, in spite of
the similarities of these anemias, it is
likely that estrogen is not the sole etiologic
agent in the anemia of pregnancy.
Progesterone alone at dosages of 4 mg.
daily has little effect on the plasma volume
or the thiocyanate space. This steroid does
exert a significant influence on the action
of estradiol on the blood and plasma volume, but it is to be noted that rather large
dosages of estradiol were still needed to induce a significant hypervolemia and that
the effect depends on the ratio of the concentration of the two hormones. The hypervolemia induced by the treatment with 4
mg. progesterone in combination with 0.1
mg. estradiol was greater than that caused
by the estradiol alone, whereas the treatment with 4 mg. progesterone in combination with 1.0 mg. estradiol resulted in an
inhibition of the estrogenic activity (Fig.
16.261. Thus progesterone may play a dual
role in the water metabolism of the gravid
female. In the presence of low titers of
estrogen, progesterone augments its action
which may be a means of insuring an adequate fluid retention to provide for the
fluid requirements of the fetus. However,
if the titers of the estrogens and possibly of
other steroids affecting salt and water metabolism became excessively high, the progesterone may provide a protective measure
by inhibiting the activity of these substances. This concept is in accord with reports describing the diuretic action of progesterone in the iiypophysectomized rat
(Selye and Bassett, 1940) and the inhibition of the salt- and water-retaining action
of DOCA and cortisone by progesterone
(Landau, Bergenstal, Lugibihl and Kascht,
1955).
G lOmg Estradiol
X Olmg Estradiol
A lOmg Estrodiol + 4 mg Progesterone
A I mg Estrodiol -f 4 mg Progesterone
TREATMENT PERIOD
-5 5 10 15 20 25
DAYS OF TREATMENT
Fig. 16.26. Changes in the blood volume of
the ovariectomized rabbit treated with 0.1 mg.
and 1.0 mg. estradiol daily and with a combination
of the two estrogen treatments and 4 mg. progesterone. (From L. M. Horger and M. X. Zarrow,
Am. J. Pliysiol., 189, 407, 1957.)
It is also of interest that no anemia was
observed in animals treated with various
combinations of estrogen and progesterone
(Horger and Zarrow, 1957). Progesterone
elicits an increase in the cell volume which
api^roximates that of the plasma volume.
Because no erythrocyte counts were made in
this study, it is not possible to state whether
this increase in the cell volume is caused
by a macrocytosis or an increase in the number of erythrocytes. Vollmer and Gordon
( 1941 ) reported that progesterone caused an
increase in the erythrocyte count of the
rat but that the action was inconsistent.
Hence it is possible that the increase in
the cell volume is due to an enhancement of
hematopoiesis by the progesterone. This
possibility is not inconsistent with the
absence of an increase in the reticulocyte
count in response to these treatments since
a reticulocytosis usually occurs only after
an intense stimulation of the hematopoietic
tissue such as by hemorrhage.
In view of the previous discussion, it is
improbable that the anemia of pregnancy
is due entirely to the interaction of estrogen
and progesterone. These hormones appear to
992
SPERM, OVA, AND PREGNANCY
play an important role in the salt and
water metabolism of the gravid female.
Furthermore, it is noted that the cow
exhibits a hypervolemia but no anemia
during pregnancy (Reynolds, 1953) and
that a similar condition is produced in the
rabbit by the treatment with various combinations of these steroids. Hence the interaction of estrogen and progesterone may be
responsible for this species difference.
Inasmuch as no antidiuretic hormone
(ADH) could be detected in any of the
plasma samples, it is apparent that the
plasma titers of ADH did not rise above
10 fiV. per ml. during the experimental
period. However, in view of the increased
ability of the blood to inactivate ADH during pregnancy (McCartney, Vallach and
Pottinger, 1952; Croxatto, Vera and Barnafi, 1953), there may be an increased
rate of turnover of ADH during gestation.
Consequently the data obtained in this
study neither substantiate nor eliminate
ADH as an etiologic agent in the anemia of
pregnancy.
A number of investigators have attributed
the hypervolemia of pregnancy to structural
changes in the circulatory system. Burwell
(1938) observed a marked similarity between the circulatory changes observed in
pregnancy and those observed in a patient
with an arteriovenous fistula. He noted that
in both conditions there is an increase in
the blood volume, cardiac output, pulse
rate, pulse pressure, and an increased venous
pressure near the opening of the fistula. He
concluded that the changes in the circulation of the pregnant woman are caused by
an arteriovenous leak through the placenta
and the obstruction of the venous return
by the enlarged uterus. Bickers (1942) correlated the intensity of the edema of the
right or left leg with the location of the
placenta in the uterus. The edema was observed to be consistently greater on the same
side as the location of the placenta whereas
the edema of the legs was equal when implantation occurred on the anterior or posterior wall of the uterus. However, when
the uterus was lifted off the interior vena
cava, there was no precipitous drop in the
venous pressure in the femoral vein. Thus
this study supports the arteriovenous shunt
theory of Burwell.
One objection to this theory is that it i
does not account for the decrease in the i
blood volume during the 10th lunar month j
of pregnancy. However, it has been reported i
that during the latter part of pregnancy
there is an increase in the resistance to the
flow of blood through the placenta. This increase is due to the increased number of
villi and to the anastomizing of the villi in
the placenta. Since an increase in the peripheral resistance to blood flow results in
a hemoconcentration, this would account for
the decrease in the blood volume during
the last lunar month of gestation (Kline,
1951; McGaughey, 1952).
Other objections to the arteriovenous
shunt theory were reported by Kellar ( 1950)
who found that blood flow through the
placenta is sluggish rather than rapid as
in an arteriovenous shunt. He also observed
that the uterine venous blood is not exceedingly rich in oxygen as is the venous
return of an arteriovenous aneuryism and
lie concluded that, although the uterus is
an area of decreased resistance to blood
flow, the effect is not entirely due to the
placenta. He suggested that thyroxine may
be partially responsible for the expansion of
the blood volume since mild thyrotoxicosis
is common in pregnancy. This concept is
supported by the observation that there
is a tendency for vasodilation in the upper
extremities during the latter months of gestation (Burt, 1950). Furthermore, the basal
metabolic rate increases during this period
(Sandiford and Wheeler, 1924; Rowe and
Boyd, 1932). It is to be noted, however,
that the changes in the blood flow in the
extremities are closely correlated with the
cardiovascular changes occurring during
pregnancy but not with the changes in the
hormonal levels in the blood (Herbert,
Banner and Wakim, 1954 ) .
Since there is no disproportionate increase in the thiocyanate space of the rabbit
during pregnancy, the increase in the blood
volume can best be explained on the basis
of cardiovascular changes during the latter
part of gestation. However, in view of the
previous discussion, it is extremely improbable that this hypervolemia is induced
by any one factor. Rather, it is more probable that the condition is produced by a
multiplicity of factors. On the basis of the
GESTATION
998
previous reports, it is evident that the
placenta, due to its similarity to an arteriovenous aneuryism, is partially responsible
for the hypervolemia. The marked increase
in the uterine size and vascularity during
pregnancy (Barcroft and Rothschild, 1932)
will also account for a considerable amount
of the increase in the blood volume. In addition, the tendency for dilation of the
peripheral blood vessels may account for
another portion of the increase in the blood
volume. Thus, in general, the hypervolemia
of pregnancy can be attributed primarily
to structural changes in the circulatory system.
It cannot be denied, however, that the
cardiovascular system is influenced by the
changes in the endocrine balance during
gestation. It is well known that the placenta
elaborates large amounts of sex steroids
and corticoids. It has also been suggested
that there is an increased production of
thyroxine and ADH at this time. In addition, water-soluble extracts of the pregnant sow's ovaries have been shown to
cause water retention and anemia in the
rabbit (Zarrow and Zarrow, 1953). The
resultant hormonal balance becomes somewhat precarious as the additional secretions
of the glands tend to build up the blood
titers of the sex steroids and other substances which influence water metabolism.
It is possible that when a proper balance of
these factors is maintained, the pregnancy
is normal and the various requirements of
the fetus are provided without disrupting
the distribution of the body fluids outside
of the vascular system. However, if the
balance is not maintained, the animal tends
to accumulate fluid, and edema and other
pathologic complications result.
VIII. Plasma Proteins
It has long been known that the plasma
proteins play a significant role in the fluid
balance of the organism and as such are
also involved in water balance during pregnancy (]\Iack, 1955). In addition, the
plasma proteins are of importance in many
other functions, such as heat and energy
source and replacement of tissue in which
function they act as a protein source whenever needed and form the metabolic pool.
These proteins are synthesized in general.
in the liver and reticuloendothelial system
and may be classified as albumins or globulins although many different entities of
these two classifications are known to
exist.
The maintenance and stabilization of
blood volume and the equilibrium of fluid
exchange between the extravascular and
intravascular compartments is a function
of the albumin fraction of special significance in i)regnancy, in addition to its other
functions of acting as carrier for other
substances and sui^jilying of nutrients. The
regulation of blood volume by albumin
depends on its osmotic action and is of
much greater significance than the globulins. Approximately 4.6 gm. albumin and
3.17 gm. globulin per 100 ml. of plasma
are found in the normal, nonpregnant
woman. At least four types of glol)ulins are
present in the plasma among which are
found the lipoproteins, prothrombin, fibrinogen, antibodies, and several hormones.
Although both the plasma proteins and
albumin drop during pregnancy, this does
not necessarily indicate a drop in the total
available albumin protein. An increase in
the plasma volume compartment of 25 per
cent as seen in pregnancy could easily
result in an increase in the total amount
of circulating protein. However, as the
total circulating blood volume increases in
pregnancy, the albumin fraction and yglobulin seem to be diluted whereas the
other globulins become more concentrated.
Nevertheless, the globulins cannot compensate for the albumin loss and the total
protein decreases. Mack (1955) has listed
several possible explanations for the above
paradox : ( 1 ) the small albumin molecule
may diffuse more freely into tissues and
across placental membrane, and (2) albumin synthesis cannot keep pace with utilization.
Innumerable studies on the plasma i^-oteins of women during pregnancy have I'evealed markedly consistent changes in the
albumin-globulin ratio of the plasma. The
concentration of total protein and albumin
decreases while the total globulin increases.
The trend is apparent by the first trimester
and continues throughout gestation. A return to the nonpregnant pattern is seen
shortly after parturition. The total protein
994
SPERM, OVA, AND PREGNANCY
dropped 13 per cent and the albumin 26
per cent. The various globulin fractions
showed a rise except for the y-globulin
(Mack, 1955). As a result of these changes,
the albumin-globulin ratio declines
throughout pregnancy and shows the well
known reversal (Fig. 16.27) and recovery
to normal by 6 weeks postpartum.
Although it is obvious that the albumin
fraction is important in maintaining the
Non- First Second Third Delivery 5-6 6-12
pregnant trimester days weeks
Dostpartum
Fig. 16.27. Progressive decreu.se in the albuminglobulin ratio of the plasma during pregnancy in
women. (From H. C. Mack, The Plasma Proteins
in Pregnancy, Charles C Thomas, Springfield, 111..
1955.)
E 150
100
2nd
Z'<^ POST
PARTUM
Fig. 16.28. Changes in the glomerular filtration
rate throughout pregnancy in the woman. (From
W. J. Dignam, P. Titus and N. S. Assali, Proc.
Soc. Exper. Biol. & Med., 97, 512, 1958.)
blood fluid compartment, the changed
albumin-globulin ratio cannot solely account for the retention of water and edema
present in pregnancy. Although it has been
argued that the hypoalbuminemia through
diminished colloid osmotic pressure is the
cause of water retention in the tissues,
the occurrence of the postpartum diuresis at
the time when the albumin is lowest would
tend to indicate some other mechanism
(Dieckmann and Wegner, 1934a-d). Additional mechanisms, such as changes in the
hormone level, especially the sex steroids
and adrenal corticoids, may be responsible.
IX. Renal Function
Studies on renal function during pregnancy have resulted in contradictory reports. The earlier investigations failed to
show any effect of pregnancy on renal function (Chesley and Chesley, 1941; Welsh,
Wellen and Taylor, 1942; Dill, Isenhour,
Cadden and Schaffer, 1942) , whereas recent
studies indicate a marked change in renal
function during gestation (Bucht, 1951;
Dignam, Titus and Assali, 1958). Part of
the explanation for the divergent results
could be the type of patient studied, the
periods when studied, and the types of
controls. Dignam, Titus and Assali studied
both the renal plasma flow and glomerular
filtration rate in various patients throughout gestation and immediately following
l^arturition. Care was taken to select individuals without any history of cardiovascular or renal disease. Both the renal
plasma flow and the glomerular filtration
rate (Fig. 16.28) were increased throughout gestation. The initial rise was extremely marked during the 1st and 2nd
trimesters of pregnancy. A slight rise was
noted during the 3rd trimester and a return to normal by 6 to 8 weeks postpartum.
Recently, de Alvarez (1958) reported a
50 to 60 per cent rise in the glomerular
filtration rate and a 60 per cent rise in the
renal plasma flow during the 1st trimester
of pregnancy in the human being. This is
in agreement with the findings of Dignam,
Titus and Assali (1958). However, de Alvarez reported, in addition, a progressive
decline in both the glomerular filtration
rate and renal plasma flow during the 2nd
and 3rd trimesters. The filtration factor
GESTATION
995
(glomerular filtration rate divided by the
renal plasma flow) remained low in the first
2 trimesters and increased in the last trimester. This is evidence for an increase in
tubular resorption of water and electrolyte.
It can only be concluded, therefore, that
kidney function is altered during pregnancy, especially the 1st trimester. Results
from investigations involving the 2nd and
3rd trimesters are contradictory. De Alvarez concludes that the changes in renal
hemodynamics during pregnancy are mediated by the endocrine system because the
alterations in renal function seem to be
related to the sodium and water retention.
If the changes are progressive throughout
gestation, it would be possible to correlate
the phenomenon with a number of hormones that increase during pregnancy. On
the other hand, if the phenomenon is transient, i.e., only during the 1st trimester, then
the phenomenon can only be correlated with
HCG.
X. Enzymes
A. HISTAMIXASE
The presence of histaminase or diamine
oxidase in tissues of the body has been
known for some time. As yet the enzyme
lias not been crystallized but is believed to
he a flavoprotein (Swedin, 1943). The enzyme is not specific for histamine because it
inactivates other diamines such as cadaverine and putrescine. Histaminase determinations, in general, are based on incubation of
the test material with histamine dihydrochloride for a fixed period of time and the
bioassay of the residual histamine carried
out on an isolated strip of guinea pig intestine.
Histaminase has been found in the
l)lasma of men and women with an increase
(luring ])regnancy from a value of between
0.003 and 0.008 /^g. per ml. per hr. to a
value of between 3.5 and 10 at parturition
(Ahlmark, 1944, 1947). This has been confirmed by Swanberg (1950), who determined the histaminolytic activity in pei-iplK>ral blood throughout pregnancy (Fig.
16.29). A marked rise is observed from
the 10th to the 20th week of pregnancy,
and thereafter the concentration plateaus
until after parturition.
20 30 40 weeks
Fif!. 16.29. Tlie histaminase activity of the
peripheral blood of the human female during
pregnancy (•) and at parturition (®). (From
H. Swanberg, Acta scandinav., Suppl. 79, 23,
1950.)
Both the maternal placenta and the
decidual tissue have been identified as
major sites for formation of the enzyme.
Danforth and Gorham (1937) reported the
presence of histaminase in the placenta
of a series of patients at term. This was
confirmed by Swanberg (1950) who, in
addition, separated the placenta by a series
of slices parallel to the surface of the organ
and reported that the layer adjacent to
the uterine wall, consisting of practically
only the thin decidual membrane, contained
a mean value of 614 /xg. per gm. per hr. of
histaminase as compared to 38 for the fetal
portion of the placenta. Confirmation of the
concept that the maternal placenta is the
main source of histaminolytic activity can
be obtained from the finding of histaminase
in decidual tissue of nonpregnant females
and in the maternal placentas of animals.
In cases in which maternal and fetal
placentas can be separated easily, the
maternal placenta contained from 14- to
100-fold the activity seen in the fetal
placenta. Comparison of the histaminolytic
activity in the decidual tissue of the sterile
horn and the control pregnant horn of the
uterus of a rabbit revealed 319 fig. per gm.
per hr. and 222 fxg. per gm. per hr., respectively. Treatment with progesterone or induction of jiseudopregnancy caused a
marked rise in the histaminase of the
endometrium to upwards of 1000 fig. per
gm. per hr. Nonetheless, histaminase was
996
SPERM, OVA, AND PREGNANCY
not observed in the blood plasma of the
progesterone treated rabbits whereas progesterone treatment of two nonpregnant
women caused a marked rise iti plasma
histaminase.
The physiologic significance of histaminase is still unknown. A consideration of
this problem must take into account not
only the action of the enzyme and changes
in its concentration under different physiologic conditions, but also the species problem. In regard to the latter point, the data
are extremely inadequate. Only two species
have been studied in any detail and these
are the human being and the ral)bit. One
can conclude from the available data that
histaminase is produced by the maternal
placenta, decidua, and uterine endometrium.
It increases with pregnancy in these tissues
and its concentration may be correlated
with the progestational hormone. It increases in the blood of tlie human being,
I'at, and guinea pig during pregnancy but
nut in the cat or rabbit (Swanberg, 1950;
Carlsten, 1950). The obvious hypothesis
that histaminase })rotects the uterus from
the stimulating action of histamine has not
been confirmed. But it is somewhat jiai'adoxical to note that urinary histamine also
increases during pregnancy. Kahlson,
Rosengren and Westling (1958) reported a
daily 24-hour excretion of 18 to 43 /xg.
of histamine during the first 2 weeks of
pregnancy in the human being. A marked
increase was noted on the 15th day with a
peak of 123 to 835 /xg. per 24 hr. at the
peak of excretion which occurred 1 to 2
days before parturition. As yet no role
can be attributed to this substance. It is
of interest that the increased histaminase
present during pregnancy can serve the role
of protecting the uterus from the musclecontracting action of this substance. Because the amount of urinary histamine excreted is correlated with the number of
young and no changes are apparent in the
concentration of histamine in the tissues
during pregnancy, it would seem that the
excessive formation of histamine during the
last trimester of pregnancy takes place in
the uterus and its contents and the basic
action of histaminase is protective.
It was shown recently that the excessive
formation of histamine during the last tri
mester of pregnancy in the rat is due to
an increase in the rate of histidine decarboxylase activity (Kahlson, Rosengren,
Westling and White, 1958). Inasmuch as
removal of the fetuses without other interference with the pregnancy abolishes the
increased urinary histamine, it can l)e concluded that the site of formation is in the
fetus. This histamine could escape into the
maternal circulation and eventually be
eliminated via the kidneys.
Roberts ( 1954) reported that aminoguanidine leads to a general disturbance of
pregnancy in the rat; large doses tended to
jiroduce death of the mother and smaller
doses tended to kill all or part of the litters
and some of the mothers. Again one could
conclude a protective action on the part of
histaminase dui'ing tlie latter i)art of i^regnancy.
B. CARBONIC ANHYDRA.se
Carbonic anhydrase was discovered by
Aleldrum and Roughton in 1933 and soon
shown to catalyze the following reaction,
H,CO, z:± CO. + HoO. The enzyme was
found to occur in many tissues and was
generally located within the cell especially
in cells possessing a secretory function. The
discovery by Lutwak-Mann and Laser
(1954) that carbonic anhydrase is present
in tlu' uterine mucosa led to a thorougli
study of the changes in the concentration
of the enzyme and the factors controlling
its presence (Lutwak-Mann, 1955; Lutwak-Mann and Adams, 1957). The enzyme
has been found to be present in the reproductive tract of a wide variety of
mammals. In general, the uterine endometrium, placenta, and Fallopian tubes are
the main loci of activity although there
are marked differences among different
species. Carbonic anhydrase activity was
found consistently in all the animals studied
such as the rat, hamster, guinea pig, rabbit,
pig, and ewe. No activity was noted in the
uterine mucosa of the nonpregnant animal
except the ewe and the rabbit. In several
species, such as the cow, human being,
and pig, carbonic anhydrase was also found
in the Fallopian tube.
A marked rise in carbonic anhydrase of
the endometrium of the rabbit was noted
during the first trimester of pregnancy
GESTATION
997
(Fig. 16.30). The value rose from a prepregnancy level of 20 enzyme units (E.U.)
per gm. of fresh tissue to a maximum of 100
E.U. per gm. at approximately the 8th day
of pregnancy. This level was maintained
until the 12th day and then declined to
approximately the prepregnancy level by
about the 20th day. Examination of the
placentas at this time revealed marked activity, 68 E.U. per gm. of maternal placenta
and 25 E.U. per gm. of fetal placenta. The
curve for the concentration of carbonic
anhydrase in the uterine mucosa during
pscudopregnancy is essentially the same
as that seen during pregnancy, although
some minor differences exist.
It is obvious from the above data and
from the evidence involving the increased
concentration of carbonic anhydrase in the
uterine mucosa following treatment with
progesterone, that the enzyme is probably
under the control of the luteoid hormone.
Indeed, an excellent correlation has been
shown between the degree of {progestational
proliferation in the uterus and the concentration of carbonic anhydrase. In the ewe,
however, the carbonic anhydrase of the
uterus is independent of the ovary. A possible explanation for this discrepancy between the two species has been offered on
the basis of differences in the blood level
of progesterone. However, no explanation is
forthcoming for the failure to maintain
the carbonic anhydrase level throughout
pregnancy in the rabbit, even though the
circulating progesterone remains liigh.
The significance of this enzyme in the
physiology of reproduction is still unknown.
From the data on the rabbit, it miglit be
inferred that the carbonic anhydrase contributes to the maintenance of bicarbonate
in the blastocyst fluid. The universal presence of the enzyme in placental tissue could
also lead to the assumption that carbonic
anhydrase is involved in fetal metabolism.
Lutwak-Mann (1955) indicates that the
enzyme might be involved in the transmission of calcium across the placenta.
^^'hether carbonic anhydrase is essential for
fetal (Icvelopment and successful pregnancy
is still unanswered. Treatment with carbonic anhydrase inhibitors (Diamox)
failed to affect adversely the pregnancy or
fetuses in pregnant rats even though no
Fid. 16.30. Carhonic anhydrase activity in the
uteiu.s of the rabbit during pregnancy, i),seudopregnancy, large doses of gonadotrophin, and
pregnant mare's serum (PMS). Pregnancy,
• •; i^seudopregnancy, O O; gonadotrophin, D D: PMS, x" X. (From C. Lut
\val<-Mann. .1. Kndocrinol.. 13, 26. 1955.)
enzyme acti\'ity was present either in the
matei'nal blood or placenta.
XI. Factors in the Maintenance
of Gestation
A. THYROID GLAND
Several recent reviews have pointed out
that the extract role of the thyroid gland in
reproductive physiology is still in need of
elucidation (Peterson, Webster, Rayner and
Young, 1952; Reineke and Soliman, 1953).
Numerous investigations over the past half
century have definitely indicated that the
thyroid gland is involved in reproduction
but the site and manner of action are still
not well known. In addition, contradictory
reports indicate that each species and even
each strain may have to be studied independently (Alaqsood, 1952). Some evidence foi- the involvement of the thyroid
gland in gestation has already been considered. The increase in FBI at the onset of
jiregnancy and the incidence of miscarriage
in the human female when the FBI fails to
rise tend to involve the thyroid hormone in
the maintenance of pregnancy. Habitual
abortion in women is usually associated
with t'ithcr hypo- or hyperthyroidism
(Litzenberg, 1926). Litzenberg and Carey
(1929 » I'eported that in 70 married women
with low basal metabolic rates appi'oximately 45 per cent had one or more abor
998
SPERM, OVA, AND PREGNANCY
tions or stillbirthsrtf one eliminates the
sterile woman from the group, the figure for
women showing abortion or stillborn rises
to approximately 35 per cent. However, the
results are still controversial both with
regard to data obtained within a single
species and from different species.
Hypothyroidism in the rat induced by
the prolonged administration of thiouracil
resulted in a resorption of the fetus in 100
per cent of the cases (Jones, Delfs and
Foote, 1946). Rogers (1947) reported a reduction in litter size following sulfaguanidine and Krohn and White (1950) reported
a reduction in litter size following thyroidectomy in the rat. Thyroidectomy early in
pregnancy caused a resorption of the
fetuses and if performed at a later stage
in pregnancy resulted in the birth of stillborn young (Chu, 1945). Following the induction of pregnancy in thyroidectomized
rabbits, either a resorption of the young or
abortion or prolongation of gestation was
noted and the newborn young were usually
dead. Chu concluded that the thyroid hormone was concerned with the vitality and
growth of the embryos during gestation. In
the pig the average duration of pregnancy
was 114 days for normal gilts and 124.5
days for thiouracil-treated animals. In addition, the controls farrowed an average of
8.67 pigs per litter compared with 3.25 per
litter for the thiouracil-treated .sows ( Lucas,
Brunstad and Fowler, 1958 ) . The difference
was significant in both instances. Bruce and
Fio. 16.31. Tlie effect of tliyioid deficiency on
litter size. O, 422 litters from tliyroid-defieient
mice; •, 423 litters from normal control mothers.
(From H. M. Bruce and H. A. Sloviter, J. Endocrinol., 15, 72. 1957.)
Sloviter (1957) pointed out that part of
the conflicting reports on the role of the
thyroid in gestation might be due to the
different methods used in producing a
thyroid-deficient state. Surgical removal of
the gland generally results in the loss of
the parathyroids which may be also important in the maintenance of gestation
(Krichesky, 1939), although adequate information is lacking. The use of antithyroidal substances offers more serious objections because these drugs not only pass
through the placenta but they are nonspecific and interfere with other glands such
as the adrenal cortex (Zarrow and Money,
1949; McCarthy, Corley and Zarrow, 1958),
with nutrition, and with the general status
of the animal. Consequently, Bruce and
Sloviter preferred to establish a thyroidectomized state in mice by the use of radioactive iodine after establishing the dose
necessary to induce total destruction of the
thyroid without damage to the jiarathyroid
or ganiete.s.
Although ( lorbman ( 1950 ) rei)orted a
complete loss of reproductive activity in the
mouse following treatment with P'*\ Bruce
and Sloviter (1957) reported no effect on
the ability of the mouse to conceive or bear
young. This discrepancy could be due in
part to the strain differences in the sensitivity of the ovary to the I^^^. Bruce and
Sloviter (1957), however, noted a decrease
in the average litter size of thyroid-deficient
mice (Fig. 16.31 ). The data indicate a maximum of 6 young per litter in thyroid-deficient mice versus 10 young per litter for
the normal mice. It is apparent that the
entire curve for the litter size of thyroiddeficient mice is shifted toward a smaller
size. This has also been observed in the rat
following thyroidectomy (Nelson and Tobin, 1937). The thyroid-deficient mice also
showed a prolongation of gestation as reported in rats, guinea pigs, and sows. Of
the thyroid-deficient rats, 46 per cent
showed a gestation period of more than 19
days whereas only 15 per cent of the normal
controls showed a gestation period of more
than 19 days whereas only 15 per cent of
the normal controls showed a gestation period of more than 19 days (Table 16.9).
Analysis of the data based on grouping
according to litter size showed clearly an
GESTATION
999
eifect of litter size on length of gestation.
The smaller litter size gave a higher inci(l(>nce of prolonged gestation.
Studies on oxygen consumption in the
guinea i)ig revealed a slight but significant
rise of 8 per cent at the end of gestation
(Hoar and Young 1957). The increase in
oxygen consumption is consistent but slight
for the first 60 days of pregnancy after
which the significant increase occurs (Fig.
16.32). The rise continued until 5 days
postpartum and then fell rapidly. In a second set of experiments oxygen consumption
was measured in control, thyroidectomized,
and thyroxine-injected, pregnant guinea
pigs. Measurements were taken at the
time of mating and at parturition. In all
three instances, an increase in the oxygen
consumption was noted at parturition as
compared with the values at the time of
TABLE 16.9
Ejfcct of thyroid-deficiency and litter size on length
of gestation in mice
(From H. M. Bruce and H. A. Sloviter, J.
Endocrinol., 15, 72, 1957.)
Thyroid-deficient
Control
No. of
Young In
Litter
No. of
pregnancies
> 19 days
No. of
pregnancies
> 19 days
No.
Per
cent
No.
Per
cent
1-5
6-9
10-14
Totals
36
40
20
96
24
13
7
44
67
33
35
46
16
38
28
82
7
3
2
12
44
7
8
15
mating (Fig. 16.33). Again the control
guinea pigs showed a 7.9 per cent gain in
oxygen consumption by the end of pregnancy, but both the thyroidectomized pregnant guinea pigs and the thyroxinc-treated
guinea pigs also showed an increase in oxygen consumption of 11.9 and 16.2 per cent,
respectively. The increase in oxygen consumption was not paralleled by increases in
heart rate; actually the heart rate decreased
in several instances. In addition, neither
the weight of the thyroid gland nor the
histology of the gland was changed during
pregnancy. It is obvious then that an explanation for the rise in oxygen consumption during pregnancy may not involve the
thyroid gland. On the basis of changes in
its appearance. Hoar and Young (1957)
suggested the possibility that the adrenal
cortex is involved and that the increased
oxygen consumption is due to an increased
release of adrenal corticoids. More evidence
is needed before this suggestion can be fully
accepted.
Further work from the same laboratory
has led to the concept that one locus of
action of thyroxine during pregnancy is at
parturition (Hoar, Goy and Young, 1957).
These investigators used an inbred strain
of guinea pigs that is characteristically hypothyroid and a genetically heterogeneous
stock in which the level of thyroid activity
is presumed to be higher. It had been previously shown that pregnancy wastage was
high in the hypothyroid guinea pigs. Treatment with thyroxine reduced the percentage
of stillborn from 40 to 13.6 in the hypothy
c 80
o
♦a.
£70
c
o
^60
c
o»
>«50
40
71.012.2
58.7±1.4
59.411.3
59.6±l.5
-— *--63!410.9 63.811.9
^
Parturition
Length of
Gestation
in Days
10
20
30
40
50
60
70
80
Fig. 16.32. Oxvgen consumption in the guinea pig during gestation. (From K. M. Hoar
and W. C. Young, Am. J. Physiol., 190, 425, 1957.)
1000
SPERM, OVA, AND PREGNANCY
100
40
%(.._»( Thyroxin-injected females
C— ^Control females 92.2±2.2
X — -XThyroidectomized
females _.
.^¥.
63.4±0.9
59.6+1.8
o 5o[.53.3±i.4 Parturition
Length of Gestation in Days
10
20
30
40
50
60
70
80
Fig. 16.33. Oxygen con.suinption in the pregnant guinea pig treated with thyroxine or
thvroiclpctomized before mating. (From R. M. Hoar and W. C. Young, Am. J. PhysioL,
190, 425, 1957.)
roid guinea pig!>, i.e., to a level !?een in the
untreated heterogeneous group. Treatment
of the heterogeneous group with thyroxine
not only failed to reduce the percentage
of stillborn but actually increased the abortion rate particularly in the 2nd and 3rd
trimesters. The most consistent result, however, was a decrease in length of gestation
following treatment with thyroxine, and an
increase following thyroidectomy. From
these experiments it was concluded that
the thyroid hormone facilitates parturition
and need be present only late in gestation
to exert its action.
It is apparent that in some species the
thyroid hormone is involved directly in
pregnancy. In the absence of the hormone,
certain species tend to resorb or to abort;
or if pregnancy is maintained gestation
tends to be lengthened. This is probably
due to an interference with the mechanism
of parturition. In certain species such as
the guinea pig only a parturitional problem
has been demonstrated; in others an entire
galaxy of symptoms may be present. Reduction in the size, number, and viability
of the young give added emphasis to an essential role for thyroxine in the phenomenon of gestation.
B. ADRENAL CORTEX
Removal of the adrenal cortex without
further treatment invariably leads to dis
turbances in rejM'oductive i)hysiology and
the termination of pregnancy. Although the
early results were controversial in that
some investigators reported that adrenalectomy failed to affect gestation in the rat
(Lewis, 1923; Ingle and Fisher, 1938),
others reported that adrenalectomy led to
abortion (Wyman, 1928; Dessau, 1937) or
to some other disturbance of gestation (McKcown and Spurrel, 1940). Davis and
Plotz (1954) adrenalectomized two groups
of pregnant rats on the 4th to 6th and the
14th to 16th day of pregnancy. Abortion
occurred in all 12 rats adrenalectomized
during the first half of pregnancy whereas
only 1 of the 12 adrenalectomized during
the second half of pregnancy aborted. However, even in those adrenalectomized during
the second half of gestation, an effect on
jiregnancy was observed. A significantly
higher incidence of stillborn and sickly
young (14.4 per cent) and a marked decrease in the weight of the fetuses were
noted (Table 16.10).
Early results indicated that extracts of
the adrenal cortex could readily replace
the absent adrenal gland and maintain successful pregnancies. Within recent years it
has been demonstrated that many steroids
such as cortisone and 9a-chlorohydrocortisone at 10 /xg. per day (Llaurado, 1955)
permit fecundation and successful maintenance of pregnancy. Successful maintenance
GESTATION
1001
TABLE Ki.lO
Effects of adrenalectomy on the character of the litter,
and on fetal body weight and adrenal weight
(From M. E. Davis and E. J. Plotz,
Endocrinology, 54, 384, 1954.)
Pregnant
Controls
Adrenalectomy
2nd Half of
Pregnancy
Percentage
versus
Pregnant
Controls
No. of litters ....
21
11
Dead and
"sickly" young.
Vigorous voung . . .
5
182
13
78
<0.01
Fetal hodv weight
(gm.)
6.13
5.22
<0.01
(=b0.10)«
(±0.30)"
Fetal adrenal
weight (mg.). . . .
0.49()
0.554
>0.3
(±0.017)"
(±0.041)«
Fetal l)()dv weight/
fetal adrenal
weight X 1000. . .
12.35
9.42
<0.01
(0.34±)''
(±0.31)"
Calculation of standard error of the mean:
S.E.
/ Ed-^
]/ nin
1)
of a i)ivgnancy has also been reported in
an adrenalectomized human female maintained on hydrocortisone 9a-fliiorohydrocortisone (Laidlaw, Cohen and Gornal,
1958). In this instance measurements of
urine excretion of aldosterone revealed an
increase to 4.4 fxg. per 24 hours during the
last trimester of pregnancy and a postpartum value of 0.5 fig. Inasmuch as the value
is only 1/10 of that seen in a normal pregnancy the authors concluded that the adrenal cortex of the mother is the major
source of aldosterone during pregnancy and
that a high output is not a major prerequisite for a normal pregnancy.
Treatment with either 0.9 per cent saline
drinking water or with cortisone increased
the number of successful pregnancies following adrenalectomy during the first half
of gestation. Pregnancy was normal in 8
of 11 adrenalectomized rats (Davis and
Plotz, 1954). Treatment with 2 mg. of cortisone acetate resulted in successful pregnancies in 13 of 14 rats adrenalectomized
on the 4th to 6th day of gestation and 12 of
12 rats adrenalectomized on the 14th to 16th
day of gestation. However, complete main
tenance was not obtained. The body weight
of the mothers and the weight of the fetuses
were significantly lower than in the controls, and the number of stillborn and sickly
young was increased.
A comparison of the pregnancy-maintenance activity in a number of adrenal corticoids indicated that a combination of a
glucocorticoid and mineralocorticoid provides the best protection in the adrenalectomized rat (Cupps, 1955). Nulliparous rats
were adrenalectomized, placed on treatment, and mated. Under these conditions
the adrenalectomized controls and the rats
treated with desoxycorticosterone acetate
failed to become pregnant inasmuch as no
implantation sites were obtained (Table
TABLE 16.11
Effect of adrenal steroids on reproduction in
adrenalectomized female rats
(From P. T. Cupps, Endocrinology, 57, 1, 1955.)
Daily Treatment
Control
Cortisone acetate
^i mg
Cortisone acetate
^i mg
Cortisone acetate
1'^ mg
Cortisone acetate
2,4 mg
Hydrocortisone
acetate Vi mg.. . ,
Cortisone acetate
Vi mg. plus
Desoxycorticosterone acetate ^
mg
Desoxycorticosterone acetate ^^
mg
Desoxycorticosterone acetate ^
mg
Desoxycorl icosterone acflate 1 mg ,
Adrenalectomized
control
No. of
Rats
No.
Born
Alive
(average)
Implantation
Sites
(average)
7
8.2
11.0
6
3.5'>
5.6^
6
3.6*
6.2'^
5
3.5
8.6"
5
5.8
10.0
7
5.0
8.5"
5
9.0
9.6
4
5
4
5
Weight
Change
during
Pregnancy
(average)"
gm.
46.4
-30. S''
-1.5«
17.2^
12.8^
30.7
44.6
"Weight change of mother from day of breeding to day after parturition.
" Significant at 0.05 level.
'= Significant at 0.01 level.
1002
SPERM, OVA, AND PREGNANCY
16.11). Treatment with 2.5 mg. cortisone
acetate per day was partially effective in
restoring reproductive capacity. Injections
of 1.25 mg. hydrocortisone acetate per day
gave results comparable with those obtained when cortisone was given, although
the ratio of young born alive to implantation sites indicated that hydrocortisone
acetate was more effective. It was definitely
more effective than cortisone acetate in
maintaining the body weight of the mother.
However, reproduction was completely restored to normal in the adrenalectomized
rat following treatment with desoxycorticosterone acetate and cortisone acetate.
Interference with gestation in the normal
animal has been reported by several investigators following treatment with ACTH
or adrenal corticoids (Courrier and Colonge, 1951; Robson and Sharaf, 1952;
Velardo, 1957). This is taken to indicate
that there is a finely balanced requirement
for adrenocortical hormones during gestation ; and that suboptimal or supra-optimal
amounts of the hormone interfere with
pregnancy. Courrier and Colonge found
that cortisone administered to intact rabbits in the second half of pregnancy interfered with gestation. Robson and Sharaf
treated both pregnant rabbits and mice
with ACTH and reported a marked effect
on gestation. Abortion or resorption occurred in 8 of 9 mice and in 8 of 11 rabbits.
Contamination by posterior pituitary hormones or gonadotrophins can be excluded.
A subsequent experiment with cortisone
also caused marked interference with pregnancy in the rabbit when 20 mg. were given ;
10 mg. were without effect. Administration
of cortisone to castrated or hypophysectomized pregnant rabbits maintained with
progesterone also caused damage to the
pregnancy. Since the hormone was not acting by way of the ovary or pituitary gland,
the authors felt that cortisone was acting
directly on the uterus and the uterine contents.
In the rat, however, ]Meunier, Duluc and
Mayer (1955) observed an effect on pregnancy only when cortisone acetate was injected at the time of mating. Rats injected
with 10 to 25 mg. cortisone acetate daily
for 5 to 6 days beginning on day 12 or day
14 of gestation had a normal pregnancy.
Velardo (1957) reinvestigated the problem in the rat and reported a marked reduction in litter size and an increase in the
number of stillborn following ACTH treatment. Although quantitative differences
appeared, a significant decrease in litter
size w^as observed only when the hormone
was given (1) before mating, (2) immediately after mating, or (3) between the 11th
and 15th day after mating. However, the
greatest effect was noted when the ACTH
was administered immediately after mating.
Surprisingly enough, litter size was markedly reduced only if adrenalectomy was
performed on day 7 of gestation. Adrenalectomy on day 8 to 14 of gestation had no
effect on live litter size. However, a total
of 6, 9, and 13 stillbirths were obtained
following adrenalectomy on days 8, 9, and
11. It is interesting that the number of stillbirths decreased from 21 following adrenalectomy on day 7 to none following adrenalectomy on day 14. It is apparent that
the adverse effects of adrenalectomy on
gestation decrease as pregnancy progresses.
It is also apparent from these and other
experiments that the action of ACTH is
mediated by the adrenal cortex. From these
results and others described above, it seems
likely that the adrenal corticoids may be
acting on the uterus.
Mayer and Duluc (1955) found that
adrenalectomy of the I'at on the 14th to
the 16th day of pregnancy led to variable
results. In 17 pregnant adrenalectomized
rats, gestation was terminated in 8, but
no interference was observed in 9. The rats
that failed to maintain pregnancy died
witiiin 2 to 3 days. Again it would appear
that delicate hormonal balances are involved. In a further investigation of this
problem Aschkenasy-Lelu and Aschkenasy
( 1957) reported that a diet adequate in salt
and proteins would prevent interference
with pregnancy in rats adrenalectomized
before mating. On a low protein diet, pregnancy could be maintained only in the intact rat (80 per cent) and then only if
daily injections of progesterone were given.
These authors believe that the role of the
GESTATION
100.^
adrenal corticoids in pregnancy is concerned with stimulation of appetite and
mobilization and degradation of proteins
to amino acids. The latter action would
permit the replacement of body protein in
the absence of a normal jirotcin intake.
C. PANCREAS
The impact of diabetes mellitus on the
course of pregnancy has been of interest
to the clinician for many years. In a recent
review of the subject, Reis, DeCosta and
Allweiss (1952) came to the conclusion that
"the carefully controlled diabetic aborts
no more frequently than the nondiabetic."
On the other hand, it has been well known
for many years that uncontrolled diabetes
and pregnancy are basically incompatible
(Eastman, 1946).
Studies in the rat have given controversial results with regard to the influence
of insulin on pregnancy. Davis, Fugo and
Lawrence (1947) reported that in the alloxan diabetic rat pregnancy was normal for
the first 12 days. Thereafter death of the
fetuses occurred followed by resorption.
Sinden and Longwell (1949) and Levi and
Weinberg (1949) reported no detrimental
effect from diabetes on the course of i^regnancy. The latter group obtained 12 pregnancies from 25 rats made permanently diabetic with alloxan. Eleven of the 12 rats
went to term and delivered normal fetuses
and 1 died during pregnancy. Recently,
Wells, Kim, Runge and Lazarow (1957)
reported a 14 per cent loss in fetal weight,
an increase in gestation length from a normal of 538 to 563 hours, and an increase in
fetal or neonatal mortality in the pregnant
rat made diabetic by pancreatectomy or
treatment with alloxan.
In general, the clinical data indicate that
uncontrolled diabetes has a detrimental effect on pregnancy, but that the abortion
rate in the controlled diabetics approaches
that seen in the ''normal" population. Since
the crux of the matter seems to hinge on the
severity of the diabetes, one might conclude
that the effect of insulin is an indirect one
by virtue of its action in maintaining a good
metabolic state. The conflicting reports
from animal experimentation may be due
to the differences resulting from uncontrolled environmental and dietary factors.
D. ovary: progesterone, estradiol,
AND RELAXIN
Marshall and Jolly (1905) were probably
the first to point out that ovariectomy during pregnancy leads to abortion or resorption of the fetuses in the rat. Subsequently,
a number of investigators repeated these
experiments and confirmed the findings in
all species tested thus far, provided ovariectomy is performed before implantation. Removal of the ovaries after gestation is well
under way, however, does not disturb the
course of pregnancy in all species. The human being, monkey, horse, ewe, and cow are
examples of species not dependent on the
ovary for the maintenance of pregnancy
once it has been well established. Species
such as the rabbit and the rat require the
presence of the ovary throughout pregnancy.
The importance of progesterone for i)regnancy was established by Allen and Corner
(1929) who first showed that an extract
of the corpus luteum will maintain pregnancy in the castrated rabbit. Identification
of the active substance in the extract as
progesterone led to the use of the hormone
in many other species. Allen (1937) reported
that crystalline progesterone was inferior
to the crude luteal extract in the maintenance of pregnancy in the castrated rabbit.
From these and other data, such as the enhancing action of estrogen on the progesterone-induced progestational reaction, he
inferred that a combination of estrogen and
progesterone should be superior to progesterone alone in the maintenance of pregnancy. However, he pointed out with proper
caution that the dosages would have to be
carefully regulated because estrogen could
also antagonize progesterone. Although Robson (1936) failed to enhance the action of
progesterone with estrone in the pregnant
hypophysectomized rabbit, Pincus and
Werthessen (1938) obtained enhancement
with both the androgens and estrogen.
Whereas the early work indicated that a
pregnancy maintenance dose of progesterone varied from 0.5 to 2 mg. (Allen and
Corner, 1930), later experimentation indi
1004
SPERM, OVA, AND PREGNANCY
cated that the dosage varied with the stage
of pregnancy. An adequate dose of approximately 1 mg. progesterone in the early
stages of pregnancy needs to be increased
to 5 mg. in the later stages (Allen and
Heckel, 1939; ComTier and Kehl, 1938a, b).
These investigators also revealed that an
optimal effect could be obtained by using a
progesterone-estrogen combination in the
ratio of 750 to 1. Chang (1951) transferred
ova to nonovulated intact rabbits and noted
that massive doses in the order of 25 mg.
macrocrystalline progesterone injected for
three times were required to obtain a 50 per
cent maintenance of pregnancy. He also reported that under the conditions of his experiment an initially high dose was needed
for the passage of the ova, implantation,
and early maintenance. Since then, further
experimentation, especially on other species, has revealed a significant role by estrogen in enhancing the pregnancy-maintaining action of progesterone.
A vast literature exists for the human
being on the prevention of threatened abortion by progesterone which is beyond the
scope of this review. Variation from negative results to excellent maintenance is reported. It is obvious that a great deal of
variability exists here and, to some extent,
this is explained by a need for more objective criteria in evaluating threatened abortion and the therapy (Guterman and Tulsky, 1949). It is obvious that if the
TABLE 16.12
Maintenance of pregnancy in the rat castrated on
the 12th day of gestation
(From J. Yochim and M. X. Zarrow, Fed. Proc,
18, 174, 1959.)
Progester
one
No.
Estradiol
Daily
Implantation
Site
No. of
Fetuses
No. of
Fetuses
Alive
Preg
Rats
Daily
dose
No.
daily
nancy
Index
mg.
Mg.
4
40
37
37
0.925
4
2
1
47
32
27
0.574
7
1
2
76
12
2
0.026
9
1.5
2
99
61
49
0.495
7
2
2
85
65
63
0.741
6
1
2
0.1
69
50
48
0.696
5
1.5
2
0.1
51
48
46
0.900
5
2
2
0.1
60
55
54
0.900
threatened abortion were the result of some
disturbance other than progesterone, that
progesterone therapy might be without success. Indirect evidence for the need for
progesterone to maintain a successful pregnancy in the human being and for the lack
of need for the corpus luteum once pregnancy is established has been presented by
Tulsky and Koff (1957). Corpora lutea
were removed from day 35 to day 77 of
pregnancy in 14 women. Two of the women
exhibited spontaneous abortion and a
marked drop in pregnanediol excretion. The
remaining 12 maintained a normal pregnancy and pregnanediol excretion. The data
can be interpreted to indicate a need for
progesterone during pregnancy and that
this need can be met by a nonovarian
source, i.e., the i)lacenta.
In both the rat and mouse, successful
maintenance of pregnancy after castration
has been obtained with progesterone or a
combination of progesterone and estrogen.
However, partial maintenance following
castration can be obtained in the rat under
special circumstances. Haterius (1936) removed all the fetuses except one and left all
placentas intact. Under these conditions the
remaining fetus was carried beyond term.
Alexander, Fraser and Lee (1955) found
that castration of the rat on the 9th day
resulted in 100 per cent abortion, whereas
60 per cent of the fetuses were retained until
term if castration was on the 17th day.
Dosage of progesterone as high as 5 to 10
mg. daily following castration the 9th day
gave only partial maintenance. It is possible
that better results would have followed
multiple daily injections. Yochim and Zarrow (1959) castrated rats on day 12 of gestation and obtained a pregnancy index (no.
of fetuses alive at day 20 h- no. of implantation sites at day 12) of 0.741 when 2 mg.
progesterone were gi^'en in two divided
daily doses and 0.495 when 1.5 mg. progesterone was given (Table 16.12). However,
the addition of 0.1 /^.g. estradiol daily
markedly enhanced the action of the progesterone so that a pregnancy index of 0.9, i.e.,
equivalent to the normal controls, was obtained with 1.5 mg. progesterone.
Finally, Hall (1957) has indicated that
relaxin synergizes with estradiol and progesterone in the maintenance of jiregnancy
GESTATION
1005
in the castrated mouse. One nig. progesterone per day maintained pregnancy in 83
per cent of the mice castrated on day 14 of
gestation, but 0.5 mg. maintained pregnancy in only 30 per cent of the animals.
The addition of 1.5 ^g. estradiol per day
was without effect. On the other hand, the
addition of relaxin to the estradiol and 0.5
mg. progesterone gave pregnancy maintenance in over 80 per cent of the mice as
compared with 30 per cent when progesterone alone was given.
Smithberg and Runner (1956) induced
ovulation and mating in prepubertal mice
(age 30 to 35 days) and obtained 100 per
cent implantation with 0.5 to 1 mg. progesterone daily and approximately 90 per cent
successful pregnancies when 2 mg. progesterone were given. A comparison of the
amount of progesterone required for maintenance of pregnancy in the normal and
castrated prepubertal mouse is given in Figure 16.34. In an interesting application of
the information available on the induction
of ovulation and maintenance of pregnancy,
Smithberg and Runner (1957) were able to
obtain successful pregnancies in genetically
sterile, obese mice.
Haterius (1936) observed that distortion
of the fetus occurred following ovariectomy
in the rat. This has been confirmed by
Zeiner (1943) in the rat and by Courrier
and Colonge (1950) in the rat and rabbit.
It was noted that castration greatly compressed the fetuses and eventually caused
death. Courrier and Colonge (1950) in very
elegant experiments showed that removal of
the rabbit fetus into the peritoneal cavity
prevented the distortion and death which
ordinarily followed castration. Frazer
(1955) obtained similar results in the rat
and concluded that fetal death after castration of the mother follows a rise in intrauterine pressure which is associated with an
increased tone of the circular uterine muscle fibers. Consequently the increased survival of the extra-uterine fetuses following
ovariectomy in the mother is the result of
the removal of this pressure by the circular
muscle of the uterus.
Many investigators have demonstrated
that gestation can be prolonged by inhibiting parturition. Both the injection of large
doses of progesterone or the formation of
0.25 0.5 1.0 2.0
PROGESTERONE (mg)
Fig. 16.34. Daily dose of progesterone required
to maintain pregnancy in the normal and castrated prepubertal mouse. (From M. Smithberg
and M. N. Runner. J. Exper. Zool., 133, 441,
1956.)
a new set of functional corpora lutea during
pregnancy will prevent parturition. The injection of an ovulating dose of HCG on the
25th day of pregnancy in the rabbit delayed
parturition for 15 days after the injection,
i.e., until the 40th day of gestation (Snyder,
1934). The fetuses survived in utero for
only 3 days and grew to greater than normal size during this period. The placentas
persisted until day 41 of gestation. Comparable results were obtained following
daily injections of progesterone into pregnant rabbits (Zarrow, 1947a). Haterius
(1936) obtained prolongation of pregnancy
in the castrated rat by removing all the
fetuses except one, leaving all placentas
intact. Recently a comparable experiment
was performed in tlie rabbit with intact
ovaries (Hafez, Zarrow and Pincus, 1959).
In 2 of 10 rabbits, live fetuses were obtained
l)y cesarean section on day 36. However, in
8 of the 10, delivery was delayed beyond
day 36, although some degree of fetal resorption was present in all instances. Prolongation of pregnancy in the rat was obtained by the injection of prolactin (Meites
and Shelesnyak, 1957), but only if the ovaries were present.
E. PITUITARY GLAND
In general, hypophysectomy before midpregnancy leads to resorption. This is especially true of the rat and mouse. On the
1006
SPERM, OVA, AND PREGNANCY
other hand, hypophysectomy at midpregnancy or later does not interfere in the
maintenance of gestation in these species
(Pencharz and Long, 1933; Selye, Collip
and Thompson, 1933a, b; Pencharz and
Lyons, 1934 ) . In the dog, ferret, and rabbit,
hypophysectomy leads to abortion (Aschner, 1912; McPhail, 1935a; White, 1932),
whereas the results in the cat seem contradictory (Allan and Wiles, 1932; McPhail,
1935b) .
Hypophysectomy of the rhesus monkey
does not always interfere with pregnancy.
Smith (1954) obtained normal pregnancies
in 10 of 20 hypophysectomized rhesus monkeys. The remaining animals aborted. Although more data are needed, it seems that
the pituitary gland can be removed very
early in gestation without disturbing the
pregnancy. Whereas hypophysectomy before midterm invariably leads to abortion
or resorption in the rat or mouse, 1 of the
4 monkeys hypophysectomized between the
29th and 34th day of gestation carried its
young to term. Inasmuch as Hartman and
Corner (1947) showed that the placenta secretes sufficient progesterone by the 25th
day of gestation to maintain pregnancy, it
is apparent that the placenta in the monkey
is able to maintain its endocrine secretory
activity independent of the pituitary and
at a sufficiently high level to replace the
ovary.
Little, Smith, Jessiman, Selenkow, van't
Hoff, Eglin and Moore (1958) reported a
successful pregnancy in the 37-year-old
woman hypophysectomized the 25th week
of pregnancy. The mother w^as maintained
on thyroid, cortisone, and pitressin tannate
replacement therapy. The excretion of chorionic gonadotropin and pregnandiol was
not markedly different from that seen in
normal gestation. Estrogen excretion was
slightly reduced and the 17-hydroxy corticosteroids dropped to zero when cortisone
therapy was discontinued. It would seem
that this phase of adrenocortical activity
was reduced and that ACTH or corticoidlike substances from the placenta were inadequate. No interference in aldosterone
output was observed.
Hypophysectomy on the 10th day of
gestation in mice terminated the pregnancy
in only 3 of 19 animals (Gardner and Allen,
1942). Sixteen mice carried their litters to
term although 7 of the 16 had a difficult
and prolonged parturition. Body weight
curves were normal and the corpora lutea
appeared unaffected by the loss of the pituitary gland, indicating either the independence of the corpus luteum or the presence
of a placental luteotrophin. Marked involution of the adrenal cortex was noted in all
instances.
Simultaneous measurements of the concentration of cholesterol in the adrenal
gland and ACTH in the pituitary of the rat
revealed a drop in adrenal cholesterol and
pituitary ACTH on the 15th day of gestation (Poulton and Reece, 1957). This was
followed by a marked increase of both substances on the 21st day of pregnancy and a
sharp drop at parturition. The authors concluded that a gradual increase occurs in
the secretory activity of the adrenal cortex
which reaches a peak on the 15th day of
pregnancy in the rat. Thereafter the activity decreased until parturition when a
marked increase was observed. The initial
decrease in pituitary ACTH potency followed by an increase after day 15 is interpreted as an initial increase in ACTH release followed by a decreased release. The
decrease in pituitary ACTH potency at
parturition is compatible with the marked
increase in adrenocortical activity at this
time if the decreased pituitary ACTH activity is interpreted as indicative of ACTH
release.
Maintenance of pregnancy in rats hyl')ophysectomized early in pregnancy was
obtained with prolactin by Cutuly (1942),
although Lyons, Simpson and Evans (1943)
reported negative results with a purified
prolactin. However, a partial maintenance
of pregnancy was obtained with purified
prolactin and estrone.
F. PLACENTA
The placenta is not only involved in the
synthesis of hormones during pregnancy but
also in the transfer of substances between
mother and fetus. It is obvious that the
transfer of substances is limited and the
l^lacenta does offer a barrier. This problem
bears not onlv on the matter of fetal
GESTATION
100/
nutrition, but also on the fetal environment
and as such is important in the sexual development of the fetus (see chapter by
Burns) .
The presence of estriol in the urine of
newborn male infants has led to the conclusion that estrogens can pass through the
placenta because of their low molecular
weight (Diczfalusy, Tillinger and Westman,
1957). Studies on the transfer of estrogens
across the placental barrier in the guinea
pig with C^'^-labeled estradiol revealed an
extremely rapid disappearance of radioactivity from the maternal blood following
intravenous injection of the hormone into
the mother, and the appearance of large
amounts of water-so.luble radioactivity in
the fetal plasma (Dancis, Money, Condon
and Levitz, 1958). However, no estradiol
was found in the fetal plasma. Replacement
of fetal circulation with a perfusion system
indicated that estradiol did not j^ass the
placenta although estriol was readily transferred in both directions. These authors reported that the placenta was relatively impermeable to the water-soluble estrogens
found in the urine, wliich are essentially
glucuronides.
The discovery in 1927 of large amounts
of estrogens and gonadotrophins in the
blood and urine of pregnant w^omen led to
the cjuestion as to whether the placenta is
a gland of internal secretion. This can be
answered with an uneciuivocal yes. Nevertheless, several questions are still unanswered: (1) the number of hormones produced by the placenta, (2) the quantities,
and (3) the secretory activity of the placenta in different species.
Data on the presence of gonadotrophins
in the placenta have already been discussed.
At least three different types of gonadotrophins have been extracted from the placentas of the human being, mare, and rat.
These have been defined physiologically and
appear to be different in the three species.
Cole and his co-workers have identified the
endometrial cups as the source of PJVIS in
the mare, whereas the elegant experiments
of Stewart, Sano and Montgomery (1948)
indicate that HCG in the human being is
secreted by the Langhans cells. These investigators grew human placental cells in
tissue culture and obtained ^ gonadotrophin
in the culture. They also noted a direct correlation between the growth of the Langhans cells and the production of gonadotrophic hormone (see also the discussion of
this subject in the chapter by Wislocki and
Padykula).
The initial discovery of a progressive rise
in the secretion of adrenal corticoids in
pregnancy (Venning, 1946) has been confirmed by numerous investigators. Gemzell
( 1953) attributed the steady rise to a stimulation of the adrenal glands by excessive
amounts of estrogen present during pregnancy and to hyperactivity of the fetal
adrenals. The hypertrophy of the fetal adrenal cortex in the rat following adrenalectomy of the pregnant mother was first reported by Ingle and Fisher in 1938 and
confirmed by Walaas and Walaas (1944),
and Knobil and Briggs (1955). However,
the 17-ketosteroid and corticoid level of
fetal urine is very low (Day, 1948; Jailer
and Knowlton, 1950) as are the 17-hydroxycorticosteroids in the blood of the newborn
infant (Klein, Fortunato and Papados,
1953). ACTH-like activity has been found
in extracts of the placenta (Jailer and
Knowlton, 1950; Tarantino, 1951; Opsahl
and Long, 1951) and corticoid activity has
been found in the placenta of horses and
human beings, as demonstrated by the glycogen deposition and growth-survival test
in adrenalectomized rats (Johnson and
Haines, 1952). Berliner, Jones and Salhanick (1956) isolated 17a-hydroxy corticoids from the human placenta.
Pincus (1956) reported that ACTH can
stimulate steroidocorticogenesis in the perfused placenta. Using the ascorbic acid depletion test, Assali and Hamermesz (1954)
assayed the blood in the intervillous space
and the chorionic villous tissue for ACTH.
Good activity was observed in the blood
from the intervillous spaces and in the tissue of the chorionic villi. Corticotrophic activity was also obtained by Lundin and
Holmdahl (1957) from placentas obtained
at full term, but the activity was small compared with that obtained from the pituitary
gland.
The possible role of the fetal pituitary
was investigated by Knobil and Briggs
1008
SPERM, OVA, AND PREGNANCY
(1955) who noted that hypophysectomy of
the mother prevented the fetal adrenal
weight increase observed following adrenalectomy of the pregnant mother. However,
complete atrophy of the adrenal gland was
not observed in the pregnant mother if the
conceptus was present. It was concluded
that ACTH can cross the placental barrier
and that the fetus or placenta or both produce a sufficient amount of ACTH, to influence the maternal adrenal gland in the
absence of the maternal hypophysis. It is
still questionable, however, whether these
sources, i.e., placenta and fetal pituitary,
are of sufficient magnitude to account for
the increased release of adrenal corticoids.
Hofmann, Knobil and Caton (1954) showed
that the ability of the hypophysectomized
nonpregnant rat to secrete a water load is
not greater than that of the hypophysectomized pregnant rat. Hence the contribution
of the fetal pituitary or j^lacenta to the
corticoid pool is not of sufficient magnitude
to influence water balance.
As with the gonadotrophins, the increased
amounts of estrogen ancl pregnanediol during pregnancy were thought to be derived
from the placenta. In 1933, Selye, Collip
and Thompson presented evidence to indicate that the placentas of rats jiroduce both
estrogen and gestagen. Many physiologic
data have been accumulated to prove this
point, but completely convincing evidence
was obtained only when these hormones
were identified in placental extracts and in
fluid perfused through the placenta. Diczfalusy and Lindkvist (1956) identified estradiol in the placenta and the presence of
progesterone was described by Salhanick,
Noall, Zarrow and Samuels (1952) and by
Pearlman and Cerceo (1952).
Perfusion experiments on human placentas have revealed that this organ secretes a
number of steroids (Pincus, 1956). These
include progesterone, desoxycorticosterone
Cortisol, and a number of unidentified steroids. Addition of ACTH to the perfusate
had no effect on the concentration of Cortisol, but it did increase the concentration of
the reduced corticosteroids, namely, the
tetrahydro derivatives of cortisone and Cortisol. This was interpreted as a stimulation
of the placenta by ACTH resulting in an increased release of the corticoid as demon
strated by the increase in the degradation
products.
The identification of the placenta as a
source of both sex steroids and certain gonadotrophins clarifies the manner by which
jiregnancy can be maintained in certain
species in the absence of the pituitary
gland or ovary (see sections above on ovary
and pituitary gland). Newton and Beck
(1939) and others showed the hypophysectomy of the pregnant mouse does not precipitate abortion. Studies of the ovary reveal that, if the placentas are retained, the
corpora lutea remain normal but removal
of the placentas causes immediate degeneration of the corpora lutea (Deanesly and
Newton, 1940). A comparable situation appears to exist in the rabbit and rat ; it is assumed, therefore, that the placenta takes
over control of the corpus luteum in pregnancy in those species that require the
ovary for successful gestation. In other species, such as man, sheep, cattle, and guinea
pig, it seems that the placenta can supplant
the ovary after pregnancy has progressed
to a certain stage.
G. PELVIC ADAPTATION
The discovery that pelvic changes are
under hormonal control in certain species
was the result of extensive studies on pelvic
adaptations associated with parturition (see
reviews by Allen, Hisaw and Gardner, 1939;
Hisaw and Zarrow, 1951). It has been argued that, in general, a narrow pelvis is
present in mammals living in burrows. This
would have the advantage of permitting an
animal to turn within narrow confines, but
a narrow pelvis would also interfere with
the delivery of the young at parturition. As
Hisaw pointed out in his extensive studies,
this problem has been met by special adaptations on the part of different species. This
has varied from a resorption of the cartilaginous pubic arch in the male and female
mole iScalopiis aquaticus machrinus, Raf.)
which is independent of the endocrine system (Hisaw and Zilley, 1927) to elongation
of the pubic ligament which is directly under hormonal control (Hisaw and Zarrow,
1951).
The symphysis pubis of the pocket gopher, Geomys bursarius (Shaw), behaves as
a female secondary sexual character so that
GESTATION
1009
a sex dimorphism exists in this species. The
pubic cartilages ossify in both sexes and
unite to form a complete pelvis with a rigid
symphysis pubis. At this stage, the pelvis is
too small for the passage of the young, but
with the first estrus in the female, the pubic
bones are gradually resorbed, leaving the
pelvis open ventrally. The pelvis in the male
remains intact (Hisaw, 1925). Treatment
with estrogen alone can readily bring about
the resorption of the pubic bones.
A third type of adaptive mechanism has
been described in great detail in the guinea
pig and led to the discovery of the hormone,
relaxin. A sex dimorphism of the pelvis exists in the guinea pig, as in the pocket gopher, but in addition parturition is further
facilitated by marked relaxation of the
pubic ligaments and of the sacroiliac joint.
Thus far extensive pelvic relaxation has
been described in the guinea pig (Hisaw,
1926, 1929 », mouse (Gardner, 1936; Newton
and Lits, 1938; Hall and Newton, 1946a),
women (see review by Hisaw and Zarrow,
1951), and rhesus monkey (Straus, 1932;
Hartman and Straus, 1939). No relaxation
of the pubic symphysis has been reported in
the ewe but a relaxation of the sacroiliac
joint and an elongation of the sacrosciatic
ligament was noted the 2nd to 3rd month of
gestation. These changes increased as pregnancy progressed (Bassett and Phillips,
1955). Treatment with stilbestrol alone
caused a marked loosening of the sacroiliac
joint and the sacrosciatic ligament. The addition of relaxin to the treatment was without effect (Bassett and Phillips, 1954).
The role of relaxin in the relaxation of
the pubic symphysis has been studied most
extensively in the guinea pig and mouse.
The work before 1950 was reviewed by Hisaw and Zarrow in 1951. The controversies
(de Fremery, Kober and Tausk, 1931 ; Haterius and Fugo, 1939) as to whether such
a hormone exists need not be discussed here,
in detail, except to point out that the evidence supporting this opinion is more than
adeciuate. Zarrow ( 1946, 1948) showed that
pubic relaxation could be induced by estradiol alone, by a combination of estradiol and progesterone, or by relaxin in
an estrogen primed animal (Table 16.13).
The difference in the time required to induce relaxation, i.e., 23 days for estrogen
alone, 13 days for estrogen and progesterone,
and 6 hours for relaxin, and data indicating
that progesterone caused the presence of
relaxin in the blood of guinea pig only if a
uterus was present led to the concept that
pubic relaxation may be produced independ
TABLE 16.13
Relaxation of the symphysis pubis and relaxin content of blood, urine, and uteri of castrated and castrated,
hysterectomized guinea pigs after treatment with moderate doses of estradiol and progesterone
(From M. X. Zarrow, Endocrinology, 42, 129, 1948.)
Treatment, Daily
Average Relaxation Time
Relaxin Content
No. of Guinea Pigs
Estradiol
Progesterone
Total
After progesterone
treatment
Blood serum
Urine
Uterus
MS
mg.
days
days
G.P.U./ml.
G.P.U./ml.
G.P.U./gm.
Castrated
9«
10
1 from day
11
13.5
(13-14)
3.5
0.5
0.3
10
10
10
2 from day
11
13.0
3
0.5
0.5
10
10
10
(12-14)
23.7
(16.31)
Negative at
4 ml.
Negative at
5 ml.
Negative
Castrated, hys
terectomized
11
10
1 from day
11
23.7
(17-30)
13.7
Negative at
4 ml.
Negative at
8 ml.
10
10
25.6
(18-32)
Negative at
4 ml.
Negative at
4 ml.
One guinea pig not included in the table refiuired 22 days of treatment for pubic relaxation.
1010
SPERM, OVA, AND PREGNANCY
ently by estradiol (prolonged treatment) or
relaxin (single injection). It is also possible
to conclude that the action of progesterone
is indirect and due to the formation of relaxin in the uterus (Zarrow, 1948; Hisaw,
Zarrow, Money, Talmage and Abramovitz,
1944) . In the mouse, however, progesterone
inhibits the action of relaxin on the pubic
symphysis (Hall, 1949).
Further evidence that two hormones are
involved in pubic relaxation was provided
by histologic examination of the pubic ligament. Symphyseal relaxation following estrogen appeared to be due to a resorption of
bone and a proliferation of loose fibrous
connective tissue with an increase in mucoid
alkaline phosphatase and water content
(Talmage, 1947a, 1947b, 1950; Heringa and
van der Meer, 1948). Relaxin produced a
breakdown and splitting of the collagenous
fibers into thin threads and a similar change
was noted with progesterone (Talmage,
1947a, 1950).
Histochemical and biocliemical studies of
the pubic symphysis have recently been reviewed (Frieden and Hisaw, 1933) and tend
to show that relaxin produces specific
changes. These include loss of metachromasia (Heringa and van der Meer, 1948) ,
accumulation of Evans blue m vivo, and increased solubility of the glycoproteins in
the McManus-Hotchkiss reaction, all of
wiiich indicate that a depolymerization of
the ground substance and basement membrane glycoproteins had occurred (Perl and
Catchpole, 1950) . Frieden and Hisaw (1951)
found an increase in water content of the
symphyseal tissue, but failed to find a decrease in the water-soluble hexose and hexoseamine following a single injection of relaxin. On the basis of a depolymerization
of ground substance, a decrease should have
occurred. However, repeated injections of
relaxin led to a decrease in the insoluble
hexoses and hexoseamines. In addition, consistent decreases in collagen content and
trypsin-resistant protein content were noted.
No hyaluronidase was found, but ^-glucuronidase was increased during relaxation.
Gersh and Catchpole (1949) reported the
presence of a collagenase from histochemical studies, but no confirmation has been
forthcoming. Relaxin also has a protein
anabolic effect which occurs in the absence
of pubic relaxation (Frieden, 1956). This
action was demonstrated by the increased
up-take of labeled glycine by the connective
tissue proteins of the pubic symphysis. Recent experiments indicate that relaxin not
only acts in conjunction with the female
sex steroids but can also act alone (Brennan and Zarrow, 1959). However, it is apparent that the available data are still inadequate for a clear understanding of the
mechanism of action of relaxin.
Relaxation of the pubic symphysis of the
mouse has been studied in great detail by
Hall. In a series of reports she showed that
pubic relaxation occurs in the mouse during pregnancy and following treatment with
estradiol and relaxin (Hall and Newton,
1946a, b). This was later confirmed by Kliman, Salhanick and Zarrow (1953). Contrary to the results reported following work
on the guinea pig, progesterone not only
failed to influence the effect of estrone on
the pubic symphysis of the mouse, but progesterone also inhibited the action of relaxin. It was suggested that this inhibition
is the result of an antagonism by progesterone on the action of relaxin and that a true
species difference exists (Hall, 1949, 1955).
Histologic studies revealed that changes in
the pubic symphysis during pregnancy and
after treatment with relaxin and estradiol
are similar (Hall, 1947) . These changes consist of proliferation of articular hyaline cartilage, resorption of the medial ends of the
pubes, lengthening of the pubic ligament by
formation of new cartilage, and reversion of
the cartilage to collagenous connective tissue. Hall (1956) suggested that estradiol
causes a depolymerization of the mucopolysaccharides through enzymatic action resulting in a matrix sufficiently pliable to
respond to the tensions set up by relaxin.
Evidence presented in support of this concept was the loss of metachromasia and the
increase in water. In addition, a two-step
effect was seen with relaxin: (1) complete
degradation of the matrix, and (2) the appearance of a gap in the cranial part of the
cartilage produced by stretching of the symphyseal cleft. Some data in support of the
latter part of this concept were presented by
van der Meer (1954) who showed that in
GESTATION
1011
hihition of pelvic muscle tension inhibited
relaxation in the guinea pig. In a similar
type of experiment Crelin (1954) tied together the innominate bones of a mouse before pregnancy and obtained some dorsoventral displacement of the pubic symphysis
but normal relaxation was inhibited.
H. DILATION OF THE UTERINE CERVIX
Dilation or softening of the uterine cervix
in the pregnant woman at the time of labor
has been known for a long time. This reaction has been used to determine whether
delivery can be anticipated. Within recent
years this phenomenon has been described
in a number of animals and some analysis
of the hormonal control of the reaction has
been attempted.
Relaxation of the uterine cervix of the
rat during pregnancy was first reported by
de Vaal in 1946 and confirmed by Uyldert
and de Vaal in 1947. Relaxation was measured by the insertion of a gauging pin into
a cervix that had been removed and the
diameter determined at the point where resistance is first felt. The measurements revealed a marked rise from approximately
3.5 mm. on the 17th day of pregnancy to 10
mm. at parturition. Recently, both Harkness and Harkness (1956) and Yochim and
ZaiTow (1959) have taken in vitro measurements of the relaxation of the uterine cervix
of the rat and observed marked relaxation
during the latter part of gestation and at
parturition. Yochim and Zarrow (1959) removed the cervix, suspended it from a rod
and measured the stretch due to weights
added at fixed intervals until the cervix
broke. The amount of relaxation of the
cervix was determined by the amount of
stretch obtained with a weight of 50 gm.
Under these conditions, the curve for relaxation of the cervix showed two sloi^es as
pregnancy progressed (Fig. 16.35). The initial slope between day 12 and day 20 showed
a rise of approximately 4 mm., with an extremely abrupt rise of 14 mm. on day 21.
By 24 hours after parturition the degree of
dilation had fallen to 3 mm. It is of interest
that the curve for the tensile strength of the
cervix (expressed in grams force necessary
to tear 1 mg. cervical tissue in a rat weighing 100 gm.) was the opposite to that seen
for cervical dilation. The tensile strength
fell from approximately 50 gm. force to a
low of 3 gm. at parturition and then rose
during the postpartum period. The drop in
tensile strength preceded the changes in the
dilation of the cervix and was essentially
completed 5 to 6 days before parturition or
when the abrupt increase in dilatability of
the cervix occurred.
Similar changes have been described in
the dilatability of the cervix of the mouse
(Steinetz, Beach and Kroc, 1957) with increased dilatability progressed beyond the
15th day (Fig. 16.36). The diameter of the
cervix increased from a])proximately 2 mm.
to about 5 mm. at delivery. It is apparent
that the rate of the reaction, i.e., dilation, is
much more rapid in the rat, although it is
possible that the method of measurement is
responsible for the differences.
The induction of cervical dilation by relaxin was reported by Graham and Dracy
(1953) in the cow, and by Zarrow, Sikes
and Neher (1954) in the sow and the heifer.
Treatment with stilbestrol followed by relaxin caused a dilation of the uterine cervix
of the gilt from % or % inch to 1 inch
(Zarrow, Neher, Sikes, Brennan and Bullard, 1956). Measurements were made by
the passage of aluminum rods, and, although the technique is not too exact, the
differences are significant. Stilbestrol given
alone or in combination with progesterone
had no effect on the cervical dilation. On
the other hand. Smith and Nalbandov
(1958) have recently reported that estrogen
treatment constricted the uterine cervix of
the sow and that relaxin was without effect.
A cue with respect to the mechanism of action of relaxin is given by the similarity of
the action of relaxin on the pubic symphyseal ligament and the uterine cervix. In
both instances, an increase in water content
and a marked dei)olymerizatioii occurs.
Cullen and Harkness ( 1958) observed relaxation of the uterine cervix of the rat with
estradiol alone, or with estradiol and progesterone, or with estradiol and relaxin, but
maximal dilation was obtained only with
a combination of estradiol, progesterone,
and relaxin. In general Kroc, Steinetz and
Beach (1959b) obtained comparable results
in the rat. Estrogen alone caused some in
1012
SPERM, OVA, AND PREGNANCY
14
PREGNANCY
Fig. 16.35. Dilation and ten.sile strength of the uterine cervLx of the rat during estrus,
pregnancy, and 2 days postpartum. The dihition of the cervix in mm. of stretch per 50 gm.
of added weight. The tensile strength is expressed in grams force necessary to tear 1 mg.
cervical tissue in a rat weighing 100 gm. E = estrus; P = parturition. (From J. Yochim and
M. X. Zarrow, Fed. Proc, 18, 174, 1959.)
crease in dilatability when 5 fxg. estradiol
cyclopentylpropionate were given, and a decrease when 50 /Ag. were given. Progesterone
had no consistent effect either alone or in
estrogen-primed animals. Relaxin alone
caused some softening of the cervix, but
gave a maximal effect only when given with
progesterone in estrogen-primed animals.
Normal cervical dilation was also obtained
in pregnant rats castrated the 15th day of
gestation and maintained with progesterone,
estradiol, and relaxin (Kroc, Steinetz and
Beach, 1959; Yochim and Zarrow, 1959).
Data on dilation of the uterine cervix of the
mouse are rather sparse; nevertheless, softening of the cervix with relaxin has been
reported (Kroc, Steinetz and Beach, 1959).
It is not the purpose of this review to
evaluate the data on cervical softening in
the human female. The nature of the action
of relaxin in the human female is controversial. Nevertheless, softening of the cervix
following treatment with relaxin has been
reported (Eichner, Waltner, Goodman and
Post, 1956; Stone, Sedlis and Zuckerman,
1958) although McGaughey, Corey and
Thornton (1958) reported no effect on the
cervix following relaxin.
GESTATION
1013
cr
UJ
X
t—
O . c
cr •= ,
<=• UJ -J
UJ t^'
ir ^ CD
UJ < <
> O I
^ ^ ^
111 ^
Q
O
cn 03 _i
UJ Z) <
CO Q. (J
^s >
UJ r: cr
U. 5 UJ
u. - o
o
d
z
6.0.
5.0
4.0
3.0
2.0
1.0
0.0.
o o CERVICAL DILATABILITY
(5-12 MICe/POINT)
^^ - INTERPUBIC LIGAyCNT
• • (5-12 MICE/POINT)
□ D RESPONSE TO OXYTOCIN
( 3-25 MICE/POIMT)
STANDARD ERROR OF
THE MEAN
DIESTRUS ESTRUS IS 16 17 18 19 20
CYCLE DAYS PREGNANT
1
DAYSP
4
OSTPA
RTUM
PREGNANCY
PARTURITION
Fig. 16.36. Increased length of the pubic ligament, inciea.sed cervical dilatability, and
increased responsiveness to oxytocin with the length of pregnanc.y in the mouse. L =
lactating; NL = not lactating. (From B. G. Steinetz, V. L. Beach and R. L. Kroc, Endocrinology, 61, 271, 1957.)
XII. Uterine Myometrial Activity
The classical and well known description
of uterine muscular activity has been more
than adequately reviewed by Reynolds
(1949). Since then Csapo and his colleagues
have reported a series of elegant experiments involving the action of estrogen and
progesterone on the uterine myometrium
and have evolved the concept of "i)rogesterone block" in the control of uterine activity
(1956a, 1956b). It has been shown that the
ovarian steroid hormones regulate myometrial activity and that the uterine contractions are dependent on the relative
amounts of the two hormones. Contractility
is dependent basically on the concentration
of the high energy phosphates which are
maintained by estrogen w^iich in turn is
involved in the synthesis of these substances
(Csapo, 1950; Menkes and Csapo, 1952).
Discovery of the staircase phenomenon in
the uterine myometrium similar to that exhibited by cardiac muscle led to a marked
difference between the action of estrogen
and progesterone (Csapo and Corner, 1952 ) .
With decreasing freciuency of electrical
stimidation in an isometric arrangement,
tension decreased if the uterus was dominated by estrogen and increased if it was
dominated by progesterone. Uteri from castrated rabbits were insensitive to the frequency of electrical stimulation. Thus estrogen induced a "positive staircase" response
and progesterone a "negative staircase" response, although in the latter instance some
estrogen is also present. These staircase responses have been used successfully as a
measure of hormone domination and have
been shown to be a function of the Na+ and
K+ gradients across the myometrial cell
membrane.
Uterine motility during estrus, the diestrum, and pregnancy has been described
by many investigators in great detail (for
a review see Reynolds, 1949). The diestrous
uterus shows extremely slow, feeble, uncoordinated movements. The contractions may
arise in any part of the uterus and extend
in any direction. At estrus, the uterine contractions become rhythmic and sweep over
1014
SPERM, OVA, AND PREGNANCY
STAIRCASE
Negative
9
§
o
p
o
o
o
o
ox
8
ox
o
8
ox
o
K
1^
ox
X
Transient
O
o
o
o
o
o
o
o
o
X
X
X
X
X
X
X
Positive
ox
o
X
X
X
X
X
X
X
X
X
X
ox
X
X
X
X
X
X
X
X
X
X
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Hours after mating
Fig. 16.37. Change from a positive to a negative staircase as the hormone dominance of
the myometrium moves from the estrus to the progestational state after mating. X and
O indicate the two strains of rabbits used. (From B. M. Schofield, J. Physiol., 138, 1,
1957.)
the uterine horn in a wave starting at the
tubal end. Both amplitude and rate are increased. During pregnancy the uterus becomes relatively quiescent. In general this
pattern of myometrial activity has been reproduced with both hormones, estradiol and
progesterone.
Recently Schofield 11957), using the
Csapo technique, has studied, in vivo, myometrial activity in the rabbit. In a series of
experiments she was able to show in several
strains of rabbits that, when mating occurs
during estrus, the uterine myometrium is
dominated by estrogen. Within 20 to 28
STAIRCASE
Negative
Transient
Positive
o
o
o
o
o
8x
OX
o
ox
X
X
X
o
o
o
ox
X
26 27 28 29 30 31
Day of pregnancy
32
Fig. 16.38. Change from negative through
transient to positive staircase as the hormone
dominance reverses at the end of pregnane}', indicating estrogen dominance. X and O indicate the
two strains of rabbits used. (From B. M. Schofield,
J. Physiol. 138, 1, 1957.)
hours after mating, the positive staircase
effect passes through a transient effect to a
negative effect indicating the development
of progesterone dominance (Fig. 16.37j.
This condition remained in effect throughout pregnancy until 24 hours before parturition when a reversion to estrogen domination was indicated by the positive staircase
response ( Fig. 16.38) . Thus the progesteronedominated uterus is maintained throughout
pregnancy and the uterus is nonreactive to
oxytocin. Csapo (1956a) and others have
shown that labor cannot be induced by
oxytocin in the rabbit before day 30 of gestation, but 24 hours later, on removal of the
progesterone block, 96 per cent of the rabbits delivered following treatment with oxytocin. He believes that the specific action of
progesterone involves a blocking of the excitation-contraction coupling which is a
consequence of the disturbed ionic balance
in the myometrial cell. Thus a block is set
up to the propagation of the contraction
wave which can be removed only by a decrease in the level of progesterone.
The role of the water-soluble extract, relaxin in myometrial activity, is still uncertain. That an inhibition of estrogen-induced uterine contractions is obtained in
certain species, such as the rat, mouse, and
guinea pig, with relaxin preparations is un(luestionable. However, we still have not
answered the questions as to w'hether this
hormone plays a role in uterine contractions
under normal physiologic conditions and
whether the uterine contraction-inhibiting
GESTATION
1015
substance is relaxin or a contaminant of the
relaxin extract.
Krantz, Bryant and Carr (1950) reported
than an aqueous extract of the corpus luteum would produce an inhibition or decrease of uterine activity in the guinea pig
and rabbit previously primed with estrone.
This has been amply confirmed with both
in vivo and in vitro preparations involving
spontaneous contractions measured isometrically in the guinea pig (Felton, Frieden
and Bryant, 1953; Wada and Yuhara, 1956;
JMiller, Kisley and Murray, 1957) , rat (Sawyer, Frieden and Martin, 1953; Wada and
Yuhara, 1956; Bloom, Paul and Wiqvist,
1958), and mouse (Kroc, Steinetz and
Beach, 1959). However, Miller, Kisley and
Murray (1957) failed to show any action of
relaxin on uterine motility in the rabbit
and the human being in vitro. Thus, the information on the rabbit is contradictory and
a similar situation exists with regard to the
human female for whom both positive and
negative results have been reported following treatment with relaxin for threatened
abortion (McGaughey, Corey and Thornton, 1958; Stone, Sedlis and Zuckerman,
1958; Eichner, Herman, Kritzer, Platock
and Rubinstein, 1959). In briefly summarizing the action of relaxin on the uterine
myometrium it should be pointed out that
( 1 ) relaxin inhibits uterine motility in an
estrogen-primed animal, (2) the action may
be species-limited, and (3) relaxin treatment docs not interfere with the action of
pitocin.
XIII. Parturition
A. PROGESTERONE
A number of theories have been suggested
to explain the hormonal control of parturition. The most popular is that parturition
is due to a decrease in the level of progesterone which allows oxytocin to exert its effect on the uterus. Evidence has already
been presented indicating that pregnancy
can be maintained in the castrated rabbit
by an extract of corpora lutea, or progesterone, and even prolonged in rats (Nelson,
Pfiffner and Haterius, 1930; Miklos, 1930),
mice (Mandelstamm and Tschaikowsky,
1931), and rabbits (Zarrow, 1947a). Snyder
(1934) and Koff and Davis (1937) prolonged gestation in rabbits by inducing the
formation of new corpora lutea during the
last trimester of pregnancy.
Knaus (1930) originally noted a marked
antagonism between posterior pituitary extract and the corpus luteum hormone and
Koff and Davis (1937) reported that in prolonged gestation induced by progesterone,
posterior pituitary extract was ineffective
until two days after the last injection.
Csapo (1956a) performed a series of elegant
experiments and concluded that progesterone blocks the uterine contractions, and
that premature labor could not be induced
with oxytocin before the 30th day of gestation in the rabbit except for a very small
percentage of animals. This has been confirmed by Fuchs and Fuchs (1958).
Zarrow and Neher (1955) found the serum gestagen levels in the pregnant rabbit
fell only after parturition was under way.
Hence the problem arose as to how parturition could begin while a high blood concentration of gestagen was present. A partial
answer was obtained in experiments by
Csapo (1956b) and Schofield (1957) who
showed that the progesterone-dominated
uterus of the pregnant rabbit becomes estrogen-dominated and responsive to oxytocin 24 hours before parturition. Hence the
concentration of progesterone in the serum
is meaningless by itself and it could be
theorized that the significant point is the
ratio of estrogen to progesterone. Csapo
(1956a), however, offered an alternative solution. He observed a local effect of placental progesterone on the myometrium so
that the myometrium closest to the placenta
is under a greater progesterone-dominance
than that portion of the myometrium lying
more distant. Hence the local level of progesterone would be the significant factor in
the onset of parturition and not the systemic
level.
B. OXYTOCIN
It is now generally believed that parturition is the result of the action of the posterior pituitary hormone on the myometrium of the uterus sensitized by estrogen.
The development of this hypothesis followed
from the well known fact that oxytocin pro
1016
SPERM, OVA, AND PREGNANCY
duces uterine contractions and induces labor
and delivery of the young. It is apparent,
however, that a mass of contradictory data
exist and the hypothesis is still in need of
better evidence before it can be fully accepted (for review of early literature see
Reynolds, 1949) .
Some of the evidence supporting the
above hypothesis is the fact of the presence,
to a limited degree, of a deficiency syndrome in parturition following removal of
the posterior pituitary gland. The data,
however, are still equivocal. Labor is apparently prolonged in the monkey (Smith,
1946) and guinea pig (Dey, Fisher and
Ranson, 1941 ) after total hypophysectomy.
Nevertheless, parturition will occur normally after removal of the pituitary gland
in the rabbit (Robson, 1936), cat (Allen
and Wiles, 1932), mouse (Gardner and Allen, 1942), and rat (Smith, 1932). Even
where there is some indication of interference with labor, delivery occurs. However,
the lack of consistent results and species
differences may be due to the recent finding
that the posterior pituitary hormones are
synthesized in the hypothalamus and that
removal of the posterior pituitary is only
effective under limited conditions because
the source of the hormone is still present.
These experiments have also been criticized on the ground that the anterior pituitary was also removed and hence interference with many other hormones occurred.
Additional evidence in favor of a role
for the neurohypophysis in the delivery of
the young is the increase in uterine motility
following stimuli that bring about release
of the posterior pituitary hormones, and the
lack of an effect on the uterus when release
of the hormone is blocked.
Positive evidence for the release of oxytocin at the time of parturition is still lacking as are measurements of the concentration in the blood. Fitzpatrick (1957) takes
the view that oxytocin is liberated as an essential part of normal parturition and cites
the following evidence. (1) A superficial
similarity exists between spontaneous labor
and that induced by oxytocin. Harris (1955)
also stresses the similarity in the uterine response to oxytocin and to electrical stimulation of the supraoptic hypophyseal nucleus.
(2) Mechanical dilation of the uterus or
cervix evokes an increase in uterine contractions presumably by way of a nervous
reflex release of oxytocin (Ferguson, 1941).
(3) Oxytocin is decreased in the posterior
pituitary gland of the rat and the dog after
labor (Dicker and Tyler, 1953).
Evidence from the attempts to measure
the concentration of oxytocin in body fluids
at the time of parturition is inadequate. The
early reports of higher concentrations in
the urine (Cockrill, Miller and Kurzrok,
1934) and blood (Bell and Morris, 1934;
Bell and Robson, 1935) during parturition
are questioned because of the inadequate
methods of extraction and lack of specificity
in the assay. Recently, Hawker and Robertson (1957, 1958) reinvestigated the problem
and concluded that two oxytocic substances
are present in the blood and hypothalamus
of cats, cows, and rats and blood of women.
However, they found that the concentration
of oxytocin in the blood fell during labor
from a high during pregnancy. It is apparent that this presents a paradoxical situation in view of the fact that the concentration of oxytocin is low at the time of
parturition; a time when the hormone is
supposedly exerting its greatest effect. The
situation is further complicated by the presence of two oxytocic factors and the presence of an oxytocinase in the blood and
l)lacenta (von Fekete, 1930; Page, 1946;
Woodbury, Ahlquist, Abreu, Torpin and
Watson, 1946; Hawker, 1956). Although
more work is required on this problem and
esi)ecially with regard to the specificity and
concentration of the oxytocinase, there is
some indication of a fall in enzyme level
before parturition. Tyler (1955) reported a
decrease in the blood level of the enzyme
towards the end of pregnancy and Sawyer
(1954) reported a decrease in oxytocinase
activity in rat tissues at the end of pregnancy.
C. RELAXIN
Recently, the discovery of the action of
relaxin on the pubic symphysis, uterine
cervix, and uterine motility has raised the
question of the role of this hormone in parturition. Certainly in the species that normally show pubic relaxation, relaxin would
appear to play a significant role. However,
this phenomenon is a special adaptation and
GESTATION
1017
the question of cervical dilatability becomes
more important because it seems to occur
in all species examined thus far. It would
seem that relaxin can induce cervical dilatability in conjunction with the sex steroids and that cervical dilation is a necessary
event in parturition, but whether relaxin
controls this event under physiologic conditions is still unknown and direct evidence
is unavailable. It is also apparent in some
species that relaxin can inhibit uterine contractions w'ithout interfering with the action
of oxytocin. Kroc, Steinetz and Beach
(1959) reported that relaxin actually restored responsiveness to oxytocin in mice
treated with progesterone. Again the question is raised as to whether this is merely
a good experiment or a part of the normal
physiologic events.
In a general way the events leading to
labor may be summarized as follows. As
pregnancy approaches term, the uterus becomes subject to increasing pressure from
within, due to a differential change in the
growth rates of the fetus and the uterus
(Woodbury, Hamilton and Torpin, 1938).
Concurrently, a reversal from progesterone
to estrogen domination occurs, which also
contributes to an increase in uterine tonus.
As intra-uterine tension increases, spontaneous contractions acquire a greater efficiency and forcefulness. Because the radius
of curvature in the human uterus is greater
at the fundus than at the cervix, and because the myometrium is thicker at the upper pole (by a factor of 2) the contractile
force is stronger at the fundus than at the
cervical end. This contractile gradient i^roduces a thrust toward the cervix.
Utilization of a type of strain gauge, the
tokodynamometer, has afforded information on the rate and strength of contraction
of the various parts of the parturient uterus
simultaneously (Reynolds, Heard, Bruns
and Hellman, 1948). These measurements
have indicated that, during the first stage of
labor, the fundus exerts strong contractions
of rather long duration. The corpus exhibits
less intense contractions, usually of shorter
duration, which frequently diminish in force
as labor advances. The lower uterine segment is almost inactive throughout the first
stage of parturition. According to Reynolds
(1949), both the fundus and the midportion
contract at the same time, but the fundus
remains contracted for a longer period of
time than the corpus beneath, thus building
up a force downward. If cervical dilation
has not occurred, the three areas of the
uterus will continue to contract. As cervical
dilation begins, the contractions in the midportion of the uterus decrease in intensity
and the contractions in the lower segment
disappear. Cervical dilation has been observed only when there is a preponderance
of rhythmic activity of the fundus over the
rest of the uterus.
When amniotic fluid is lost after the rupture of the membranes, the absolute tension
within the wall of the uterus is reduced so
that the ratio of force between fundus and
cervix is increased. Thus rupture of the
membranes decreases the tension in the cervix more than the fundus and the net effect
is an increased force from the fundus. This
change tends to precipitate the parturition
more rapidly.
As pregnancy nears term, both increased
tonus of the myometrium and rapid growth
of the fetus cause a rise in intra-uterine
pressure. This rise results in a decrease of
effective arterial blood pressure in the placenta. During this period also, thrombosis
is observed in many of the venous sinuses
of the placenta and many of the blood vessels become more or less obstructed by giant
cells. During parturition, the systemic blood
pressure of the mother rises with each contraction, but, due to the increased intrauterine pressure produced by the contractions, the effective maternal arterial blood
pressure in the placenta decreases to zero.
Thus maternal circulation is cut off from
the fetus.
Measurements of intra-uterine pressure
at term show that the human uterus contracts with a pressure wave which varies
from 25 to 95 mm. Hg (Woodbury, Hamilton and Torpin, 1938). The uterine wall is
subjected to an average tension of 500 gm.
per cm.- and, during delivery of the head,
may, with the aid of abdominal musculature, develop as much as 15 kg. force.
In animals giving birth to multiple young
(rat and mouse) evacuation of the horn proceeds in an orderlv fashion beginning at the
1018
SPERM, OVA, AND PREGNANCY
cervical end. As evacuation of the lowest
implantation site starts, changes occur in
the periods of contractions of segments of
uterine artery near its entrance into the
uterine wall (Knisely, 1934; Keiffer, 1919).
Gradually the constriction phase becomes
proportionately longer than the dilation
phase until the arterial lumen is obliterated.
The myometrium in the area of the constricting segments becofes more active and,
after long intense local contractions of the
uterine muscle, the fetuses and the placentas separate and are discharged through the
dilated cervix. After evacuation, a relaxation of the contracted segment of uterus
occurs and the process is repeated at the
next implantation site.
Recently, Cross (1958) re-examined the
problem of labor in the rabbit. He concluded
that (1) oxytocin in physiologic amounts
can induce labor that is comparable to the
events normally seen, (2) oxytocin is released during a normal labor, and (3) oxytocin can induce delivery without supplementary mechanisms. He noted that
straining movements involving reflex abdominal contractions initiated by distention
of the vagina and cervix aided in expulsion
of the fetus. It is also possible that this
might cause reflexly an increased secretion
of oxytocin. Other reflex mechanisms have
been suggested, but evidence is inadequate.
Cross cites a report by Kurdinowski published in 1904 in which the entire process of
labor and delivery in an isolated full-term
rabbit uterus perfused with Locke's solution
is described. In these experiments orderly
delivery of the viable fetuses was affected
by the contractile efforts of the uterus and
vagina in absence of any hormonal or nervous stimuli.
XIV. Conclusion
Although we have garnered much information, no major conclusions can be drawn
at this time concerning gestation in the
mammal. This is probably true because of
the vastness of the subject and the lack of
sufficient data, especially that of a comparative nature. It is probably fitting to
close this chapter with the final statement
written by Newton in the second edition of
Sex and Internal Secretion, "It seems rather
that the investigation of endocrine relationships during pregnancy is still in the
exploratory stage and that the time is not
ripe for systematization."
It is true that many data have been accumulated in the last two decades since the
publication of the second edition of this
book. It is also probably true that some systematization can now be started. But above
all we need more data on different species
in order to systematize fully the role of the
various hormones and glands in pregnancy
and to evaluate the metabolic and other
changes that occur at this time.
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Krichesky, B. 1939. The influence of thyroidectomv on the period of gestation in the rabbit.
Am. J. Physiol.. 126, 234.
Kroc, R. L.. Steinetz. B. G., and Beach, V. L.
1959. The effects of estrogen, progestogens
and relaxin in pregnant and nonpregnant laboratorv rotlents. Ann. New York Acad. Sc,
75, 942.
Krohn, p. L., and White, H. C. 1950. The effect of hypothyroidism on reproduction in the
female albino rat. J. Endocrinol., 6, 375.
Laidlaw, J. C, Cohen, M., and Gornal, A. G.
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Landau, R. L., Bergenstal, D. M., Lugibihl, K.,
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Pregnane!/. Siningfield. 111.: Charles C Thomas.
1026
SPERM, OVA, AND PREGNANCY
Man, E. B., Heinemanx, M., Johnson, C. E., Le.^ry,
D. C, AND Peters, J. P. 1951. The precipitable iodine of serum in normal pregnancy and
its relation to abortions. J. Clin. Invest., 30,
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M.^QSOOD, M. 1952. Thyroid functions in relation to reproduction of mammals and birds.
Biol. Rev., 27, 281.
M.^rder, S. N., AND Money, W. L. 1944. Concentration of relaxin in the blood serum of
pregnant and postpartum rabbits. Endocrinologv, 34, 115.
M.\RSHALL, F. H. A., AND JoLLY, W. A. 1905. Contributions to the physiology of mammalian reproduction. II. The ovary as an organ of internal secretion. Philos. Tr., ser. B, 198, 123.
M.\YER, G., AND DuLUC, A. -J. 1955. Surrenales
et corps jaunes on cours de la gestation et de
la lactation. Arch. Sc. Physiol., 9, 97.
McCarthy, J. L., Corley, R. C., and Zarrow, M. X.
1958. Effect of goitrogens on the adrenal
gland. Fed. Proc, 17, 424.
McCartney, C. P., Vallach, F. J., and Pottinger,
R. E. 1952. Further studies on the inactivation of pitressin antidiuretic effect by the blood
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McDonald, L. E., McNutt, 8. H., and Nichols,
R. E. 1953. On the essentiality of the bovine
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14, 539.
McGaughey, H. S. 1952. Tiie cau.^e of the blood
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Gynec, 64, 1268.
McGaughey, H. S., Corey, E. L., and Thornton,
W. N. 1958. An evaluation of the action of
relaxin on isolated human uterine muscle and
cervical tissues iii vitro. Am. J. Obst. & Gynec, 75, 23.
McKay, E., Ass.ali, N. S., .and Henley, M. 1957.
Blood levels of 17-hydroxycorticosteroids ( 17OHCS) during labor of human pregnancv.
Proc Soc Exper. Biol. & Med., 95, 653.
McKeown, T., and MacMahon, B. 1956. Sex
differences in length of gestation in mammals.
J. Endocrinol., 13, 309.
McKeown, T., and Spurrel, W. R. 1940. The results of adrenalectomv in the pregnant albino
rat. J. Phvsiol., 98, 255.
McLennan, C. E., and Corey, D. L. 1950. Pla.sma
vohnne late in pregnancy. Am. J. Obst. &
Gynec, 59, 662.
McLennan. C. E., and Thouin, L. G. 1948.  
McLennan. C. E., and Thouin, L. G. 1948.  
Blood volume in pregnancy. Am. J. Obst. &  
Blood volume in pregnancy. Am. J. Obst. &  
Line 12,992: Line 9,153:
pregnancy in the rat. Endocrinology, 40, 182.  
pregnancy in the rat. Endocrinology, 40, 182.  


Newton, W. H. 1939. Some problems of endocrine function in pregnancy. In Sex and In
Newton, W. H. 1939. Some problems of endocrine function in pregnancy. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds. Baltimore: The  
 
 
GESTATION
 
 
 
102
 
 
 
ternal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds. Baltimore: The  
Williams & Wilkins Company.  
Williams & Wilkins Company.  


Line 13,071: Line 9,221:
York: The Macmillan Company.  
York: The Macmillan Company.  


Pixfrs. G. 1943. New color reaction for certain  
Pixfrs. G. 1943. New color reaction for certain urinary 17-ketosteroids. Endocrinologv, 32,  
 
 
 
urinary 17-ketosteroids. Endocrinologv, 32,  
176.  
176.  


Line 13,153: Line 9,299:
Rogers. P. ^'. 1947. The effect of sulfaguanidine  
Rogers. P. ^'. 1947. The effect of sulfaguanidine  
on iei)ro(luction in the rat. Anat. Rec, 97, 48.  
on iei)ro(luction in the rat. Anat. Rec, 97, 48.  
1028
SPERM, OVA, AND PREGNANCY




Line 13,237: Line 9,374:
Selle, R. M. 1945. Hamster sexually mature at  
Selle, R. M. 1945. Hamster sexually mature at  
28 days of age. Science, 102, 485.  
28 days of age. Science, 102, 485.  


Selye, H., AND Bassett, L. 1940. Diuretic effect  
Selye, H., AND Bassett, L. 1940. Diuretic effect  
Line 13,315: Line 9,450:
A confirmed 310-day period of human gestation. Am. J. Obst. & Gynec, 62, 458.  
A confirmed 310-day period of human gestation. Am. J. Obst. & Gynec, 62, 458.  


Smithberg, M., and Runner, M. N. 1956. The  
Smithberg, M., and Runner, M. N. 1956. The induction and maintenance of pregnancy in  
 
 
 
GESTATION
 
 
 
1029
 
 
 
induction and maintenance of pregnancy in  
piepuberal mice. J. Exper. Zool., 133, 441.  
piepuberal mice. J. Exper. Zool., 133, 441.  


Line 13,401: Line 9,524:
TuLSKY, A. S., AND KoFF, A. K. 1957. Some observations on the role of the cor]>us hit cum in  
TuLSKY, A. S., AND KoFF, A. K. 1957. Some observations on the role of the cor]>us hit cum in  
early pregnancy. Fertil. & Steril., 8, 118.  
early pregnancy. Fertil. & Steril., 8, 118.  


Tyler, CM. 1955. The elaboration and utilization of posterior pituitary hormones. Ph.D.  
Tyler, CM. 1955. The elaboration and utilization of posterior pituitary hormones. Ph.D.  
Line 13,475: Line 9,596:


Weisman, a. I., AND Co.\tes, C. W. 1944. The  
Weisman, a. I., AND Co.\tes, C. W. 1944. The  
South Ajricnn Frog (Xenopus laeris) in  
South Ajricnn Frog (Xenopus laeris) in Pregnancy Diagnosis. New York: Clark &  
 
 
 
1030
 
 
 
SPERM, OVA, AND PREGNANCY
 
 
 
Pregnancy Diagnosis. New York: Clark &  
Fritts.  
Fritts.  


Line 13,625: Line 9,734:
vulva of young castrated sows and heifers. Am.  
vulva of young castrated sows and heifers. Am.  
J. Physiol., 179, 684.  
J. Physiol., 179, 684.  
GESTATION
1031


Zakroav, M. X., AND Zarrow, I. G. 1953. Anemia  
Zakroav, M. X., AND Zarrow, I. G. 1953. Anemia  

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Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.
Section A Biologic Basis of Sex Cytologic and Genetic Basis of Sex | Role of Hormones in the Differentiation of Sex
Section B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproduction Morphology of the Hypophysis Related to Its Function | Physiology of the Anterior Hypophysis in Relation to Reproduction
The Mammalian Testis | The Accessory Reproductive Glands of Mammals | The Mammalian Ovary | The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms | Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates | The Mammary Gland and Lactation | Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones | Nutritional Effects on Endocrine Secretions
Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy Biology of Spermatozoa | Biology of Eggs and Implantation | Histochemistry and Electron Microscopy of the Placenta | Gestation
Section E Physiology of Reproduction in Submammalian Vertebrates Endocrinology of Reproduction in Cold-blooded Vertebrates | Endocrinology of Reproduction in Birds
Section F Hormonal Regulation of Reproductive Behavior The Hormones and Mating Behavior | Gonadal Hormones and Social Behavior in Infrahuman Vertebrates | Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals | Sex Hormones and Other Variables in Human Eroticism | The Ontogenesis of Sexual Behavior in Man | Cultural Determinants of Sexual Behavior
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Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy

Gestation

M. X. Zarrow, Ph.D. Professor Of Zoology, Purdue University, Lafayette, Indiana


I. Introduction

Reproduction in the animal kingdom is accomiilished by a wide variety of methods, from simple budding and binary fission in the invertebrates to gestation in the mammal and the development of a new organ, the placenta. The development of viviparity, which covers millions of years of evolution, brought with it many new problems, and with each problem new factors came into play so that reproduction in the mammal is a highly co-ordinated series of events — a co-ordination that is both temporal and spatial, that requires certain events to occur in a proper sequential arrangement, and, above all, is dependent on the endocrine system.

It is obvious that the maintance of gestation in the mammal is a complex phenomenon. It involves directly or indirectly a major portion of the endocrine system with concomitant changes in the general metabolic state of the organism and in many of the enzymes present in the blood and the tissues. Finally, a new endocrine organ, the placenta, comes into being also to play its specific role in gestation.


II. Length of Gestation

The duration of gestation is highly variable and depends primarily on the species involved. In general, the longer the gestation, the more self-sufficient and mature are the young at the time of birth. It is obvious, however, that this is not true under all conditions. The young of the guinea pig are highly advanced at birth, although the length of gestation is approximately 69 days, whereas in the iH-iniate, with a gestation period of 6 to 9 months, depending on the species, the young are helpless at birth. A partial summary of gestation length and tlie litter size of a representative but not inclusive list of mammals is presented in Table 16.1.

The length of gestation appears to be rather constant for each species or at least within a strain. Even where the phenomenon of delayed implantation is a natural event the length of pregnancy remains constant, although the quiescent period may vary. It is, however, possible for delayed implantation to occur in a species where this does not ordinarily appear, which could lead to a marked increase in the duration of gestation. Thus, an increase of 1 to 7 days has been reported in the rat or mouse if mated while lactating (Pincus, 1936). Recently, Bruce and East (1956) examined the effect of concurrent lactation on the number and viability of the young and the length of pregnancy in the mouse. They observed a wide variation in the delay of implantation for every size of litter studied, but, in general, the delay tended to be longer for the larger suckling litters.

Smith, Albert and Wilson (1951) reported a 310-day pregnancy period in a human female. Gestation was confirmed early by pregnancy tests and a normal child with respect to body weight was born at 30 days after the expected parturition. Such phenomena seem to be rare in primates and no explanation is possible at the present time.

Although the lengths of the gestation periods are quite constant for a given strain, the length of gestation is inversely related to the litter size. This has been demonstrated in both a genetically pure strain and a heterogeneous strain of quinea pigs (Goy, Hoar and Young, 1957). An average gestation length of 69.9 days was obtained in the pure strain of guinea pigs with a litter size of 1, as compared with a gestation length of 65.3 days for a litter size of 6.

A sex difference has also been postulated in length of gestation. Although the difference is very small, e.g., only a fraction ■of a day in man, the difference is significant. Recently, McKeown and MacMahon (1956) concluded that pregnancy is longer in the cow, horse, and possibly the sheep and camel when the offspring are male, and longer in man and possibly the guinea pig when the offspring are female.


III. Normal Reproductive Potential

The reproductive potential in the primate is limited to the period from the menarche to the menopause. Hence, it is much shorter than the total life span of the female. Fertility studies as a function of age have l)ecn rather sparse for different species although it is generally agreed that fertility declines with age. A reproductive period considerably shorter than the life span of the animal has also been reported in certain strains of mice (Thung, Boot and Miihlbock, 1956) and in the rat (Ingram, Mandl and Zuckerman, 1958).

Although Slonaker (1928) showed that the rat may remain fertile for 22 months, it is known that the average size of successive litters in both rats and mice first rises to a maximum and then falls (King, 1924; Ingram, Mandl and Zuckerman, 1958). The latter have shown both a decrease in the number of fertile female rats with each successive litter and hence with age (Fig. 16.1) and a decline in the number of young with each successive litter (Fig. 16.2).

These results indicate that the reproductive potential of both the colony of rats and of the individual rat declines with age. Many factors may obviously be at work here, such as nutrition, size, and part played by the male. Ingram, Mandl and Zuckerman ( 1958 ) feel that none of the above factors is responsible for the decline in litter size and offer the following four possibilities: (1) the number of follicles which mature and ovulate declines with age, (2) the capacity of the ovum to be fertilized declines with age, (3) the number of fertilized ova that develop to term declines with age, and (4) the total number of available oocytes declines with age.

Evidence from the pig (Perry, 1954) and rat indicates that factors 2 and 3 are certainly involved. Inasmuch as the number of corpora lutea rises with age in the pig, the decline in size of litters can be


TABLE 16.1 Length uf gestation and litter size in various species of mammals



Species







No. of Young


Length of Gestation


Reference*^


Common name


Scientific name




Armadillo


Das y pus novemcinctus, L.


4


150 days


(1), (2)


Baboon


Papio hamadrys, L.


1


183 days


(1)


Baboon, chacma


Papio porcarius, B.


1


7 months


(1), (2)


Bat, common European


Vespertilio miirinus, L.


1


50 days


(1)


Bat, common pipistrello


Pipistrellus pipistrellus, S.


1


44 days


(3)


Bear, black


Euarctos americanus, P.


1-4


208 days


(1), (2>


Bush baby


Galago senegalensis, G.


1-2


4 months


(1)


Camel, batrachian


Cameliis bactrianus, L.


1


370-440 da.ys


(1)


Capuchin


Cebus apella, L.



160-170 davs


(1)


Cat


Felis catus, L.


3.8 (1-8)


56-65 days^


(4)


Cat, domestic


Felis catus, L.


4


63 days


(1)


Chimpanzee


Pan satyrus, L.


1


236.5 ± 13.3 davs


(1)


Chimpanzee


Pan satyrus, L.


1-2


226.8 ± 13.3" davs


(17)


Chinchilla


Chinchilla laniger, B.


1-4


105-111 days


(1), (2>


Chipmunk


Tanarios strialus, L.


3-5


31 davs


(1)


Cow


Bos taurus, L.


1-2


277-290 days


(1)


Cow (Jersey)


Bos taurus, L.


1-2


282.7 ± 5.4" days


(5)


Cow (Holstein-Friesian)


Bos taurus, L.


1-2


278-280 days-^


(6)


Coyote


Canis latrans, S.


5.7


60-(J5 days


(1)


Deer, Virginia


Odocoileus Virginian us, B.


2


7 months


(1)


Dog


Canis familiaris, L.


Multiple


58-63 days


(1)


Dog


Canis familiaris, L.


Multiple


61 davs


(4)


Echidna


Echidna acn.leata


1


16-28 days


(1)


Elephant



1


20 months


(8)


Elephant, Indian


Elephas maxim us, L.


1


607-641 days


(1)


Ferret


Mustela furo, L.


5-13


42 days


(1)


Ferret


Mustela furo, L.


5-13


42 days


(4)


Fox, red


Vulpes fulfa, D.


1-8


52 days


(1)


Goat


Capra hircxis, L.


1-2


21 weeks


(7)


Goat, domestic


Capra hircus, L.


1-3


146-151 days


(1)


Gopher, pocket


Geomys bursarius, S.


1-6



(1)


Ground squirrel, thirteen

Citellus tridecimlineatus, N.


5-13


28 days


(1>


lined






Guinea pig


Cavia porcellus, L.


1-6


67-68 davs


(1)


Guinea pig


Cavia porcellus, L.



65.3-70.5 days


(7)


Hamster, golden


Cricetus auratus, H.



16-19 days


(1)


Hamster, golden


Cricetus auratus, H.


5


10 days


(9)


Hare, snowshow


Lepus americanus, E.


1-7


38 days


(1)


Hedgehog, European


Erinoceus europaeus, L.


5


34-49 davs


(1)


Hippopotamus


Hippopotamus amphibius, L.


1


237 ± 12 days


(1)


Horse


Equus cabaUus, L.


1


330 davs


(1)


Hyena, spotted


Crocuta crocuta, E.


1-2


110 days


(1)


Kangaroo rat


Bettongia cuniculus, 0.


1


6 weeks


(1)


Lemur


Lemur macaco, L.


1-2


146 days


(1)


Lion


Felis leo, L.


2-6


105-113 davs


(1)


Macaque


Macaca mulatta, Z.


1


163.7 ± 8 days


(1), (10)


Macaque



1


24 weeks


(8)


Man


Homo sapiens, L.


1


280 ± 9.2 days


(1)


Marmoset


Hopale jacchus, L.


1-3


140-50 davs


(1)


Marten, pine


Martes americana, T.


3-5


220-265 days


(1^


Mink


Mustela vison, S.


4-10


39-76 days


(1)


Mink


Mustela vison


4


51 days'* (40-75)


(11), (12)


Mole, common American


Scalupus aquaticus, L.


2-5


6 weeks


(1)


Mouse


Mus musculus, L.



19-20 days


(4)


Mouse, field


Microtus pennsylvanicus , 0.


6-8



(1)


Mouse, house


Mus musculus, L.


4-5-7.5


19 days


(1)


Mouse, wild


Peromyscus maniculatus



23 davs


(13)


Mouse, wood


Peromyscus leucopus, L.


3-7


23 days


(1)


960


GESTATION


961


TABLE m.l— Continued



species







No. of Young


Length of Gestation


Reference"


Common name


Scientific name


Opossum, Australicaii


Trichosunis vulpecula, K.


1


16 days



Opossum, Virginia


Didclphis rir(/itiiana, K.


8-12


12.5-13 days


(1), (2), (8)


Otter


Lutra canadensis, S.


1-4


60 days


Pig, domestic


Sus scrofa, L.


4-12


112-115 days



Pig, wild


Sus cristatus, W.


4-6


4 months



Porcupine


ErclJiizan (lorsoliun, L.


1


16 weeks



Puma


Frlis ,„nr„ln,-, T.


1-4


90-93 days



Rabbit, domestic


Crycutaluyiis c/uiicidus. L.


Multiple


30-32 days



Rabbit, domestic


Crycotalagus cnnicuhis, L.


Multiple


31 (28-36 davs)



Racoon


Procyon lotor, L.


1-0


63 days



Rat


Rattus rattus, L.


(J. 1-9.2'

22 days



Rat


Rattus rattus, L.



21-23 days



Reindeer


Rangifer tarandus, L.


1-2


7-8 months



Rhinoceros



1


18 months



Rhinoceros, black


Rhinocerus bicornuis, L.


1


530-550 days



Seal, northern fur


CaUorhinns ursinus, L.


1


Almost 1 year



Sheep, bighorn


Ovis canadensis, L.


1


180 davs



Sheep, domestic


Ovis aries, L.


1-2


144-152 davs-^



Shrew, common


Sorex aranus, L.


6.45


13-19 davs



Shrew, short-tailed


Blarina brevicauda, S.


3-7


17-20 days



Skunk


Mephitis mephitis, S.


4-7


62 days



Squirrel, red


Sciurus hudsonicus, E.


3-()


40 days



Stoat


Mustela musleta



6 weeks'*


(14)


Tasmanian devil


Sarcophilus ursinus, K.


4


31 days



Vole


Microtus agrestis



20-22 days


(Ifi)


Weasel


Mustela nivalis


6-7


50 weeks'* (includes) lactation)


(15)


Whale, sperm


Fhyseter catadon, L.


1


1 year


(1)


Wolf, timber


Canis tycoon, S.


1-12


63 davs


(1)


Woodchuck


Marmot a nionox, L.


4.07


28 days


(1)


Zebu


Bos indicus, L.



285 davs


(1)


'- References. (1) Asdell, 1946. (2) Kenneth, 1947. (3) Deanesly and Warwick, 1939. (4) Farris, 1950. (5) Rollins, Laben and Mead, 1956. (6) Norton, 1956. (7) Goy, Hoar and Young, 1957. (8) Arey, 1946. (9) Selle, 1945. (10) Hartman, 1932. (11) Pearson and Enders, 1944. (12) Enders, 1952. (13) Svihia, 1932. (14) Deanesly, 1943. (15) Deanesly, 1944. (16) Chitty, 1957. (17) Peacock and Rogers, 1959.

  • " Standard deviation.

" Depends on the strain.

'* Excluding the quiescent period.



3l no. of litter


-1^


Fig. 16.1. Decline in litter size with birth of successive litters. • mean of 35 litters; EI mean of 14 litters; A mean of 4 litters. (From D. L. Ingram, A. M. Mandl and S. Zuckcinian. J. Endocrinol., 17, 280, 1958.)



1 2 3 4 5 6 7 8 9 10 11

Serial no. of litter

Fig. 16.2. Decline in number of fertile female rats with birth of successive litters. (From D. L, Ingram, A. M. Mandl and S. Zuckerman, J. Endocrinol., 17, 280, 1958.)


962


SPERM, OVA, AND PREGNANCY


attributed only to failure of fertilization or to fetal death. A similar increased incidence of embryonic death or failure of fertilization has been described in the aged rat, although it should be noted that the number of corpora lutea present at parturition in the rat is not an index of the number of ova released before conception because the corpora lutea in old age may persist for longer periods. Finally, a marked reduction in the number of oocytes with age has been shown in the rat, a drop from approximately 20,000 oocytes at age day 1 to approximately 2000 oocytes at age 250 to 300 days (Mandl and Zuckerman, 1951). In addition, Ingram (1958) showed that the litter size in rats declined markedly with the reduction in number of oocytes following graded doses of x-ray. This experiment tends to confirm the concept that the decline in fertility with age is due to a decline in the number of oocytes.

IV. Environment

A. CROWDING

The factors concerned in the growth and survival of a population under natural conditions may obviously involve reproduction. Variations in population level have been of great interest to the mammalogist and student of wildlife for many years. Decreased productivity in mammalian populations associated with increased density of the population has been considered a controlling factor in the regulation of wildlife population.

Experimental analysis by Christian and Lemunyan (1958) indicated that a number of factors are involved. These authors exl)osed mice to excessive crowding and noted the number of implantation sites, embryos.


and births. All the females became pregnant but only 3 of the 10 bore litters during the period of crowding or later (Table 16.2). It would seem that the crowded females were unable to maintain normal pregnancies and that the environmental situation interfered with the endocrine balance and resulted in a marked pregnancy wastage. The data reveal that in addition to a postimplantation loss, there was also a prc-implantation loss because the number of implantation scars in the uterus was markedly less in the crowded females. This could be due to a failure of the fertilized egg to implant or to a decrease in the number of available ova. Although direct data are not available, it is of interest to speculate as to whether this effect of crowding is mediated by way of the pituitaryadrenocortical axis.

B. BODY TEMPERATURE AND HYPOXIA

Disturbances in reproduction have been noted in mammals exposed to high temperatures or chronic hypoxia. It has been known for some time that women moving to the tropics show a high rate of abortion (Castellani and Chalmers, 1919). Recently Macfarlane, Pennyamt and Thrifte (1957) reported a 30 per cent reduction in human conception rates in the summer as compared with the winter in Australia. The same authors reported a marked degree of fetal resorption in rats exposed to a temperature of 35°C. This confirmed the previous observations of Sundstroem (1927) in rats and Oegle (1934) in mice where exposure to 31 °C. caused a reduction in litter size.

In a similar manner, disturbances have also been reported in reproduction following exposure to decreased oxygen tension.


TABLE 16.2 Productivity of mice crowded 10 pairs to a cage compared to their 10 control pairs Note that all of the females became pregnant but that the crowded females exhibited a marked intrauterine loss of young, reduction of implantation sites, reduction of litter size, and significant delay until the birth of first litters compared to the controls. Crowding produced a 75 per cent loss in the


number of young born.


(From J. J.


Christian


and C. D. Lemunyan, Endocrinology, 63


, 517, 1958.)



No. of Pairs


No. of Litters Born


Mean No. Days to Litter Birth


Mean No. Progeny per Litter ± S.E.


No. Females with Placental Scars


Mean No. Scars per Female


Crowded females

Isolated females


10 10


3 10


40 ± 1.0 26 ± 1.5


7.67 ± 0.33 9.00 ± 0.75


10 10


6.90 ± 1.37 11.00 ± 0.47


GESTATOON


963


Monge (1942) reported a lack of reproduction in the Spaniards for more than 50 years after residence in certain areas of Bolivia (14,000 feet or more above sea level). Many malformations have also been observed in the progency of mice, rats, and rabbits exposed to low atmosperic pressures. Exposure of mice on the 10th day of pregnancy for 2 hours to a 6 per cent oxygen-94 per cent nitrogen mixture at normal atmospheric pressure gave malformations in the young comparable with those found after exposure to a low atmospheric pressure which was equivalent to the above with respect to the number of oxygen molecules per unit of air (Curley and Ingalls, 1957). Although these malformations involved the ribs and vertebrae, it is conceivable that more extensive malformations could result in death of the fetuses leading to resorption or abortion of the young.

Vidovic (1952, 1956) made a very complete study of the effect of lowered body temperature on gestation in the rat using the technique of Giaja (1940) in which an hypoxic hypothermia is induced by cooling under reduced oxygen tension. The animal is placed in a sealed container which is surrounded by ice for a period of approximately 10 hours. Under these conditions a hypothermia of 3 to 4 hours' duration and body temperature of 14 to 18°C. can be induced. No deleterious effects were noted in the rats cooled on or before the 13th day of pregnancy. However, the induction of hypothermia after the 13th day resulted in a marked increase in the disturbance of gestation. These disturbances consisted of an increased number of resorbed fetuses, an increased ratio between stillborn and live young in that more stillborn occurred, a decreased body weight in the progency, and a delay in the onset of parturition. In addition, a marked increase in sensitivity to hypothermia was noted in the animals as pregnancy progressed. Courrier and Marois (1953) cooled pregnant rats by exposure to a temperature of 0°C. for 2 hours. Thereafter the rats were placed in cold water for 3 to 4 hours and a body temperature of 15.5 to 17°C. was obtained. Exposure to the above treatment on the 7th to the 11th day of preg


nancy had no effect on the fetuses or the pregnancy. Treatment on the 12th to the 18th day of pregnancy led to resorption and abortion of the young. The authors concluded that the degree of deleterious effects following exposure to cold varied with the length of the pregnancy.

Recently, Fernandez-Cano (1958a) exposed pregnant rats for 5 hours on 2 consecutive days to one of the following three experimental procedures: (1) an environmental temperature of 103°F. that led to an increase in body temperature to 104°F.; (2) an environmental temperature of 26°F. that led to a decrease in body temperature to 94°F.; and (3) barometric pressure of 410 mm. Hg. Both temperature changes led to a marked decrease in the number of implantations and, to a lesser extent, to some embryonic degeneration after implantation (Table 16.3). Although some deleterious action was seen before implantation, hyi^oxia was more harmful after implantation. Whereas these results are not in full agreement with Vidovic 's report, it must be remembered that Vidovic used a combination of cold and hypoxia to induce the effects that he observed. Adrenalectomy failed to increase embryonic degenerations in rats treated as above (Fernandez-Cano, 1958b). Inasmuch as adrenocorticotrophic hormone (ACTH) causes degeneration of the embryos in intact pregnant rats and not in adrenalectomized rats (Velardo, 1957 1 , it is apparent that these results are explainable on the basis of an increased release of adrenal corticoids due to the stressor and/or a direct action of the corticoids on the development of the embryo.

V. Maternal Hormone Levels during Gestation

Proof that certain hormones are necessary for a successful pregnancy came from evidence involving ablation of the source of the hormone and replacement therapy. This was followed by quantitative analyses of the concentration of the hormone in the blood and urine throughout gestation. The increasing concentrations of the hormones as pregnancy advances can be used as a second argument for the role of hormones in the development and maintenance of pregnancy (Zarrow, 1957). Changes of this


964


SPERM, OVA, AND PREGNANCY


TABLE 16.3

The effect of increase or decrease of body temperature and hypoxia on the pregnancy of the rat

(From L. Fernandez-Cano, Fertil. & Steril., 9, 45, 1958.)


Group


Control

High body temperature High body temperature High body temperature High body temperature Low body temperature . Low body temperature . Low body temperature . Low body temperature .

Hypoxia

Hypoxia

Hypoxia

Hypoxia


Days of Treatment


1-2 3-4 6-7

10-11 1-2 3-4 6-7

10-11 1-2 3-4 6-7

10-11


Total No. Corpora Lutea


166 98

117 95 89 93 98

100 91

103

108 97 94


Percentage of Degeneration


Before implantation


2.4 52 28

2

2 25 33

3

2.1 21.3 25.9


2.1


After implantation



12

3 14 10

5

4 13 12.1

2.9

3.7 25.7 65.9


Total degener


2.4 64 31 16 12 30 37 16

14.2 24.2 29.6 25.7 68.0


Means of Degeneration for Each Rat


0.2 8.3 4.6 1.9 1.3 3.5 4.5 2.0 1.6 3.1 3.8 3.1 8.0


Standard Error


0.11

1.6

2.6

0.5

1.8

1.0

1.4

0.2

0.5

0.4

0.3

1.0

1.4


Percentage against Control


>0.01 >0.01

>0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01


kind have been observed for such steroids as the estrogens, gestagens, and the 17a-hydroxycorticoids. In addition, certain nonsteroidal hormones such as the gonadotrophins human chorionic gonadotrophin (HCG) and pregnant mare's serum (PMS) and the polypeptide, relaxin, increase during gestation. Some evidence for a possible involvement of thyroxine, prolactin, and oxytocin will be included. The maximal concentration of these hormones in the blood of the female during pregnancy is given in Table 16.4.

A. ESTROGENS

The fact that large amounts of estrogen are excreted in the urine of pregnant women and mares has been known for a long time. Additional data (reviewed by Newton, 1939) indicate that this phenomenon occurs in all species studied, such as the chimpanzee, the macaque, the cow, the pig, and the rat. In general, an increasing amount of estrogen is excreted as pregnancy progresses. The estrogenic material in the urine of the pregnant woman appears mostly in the form of estriol with lesser amounts of estrone and estradiol (Fig. 16.3) . The estriol concentration increases only slightly in the urine of women for the first 100 to 125 days of pregnancy, but thereafter it increases very rapidly until parturition. Newton (1939) discussed the possible


role of estrogen in pregnancy in great detail. He first asked whether the increased urinary concentration of estrogen indicates that this hormone is acting to a lesser degree as pregnancy advances or to a greater degree. He marshaled his facts pro and con and came to the conclusion that there is an increased production of estrogen throughout pregnancy and hence an increased activity of the hormone. In his analysis of the action of estrogen, five possibilities were suggested. (1.) Estrogen is involved in the growth of the uterus in pregnancy. (2) Estrogen is involved in the increased uterine contractility and sensitivity to oxytocin necessary for parturition. (3) Estrogen is concerned with the continued secretion of progesterone by way of the pituitary glancl or acting directly on the corpus luteum. (4) Estrogen synergizes with progesterone. (5) Estrogen stimulates mammary gland growth. A 6th possibility is that estrogen reverses the progesterone block (Csapo, 1956a). Several of these possibilities will be considered later in conjunction with progesterone, the maintenance of pregnancy, and jiarturition.

B. GESTAGENS

The significance of the role of progesterone during pregnancy stemmed from the historic work of Fraenkel who proved the validity of Gustav Born's suggestion that


GESTATION


965


TABLE 16.4 Maximal hormone levels in the blood during pregnancy


Hormone


Species


Type of Assay


Hormone Amt./ml. Plasma


Reference


Estriol


Man


Chem.


0.0914 Mg.


Aitkin and Preedy, 1957



Estriol


Man


Chem.


0.066 Mg •


Loraine, 1957



PJstrone


Man


Chem.


0.0647 Mg.


Aitkin and Preedy, 1957



E.strone


Man


Chem.


0.0305 Mg.


Loraine, 1957



]stnuli()l


Man


Chem.


0.0144 Mg.


Aitkin and Preedy, 1957



K.-^tradiol


Man


Chem.


0.0105 Mg

Loraine, 1957



( lestajfen


Rabbit


Biol.


10 Mg."


Zarrow and Neher, 1955



Cicstugen


Mouse


Biol.


SMg."


Forbes and Hooker, 1957



(Jostagen


Ewe


Biol.


12 Mg."


Neher and Zarrow, 1954



Progesterone ....


Ewe


Chem.


0.0033 Mg.


Short, 1957



Progesterone ....


Ewe (ovarian vein


Chem.


2Mg.


Edgar and Ronaldson, 1958



Progesterone ....


Cow


Chem.


0.0086 Mg.


Short, 1958b



Gestagen


Man


Biol.


2Mg."


Forbes, 1951



Gestagen


Man


Biol.


25Mg-"


Fujii, Hoshino, Aoki and Yao, 1956



Progesterone ....


Man


Chem.


0.239 Mg.


Oertel, Weiss and Eik-Nes, 1959



Prog(^stei'one ....


Man


Chem.


0.142 Mg.


Zander and Simmer, 1954



Progesterone . . . .


Sow


Chem.


0.0034 Mg.


Short, 1957



Progesterone . . . .


Goat


Chem.


0.0071 Mg.


Short, 1957



Rela.\in


Guinea pig


Biol.


0.5G.P.U.


Zarrow, 1947a



Rela.xin


Rabbit


Biol.


10 G.P.U."


Marder and Money, 1944



Relu.xin


Man


Biol.


2 G.P.U."


Zarrow, Holmstrom and Salhanick,


1955


Relaxin


Sow


Biol.


2G.P.U.


Hisaw and Zarrow, 1951



Rela.xin


Cow


Biol.


4 G.P.U.


Wada and Yuhara, 1955



Hydrocortisone. .


Man


Chem.


0.22 Mg.


Gemzell, 1953



Thyroxine


Man


Chem.


0.83 Mg.^


Peters, Man and Heinemann, 1948



STH


Rat Man Man Horse


Biol. Biol. Biol. Biol.


3.5-7-^ 120 I.U. 70 I.U. 50 I.U.


Conlopoulos and Simjjson, 1957 Haskiiis and Slierman. 1952 Wilson, Allien and Randall, 1949 Cole and Saunders, 1935



HOG



HCG



PMS





" Expressed as equivalents of progesterone.

Guinea pig units.

"■ Protein-bound iodine.

'Vg. equivalent of a purified bovine growth-promoting substance.


the corpus luteum is necessary for the maintenance of pregnancy. Fraenkel demonstrated at the turn of the century that the corpus luteum of the rabbit is essential for the maintenance of pregnancy in the rabbit (Fraenkel and Cohn, 1901; Fraenkel, 1903; Fraenkel, 1910). These observations were confirmed by Hammond and Marshall (1925.) who found that castration before the 20th day of pregnancy led to the termination of pregnancy in 24 hours. Castration later in pregnancy resulted in abortion approximately 2 days after the operation. In 1928, Corner showed that an extract of the corpus luteum could induce a progestational endometrium in the castrated rabbit. This was soon followed by the demonstration that this extract could induce implantation of the fertilized egg in the rabbit and maintain pregnancy in the castrated animal (Allen and Corner, 1929; 1930). Purification of the extract of the corpus luteum led to the chemical identification of the active substance by Butenandt, Westphal and Cobler in 1934, and in the following year Allen, Butenandt, Corner and Slotta (1935) agreed to the name pi^ogesterone for this hormone of the corpus luteum.


These events were soon followed by tlu' discovery that progesterone is excreted in the urine as tlie glucuronide of pregnanediol and i)regnanolone, metabolites of progesterone. Studies of urinary products of progesterone were immediately undertaken and a marked increase in urinary pregnancdiol was observed in the human female throughout pregnancy, especially in the second half (Fig. 16.3).

The discovery by Hooker and Forbes (1947) of a new assay for progesterone sensitive to a concentration of 0.3 /^g. per ml. led to many studies on the blood levels of this hormone during gestation. Subsequent studies revealed a lack of specificity for the assay (Zarrow, Neher, Lazo-Wasem and Salhanick, 1957; Zander, Forbes, von Miinstermann and Neher, 1958) and a discrepancy between the values obtained by chemical and biologic techniques. It is obvious that the bioassay data possess significance but a final evaluation can be made only when the identity of the compound or compounds measured in the blood of the animals by the Hooker-Forbes test has been established.


Fig. 16.3. Urinary excretion of estrogens and pregnanediol throughout gestation in the human being. (From E. Venning, Macy Foundation, Conferences on Gestation, 3, 1957.)


The concentration of gestagen in the blood of pregnant sheep (Neher and Zarrow, 1954) , women (Forbes, 1951 ; Schultz, 1953; Fujii, Hoshino, Aoki and Yao, 1956), rabbits (Zarrow and Neher, 1955), and mice (Forbes and Hooker, 1957) has been determined by the Hooker-Forbes test and expressed as /^g. equivalents of progesterone. The data obtained from pregnant women by the different investigators are in marked disagreement. Whereas both Forbes (1951) and Schultz (1953) failed to observe any significant rise in blood gestation of pregnant women throughout gestation, Fujii, Hoshino, Aoki and Yao (1956) obtained a conspicuous rise during this period. The data reported by Forbes (1951) indicate an extremely low level for protein-bound progesterone (0.5 /^g. per ml. plasma or less) and a maximum of 2 /xg. per ml. free progesterone (Fig. 16.4). The concentration of the hormone in the blood showed a series of irregular peaks throughout gestation and varied from less than 0.3 /xg. to 2 /xg. per ml. plasma. In general, these results were confirmed by Schultz (1953) who assayed the blood from 46 women at 6 to 17 weeks of pregnancy. Again the results failed to reveal any consistent change with the length of pregnancy. Both investigators (Forbes, 1951; Schultz, 1953) were led to question the importance of progesterone during gestation in the primate. Fujii, Hoshino, Aoki and Yao (1956), on the other hand, reported a significant increase in the level of circulating progesterone throughout gestation. Again these investigators used the Hooker-Forbes assay but indicated that the plasma was not treated in any way except for dilution before the assay. The results obtained by this latter group revealed a rise from a level of 6 /*g. progesterone per ml. plasma during the luteal phase of the cycle to a high of 25 /i,g. during: the last trimester of pregnancy (Fig. 16.5). The concentration showed a steady increase from the 4th to the 24th week of pregnancy,, and a plateau from the 24th week until term. A sharp drop occurred within 12 to 24 hours after parturition with zero values noted by 72 hours postpartum. Analysis of the urine for pregnanediol showed a rather good correlation between the two curves although the plasma levels rose sooner than the urinary pregnanediol.

The curve for the concentration of progesterone in the pregnant mouse is markedly different from those reported for other species (Forbes and Hooker, 1957). Again the Hooker-Forbes assay was used



Fig. 16.4. Free and bound ge.stagen in the plasma of the pregnant human female. (From




Fig. 16.5. Concentration of gestagen in the blood plasma and pregnanediol in the uterine of the pregnant human female. Gestagen levels were determined by the HookerForbes test. (From K. Fujii, K. Hoshino, I. Aoki and J. Yao, Bull. Tokyo Med. & Dent. Univ., 3, 225, 1956.)


as with the other species and the values expressed as activity equivalent to progesterone. The values for the bound action were consistently low and, in general, less than 1 fjig. per ml. plasma (Fig. 16.6). The concentration of the free hormone showed marked variations on the first day or so of pregnancy. Actually a variation from 1 fig. per ml. plasma to 8 fig. per ml. plasma was seen on day 0. This type of fluctuation has also been seen in the rabbit and is without explanation at the present time. However, such marked variations disappeared by the 4th day of pregnancy and the results became much more consistent. The average curve for the concentration of gestagen in the blood of the pregnant mouse showed two peaks, one the 7th to the 9th day and a second the 15th day. The concentration increased from 2 fxg. per ml. plasma the 4th day of gestation to an average of approximately 8 fig. the 7th day. This level was maintained until day 9 and fell thereafter with a second peak occurring on day 15 and an immediate drop on day 16. Thereafter the levels remained low throughout the remainder of pregnancy.

Although it may be assumed that the initial peak in the concentration of the gestagen is due to an increased activity



Fig. 16.6. Concentration of free and bound gestagen in the plasma of the pregnant mouse. Gestagen levels were determined by the Hooker-Forbes test. (From T. R. Forbes and C. W. Hooker, Endocrinology, 61, 281, 1957.)



on the part of the corpora lutea, an explanation of the second peak and the drop between the two peaks offers more difficulty. The latter may reflect a diminished luteal activity. This could be assumed on the grounds that the corpus luteum is the only source of gestagen during this period of gestation and that the luteal cells show cytologic signs of regressive changes, although the drop in serum progestogen antidates the cytologic changes by several days. An explanation for the second peak would probably involve increased secretory activity !)y the placenta. Progestational activity has l)een found in i:)lacental extracts and progesterone has been isolated from the placentae of human beings and mares (Salhanick, Noall, Zarrow^ and Samuels, 1952; Pearlman and Cerceo, 1952; Zander, 1954; Short, 1956). Thus, the drop in serum gestagen seen on day 10 could be due to loss in the activity of the corpora lutea and the second rise as a contribution from the placentas. It is of interest that the low levels on days 10 to 13 and between day 16 to term appear to have no counterpart in other species. The physiologic significance of this is still unknown and will require further work on additional species and on the mouse before an explanation is forthcoming. It is of interest that the concentration of gestagen in the blood dur


ing the first 12 days of pregnancy corresponds with the intensity of the response to progesterone exhibited by the endometrium during the same period (Atkinson and Hooker, 1945). This w^ould suggest that the serum gestagen levels reflect the physiologic state of the animal.

Serum gestagen levels in the rabbit reveal a curve of increasing concentration throughout pregnancy (Zarrow and Neher, 1955). Initial values of 0.3 to 1 yug. per ml. serum were noted at the time of mating, with a sharp rise beginning on the 4th day of gestation. The concentration rose to a level of 6 to 8 /xg. per ml. by the 12th day and thereafter showed only a slight rise to a maximal concentration of 8 to 10 /xg. per ml. serum at parturition (Fig. 16.7). No drop in serum hormone level was observable at parturition or 1 hour later. The first significant drop occurred at 6 to 12 hours postpartum when the gestagen level had decreased 50 per cent. It is of interest that the serum progestagen levels did not fall until after the conceptus had been expelled.

castrated the 12th, of gestation aborted following removal of the ovaries (Zarrow and Neher, 1955). In all instances the serum gestagen levels fell before the abortion. Figure 16.8 shows the


Pregnant rabbits 19th, or 24th day within 1 to 3 days


GESTATION


969


± 5


3


NORMAL PREGNANCY RABBIT 25 o RABBIT 26 • RABBIT 31 e - RABBIT 27 x RABBIT 30 ^



Fig. 16.7. Concentration of gestagen in the blood of the normal pregnant rabbit as determined by the Hooker-Forbes test. (From M. X. Zarrow and G. M. Neher, Endocrinology, 56, 1, 1955.)







Fig. 16.8. The effect of castration on serum progestogen levels and maintenance of gestathe rabbit. Gestagen levels were determined by the Hooker-Forbes test. (From M. X. and G. M. Xeher. Endocrinology, 56, 1, 1955.)


changes in serum gestagen levels before and after castration of a pregnant rabbit. The concentration increased from a level of 0.3 ing. per ml. at day to 10 /xg. per ml. on day 24 when the rabbit was castrated. A 60 per cent drop in serum gestagen level is seen 12 hours after castration with a fur


ther drop at the 36th hour, when the animal aborted.

Studies on the concentration of serum gestagen in the pregnant ewe (Neher and Zarrow, 1954) permit a comparison with the results obtained in the rabbit. Such a comparison is extremely valuable in view



Fig. 16.9. Concentration of gestagen in the blood of the pregnant ewe. Gestation levels were determined bv the Hooker-Forbes test. (From G. M. Neher and M. X. Zarrow, J. Endocrinol., 11,323,1954.)


of the fact that castration of the rabbit invariably leads to abortion whereas castration of the pregnant ewe does not do so if the ovaries are removed during the second half of pregnancy. Again the progesterone determinations were carried out on untreated serum and the samples assayed by the Hooker-Forbes technique using progesterone as a standard. An initial rise in the serum gestagen level occurred soon after mating and seemed to level off at a concentration of 6 ixg. per ml. approximately the 50th day of gestation (Fig. 16.9). Thereafter, the concentration remained unchanged for approximately 50 days, when a second rise to a level of 8 to 12 fj.g. occurred. These levels remained unchanged until at least 30 minutes after parturition was complete.

Castration at various times after the 66th day of pregnancy failed to influence the concentration of circulating gestagen or interfere with the pregnancy. The data in Figure 16.10 show a normal concentration of 8 to 10 fxg. gestagen from the 114th day of gestation to parturition although the animal was ovariectomized the 114th day. Pregnancy was normal in all castrated ewes and the expected drop in scrum gestagen was observed following parturition.

It can now be stated that the human being, the monkey, the ewe, the rabbit, the mouse, and probably the guinea pig (Herrick, 1928; Ford, Webster and Young, 1951) have met the problem of a second source of progesterone supply with varying degrees of success. In the ewe, placental replacement of the ovary as a source of progesterone can be considered as complete by approximately the 66th day of pregnancy. Castration at this time will neither interfere with the pregnancy nor with the concentration of the hormone in the blood. In the monkey, castration as early as the 25th day of gestation (Hartman, 1941) does not interfere with pregnancy and in the human being castration as early as the 41st day after the last menstrual period may not interfere with pregnancy (Melinkoff, 1950; Tulsky and Koff, 1957). One may conclude, therefore, that the placenta can adequately take on the role of the ovary in this regard. On the other hand, aspects of the situation in the human female are still puzzling, especially the blood gestagen values; but despite this ambiguity



Fig. 16.10. The effect of castration on gestagen levels in the pregnant ewe. Gestagen le\els were obtained by the Hooker-Forbes test. Note that castration failed to interfere with the pregnancy or the level of gestagen in the blood. (From G. M. Neher and M. X. Zarrow, J. Endocrinol., 11, 323, 1954.)


it might be concluded that here also the placenta has successfully replaced the ovary. In the rabbit, on the other hand, castration at any time during pregnancy vvill cause a decrease in the level of the circulating hormone and terminate the pregnancy. Hence, in this species, the placenta has failed to replace completely the ovary. The mouse is another instance in which castration leads to abortion so that one can assume a failure on the part of the placenta to replace the endocrine activity of the ovary. In this case, however, the second peak of circulating gestagen has been ascribed to the placenta and this presents the possibility of a partial replacement of the ovary by the placenta but a replacement that is not adequate since pregnancy is terminated by ovariectomy.

As indicated above, a marked discrepancy exists between the bioassays and the chemical determinations of gestagens in the blood and other tissues. The chemical determinations of progesterone invariably give results that are far lower than those obtained by bioassay methods. Edgar and Ronaldson (1958) found a maximal concentration of approximately 2 /xg. progesterone per ml. ovarian venous blood during


gestation in the ewe. This concentration was no higher than that seen in the ewe during a normal estrous cycle. The maximal level reached during the estrous cycle was maintained when pregnancy supervened and remained fairly constant until the last month of pregnancy. Thereafter the concentration fell and no progesterone was detectable at 15 days prepartum (Fig. 16.11). Inasmuch as no progesterone was found in the peripheral blood of the ewe, this poses again the following question: What was being measured in the peripheral blood by the bioassay procedure? In addition, a second question is posed by the earlier discussion on the need of the ovary in the maintenance of pregnancy as to the relative contributions of the ovary and the placenta to the concentration of this hormone in the body.

That the biologic methods are measuring more than progesterone is obvious from the many reports emphasizing the high levels obtained by bioassay and the low levels obtained by chemical techniques. In addition to the above data. Short (1957, 1958a, 1958b) reported the presence of progesterone in the peripheral blood of the pregnant


Fig. 16.11. The con«?ntiation of progesterone in the ovarian venous blood of the pregnant ewe. Progesterone was determined by chemical methods. (From D. G. Edgar and J. W. Ronaldson, J. Endocrinol., 16, 378, 1958.)


0.0098 ixg. per ml. plasma. It is of interest that the level remained constant from the 32nd to about the 256th day of pregnancy and then decreased several days before parturition. In the human being values of 0.17 to 0.44 fig. per ml. during the final trimester of pregnancy have recently been reported (Oertel, Weiss and Eik-Nes ( 1959 ) . Numerous investigators have suggested that the discrepancy between the chemical and biologic assays is due to the presence of unknown gestagens in the blood. This has been validated in part by the discovery of 2 metabolites in the blood of the pregnant human female (Zander, Forbes, Neher and Desaulles, 1957). They have been identified as 20a-hydroxypregn-4-en-3-one and 20^-hydroxypregn-4-en-3-one and have been shown to be active in both the Clauberg and Hooker-Forbes tests (Zander, Forbes, von IMiinstermann and Neher 1958 ) . The 20/?-epimer was twice as active as progesterone in the Hooker-Forbes test and the 20a-epimer one-fifth as active. It is likely that more unidentified gestagens occur in the blood and other tissues.


C. Sources of Gestagens

The second question asked above concerning the role of the placenta versus the ovary as a source of progesterone probably cannot be answered in a simple manner. Wide differences exist between species (1) in the need of the ovary for maintenance of pregnancy, (2) in the concentration of the hormone in peripheral blood, (3) in the activity of the placenta in secreting progesterone, and (4) in the presence of extraovarian and extraplacental sources of the hormone.

The presence of progesterone in the placenta of the human being has been confirmed (Salhanick, Noall, Zarrow and Samuels, 1952; Pearlman and Cerceo, 1952) and a high output of progesterone demonstrated. Zander and von ]\Iiinstermann (1956) and Pearlman (1957) independently reported the production of approximately 250 mg. progesterone into the peripheral circulation every 24 hours. This and other evidence tends to prove that the placenta is the major source of progesterone in the human species. However, with respect to other species, progesterone has been found only in the placenta of the mare (Short, 1957) although in amounts much less than in the human being. Placentas of the cow, ewe, sow, or bitch were all negative. Although the placenta of the mare contains progesterone and castration does not lead to abortion after day 200 of gestation, no progesterone was found in the peripheral blood or uterine vein blood. The ewe offers an even more intriguing problem inasmuch as (1) a discrepancy exists between the biologic and chemical values for progesterone in the peripheral blood, (2) the placentas contain no progesterone, and (3) no {progesterone is found in the uterine vein blood (Edgar, 1953). This has led to the conclusion that the maintenance of pregnancy in the ewe may be dependent on an extra-ovarian, extraplacental source of progesterone.

If such a conclusion is correct, and it must be added that the evidence is still tenuous, then the adrenal cortex must be considered as a possible source. Beall and Reichstein isolated a small amount of progesterone from the adrenal cortex in 1938 and Heehter, Zaffaroni, Jacobson, Levy, Jeanloz, Schenker and Pincus (1951) demonstrated from perfusion experiments that progesterone is an important intermediate metabolite in the synthesis of the adrenal corticoids. In addition, it has long been known that desoxycorticosterone possesses progesterone-like activity (Courrier, 1940) which is due to a conversion of the desoxycorticosterone molecule to a gestagen. This has been shown by experiments in vivo in the monkey (Zarrow, Hisaw and Bryans, 1950), rat, and rabbit (Lazo-Wasem and Zarrow, 1955), and by an incubation experiment with rat tissue (Lazo-Wasem and Zarrow, 1955). In addition, Zarrow and Lazo-Wasem reported the release of a gestagen from the adrenal cortex of the rat and rabbit following treatment with ACTH. The substance was obtained from the peripheral blood and measured by the Hooker-Forbes test, but it was not identified chemically. This was followed by the finding that pregnanediol is present in the urine of ovariectomized women, but not ovariectomized, adrenalectomized women (Klopper, Strong and Cook, 1957), and by the finding that progesterone is present in the adrenal venous blood of the cow, sow, and ewe (Balfour, Comline and Short, 1957). In all instances the concentration of progesterone in the adrenal venous blood was 10 to 100 times greater than the concentration in the arterial blood. Thus the total evidence that the adrenal cortex can secrete progesterone is more than adequate. The question remains as to whether the adrenal cortex contributes to the progesterone pool of the body during pregnancy and whether a species difference exists here.

D. Relaxin

The initial discovery by Hisaw (1926, 1929) of the presence of an active substance in the blood and ovaries responsible for relaxation of the pubic symphysis of the guinea pig has led in recent years to a consideration of this substance as a hormone of pregnancy (Hisaw and Zarrow, 1951). Some doubt as to the existence of relaxin was raised in the 1930's by investigators who were able to show that pubic relaxation in the guinea pig could be obtained with estrogen alone or estrogen and progesterone (de Fremery, Kober and Tausk, 1931; Courrier, 1931; Tapfer and Haslhofer, 1935; Dessau, 1935; Haterius and Fugo, 1939). This matter was resolved by the demonstration that pubic relaxation in the guinea pig following treatment with the steroids or relaxin differed in ( 1 ) time required for relaxation to occur, (2) histologic changes in the pubic ligament, and (3) treatment with estrogen and progesterone which induced the formation of relaxin (Zarrow, 1948; Talmage, 1947a, 1947b). Subsequent discoveries of additional biologic activities possessed by relaxin and further purification of the hormone has led to the conclusion that relaxin is an active substance in the body, and that it plays a significant role during parturition. The hormone has been found in the blood or other tissues of the dog, cat, rabbit, sheep, cow, rat, and man. The specific action of this hormone varies with the species involved. Still unsolved is the question as to whether the water-soluble extract obtained from the ovary and referred to as relaxin is a single substance or a group of active substances (Friedcn and Hisaw, 1933; Sher and Martin, 1956).

The concentration of relaxin in the blood increases as pregnancy progresses until a plateau is reached. This has been demonstrated in the rabbit (Marder and Money, 1944), guinea pig (Zarrow, 1947), cow (Wada and Yuhara, 1955), and human being (Zarrow, Holmstrom and Salhanick, 1955). Relaxin has also been found to increase in the ovary of the sow (Hisaw and Zarrow, 1949). In general, the shape of the curve for the concentration of relaxin in the blood as a function of the length of pregnancy has been more or less the same for all species studied. Figure 16.12 indicates that the concentration of relaxin in the blood of the pregnant rabbit rises from a level of 0.2 guinea pig unit (G.P.U.) per ml. for the first trimester of pregnancy, i.e.. until day 12, to a level of 10 G.P.U. per ml.


on day 24. This concentration was then maintained until parturition. After delivery of the young, the concentration of the hormone decreased 80 per cent in 6 hours. On the 3rd day postpartum no hormone could be detected.

As indicated above, the concentration of relaxin in the blood of the pregnant cow and human being showed approximately the same type of curve. In the cow the concentration rose gradually from a level of 1 G.P.U. per ml. to a maximum of approximately 4 G.P.U. at 6 months (Fig. 16.13). Thereafter the level remained unchanged until parturition, wdien the level dropped at a rate comparable to that seen in the rabbit. The curve for the concentration of relaxin in the blood serum of the pregnant woman followed the general pattern described above (Fig. 16.14). The concentration rose from a level of 0.2 G.P.U. per ml. the 6th week of i:)regnancy to a maximum of 2 G.P.U. the 36th week. Thereafter the level remained unchanged until delivery. Again the postpartum fall was precipitous and the hormone was not detectable at 24





Fig. 16.12. Concentration of relaxin in the blood of the rabbit during pregnancy. Parturition (P) occurred 32 days after mating. Guinea pig units (G.P.U.) of relaxin are plotted against days pregnant. (From S. N. Marder and W. L. Money, Endocrinology, 34, 115, 1944.)



Fig. 16.13. Concentration of I'elaxin in the blood of the cow during pregnancy. Partiuition is indicated by P. (From H. Wada and M. Yuhara, Jap. J. Zootech. Sc, 26, 12, 1955.)



Fig. 16.14. The concentration of relaxin in the blood serum of normal pregnant women. (From M. X. Zarrow, E. G. Holmstrom and H. A. Salhanick, Endocrinology. 15, 22. 1955.)


hours postpartum. Studies in the guinea pig revealed a marked rise in relaxin on day 21 of gestation to a maximal concentration of 0.5 G.P.U. per ml. serum on day 28 (Zarrow, 1948). Thereafter the level remained unchanged for approximately 4 weeks. Contrary to the results obtained in the rabbit, cow, and human being a drop in the concentration of the hormone in the pregnant guinea pig was noted before parturition. The concentration of relaxin fell to 0.33 G.P.U. per ml. on the 63rd day of gestation and then dropped to nondetectable levels within 48 hours postpartum.

Although no studies have been carried out on the blood levels of relaxin in the sow as a function of the length of pregnancy, analysis of the ovary for relaxin has revealed a situation comparable to that reported for the blood in other species. The concentration rose from 5 G.P.U. per gm. ovarian tissue during the luteal phase of the cycle to approximately 10,000 G.P.U. per gm. fresh ovarian tissue by the time a fetal length of 5 inches had been reached (Hisaw and Zarrow, 1949).


E. Sources of Relaxin

The ovaries, placentas, and uteri are possible sources of relaxin in different species. It seems from the extremely high concentration in the ovary of the sow during pregnancy that this organ is the major site of relaxin synthesis at this time. However, studies on other species indicate that both the placenta and uterus may be involved.

Treatment of castrated, ovariectomized rabbits with estradiol and progesterone stimulated the appearance of relaxin in the blood of the rabbit as indicated by the ability of the blood to induce relaxation of the pubic symphysis of estrogen-primed guinea pigs (Hisaw, Zarrow, Money, Talmage and Abramovitz, 1944). Similar experiments on castrated, hysterectomized rabbits failed to reveal the presence of the hormone in the blood of the treated animals. Treatment with estradiol alone also failed to stimulate the release of relaxin. It is obvious then that, if the bioassay is specific for relaxin, the uterus is a definite source of this hormone. Comparable results were also obtained in the guinea pig (Zarrow, 1948). Treatment with estradiol and progesterone caused pubic relaxation and the presence of relaxin in the l)lood after approximately 3 days of treatment with progesterone. In the absence of the uterus relaxin was not demonstrable in the blood.

The concentration of relaxin in the blood of the rabbit castrated the 14th day of i^regnancy and maintained with progesterone remained unaffected by removal of the ovaries, provided the pregnancy was maintained (Zarrow and Rosenberg, 1953). Figure 16.15 shows a typical curve for the relaxin content of the blood of such an animal. The concentration of the hormone rose between days 12 and 24 to a maximal concentration of 10 G.P.U. per ml. and was maintained till the time of normal parturition. It is of interest that in those instances in which the placentas were not maintained in good condition, the concentration of the hormone fell. Analysis of the reproductive tract revealed concentrations of 5 G.P.U. per gm. fresh ovarian tissue during pseudopregnancy and approximately 25 G.P.U. during the last trimester of gestation. The uterus contained 50 G.P.U. per gm. fresh tissue during pseudopregnancy and an equal concentration the first 24 days of pregnancy. The 26th day of pregnancy the concentration fell to 15 G.P.U. per gm. The highest concentration was in the placenta which contained from 200 to 350 G.P.U. per gm. Some evidence indicated that after treatment with estradiol minimal amounts of relaxin, i.e., 5 G.P.U. per gm., were present in the vaginal tissue (Table 16.5).

F. Adrenal Cortex

1. Hydrocortisone

Initial studies on the possible role of the adrenal cortex in gestation involved the determination of the two urinary metabolites of the gland, i.e., the 17-ketosteroids and the corticoids. Inasmuch as the 17-ketosteroids are believed to be associated with the androgenic activity of the adrenal cortex, bioassays for adrenogenic activity in the urine were carried out. Dingemanse, Borchart and Laqueur (1937) found no increase in urinary androgen by the 6th to the 8th month of pregnancy whereas Hain (1939) reported that pregnant women secreted even less androgen than nonjircgnant women. Pincus and Pearlman (1943) found no change in the urinary 17-ketosteroids of the pregnant and nonpregnant woman although Dobriner (1943), by the use of chromatograi)hic separation, showed a marked decrease in androsterone. Venning (1946) found no change in the urinary ketosteroids as measured by the antimony trichloride reagent described by Pincus (1943), but the ketosteroids measured by the Zimmerman reagent (dinitrobenzene) showed a significant rise in the latter part of pregnancy. The discrepancy between the two determinations can be explained by the fact that other ketonic substances besides 17-ketosteroids give a color in the Zimmerman reaction. These are the 20-ketosteroids and to a limited extent the 3-ketosteroids. V^enning (1946) believes most of this in



Fig. 16.15. Concentration of relaxin in the blood of a pregnant rabbit castrated the 14th day of gestation and maintained with 4 nig. progesterone daily until the 32nd day. Postmortem examination revealed 8 placentas and 2 dead fetuses. (From M. X. Zarrow and B. Rosenberg, Endocrinology, 53, 593, 1953.)


TABLE 16.5

Relaxin content of the blood serum and tissue of the reproductive tract of the rabbit

(From M. X. Zarrow and B. Rosenberg, Endocrinology, 53, 593, 1953.)



No. of Rabbits


Relaxin Concentration in G.P.U.


Treatment


Per ml. serum


Per gm fresh tissue



Ovary


Uterus


Placenta whole


Placenta fetal


Placenta maternal


Pseudopregnant

Chorionic gonadotrophin . . .


3

4 3 2 2

2

1


0.2-0.3 0.2 1.0 10.0 10.0 10.0 10.0


5 5 30 25 20 25 25


50 50 50 50 30 15


75 50 50

75


10 20 25



Pregnant 24 davs


250


Pregnant 25 days

Pregnant 26 days


350 200


Pregnant 28 days



crease in ketosteroid excretion during pregnancy is the result of increased output of the stereoisomers of pregnanolone:

Measurement of urinary glucocorticoids by the glycogen deposition test showed an initial increase in the first trimester of pregnancy in the human being. After the initial rise, the urinary excretion level returned to normal with a second increase the 140th to 160th day of pregnancy. Values of 200 to 300 /xg. equivalent of 17,hydroxy11-dehydrocorticosterone per 24 hours of urine were obtained at days 200 to 240. In most instances the urinary outj^ut fell several weeks before parturition. ' Analysis of the blood levels for 17ahydroxycorticosterone in the jiregnant wo


man confirmed the results obtained with the urine (Gemzell, 1953; Seeman, Varangot, Guiguet and Cedard, 1955). Gemzell ( 1953) reported a rise from approximately 5 /xg. per 100 ml. plasma to an average of approximately 22 fxg. per cent (Fig. 16.16). A further rise to 36 /xg. per cent was noted at the time of labor. This has been confirmed by McKay, Assali and Henley (1957) who found an average rise of approximately 40 /xg. per cent during labor lasting more than 6 hours. Although McKay, Assali and Henley reported values still well above normal on the 4th to 6th



Fig. 16.16. Correlation between the concentration of 17-hydroxycorticosteroids in the blood of pregnant women and the duration of pregnancy (in weeks). Conception at zero time. (From C. A. Gemzell, J. Clin. Endocrinol.,13, 898, 1953.)



The mechanism whereby labor induces a marked stimulation of the adrenal cortex is still obscure. It is possible that labor is a stressful state and the stress induced by both the pain and the muscular work act to stimulate the increased release of ACTH resulting in increased adrenocortical activity. Some confirmation of this may be obtained from the fact that significant in(■i'eas(> in }ilasma 17a-hydroxycorticoids is noted only if the labor lasts more than 6 hours.

Analysis of the rise in plasma levels of hydrocortisone during pregnancy has suggested that the phenomenon is not simply the result of an increased rate of secretion from the adrenal cortex, but rather the result of an increased retention and an alteration in the metabolism of the hormone < Cohen, Stiefel, Redely and Laidlaw, 1958).

2. Aldosterone

The isolation for aldosterone by Simi)son, Tait, Wettstein, Neher, von Euw, Schindler and Reichstein (1954) and its identification as the hormone regulating fluid and mineral metal)olism stimulated marked interest in the role of this hormone. Among the items of interest was its significance in pregnancy and in the toxemia of pregnancy. Early studies by Chart, Shipley and Gordon ( 1951 1 revealed the presence of a sodium retention factor in the urine that increased from a normal pregnancy value of 36 to 106 fxg. equivalent of desoxycorticosterone acetate (DOCA) per 24 hours to a maximum of 1008 |U.g. equivalent in pregnancy toxemia. These results were confirmed by Venning, Simpson and Singer (1954) and by Gordon, Chart, Hagedorn and Shipley (1954). In addition a slight increase in the sodium retaining factor was observed in gravid women as compared to nongravid women.

The discovery that the greater part of the aldosterone in urine is present in the conjugated fraction led to a repetition of the above work using both acid hydrolysis and incubation with /3-glucuronidase (Venning and Dyrenfurth, 1956; Venning, Primrose, Caligaris and Dyrenfurth, 1957). The results show little change in the excretion of free aldosterone throughout pregnancy, but the glucuronidase and acid-liydrolyzed


fractions increased markedly (Fig. 16.17). The urinary excretion values increased from a prepregnancy normal of 1 to 6 /xg. aldosterone (average for women was 3.8 ± 14 fig. per 24 hours; Venning, Dyrenfurth and Giroud, 1956) to approximately 25 /xg. per 24 hours. The first significant rise occurred about the third month of gestation and an increased concentration was obtained until after parturition, when there was a rapid fall to the nonpregnant values.


G. Thyroid Gland

Clinical data have long indicated a possible involvement of the thyroid gland in gestation (Salter, 1940). In regions where the iodine supply is low this is demonstrated by an enlargement of the thyroid during pregnancy. The formation of a goiter has been interpreted as evidence for an increased need for iodine during gestation. Scheringer (1930) and Bokelmann and Scheringer ( 1930) reported a rise in the iodine content of the blood of pregnant women during the first trimester of pregnancy with a peak at the seventh month. The increased concentration is maintained until shortly after parurition. In the goat, however, Leitch (1927) reported no change in serum iodine during gestation until just before parturition. Analysis of umbilical vein blood revealed values that were normal, i.e., lower than in the mother (Leipert, 1934). Increased thyroid secretion (Scheringer, 1931 ) and increased urinary excretion of iodine have been reported in pregnant women (Nakamura, 1932; 1933). However, Salter (1940) concluded in his review that no reliable evidence of increased thyroid hormone levels in the blood during jiregnancy is available.

Peters, Man and Heinemann (1948) reported a range of 4 to 8 fig. per cent of serum-precipitable iodine in the normal, nonpregnant woman with a rise to 8.3 fig. per cent (range 6 to 11.2 fig. per cent) in the pregnant woman (Fig. 16.18). It is of interest that the elevation in the proteinbound iodine (PBI) does not follow the course of changes in the basal metabolic rate. Whereas the former is already high by the second month of pregnancy the basal metabolic rate rises gradually after approximately the 4th month of pregnancy (Rowe and Boyd, 1932; Javert, 1940). No other sym})toms of hyperthyroidism are seen in pregnancy which leads to the question of the significance of the rise in protein-bound iodine. A somewhat comparable paradox exists in the guinea pig in which a rise in the rate of oxygen consumption during pregnancy is not accompanied by an increase in heart rate (Hoar and Young, 1957).

Recently, AVerner (1958) rcj^orted a decrease in the I^-^^ up-take after treatment with triiodothyronine in both the normal and pregnant woman. From this and other data he ruled out any abnormal pituitarythyroid relationship or marked secretion of thyroid-stimulating hormone (TSH) by the placenta and concluded that the increased PBI in pregnancy is due to an increased binding capacity of the serum protein.

Feldman ( 1958) failed to find any increase in the level of serum-hutanol-extracted iodine throughout pregnancy in the rat. Actually the values were consistently lower than in the controls and similarly the total amount of PBI in the thyroid of the pregnant rat was consistently lower. He did find an increase in the rate of excretion of V-\ a diminished up-take of I^^^ by the thyroid, and a decreased half-life for thyroxine in the pregnant rats. It is obvious that these results are quite dissimilar from those obtained in the pregnant women. One can only conclude at this time that pregnancy has an effect on iodine metabolism and a species difference exists.

H. Growth Hormone

Although it has been possible to demonstrate the presence of growth-promoting substance (STH) in the blood plasma, there are few data bearing on the identity of the substance and few ciuantitative measurements. Westman and Jacobsohn ( 1944) first showed the ]irescnce of a growth-]5romoting sub



Fig. 16.17. Urinary excretion of aldosterone throughout pregnancy in the human being. O, free fraction only; •, free and acid-hydrolyzed fraction; O, free, enzyme and acidhA'drolyzed. (From E. H. Venning and I. Dyrenfurth. J. Clin. Endocrinol,, 16, 426, 1956.)


stance in the blood by cross transfusion between a normal and hypophysectomized rat united in parabiosis. Gemzell, Heijkenskjold and Strom (1955), using the technique of adding exogenous growth hormone to the sample of blood, failed to find any growthjiromoting substance in 23-ml. equivalents of blood. However, retroplacental blood from human beings gave a positive response at a level of 7- to 15-ml. equivalents of plasma without the addition of exogenous STH. Increase in the width of the proximal tibial epiphysis of the rat was used as an end l)oint. A comparable concentration of 650 fxg. eciuivalent of the standard STH per 100 ml. plasma was also found in the blood from the umbilical cord.

Contopoulos and Simpson (1956, 1957) measured the STH of the plasma in the pregnant rat, using the tibial cartilage, tail length, and body weight increase. No significant increase in plasma STH was noted on the 5th day of pregnancy, however, a significant rise was observed by the 9th day. An estimated 3-fold increase in plasma STH during pregnancy was reported from calculations on both the tibial cartilage and the tail length tests. No changes were reported in the STH activity of the pituitary gland throughout pregnancy. Recently, the persistence of greater than normal amounts of growth-promoting activity was reported in the plasma of pregnant rats after hypophysectomy. Since the fetal pituitary probably does not contribute to the STH pool of the mother, at least in early pregnancy, it is likely that the placenta may be a source of the hormone.

I. Prolactin

Few data are available on the concentration of prolactin during gestation. This has been due, in part, to the minute amounts of the hormone present in the urine and blood and to the inadequacy of the available assays. Although Hoffmann ( 1936 ) failed to find any prolactin in the urine of women before parturition, Coppedge and Segaloff (1951) and Fujii and Schimizu (1958) reported measurable amounts of prolactin in the urine of pregnant women. Coppedge and Segaloff reported a gradual rise in the excretion of prolactin throughout pregnancy and a gradual decline following parturition even though lactation was maintained. The number of observations, however, was limited and the authors point out that the results were ecjuivocal. Fujii and Shimizu observed an initial drop in the prolactin output during the first month of pregnancy followed by a rise to approximately 32 P.C.U. (one pigeon crop sac unit is equivalent to 0.3 I.U.) per 24 hours during the second trimester of pregnancy in women. (Fig. 16.19). This was followed by a drop to approximately 10 P.C.U. per^24 hours between the 30th and 38th wrecks of pregnancy and a marked rise to 64 P.C.U. per 24 hours during the lactation period.


FiG. 16.18. The level of protein-bound iodine in the pregnant woman. (From J. P. Peters, E. B. Man and M. Heinemann, in The Normal and Pathologic Physiology oj Pregnancy, The Williams & Wilkins Co., 1948.)



Fig. 16.19. Urinary excretion of prolactin throughout gestation in the human being. One pigeon crop unit (P.C.U.) is equivalent to 0.3 I.U. (From K. Fujii and A. Shimizu, Bull. Tokyo Med. & Dental Univ., 5, 33, 1958.)


J. Placental Gonadotrophins

Placental gonadotrophins have been found in the monkey, chimpanzee, human being, mare, and rat (Hisaw and Astwood, 1942). The physiologic activities of these placental hormones differ among the three groups of niannnals and appear to represent divergent evolutionary steps in the adoption of pituitary function by the placenta. The physiologic properties of the placental gonadotrophins differ not only among themselves but also from the pituitary gonadotrophins. The gonadotrophin from the rat placenta (luteotrophin) has been shown to be leuto


trophic with the ability to maintain a functional corpus luteum in the hypophysectomized rat (Astwood and Greep, 1938). The hormone has no effect on follicular growth or ovulation. Its function appears to be that of maintaining the secretory activity of the corjius luteum in the rat from the 10th day of pregnancy to term.

The placental hormones of the human being (HCG) and the mare (PMS) have been studied in much greater detail. These two hormones differ markedly in both chemical and physiologic properties. The presence of HCG in the urine and the absence of P]\IS in the urine would alone indicate a marked difference in the size of the two molecules. Physiologically, PMS is highly active in producing follicular growth and some luteinization, whereas HCG has no effect on follicular growth but will induce ovulation and a delay in the onset of menstruation. This would indicate a luteotrophic action. Although chorionic gonadotrophin has been reported in the macaque (Hamlett, 1937) between the 18th and 25th day of pregnancy, and in the chimpanzee from the 25th to the 130th day of gestation (Zuckerman, 1935; Schultz and Snyder, 1935), little work has been done on the characterization and identification of these substances except in man and horse.



Fig. 16.20. The relative time of appearance of placental gonadotropliins in the pregnant mare and the woman. (From E. T. Engle, in Sex and Internal Secretions, 2nd ed., The Williams & Wilkins Company, Baltimore, 1939.)


It is of some interest to note that the appearance of the placental gonadotrophins in the blood and urine of horse and man occurs at approximately the same relative time in pregnancy (Fig. 16.20). The role played by these hormones in gestation is still not clear, but it is significant that their appearance corresponds with the time of implantation of the blastocyst and their disappearance roughly with the time when ovariectomy no longer interferes with the maintenance of the pregnancy.

K. Human Chorionic Gonndotrophin (HCG)

The discovery of the presence of a gonadotrophic hormone in human pregnancy urine by Aschheim and Zondek (1927j was soon followed by a description of its biologic activity and quantitative determinations of its concentration in the urine throughout pregnancy (Ascheim and Zondek, 1928). Recently a number of investigators have determined the titer of chorionic gonadotrophin in the serum of pregnant women. These curves agree very well with the values obtained from the urine. Figure 16.21 is a typical curve for the concentration of chorionic gonadotrophin in the blood of pregnant women (Haskins and Sherman, 1952). A peak value of 120 I.U. per ml. of serum was obtained on the 62nd day after the last menses and a rapid decline was noted to a low of approximately 10 I.U. per ml. of serum on day 154. A subsequent rise to 20 I.U. was noted by day 200 and this was maintained until the end of pregnancy. These results are in excellent argeement with those reported by Wilson, Albert and Randall (1949) using the ovarian hyperemia test in the immature rat. These authors obtained a peak concentration of approximately 70 I.U. per ml. of serum on the 55th day after the last menses. A gradual decrease occurred thereafter to a low of approximately 20 I.U. per ml. of serum which remained unchanged from day 100 to parturition although the data indicate a slight rise towards the end of pregnancy.




Fig. 16.21. Concentration of human chorionic gonadotiophin in the blood of the normal pregnant woman. The hormone levels were determineti b>' the male frog test. (From A. L. Haskins and A. I. Sherman. J. CUn. Endocrinol., 12, 385, 1952.)


The significance of the excretion pattern and concentration of the hormone in the serum is still a matter of conjecture. Browne, Henry and Venning (1938) suggested that the peak level of chorionic gonadotrophin in the blood reflects an increased production and a physiologic need in order to maintain a functional corpus luteum during early pregnancy. Recent evidence has tended to confirm this opinion in that HCG has been found to be active in the maintenance of the secretory activity of the corjius luteum in the primate (Hisaw, 1944; Brown and Bradbury, 1947; Bryans, 1951 j. In addition, histologic studies reveal a direct proportion between the number of Langhans' cells and the amount of hormone excreted (Stewart, Sano and Montgomery, 1948; Wislocki, Dempsey and Fawcett, 1948) .

The possibility that the kidney plays a role in the changes in the concentration of HCG was investigated by Gastineau, Albert and Randall (1948) . The renal clearance was relatively constant throughout all stages of pregnancy although the urine and serum concentrations of the hormone varied as much as 20- fold. In addition, the mean, renal clearance found during pregnancy was not markedly different from that found in cases of hydatiform mole and testicular chorioma. Inasmuch as the renal elimination of the hormone remained constant, it was obvious that two possible explanations existed : these were (1) changes in the secretion rate, and (2) changes in extrarenal disposal of the hormone. Studies on the latter were contradictory. Whereas Friedman and Weinstein (1937) and Bradbury and Brown (1949) reported an excretion of 20 per cent and higher of HCG following the injection of HCG, Johnson, Albert and Wilson (1950) found an excretion of 5.8 per cent in pregnant women during the immediate postpartum period. Zondek and Sulman (1945) reported a 5 to 10 i)er cent elimination of HCG in the urine of animals. Thus Bradbury and Brown felt that there is relatively little destruction or utilization of the hormone in the body; Wilson, Albert and Randall ( 1949) believed that 94 per cent of the circulating hormone is affected by extrarenal factors and that the fluctuating character of hormonal level in serum or urine depends entirely on changes in rate of hormone production.

An analysis of the distribution of chorionic gonadotrophin in the mother and fetus led Bruner (1951) to conclude that the ratio of maternal blood to urinary gonadotrophin is not constant although the ratio of gonadotrophin in the chorion to maternal blood is constant. Consequently, she concluded that the concentration of gonadotrophin in the urine does not depend entirely on the rate of production of the hormone and that the method of gonadotrophin elimination changes during pregnancy. She also pointed out that a significant amount of chorionic gonadotrophin is found in the fetus and that this is due to the fact that, although the chorion releases the hormone into the maternal blood, secondarily some of it passes the placental barrier and enters the fetal system across the wall of the chorionic vesicle.

2. Equine Goyiadotrophin (PAIS)

The presence of a gonadotrophin in the blood of the pregnant mare was first described by Cole and Hart in 1930. The hormone appears in the blood about the 40th day of pregnancy and increases rapidly to a concentration of 50 to 100 rat units (R.U.) per ml. by the 60th day of pregnancy (Cole and Saunders, 1935). This concentration is maintained for approximately 40 to 65 days. By day 170 it has fallen to a nondetectable level (Fig. 16.22).

Catchpole and Lyons (1934) suggested that the placenta is the source of the gonadotrophin and indicated that the chorionic epithelium is the probable source. Cole and



Fk;. 16.22. Tlie concentration of i)rognant mare's serum in the blood of the mare throug out pregnancy. (From H. H. Cole and F. J. Saunders, Endocrinology. 19, 199, 1935.)


Goss (1943), on the other hand, concluded that the endometrial cups are the source of the hormone. Recent evidence tends to confirm the endometrial cups as the source of the hormone (Clegg, Boda and Cole, 1954). The endometrial cups form in the endometrium opposite the chorion in the area where the allantoic blood vessels fan out. The cups develop precisely at the time when the hormone is first obtained in the serum of the pregnant mares and desquamation of the enclometrial cups is complete at the time of the disappearance of the hormone from the maternal blood. Analyses of the cups for gonadotrophin content reveal a correlation between the concentration of the hormone in the maternal blood and the concentration in the endometrial cups. Finally, histochemical stains for glycoprotein indicate the presence of this substance only in the epithelial cells lining the uterine lumen and the uterine glands in the cup area ( for complete discussion of the subject see the chapter by Wislocki and Padykulal.


VI. Pregnancy Tests

The discovery of gonadotrophic activity in the urine of pregnant women by Aschheim and Zondek in 1927 led to introduction of the first valid test for pregnancy (Aschheim and Zondek, 1928). These investigators used the innnature mouse and reported the presence of corpora hemorrhagica as indicative of the presence of a gonadotrophin in the urine and a positive reaction for pregnancy. The Aschheim-Zondek test for pregnancy was the first successful test of its kind and has been used both as a qualitative and quantitative test. In the latter instance, a serial dilution of the urine is made in order to obtain the minimal effective dose.

It is not too surprising that many tests for pregnancy have been described. In general, all of the successful tests involve the detection of chorionic gonadotrophin in the urine, and to some extent in the blood. The changes that have appeared in the development of new pregnancy tests have been those concerned with the use of different species of animals, the rapidity with which the test could be completed, and convenience to the laboratory. Thus the Friedman test (Friedman, 1929; Friedman and Lapham, 1931 ) followed soon after the Aschheim-Zondek test and in turn was succeeded by several newer tests.

Ap])roximately five reliable tests are now available (Table 16.6). All are concerned with the detection of HCG and have an accuracy of 98 to 100 per cent. The AschheimZondek suffers from a time requirement of 96 hours and was largely supplanted by the Friedman test that used the isolated rabbit and required only 48 hours. Within recent years several new tests have been reported using the frog, toad, and immature rat. Frank and Berman (1941) first noted the occurrence of hyperemia in the ovary of the immature rat, following the injection of HCG. Albert (1949) reported excellent results with the use of this test in 1000 cases. Comparison of the rat hyperemia test with the Friedman test was on the whole very good and revealed the same order of accuracy for both tests. The Friedman test, however, will detect about 5 I.U. of HCG which would mean a concentration of 500 I.U. of HCG per 24-hour output of urine ( assuming a 24-hour urine output of 1500 ml.). Positive results in the rat test require a 24-hour output of 1000 I.U., indicating that the ovarian hyperemia test in the rat is about one-half as sensitive as the Friedman test. Nevertheless, the rat



TABLE 16.6 Pregnancy tests with an accuracy


oj 98 to 100 per cent


Animal


Sex


Observed End Point


Time


Reference


Immature mouse Isolated rabbit Xenopus laevis Bufo arenarum Immature rat


F F F M F


Corpora hemorrhagica Corpora hemorrhagica Extrusion of ova Extrusion of sperm Hyperema of ovary


hr.

96

48 8-12 2-4

4


Aschheim and Zondek, 1928 Friedman and Lapham, 1931 Shapiro and Zwarenstein, 1934a Galli-Mainini, 1947 Frank and Berman, 1941



test requires only 4 hours and a larger number of animals can be utilized, thus decreasing the error due to use of inadequate numbers of animals. Comparison of the rat hyperemia and the Friedman tests revealed that the former is slightly more accurate but a little less sensitive (Albert, 1949).

Within two years after the publication of the Friedman test for pregnancy, Shapiro and Zwarenstein (1934a, 1934b, 1935) and Bellerby (1934) reported the use of the African toad {Xenopus laevis, D) in the diagnosis of pregnancy. Again the test was based on the ability of HCG to induce the extrusion of ova by the frog following the injection of the urine into the dorsal lymph sac. Extrusion of the ova occurred in 6 to 15 hours and the test was shown to compare favorably with both the Aschheim-Zondek and Friedman tests, although it did not give tiie graded response seen with the A-Z test (Crew, 1939). Weisman and Coates (1944) found an accuracy of 98.9 per cent with the Xenopus test over a 5-year period during which 1000 clinical cases were examined.

Galli-Mainini (1947) first reported the use of the male batrachian in the diagnosis of pregnancy and Robbins, Parker and Bianco (1947) simultaneously reported the release of sperm by Xenopus following treatments with gonadotrophins. Galli-Mainini (1948» pointed out that this reaction is not restricted to a single toad, but would ]irobably be found in many frogs and toads. He added that care should be used to employ animals with a continuous spermatogenesis. This was immediately confirmed by reports from different countries using various species of frogs and toads endogenous to the areas. Immediate use of Rana pipiens was reported in the United States and this species became very popular in that country ( Wiltberger and :\Iiller, 1948) .

The advantages of the sperm-release test are the time requirements, simplicity, end point, and opportunity to use many animals. On the other hand, the reaction is all or none and shows no gradation in degree of reaction. In general, the urine is injected into the dorsal lymph sac and the cloaca aspirated for sperm 1 to 3 hours later. Although this is the most recent of the pregnancy tests, many reports have appeared and some evaluation as to accuracy may be attempted. Galli-IVIainini (1948) reported an accuracy of 98 to 100 per cent in a summary of more than 3000 tests and 100 per cent accuracy for negative results in more than 2000 controls. Robbins (1951 ) reported an accuracy of 89.5 per cent in the first trimester of pregnancy. Pollak (1950) indicated that as many as 20 per cent of the negative tests obtained in the summer were false. This suggested the existence of a refractory state at this season. Bromberg, Brzezinski, Rozin and Sulman (1951) reported on a comparison of several tests including 700 cases. An accuracy of 85 per cent was obtained with the male frog test, 99 per cent with the rat hyperemia test, 98.5 per cent with the Friedman test and 98 per cent with the Aschheim-Zondek test. The authors indicate that the 15 per cent failures to get a positive reaction in the frog could be due in part to the poor sensitivity of the animal which could only be overcome by concentrating and detoxifying the urine. Comparison of the minimal amounts of HCG to elicit a positive reaction are Vs I.U. for the rat hyperemia test, 1 I.U. for the Aschheim-Zondek and Hyla tests, 2 I.U. for the Rana and Bufo tests, and 5 I.U. for the Friedman test. Reinhart, Caplan and Shinowara (1951) reported an accuracy of 99 per cent with 840 urine specimens; only 3 false negatives were noted in 346 specimens from known pregnant women and no false positives noted in 125 nonpregnant women. The authors attribute the high degree of accuracy to standardization of the procedure by which extraneous factors were eliminated. These include ( 1 ) the use of 2 or more 30- to 40-gm. frogs for each test, (2) elimination of all animals suffering from red leg and other diseases, (3) adequate time for sperm release, (4) concentration of the urine, (5) maintenance of frogs at 15 to 22°C., and (6) during the summer the injection of an increased volume of urine and an increase to 4 hours in the period for sperm release. The maintenance of frogs in a hibernating state by keeping them in a refrigerator at 38°F. has been rei:)orted to insure a high degree of sensitivity regardless of the season (Allison, 1954). Although it is obvious that more data are needed, the present results are very promising for the "frog-sperm" test and if the seasonal effect can be eliminated, this test will be the equal of the other four. VII. Water and Electrolyte Balance The changes in the various components of the blood during pregnancy have been described in a number of species (Tables 16.7 and 16.8). It is generally agreed that a marked increase in the blood and plasma volume and a decrease in the relative amounts of erythrocytes and hemoglobin occur during the last trimester. Inasmuch as the increase in the plasma volume in man exceeds the concurrent increase in the total cell volume, the resultant hemodilution produces an anemia which has been described as the "physiologic anemia of jM-egnancy.


TABLE l(i.7 The average percentage of change in the constituents of the blood and in the extracellular fluid volume during

normal pregnancy in man


Extracellular space


Adams, 1954.


Bibb. 1941

Caton, Robv, Reid and Gibson, 2nd, "l949

Caton, Roby, Reid, Caswell. Maletskos, Fluharty and Gibson, 1951


Chesley, 1943

Dieckmann and Wegner,

1934a, b, c, d

Ferguson, 1950


Freis and Kenny, 1948

Friedman, Goodfriend, Berlin

and Goldstein, 1951

Ganguli, 1954


Gemzell, Robbe and Sjostrand, 1954


Hamilton and Higgins, 1949. .

Jarosova and Daum, 1951 ...

Lambiotte-Escoffier, Moore and Tavlor, Jr.. 1953

Lund, 1951

McLennan and Corey, 1950..

McLennan and Thouin, 1948. .

Miller, Keith and Rownetree, 1915

Mukherjee and Mukherjee, 1953

Roscoe and Donaldson, 1946.

Thompson, Hersheimer, Gibson and Evans, Jr., 1938 . . .

Tysoe and Lowenstein, 1950 .

White, 1950


59


25


15


Blood volume


49 None


45


23


30


59


32


Plasma volume


22


49


55


25


32


Total hemoglobin


20


59


20


Hematocrit


10


15


10


12


16


RBC

count


14


21


22


Gram % hemoglobin


25


15

25% in 50% of patients


10


Time of Determinat in Pregnancy


6th week antepartum 3rd trimester

3rd trimester


60 days antepar

tum 3rd trimester

3rd trimester


3rd trimester

At term

Throughout pregnancy

8th week antepartum

3 months antepartum

9th monlli

10th lunar month 3rd trimester 10th lunar month At term

3rd trimester

3rd trimester 3rd trimester

9th lunar month

At term

3rd trimester


GESTATION


989


TABLE 16.8


The


average percentage of cl


aiu/e in


the constituents


of the blood and


in the extracellular fluid



volume during norn


al pregnancy in


variouf


laboratory and domestic animals



Increase


Decrease



Animals





Total hemoglobin





Author



Blood


Pasma


Cell


Hema

RBC


Gram %




volume


volume


volume


tocrit


count


hemoglobin




%


%


%


%


%


%




Rat







40


20-40


Beard and Mvers, 1933



41




20


10 29


18 30 25


7 31 33


Bond, 1958 Newcomer, 1947 van Donk, Feldman and Steenbock, 1939


Rabbit


50





17


20


10-20 17


Salvesen, 1919

Zarrow and Zarrow, 1953



6


12






12


13


Horger and Zarrow, 1957


Sheep


25


22


10





Initial decrease, normal at term


Barcroft, Kenned}- and Mason, 1939


Cow


Slight


Slight








Reynolds, 1953


Comparable changes were observed in the blood constituents and plasma volumes of the rat and rabbit during the latter third of gestation. Although there is no increase in the total cell volume, the resultant "physiologic" anemia of pregnancy in the rabbit follows the same general pattern as that rei)orted in the human being (Horger and Zarrow, 1957).

A significant decrease in the erythrocyte number, hemoglobin concentration, and hematocrit, and an increase in the blood volume have been noted in the rat during pregnancy (Table 16.8). However, the increase in blood volume is correlated with an increase in body weight and the ratio of blood volume to body weight remains unchanged (Bond, 1948). Calculation of the total number of erythrocytes and grams of hemoglobin actually showed an increase in these constituents during gestation, indicating that the anemia of pregnancy in the rat is due to a hemodilution in which the blood volume increases proportionately faster than the number of erythrocytes.

Comparable results were also reported in the rabbit (Zarrow and Zarrow, 1953). A marked drop in the relative number of circulating erythrocytes and percentage of hemoglobin is seen invariably towards the end of gestation (Fig. 16.23). A marked fall in hematocrit occurs concomitantly with the fall in the two blood constituents along with an increase in the reticulocytes. The time of onset of the increase in reticulocytes is variable and seems to occur during the second trimester of gestation. Their number returns to normal before parturition in spite of the increasing severity of the anemia. A second rise in the reticulocytes is seen during the first week postpartum. All the other constituents return to normal values during the first or second week postpartum.



Fig. 16.23. Changes in the relative number of circulating erythrocytes, reticulocytes, percentage of hemoglobin, hematocrit, and nonprotein nitrogen of the blood of the rabbit during pregnancy and after parturition. (From M. X. Zarrow and I. G. Zarrow, Endocrinology, 52, 424, 1953.)




Disagreement exists as to whether there is a change in the volume of the extracellular fluid compartment during pregnancy in the human being. Whereas certain investigators have reported rather marked increases in the extracellular space (Chesley and Chesley, 1941; Chesley, 1943; Freis and Kenny, 1948; Caton, Roby, Reid and Gibson, 1949; Friedman, Goodfriend, Berlin and Goldstein, 1951; Jarosova and Damn, 1951), others have reported that the changes in this fluid compartment are proportional to changes in the body weight (LambiotteEscofiier, Moore and Taylor, 1953; Seitchik and Alper, 1954). The results obtained in the rabbit support the findings of the latter authors as no disproportionate increase in the thiocyanate space was observed during pregnancy in the rabbit. The slight increase that occurred during the last trimester of gestation was in good agreement with the fluid accumulation by the developing fetus. Similarly the increase in blood volume in the rat is correlated with increase in body weight.



Fig. 16.24. Changes in blood plasma and total erythrocyte volume in the ovariectomized rabbit treated with 1 mg. estradiol daily. (From L. M. Horger and M. X. Zarrow, Am. J. Physiol., 189, 407, 1957.)



Fig. 16.25. Changes in the blood plasma and total erythrocyte volume during pregnancy. (From L. M. Horger and M. X. Zarrow, Am. J. Physiol., 189, 407, 1957.)


Thus the anemia of pregnancy as observed in the rabbit and rat is very similar to that reported for man. It can be characterized as a normochromic and normocytic anemia. Although a decrease in the relative concentrations of hemoglobin and erythrocytes occurs, the total amounts of these components of the blood remain unchanged. Consequently, the anemia of pregnancy is due to a hemodilution.

The anemia induced by treatment with estradiol is similar to the anemia of pregnancy in many respects. Witten and Bradbury (1951) treated 16 women with 5 mg. estrone or 0.4 mg. estradiol dipropionate and noted an erythrocyte drop of 14.8 per cent, a hemoglobin drop of 8.5 per cent, a hematocrit drop of 15 per cent, and a blood volume increase. Treatment of the castrated rabbit with 1 mg. estradiol daily caused a 20 per cent decrease in both erythrocyte count and hemoglobin with no significant changes in total hemoglobin or number of erythrocytes. Estradiol also caused an increase in plasma and blood volume (Fig. 16.24) which was comparable to that seen during pregnancy (Fig. 16.25), but no significant change in cell voluiiic was obtained. The estradiol-induced anemia is both normochromic and normocytic and is caused by a htMnodilution. However, in addition to the ciianges in the blood and plasma volumes, estradiol induces a significant increase in the thiocyanate space. Furthermore, only the massive dosage of 1.0 mg. estradiol per day elicits an anemia comjKirable to that observed in pregnancy. This dosage level is probably toxic since there is a decrease in the body weight of most rabbits which received this treatment. Thus, in spite of the similarities of these anemias, it is likely that estrogen is not the sole etiologic agent in the anemia of pregnancy.

Progesterone alone at dosages of 4 mg. daily has little effect on the plasma volume or the thiocyanate space. This steroid does exert a significant influence on the action of estradiol on the blood and plasma volume, but it is to be noted that rather large dosages of estradiol were still needed to induce a significant hypervolemia and that the effect depends on the ratio of the concentration of the two hormones. The hypervolemia induced by the treatment with 4 mg. progesterone in combination with 0.1 mg. estradiol was greater than that caused by the estradiol alone, whereas the treatment with 4 mg. progesterone in combination with 1.0 mg. estradiol resulted in an inhibition of the estrogenic activity (Fig. 16.261. Thus progesterone may play a dual role in the water metabolism of the gravid female. In the presence of low titers of estrogen, progesterone augments its action which may be a means of insuring an adequate fluid retention to provide for the fluid requirements of the fetus. However, if the titers of the estrogens and possibly of other steroids affecting salt and water metabolism became excessively high, the progesterone may provide a protective measure by inhibiting the activity of these substances. This concept is in accord with reports describing the diuretic action of progesterone in the iiypophysectomized rat (Selye and Bassett, 1940) and the inhibition of the salt- and water-retaining action of DOCA and cortisone by progesterone (Landau, Bergenstal, Lugibihl and Kascht, 1955).


Fig. 16.26. Changes in the blood volume of the ovariectomized rabbit treated with 0.1 mg. and 1.0 mg. estradiol daily and with a combination of the two estrogen treatments and 4 mg. progesterone. (From L. M. Horger and M. X. Zarrow, Am. J. Pliysiol., 189, 407, 1957.)

It is also of interest that no anemia was observed in animals treated with various combinations of estrogen and progesterone (Horger and Zarrow, 1957). Progesterone elicits an increase in the cell volume which api^roximates that of the plasma volume. Because no erythrocyte counts were made in this study, it is not possible to state whether this increase in the cell volume is caused by a macrocytosis or an increase in the number of erythrocytes. Vollmer and Gordon ( 1941 ) reported that progesterone caused an increase in the erythrocyte count of the rat but that the action was inconsistent. Hence it is possible that the increase in the cell volume is due to an enhancement of hematopoiesis by the progesterone. This possibility is not inconsistent with the absence of an increase in the reticulocyte count in response to these treatments since a reticulocytosis usually occurs only after an intense stimulation of the hematopoietic tissue such as by hemorrhage.

In view of the previous discussion, it is improbable that the anemia of pregnancy is due entirely to the interaction of estrogen and progesterone. These hormones appear to play an important role in the salt and water metabolism of the gravid female. Furthermore, it is noted that the cow exhibits a hypervolemia but no anemia during pregnancy (Reynolds, 1953) and that a similar condition is produced in the rabbit by the treatment with various combinations of these steroids. Hence the interaction of estrogen and progesterone may be responsible for this species difference.

Inasmuch as no antidiuretic hormone (ADH) could be detected in any of the plasma samples, it is apparent that the plasma titers of ADH did not rise above 10 fiV. per ml. during the experimental period. However, in view of the increased ability of the blood to inactivate ADH during pregnancy (McCartney, Vallach and Pottinger, 1952; Croxatto, Vera and Barnafi, 1953), there may be an increased rate of turnover of ADH during gestation. Consequently the data obtained in this study neither substantiate nor eliminate ADH as an etiologic agent in the anemia of pregnancy.

A number of investigators have attributed the hypervolemia of pregnancy to structural changes in the circulatory system. Burwell (1938) observed a marked similarity between the circulatory changes observed in pregnancy and those observed in a patient with an arteriovenous fistula. He noted that in both conditions there is an increase in the blood volume, cardiac output, pulse rate, pulse pressure, and an increased venous pressure near the opening of the fistula. He concluded that the changes in the circulation of the pregnant woman are caused by an arteriovenous leak through the placenta and the obstruction of the venous return by the enlarged uterus. Bickers (1942) correlated the intensity of the edema of the right or left leg with the location of the placenta in the uterus. The edema was observed to be consistently greater on the same side as the location of the placenta whereas the edema of the legs was equal when implantation occurred on the anterior or posterior wall of the uterus. However, when the uterus was lifted off the interior vena cava, there was no precipitous drop in the venous pressure in the femoral vein. Thus this study supports the arteriovenous shunt theory of Burwell.


One objection to this theory is that it i

does not account for the decrease in the i

blood volume during the 10th lunar month j

of pregnancy. However, it has been reported i

that during the latter part of pregnancy there is an increase in the resistance to the flow of blood through the placenta. This increase is due to the increased number of villi and to the anastomizing of the villi in the placenta. Since an increase in the peripheral resistance to blood flow results in a hemoconcentration, this would account for the decrease in the blood volume during the last lunar month of gestation (Kline, 1951; McGaughey, 1952).

Other objections to the arteriovenous shunt theory were reported by Kellar ( 1950) who found that blood flow through the placenta is sluggish rather than rapid as in an arteriovenous shunt. He also observed that the uterine venous blood is not exceedingly rich in oxygen as is the venous return of an arteriovenous aneuryism and lie concluded that, although the uterus is an area of decreased resistance to blood flow, the effect is not entirely due to the placenta. He suggested that thyroxine may be partially responsible for the expansion of the blood volume since mild thyrotoxicosis is common in pregnancy. This concept is supported by the observation that there is a tendency for vasodilation in the upper extremities during the latter months of gestation (Burt, 1950). Furthermore, the basal metabolic rate increases during this period (Sandiford and Wheeler, 1924; Rowe and Boyd, 1932). It is to be noted, however, that the changes in the blood flow in the extremities are closely correlated with the cardiovascular changes occurring during pregnancy but not with the changes in the hormonal levels in the blood (Herbert, Banner and Wakim, 1954 ) .

Since there is no disproportionate increase in the thiocyanate space of the rabbit during pregnancy, the increase in the blood volume can best be explained on the basis of cardiovascular changes during the latter part of gestation. However, in view of the previous discussion, it is extremely improbable that this hypervolemia is induced by any one factor. Rather, it is more probable that the condition is produced by a multiplicity of factors. On the basis of the previous reports, it is evident that the placenta, due to its similarity to an arteriovenous aneuryism, is partially responsible for the hypervolemia. The marked increase in the uterine size and vascularity during pregnancy (Barcroft and Rothschild, 1932) will also account for a considerable amount of the increase in the blood volume. In addition, the tendency for dilation of the peripheral blood vessels may account for another portion of the increase in the blood volume. Thus, in general, the hypervolemia of pregnancy can be attributed primarily to structural changes in the circulatory system.

It cannot be denied, however, that the cardiovascular system is influenced by the changes in the endocrine balance during gestation. It is well known that the placenta elaborates large amounts of sex steroids and corticoids. It has also been suggested that there is an increased production of thyroxine and ADH at this time. In addition, water-soluble extracts of the pregnant sow's ovaries have been shown to cause water retention and anemia in the rabbit (Zarrow and Zarrow, 1953). The resultant hormonal balance becomes somewhat precarious as the additional secretions of the glands tend to build up the blood titers of the sex steroids and other substances which influence water metabolism. It is possible that when a proper balance of these factors is maintained, the pregnancy is normal and the various requirements of the fetus are provided without disrupting the distribution of the body fluids outside of the vascular system. However, if the balance is not maintained, the animal tends to accumulate fluid, and edema and other pathologic complications result.


VIII. Plasma Proteins

It has long been known that the plasma proteins play a significant role in the fluid balance of the organism and as such are also involved in water balance during pregnancy (]\Iack, 1955). In addition, the plasma proteins are of importance in many other functions, such as heat and energy source and replacement of tissue in which function they act as a protein source whenever needed and form the metabolic pool. These proteins are synthesized in general in the liver and reticuloendothelial system and may be classified as albumins or globulins although many different entities of these two classifications are known to exist.


The maintenance and stabilization of blood volume and the equilibrium of fluid exchange between the extravascular and intravascular compartments is a function of the albumin fraction of special significance in i)regnancy, in addition to its other functions of acting as carrier for other substances and sui^jilying of nutrients. The regulation of blood volume by albumin depends on its osmotic action and is of much greater significance than the globulins. Approximately 4.6 gm. albumin and 3.17 gm. globulin per 100 ml. of plasma are found in the normal, nonpregnant woman. At least four types of glol)ulins are present in the plasma among which are found the lipoproteins, prothrombin, fibrinogen, antibodies, and several hormones.


Although both the plasma proteins and albumin drop during pregnancy, this does not necessarily indicate a drop in the total available albumin protein. An increase in the plasma volume compartment of 25 per cent as seen in pregnancy could easily result in an increase in the total amount of circulating protein. However, as the total circulating blood volume increases in pregnancy, the albumin fraction and yglobulin seem to be diluted whereas the other globulins become more concentrated. Nevertheless, the globulins cannot compensate for the albumin loss and the total protein decreases. Mack (1955) has listed several possible explanations for the above paradox : ( 1 ) the small albumin molecule may diffuse more freely into tissues and across placental membrane, and (2) albumin synthesis cannot keep pace with utilization.


Innumerable studies on the plasma i^-oteins of women during pregnancy have I'evealed markedly consistent changes in the albumin-globulin ratio of the plasma. The concentration of total protein and albumin decreases while the total globulin increases. The trend is apparent by the first trimester and continues throughout gestation. A return to the nonpregnant pattern is seen shortly after parturition. The total protein dropped 13 per cent and the albumin 26 per cent. The various globulin fractions showed a rise except for the y-globulin (Mack, 1955). As a result of these changes, the albumin-globulin ratio declines throughout pregnancy and shows the well known reversal (Fig. 16.27) and recovery to normal by 6 weeks postpartum.



Fig. 16.27. Progressive decreu.se in the albuminglobulin ratio of the plasma during pregnancy in women. (From H. C. Mack, The Plasma Proteins in Pregnancy, Charles C Thomas, Springfield, 111.. 1955.)



Fig. 16.28. Changes in the glomerular filtration rate throughout pregnancy in the woman. (From W. J. Dignam, P. Titus and N. S. Assali, Proc. Soc. Exper. Biol. & Med., 97, 512, 1958.)


Although it is obvious that the albumin fraction is important in maintaining the blood fluid compartment, the changed albumin-globulin ratio cannot solely account for the retention of water and edema present in pregnancy. Although it has been argued that the hypoalbuminemia through diminished colloid osmotic pressure is the cause of water retention in the tissues, the occurrence of the postpartum diuresis at the time when the albumin is lowest would tend to indicate some other mechanism (Dieckmann and Wegner, 1934a-d). Additional mechanisms, such as changes in the hormone level, especially the sex steroids and adrenal corticoids, may be responsible.

IX. Renal Function

Studies on renal function during pregnancy have resulted in contradictory reports. The earlier investigations failed to show any effect of pregnancy on renal function (Chesley and Chesley, 1941; Welsh, Wellen and Taylor, 1942; Dill, Isenhour, Cadden and Schaffer, 1942) , whereas recent studies indicate a marked change in renal function during gestation (Bucht, 1951; Dignam, Titus and Assali, 1958). Part of the explanation for the divergent results could be the type of patient studied, the periods when studied, and the types of controls. Dignam, Titus and Assali studied both the renal plasma flow and glomerular filtration rate in various patients throughout gestation and immediately following l^arturition. Care was taken to select individuals without any history of cardiovascular or renal disease. Both the renal plasma flow and the glomerular filtration rate (Fig. 16.28) were increased throughout gestation. The initial rise was extremely marked during the 1st and 2nd trimesters of pregnancy. A slight rise was noted during the 3rd trimester and a return to normal by 6 to 8 weeks postpartum.

Recently, de Alvarez (1958) reported a 50 to 60 per cent rise in the glomerular filtration rate and a 60 per cent rise in the renal plasma flow during the 1st trimester of pregnancy in the human being. This is in agreement with the findings of Dignam, Titus and Assali (1958). However, de Alvarez reported, in addition, a progressive decline in both the glomerular filtration rate and renal plasma flow during the 2nd and 3rd trimesters. The filtration factor (glomerular filtration rate divided by the renal plasma flow) remained low in the first 2 trimesters and increased in the last trimester. This is evidence for an increase in tubular resorption of water and electrolyte. It can only be concluded, therefore, that kidney function is altered during pregnancy, especially the 1st trimester. Results from investigations involving the 2nd and 3rd trimesters are contradictory. De Alvarez concludes that the changes in renal hemodynamics during pregnancy are mediated by the endocrine system because the alterations in renal function seem to be related to the sodium and water retention. If the changes are progressive throughout gestation, it would be possible to correlate the phenomenon with a number of hormones that increase during pregnancy. On the other hand, if the phenomenon is transient, i.e., only during the 1st trimester, then the phenomenon can only be correlated with HCG.

X. Enzymes

A. Histamixase

The presence of histaminase or diamine oxidase in tissues of the body has been known for some time. As yet the enzyme lias not been crystallized but is believed to he a flavoprotein (Swedin, 1943). The enzyme is not specific for histamine because it inactivates other diamines such as cadaverine and putrescine. Histaminase determinations, in general, are based on incubation of the test material with histamine dihydrochloride for a fixed period of time and the bioassay of the residual histamine carried out on an isolated strip of guinea pig intestine.

Histaminase has been found in the l)lasma of men and women with an increase (luring ])regnancy from a value of between 0.003 and 0.008 /^g. per ml. per hr. to a value of between 3.5 and 10 at parturition (Ahlmark, 1944, 1947). This has been confirmed by Swanberg (1950), who determined the histaminolytic activity in pei-iplK>ral blood throughout pregnancy (Fig. 16.29). A marked rise is observed from the 10th to the 20th week of pregnancy, and thereafter the concentration plateaus until after parturition.


Fig. 16.29. Tlie histaminase activity of the peripheral blood of the human female during pregnancy (•) and at parturition (®). (From H. Swanberg, Acta scandinav., Suppl. 79, 23, 1950.)

Both the maternal placenta and the decidual tissue have been identified as major sites for formation of the enzyme. Danforth and Gorham (1937) reported the presence of histaminase in the placenta of a series of patients at term. This was confirmed by Swanberg (1950) who, in addition, separated the placenta by a series of slices parallel to the surface of the organ and reported that the layer adjacent to the uterine wall, consisting of practically only the thin decidual membrane, contained a mean value of 614 /xg. per gm. per hr. of histaminase as compared to 38 for the fetal portion of the placenta. Confirmation of the concept that the maternal placenta is the main source of histaminolytic activity can be obtained from the finding of histaminase in decidual tissue of nonpregnant females and in the maternal placentas of animals. In cases in which maternal and fetal placentas can be separated easily, the maternal placenta contained from 14- to 100-fold the activity seen in the fetal placenta. Comparison of the histaminolytic activity in the decidual tissue of the sterile horn and the control pregnant horn of the uterus of a rabbit revealed 319 fig. per gm. per hr. and 222 fxg. per gm. per hr., respectively. Treatment with progesterone or induction of jiseudopregnancy caused a marked rise in the histaminase of the endometrium to upwards of 1000 fig. per gm. per hr. Nonetheless, histaminase was not observed in the blood plasma of the progesterone treated rabbits whereas progesterone treatment of two nonpregnant women caused a marked rise iti plasma histaminase.

The physiologic significance of histaminase is still unknown. A consideration of this problem must take into account not only the action of the enzyme and changes in its concentration under different physiologic conditions, but also the species problem. In regard to the latter point, the data are extremely inadequate. Only two species have been studied in any detail and these are the human being and the ral)bit. One can conclude from the available data that histaminase is produced by the maternal placenta, decidua, and uterine endometrium. It increases with pregnancy in these tissues and its concentration may be correlated with the progestational hormone. It increases in the blood of tlie human being, I'at, and guinea pig during pregnancy but nut in the cat or rabbit (Swanberg, 1950; Carlsten, 1950). The obvious hypothesis that histaminase })rotects the uterus from the stimulating action of histamine has not been confirmed. But it is somewhat jiai'adoxical to note that urinary histamine also increases during pregnancy. Kahlson, Rosengren and Westling (1958) reported a daily 24-hour excretion of 18 to 43 /xg. of histamine during the first 2 weeks of pregnancy in the human being. A marked increase was noted on the 15th day with a peak of 123 to 835 /xg. per 24 hr. at the peak of excretion which occurred 1 to 2 days before parturition. As yet no role can be attributed to this substance. It is of interest that the increased histaminase present during pregnancy can serve the role of protecting the uterus from the musclecontracting action of this substance. Because the amount of urinary histamine excreted is correlated with the number of young and no changes are apparent in the concentration of histamine in the tissues during pregnancy, it would seem that the excessive formation of histamine during the last trimester of pregnancy takes place in the uterus and its contents and the basic action of histaminase is protective.

It was shown recently that the excessive formation of histamine during the last trimester of pregnancy in the rat is due to an increase in the rate of histidine decarboxylase activity (Kahlson, Rosengren, Westling and White, 1958). Inasmuch as removal of the fetuses without other interference with the pregnancy abolishes the increased urinary histamine, it can l)e concluded that the site of formation is in the fetus. This histamine could escape into the maternal circulation and eventually be eliminated via the kidneys.

Roberts ( 1954) reported that aminoguanidine leads to a general disturbance of pregnancy in the rat; large doses tended to jiroduce death of the mother and smaller doses tended to kill all or part of the litters and some of the mothers. Again one could conclude a protective action on the part of histaminase dui'ing tlie latter i)art of i^regnancy.

B. Carbonic Anhydrase

Carbonic anhydrase was discovered by Aleldrum and Roughton in 1933 and soon shown to catalyze the following reaction, H,CO, z:± CO. + HoO. The enzyme was found to occur in many tissues and was generally located within the cell especially in cells possessing a secretory function. The discovery by Lutwak-Mann and Laser (1954) that carbonic anhydrase is present in tlu' uterine mucosa led to a thorougli study of the changes in the concentration of the enzyme and the factors controlling its presence (Lutwak-Mann, 1955; Lutwak-Mann and Adams, 1957). The enzyme has been found to be present in the reproductive tract of a wide variety of mammals. In general, the uterine endometrium, placenta, and Fallopian tubes are the main loci of activity although there are marked differences among different species. Carbonic anhydrase activity was found consistently in all the animals studied such as the rat, hamster, guinea pig, rabbit, pig, and ewe. No activity was noted in the uterine mucosa of the nonpregnant animal except the ewe and the rabbit. In several species, such as the cow, human being, and pig, carbonic anhydrase was also found in the Fallopian tube.

A marked rise in carbonic anhydrase of the endometrium of the rabbit was noted during the first trimester of pregnancy


(Fig. 16.30). The value rose from a prepregnancy level of 20 enzyme units (E.U.) per gm. of fresh tissue to a maximum of 100 E.U. per gm. at approximately the 8th day of pregnancy. This level was maintained until the 12th day and then declined to approximately the prepregnancy level by about the 20th day. Examination of the placentas at this time revealed marked activity, 68 E.U. per gm. of maternal placenta and 25 E.U. per gm. of fetal placenta. The curve for the concentration of carbonic anhydrase in the uterine mucosa during pscudopregnancy is essentially the same as that seen during pregnancy, although some minor differences exist.

It is obvious from the above data and from the evidence involving the increased concentration of carbonic anhydrase in the uterine mucosa following treatment with progesterone, that the enzyme is probably under the control of the luteoid hormone. Indeed, an excellent correlation has been shown between the degree of {progestational proliferation in the uterus and the concentration of carbonic anhydrase. In the ewe, however, the carbonic anhydrase of the uterus is independent of the ovary. A possible explanation for this discrepancy between the two species has been offered on the basis of differences in the blood level of progesterone. However, no explanation is forthcoming for the failure to maintain the carbonic anhydrase level throughout pregnancy in the rabbit, even though the circulating progesterone remains liigh.

The significance of this enzyme in the physiology of reproduction is still unknown. From the data on the rabbit, it miglit be inferred that the carbonic anhydrase contributes to the maintenance of bicarbonate in the blastocyst fluid. The universal presence of the enzyme in placental tissue could also lead to the assumption that carbonic anhydrase is involved in fetal metabolism. Lutwak-Mann (1955) indicates that the enzyme might be involved in the transmission of calcium across the placenta. ^^'hether carbonic anhydrase is essential for fetal (Icvelopment and successful pregnancy is still unanswered. Treatment with carbonic anhydrase inhibitors (Diamox) failed to affect adversely the pregnancy or fetuses in pregnant rats even though no enzyme acti\'ity was present either in the matei'nal blood or placenta.



Fig. 16.30. Carhonic anhydrase activity in the uteiu.s of the rabbit during pregnancy, i),seudopregnancy, large doses of gonadotrophin, and pregnant mare's serum (PMS). Pregnancy, • •; i^seudopregnancy, O O; gonadotrophin, D D: PMS, x" X. (From C. Lut \val<-Mann. .1. Kndocrinol.. 13, 26. 1955.)



XI. Factors in the Maintenance of Gestation

A. Thyroid Gland

Several recent reviews have pointed out that the extract role of the thyroid gland in reproductive physiology is still in need of elucidation (Peterson, Webster, Rayner and Young, 1952; Reineke and Soliman, 1953). Numerous investigations over the past half century have definitely indicated that the thyroid gland is involved in reproduction but the site and manner of action are still not well known. In addition, contradictory reports indicate that each species and even each strain may have to be studied independently (Alaqsood, 1952). Some evidence foi- the involvement of the thyroid gland in gestation has already been considered. The increase in FBI at the onset of jiregnancy and the incidence of miscarriage in the human female when the FBI fails to rise tend to involve the thyroid hormone in the maintenance of pregnancy. Habitual abortion in women is usually associated with t'ithcr hypo- or hyperthyroidism (Litzenberg, 1926). Litzenberg and Carey (1929 » I'eported that in 70 married women with low basal metabolic rates appi'oximately 45 per cent had one or more abortions or stillbirthsrtf one eliminates the sterile woman from the group, the figure for women showing abortion or stillborn rises to approximately 35 per cent. However, the results are still controversial both with regard to data obtained within a single species and from different species.


Hypothyroidism in the rat induced by the prolonged administration of thiouracil resulted in a resorption of the fetus in 100 per cent of the cases (Jones, Delfs and Foote, 1946). Rogers (1947) reported a reduction in litter size following sulfaguanidine and Krohn and White (1950) reported a reduction in litter size following thyroidectomy in the rat. Thyroidectomy early in pregnancy caused a resorption of the fetuses and if performed at a later stage in pregnancy resulted in the birth of stillborn young (Chu, 1945). Following the induction of pregnancy in thyroidectomized rabbits, either a resorption of the young or abortion or prolongation of gestation was noted and the newborn young were usually dead. Chu concluded that the thyroid hormone was concerned with the vitality and growth of the embryos during gestation. In the pig the average duration of pregnancy was 114 days for normal gilts and 124.5 days for thiouracil-treated animals. In addition, the controls farrowed an average of 8.67 pigs per litter compared with 3.25 per litter for the thiouracil-treated .sows ( Lucas, Brunstad and Fowler, 1958 ) . The difference was significant in both instances. Bruce and



Fig. 16.31. Tlie effect of tliyioid deficiency on litter size. O, 422 litters from tliyroid-defieient mice; •, 423 litters from normal control mothers. (From H. M. Bruce and H. A. Sloviter, J. Endocrinol., 15, 72. 1957.)


Sloviter (1957) pointed out that part of the conflicting reports on the role of the thyroid in gestation might be due to the different methods used in producing a thyroid-deficient state. Surgical removal of the gland generally results in the loss of the parathyroids which may be also important in the maintenance of gestation (Krichesky, 1939), although adequate information is lacking. The use of antithyroidal substances offers more serious objections because these drugs not only pass through the placenta but they are nonspecific and interfere with other glands such as the adrenal cortex (Zarrow and Money, 1949; McCarthy, Corley and Zarrow, 1958), with nutrition, and with the general status of the animal. Consequently, Bruce and Sloviter preferred to establish a thyroidectomized state in mice by the use of radioactive iodine after establishing the dose necessary to induce total destruction of the thyroid without damage to the parathyroid or ganiete.s.

Although ( lorbman ( 1950 ) rei)orted a complete loss of reproductive activity in the mouse following treatment with P'*\ Bruce and Sloviter (1957) reported no effect on the ability of the mouse to conceive or bear young. This discrepancy could be due in part to the strain differences in the sensitivity of the ovary to the I^^^. Bruce and Sloviter (1957), however, noted a decrease in the average litter size of thyroid-deficient mice (Fig. 16.31 ). The data indicate a maximum of 6 young per litter in thyroid-deficient mice versus 10 young per litter for the normal mice. It is apparent that the entire curve for the litter size of thyroiddeficient mice is shifted toward a smaller size. This has also been observed in the rat following thyroidectomy (Nelson and Tobin, 1937). The thyroid-deficient mice also showed a prolongation of gestation as reported in rats, guinea pigs, and sows. Of the thyroid-deficient rats, 46 per cent showed a gestation period of more than 19 days whereas only 15 per cent of the normal controls showed a gestation period of more than 19 days whereas only 15 per cent of the normal controls showed a gestation period of more than 19 days (Table 16.9). Analysis of the data based on grouping according to litter size showed clearly an effect of litter size on length of gestation. The smaller litter size gave a higher inci(l(>nce of prolonged gestation.

Studies on oxygen consumption in the guinea i)ig revealed a slight but significant rise of 8 per cent at the end of gestation (Hoar and Young 1957). The increase in oxygen consumption is consistent but slight for the first 60 days of pregnancy after which the significant increase occurs (Fig. 16.32). The rise continued until 5 days postpartum and then fell rapidly. In a second set of experiments oxygen consumption was measured in control, thyroidectomized, and thyroxine-injected, pregnant guinea pigs. Measurements were taken at the time of mating and at parturition. In all three instances, an increase in the oxygen consumption was noted at parturition as compared with the values at the time of

TABLE 16.9

Ejfcct of thyroid-deficiency and litter size on length

of gestation in mice

(From H. M. Bruce and H. A. Sloviter, J. Endocrinol., 15, 72, 1957.)



Thyroid-deficient


Control


No. of

Young In

Litter


No. of pregnancies


> 19 days


No. of pregnancies


> 19 days


No.


Per cent


No.


Per

cent


1-5

6-9 10-14

Totals


36 40 20

96


24

13

7

44


67 33 35

46


16

38

28

82


7 3 2

12


44

7 8

15


mating (Fig. 16.33). Again the control guinea pigs showed a 7.9 per cent gain in oxygen consumption by the end of pregnancy, but both the thyroidectomized pregnant guinea pigs and the thyroxinc-treated guinea pigs also showed an increase in oxygen consumption of 11.9 and 16.2 per cent, respectively. The increase in oxygen consumption was not paralleled by increases in heart rate; actually the heart rate decreased in several instances. In addition, neither the weight of the thyroid gland nor the histology of the gland was changed during pregnancy. It is obvious then that an explanation for the rise in oxygen consumption during pregnancy may not involve the thyroid gland. On the basis of changes in its appearance. Hoar and Young (1957) suggested the possibility that the adrenal cortex is involved and that the increased oxygen consumption is due to an increased release of adrenal corticoids. More evidence is needed before this suggestion can be fully accepted.


Further work from the same laboratory has led to the concept that one locus of action of thyroxine during pregnancy is at parturition (Hoar, Goy and Young, 1957). These investigators used an inbred strain of guinea pigs that is characteristically hypothyroid and a genetically heterogeneous stock in which the level of thyroid activity is presumed to be higher. It had been previously shown that pregnancy wastage was high in the hypothyroid guinea pigs. Treatment with thyroxine reduced the percentage of stillborn from 40 to 13.6 in the hypothy




Fig. 16.32. Oxvgen consumption in the guinea pig during gestation. (From K. M. Hoar and W. C. Young, Am. J. Physiol., 190, 425, 1957.)



Fig. 16.33. Oxygen con.suinption in the pregnant guinea pig treated with thyroxine or thvroiclpctomized before mating. (From R. M. Hoar and W. C. Young, Am. J. PhysioL, 190, 425, 1957.)


roid guinea pig!>, i.e., to a level !?een in the untreated heterogeneous group. Treatment of the heterogeneous group with thyroxine not only failed to reduce the percentage of stillborn but actually increased the abortion rate particularly in the 2nd and 3rd trimesters. The most consistent result, however, was a decrease in length of gestation following treatment with thyroxine, and an increase following thyroidectomy. From these experiments it was concluded that the thyroid hormone facilitates parturition and need be present only late in gestation to exert its action.

It is apparent that in some species the thyroid hormone is involved directly in pregnancy. In the absence of the hormone, certain species tend to resorb or to abort; or if pregnancy is maintained gestation tends to be lengthened. This is probably due to an interference with the mechanism of parturition. In certain species such as the guinea pig only a parturitional problem has been demonstrated; in others an entire galaxy of symptoms may be present. Reduction in the size, number, and viability of the young give added emphasis to an essential role for thyroxine in the phenomenon of gestation.


B. Adrenal Cortex

Removal of the adrenal cortex without further treatment invariably leads to disturbances in rejM'oductive i)hysiology and the termination of pregnancy. Although the early results were controversial in that some investigators reported that adrenalectomy failed to affect gestation in the rat (Lewis, 1923; Ingle and Fisher, 1938), others reported that adrenalectomy led to abortion (Wyman, 1928; Dessau, 1937) or to some other disturbance of gestation (McKcown and Spurrel, 1940). Davis and Plotz (1954) adrenalectomized two groups of pregnant rats on the 4th to 6th and the 14th to 16th day of pregnancy. Abortion occurred in all 12 rats adrenalectomized during the first half of pregnancy whereas only 1 of the 12 adrenalectomized during the second half of pregnancy aborted. However, even in those adrenalectomized during the second half of gestation, an effect on jiregnancy was observed. A significantly higher incidence of stillborn and sickly young (14.4 per cent) and a marked decrease in the weight of the fetuses were noted (Table 16.10).

Early results indicated that extracts of the adrenal cortex could readily replace the absent adrenal gland and maintain successful pregnancies. Within recent years it has been demonstrated that many steroids such as cortisone and 9a-chlorohydrocortisone at 10 /xg. per day (Llaurado, 1955) permit fecundation and successful maintenance of pregnancy. Successful maintenance


GESTATION


1001


TABLE Ki.lO

Effects of adrenalectomy on the character of the litter,

and on fetal body weight and adrenal weight

(From M. E. Davis and E. J. Plotz,

Endocrinology, 54, 384, 1954.)



Pregnant Controls


Adrenalectomy 2nd Half of Pregnancy


Percentage versus Pregnant Controls


No. of litters ....


21


11



Dead and





"sickly" young. Vigorous voung . . .


5

182


13

78


<0.01


Fetal hodv weight





(gm.)


6.13


5.22


<0.01



(=b0.10)« 


(±0.30)"



Fetal adrenal





weight (mg.). . . .


0.49()


0.554


>0.3



(±0.017)"


(±0.041)« 



Fetal l)()dv weight/





fetal adrenal





weight X 1000. . .


12.35


9.42


<0.01



(0.34±)


(±0.31)"



Calculation of standard error of the mean:


S.E.


/ Ed-^

]/ nin


1)


of a pregnancy has also been reported in an adrenalectomized human female maintained on hydrocortisone 9a-fliiorohydrocortisone (Laidlaw, Cohen and Gornal, 1958). In this instance measurements of urine excretion of aldosterone revealed an increase to 4.4 fxg. per 24 hours during the last trimester of pregnancy and a postpartum value of 0.5 fig. Inasmuch as the value is only 1/10 of that seen in a normal pregnancy the authors concluded that the adrenal cortex of the mother is the major source of aldosterone during pregnancy and that a high output is not a major prerequisite for a normal pregnancy.

Treatment with either 0.9 per cent saline drinking water or with cortisone increased the number of successful pregnancies following adrenalectomy during the first half of gestation. Pregnancy was normal in 8 of 11 adrenalectomized rats (Davis and Plotz, 1954). Treatment with 2 mg. of cortisone acetate resulted in successful pregnancies in 13 of 14 rats adrenalectomized on the 4th to 6th day of gestation and 12 of 12 rats adrenalectomized on the 14th to 16th day of gestation. However, complete maintenance was not obtained. The body weight of the mothers and the weight of the fetuses were significantly lower than in the controls, and the number of stillborn and sickly young was increased.


A comparison of the pregnancy-maintenance activity in a number of adrenal corticoids indicated that a combination of a glucocorticoid and mineralocorticoid provides the best protection in the adrenalectomized rat (Cupps, 1955). Nulliparous rats were adrenalectomized, placed on treatment, and mated. Under these conditions the adrenalectomized controls and the rats treated with desoxycorticosterone acetate failed to become pregnant inasmuch as no implantation sites were obtained (Table

TABLE 16.11

Effect of adrenal steroids on reproduction in adrenalectomized female rats

(From P. T. Cupps, Endocrinology, 57, 1, 1955.)


Daily Treatment


Control

Cortisone acetate ^i mg

Cortisone acetate ^i mg

Cortisone acetate 1'^ mg

Cortisone acetate 2,4 mg

Hydrocortisone acetate Vi mg.. . ,

Cortisone acetate Vi mg. plus

Desoxycorticosterone acetate ^ mg

Desoxycorticosterone acetate ^^ mg

Desoxycorticosterone acetate ^ mg

Desoxycorl icosterone acflate 1 mg ,

Adrenalectomized control


No. of Rats


No. Born Alive

(average)


Implantation Sites (average)


7


8.2


11.0


6


3.5'>


5.6^


6


3.6*


6.2'^


5


3.5


8.6"


5


5.8


10.0


7


5.0


8.5"


5


9.0


9.6


4





5





4





5





Weight Change during Pregnancy (average)"


gm. 46.4

-30. S

-1.5«  17.2^ 12.8^ 30.7


44.6


"Weight change of mother from day of breeding to day after parturition. " Significant at 0.05 level. '= Significant at 0.01 level.


1002


SPERM, OVA, AND PREGNANCY


16.11). Treatment with 2.5 mg. cortisone acetate per day was partially effective in restoring reproductive capacity. Injections of 1.25 mg. hydrocortisone acetate per day gave results comparable with those obtained when cortisone was given, although the ratio of young born alive to implantation sites indicated that hydrocortisone acetate was more effective. It was definitely more effective than cortisone acetate in maintaining the body weight of the mother. However, reproduction was completely restored to normal in the adrenalectomized rat following treatment with desoxycorticosterone acetate and cortisone acetate.

Interference with gestation in the normal animal has been reported by several investigators following treatment with ACTH or adrenal corticoids (Courrier and Colonge, 1951; Robson and Sharaf, 1952; Velardo, 1957). This is taken to indicate that there is a finely balanced requirement for adrenocortical hormones during gestation ; and that suboptimal or supra-optimal amounts of the hormone interfere with pregnancy. Courrier and Colonge found that cortisone administered to intact rabbits in the second half of pregnancy interfered with gestation. Robson and Sharaf treated both pregnant rabbits and mice with ACTH and reported a marked effect on gestation. Abortion or resorption occurred in 8 of 9 mice and in 8 of 11 rabbits. Contamination by posterior pituitary hormones or gonadotrophins can be excluded. A subsequent experiment with cortisone also caused marked interference with pregnancy in the rabbit when 20 mg. were given ; 10 mg. were without effect. Administration of cortisone to castrated or hypophysectomized pregnant rabbits maintained with progesterone also caused damage to the pregnancy. Since the hormone was not acting by way of the ovary or pituitary gland, the authors felt that cortisone was acting directly on the uterus and the uterine contents.

In the rat, however, ]Meunier, Duluc and Mayer (1955) observed an effect on pregnancy only when cortisone acetate was injected at the time of mating. Rats injected with 10 to 25 mg. cortisone acetate daily for 5 to 6 days beginning on day 12 or day 14 of gestation had a normal pregnancy.


Velardo (1957) reinvestigated the problem in the rat and reported a marked reduction in litter size and an increase in the number of stillborn following ACTH treatment. Although quantitative differences appeared, a significant decrease in litter size w^as observed only when the hormone was given (1) before mating, (2) immediately after mating, or (3) between the 11th and 15th day after mating. However, the greatest effect was noted when the ACTH was administered immediately after mating. Surprisingly enough, litter size was markedly reduced only if adrenalectomy was performed on day 7 of gestation. Adrenalectomy on day 8 to 14 of gestation had no effect on live litter size. However, a total of 6, 9, and 13 stillbirths were obtained following adrenalectomy on days 8, 9, and 11. It is interesting that the number of stillbirths decreased from 21 following adrenalectomy on day 7 to none following adrenalectomy on day 14. It is apparent that the adverse effects of adrenalectomy on gestation decrease as pregnancy progresses. It is also apparent from these and other experiments that the action of ACTH is mediated by the adrenal cortex. From these results and others described above, it seems likely that the adrenal corticoids may be acting on the uterus.

Mayer and Duluc (1955) found that adrenalectomy of the I'at on the 14th to the 16th day of pregnancy led to variable results. In 17 pregnant adrenalectomized rats, gestation was terminated in 8, but no interference was observed in 9. The rats that failed to maintain pregnancy died witiiin 2 to 3 days. Again it would appear that delicate hormonal balances are involved. In a further investigation of this problem Aschkenasy-Lelu and Aschkenasy ( 1957) reported that a diet adequate in salt and proteins would prevent interference with pregnancy in rats adrenalectomized before mating. On a low protein diet, pregnancy could be maintained only in the intact rat (80 per cent) and then only if daily injections of progesterone were given. These authors believe that the role of the adrenal corticoids in pregnancy is concerned with stimulation of appetite and mobilization and degradation of proteins to amino acids. The latter action would permit the replacement of body protein in the absence of a normal jirotcin intake.


C. Pancreas

The impact of diabetes mellitus on the course of pregnancy has been of interest to the clinician for many years. In a recent review of the subject, Reis, DeCosta and Allweiss (1952) came to the conclusion that "the carefully controlled diabetic aborts no more frequently than the nondiabetic." On the other hand, it has been well known for many years that uncontrolled diabetes and pregnancy are basically incompatible (Eastman, 1946).

Studies in the rat have given controversial results with regard to the influence of insulin on pregnancy. Davis, Fugo and Lawrence (1947) reported that in the alloxan diabetic rat pregnancy was normal for the first 12 days. Thereafter death of the fetuses occurred followed by resorption. Sinden and Longwell (1949) and Levi and Weinberg (1949) reported no detrimental effect from diabetes on the course of i^regnancy. The latter group obtained 12 pregnancies from 25 rats made permanently diabetic with alloxan. Eleven of the 12 rats went to term and delivered normal fetuses and 1 died during pregnancy. Recently, Wells, Kim, Runge and Lazarow (1957) reported a 14 per cent loss in fetal weight, an increase in gestation length from a normal of 538 to 563 hours, and an increase in fetal or neonatal mortality in the pregnant rat made diabetic by pancreatectomy or treatment with alloxan.

In general, the clinical data indicate that uncontrolled diabetes has a detrimental effect on pregnancy, but that the abortion rate in the controlled diabetics approaches that seen in the normal" population. Since the crux of the matter seems to hinge on the severity of the diabetes, one might conclude that the effect of insulin is an indirect one by virtue of its action in maintaining a good metabolic state. The conflicting reports from animal experimentation may be due to the differences resulting from uncontrolled environmental and dietary factors.


D. Ovary: Progesterone, Estradiol, and Relaxin

Marshall and Jolly (1905) were probably the first to point out that ovariectomy during pregnancy leads to abortion or resorption of the fetuses in the rat. Subsequently, a number of investigators repeated these experiments and confirmed the findings in all species tested thus far, provided ovariectomy is performed before implantation. Removal of the ovaries after gestation is well under way, however, does not disturb the course of pregnancy in all species. The human being, monkey, horse, ewe, and cow are examples of species not dependent on the ovary for the maintenance of pregnancy once it has been well established. Species such as the rabbit and the rat require the presence of the ovary throughout pregnancy.

The importance of progesterone for i)regnancy was established by Allen and Corner (1929) who first showed that an extract of the corpus luteum will maintain pregnancy in the castrated rabbit. Identification of the active substance in the extract as progesterone led to the use of the hormone in many other species. Allen (1937) reported that crystalline progesterone was inferior to the crude luteal extract in the maintenance of pregnancy in the castrated rabbit. From these and other data, such as the enhancing action of estrogen on the progesterone-induced progestational reaction, he inferred that a combination of estrogen and progesterone should be superior to progesterone alone in the maintenance of pregnancy. However, he pointed out with proper caution that the dosages would have to be carefully regulated because estrogen could also antagonize progesterone. Although Robson (1936) failed to enhance the action of progesterone with estrone in the pregnant hypophysectomized rabbit, Pincus and Werthessen (1938) obtained enhancement with both the androgens and estrogen. Whereas the early work indicated that a pregnancy maintenance dose of progesterone varied from 0.5 to 2 mg. (Allen and Corner, 1930), later experimentation indicated that the dosage varied with the stage of pregnancy. An adequate dose of approximately 1 mg. progesterone in the early stages of pregnancy needs to be increased to 5 mg. in the later stages (Allen and Heckel, 1939; ComTier and Kehl, 1938a, b). These investigators also revealed that an optimal effect could be obtained by using a progesterone-estrogen combination in the ratio of 750 to 1. Chang (1951) transferred ova to nonovulated intact rabbits and noted that massive doses in the order of 25 mg. macrocrystalline progesterone injected for three times were required to obtain a 50 per cent maintenance of pregnancy. He also reported that under the conditions of his experiment an initially high dose was needed for the passage of the ova, implantation, and early maintenance. Since then, further experimentation, especially on other species, has revealed a significant role by estrogen in enhancing the pregnancy-maintaining action of progesterone.

A vast literature exists for the human being on the prevention of threatened abortion by progesterone which is beyond the scope of this review. Variation from negative results to excellent maintenance is reported. It is obvious that a great deal of variability exists here and, to some extent, this is explained by a need for more objective criteria in evaluating threatened abortion and the therapy (Guterman and Tulsky, 1949). It is obvious that if the

TABLE 16.12 Maintenance of pregnancy in the rat castrated on

the 12th day of gestation

(From J. Yochim and M. X. Zarrow, Fed. Proc,

18, 174, 1959.)



Progester







one







No.



Estradiol Daily


Implantation Site


No. of Fetuses


No. of

Fetuses

Alive


Preg

Rats


Daily dose


No. daily


nancy Index



mg.



Mg.






4





40


37


37


0.925


4


2


1



47


32


27


0.574


7


1


2



76


12


2


0.026


9


1.5


2



99


61


49


0.495


7


2


2



85


65


63


0.741


6


1


2


0.1


69


50


48


0.696


5


1.5


2


0.1


51


48


46


0.900


5


2


2


0.1


60


55


54


0.900


threatened abortion were the result of some disturbance other than progesterone, that progesterone therapy might be without success. Indirect evidence for the need for progesterone to maintain a successful pregnancy in the human being and for the lack of need for the corpus luteum once pregnancy is established has been presented by Tulsky and Koff (1957). Corpora lutea were removed from day 35 to day 77 of pregnancy in 14 women. Two of the women exhibited spontaneous abortion and a marked drop in pregnanediol excretion. The remaining 12 maintained a normal pregnancy and pregnanediol excretion. The data can be interpreted to indicate a need for progesterone during pregnancy and that this need can be met by a nonovarian source, i.e., the i)lacenta.


In both the rat and mouse, successful maintenance of pregnancy after castration has been obtained with progesterone or a combination of progesterone and estrogen. However, partial maintenance following castration can be obtained in the rat under special circumstances. Haterius (1936) removed all the fetuses except one and left all placentas intact. Under these conditions the remaining fetus was carried beyond term. Alexander, Fraser and Lee (1955) found that castration of the rat on the 9th day resulted in 100 per cent abortion, whereas 60 per cent of the fetuses were retained until term if castration was on the 17th day. Dosage of progesterone as high as 5 to 10 mg. daily following castration the 9th day gave only partial maintenance. It is possible that better results would have followed multiple daily injections. Yochim and Zarrow (1959) castrated rats on day 12 of gestation and obtained a pregnancy index (no. of fetuses alive at day 20 h- no. of implantation sites at day 12) of 0.741 when 2 mg. progesterone were gi^'en in two divided daily doses and 0.495 when 1.5 mg. progesterone was given (Table 16.12). However, the addition of 0.1 /^.g. estradiol daily markedly enhanced the action of the progesterone so that a pregnancy index of 0.9, i.e., equivalent to the normal controls, was obtained with 1.5 mg. progesterone.

Finally, Hall (1957) has indicated that relaxin synergizes with estradiol and progesterone in the maintenance of pregnancy in the castrated mouse. One nig. progesterone per day maintained pregnancy in 83 per cent of the mice castrated on day 14 of gestation, but 0.5 mg. maintained pregnancy in only 30 per cent of the animals. The addition of 1.5 ^g. estradiol per day was without effect. On the other hand, the addition of relaxin to the estradiol and 0.5 mg. progesterone gave pregnancy maintenance in over 80 per cent of the mice as compared with 30 per cent when progesterone alone was given.

Smithberg and Runner (1956) induced ovulation and mating in prepubertal mice (age 30 to 35 days) and obtained 100 per cent implantation with 0.5 to 1 mg. progesterone daily and approximately 90 per cent successful pregnancies when 2 mg. progesterone were given. A comparison of the amount of progesterone required for maintenance of pregnancy in the normal and castrated prepubertal mouse is given in Figure 16.34. In an interesting application of the information available on the induction of ovulation and maintenance of pregnancy, Smithberg and Runner (1957) were able to obtain successful pregnancies in genetically sterile, obese mice.

Haterius (1936) observed that distortion of the fetus occurred following ovariectomy in the rat. This has been confirmed by Zeiner (1943) in the rat and by Courrier and Colonge (1950) in the rat and rabbit. It was noted that castration greatly compressed the fetuses and eventually caused death. Courrier and Colonge (1950) in very elegant experiments showed that removal of the rabbit fetus into the peritoneal cavity prevented the distortion and death which ordinarily followed castration. Frazer (1955) obtained similar results in the rat and concluded that fetal death after castration of the mother follows a rise in intrauterine pressure which is associated with an increased tone of the circular uterine muscle fibers. Consequently the increased survival of the extra-uterine fetuses following ovariectomy in the mother is the result of the removal of this pressure by the circular muscle of the uterus.


Fig. 16.34. Daily dose of progesterone required to maintain pregnancy in the normal and castrated prepubertal mouse. (From M. Smithberg and M. N. Runner. J. Exper. Zool., 133, 441, 1956.)

Many investigators have demonstrated that gestation can be prolonged by inhibiting parturition. Both the injection of large doses of progesterone or the formation of a new set of functional corpora lutea during pregnancy will prevent parturition. The injection of an ovulating dose of HCG on the 25th day of pregnancy in the rabbit delayed parturition for 15 days after the injection, i.e., until the 40th day of gestation (Snyder, 1934). The fetuses survived in utero for only 3 days and grew to greater than normal size during this period. The placentas persisted until day 41 of gestation. Comparable results were obtained following daily injections of progesterone into pregnant rabbits (Zarrow, 1947a). Haterius (1936) obtained prolongation of pregnancy in the castrated rat by removing all the fetuses except one, leaving all placentas intact. Recently a comparable experiment was performed in tlie rabbit with intact ovaries (Hafez, Zarrow and Pincus, 1959). In 2 of 10 rabbits, live fetuses were obtained l)y cesarean section on day 36. However, in 8 of the 10, delivery was delayed beyond day 36, although some degree of fetal resorption was present in all instances. Prolongation of pregnancy in the rat was obtained by the injection of prolactin (Meites and Shelesnyak, 1957), but only if the ovaries were present.


E. Pituitary Gland

In general, hypophysectomy before midpregnancy leads to resorption. This is especially true of the rat and mouse. On the other hand, hypophysectomy at midpregnancy or later does not interfere in the maintenance of gestation in these species (Pencharz and Long, 1933; Selye, Collip and Thompson, 1933a, b; Pencharz and Lyons, 1934 ) . In the dog, ferret, and rabbit, hypophysectomy leads to abortion (Aschner, 1912; McPhail, 1935a; White, 1932), whereas the results in the cat seem contradictory (Allan and Wiles, 1932; McPhail, 1935b) .

Hypophysectomy of the rhesus monkey does not always interfere with pregnancy. Smith (1954) obtained normal pregnancies in 10 of 20 hypophysectomized rhesus monkeys. The remaining animals aborted. Although more data are needed, it seems that the pituitary gland can be removed very early in gestation without disturbing the pregnancy. Whereas hypophysectomy before midterm invariably leads to abortion or resorption in the rat or mouse, 1 of the 4 monkeys hypophysectomized between the 29th and 34th day of gestation carried its young to term. Inasmuch as Hartman and Corner (1947) showed that the placenta secretes sufficient progesterone by the 25th day of gestation to maintain pregnancy, it is apparent that the placenta in the monkey is able to maintain its endocrine secretory activity independent of the pituitary and at a sufficiently high level to replace the ovary.

Little, Smith, Jessiman, Selenkow, van't Hoff, Eglin and Moore (1958) reported a successful pregnancy in the 37-year-old woman hypophysectomized the 25th week of pregnancy. The mother w^as maintained on thyroid, cortisone, and pitressin tannate replacement therapy. The excretion of chorionic gonadotropin and pregnandiol was not markedly different from that seen in normal gestation. Estrogen excretion was slightly reduced and the 17-hydroxy corticosteroids dropped to zero when cortisone therapy was discontinued. It would seem that this phase of adrenocortical activity was reduced and that ACTH or corticoidlike substances from the placenta were inadequate. No interference in aldosterone output was observed.

Hypophysectomy on the 10th day of gestation in mice terminated the pregnancy in only 3 of 19 animals (Gardner and Allen, 1942). Sixteen mice carried their litters to term although 7 of the 16 had a difficult and prolonged parturition. Body weight curves were normal and the corpora lutea appeared unaffected by the loss of the pituitary gland, indicating either the independence of the corpus luteum or the presence of a placental luteotrophin. Marked involution of the adrenal cortex was noted in all instances.

Simultaneous measurements of the concentration of cholesterol in the adrenal gland and ACTH in the pituitary of the rat revealed a drop in adrenal cholesterol and pituitary ACTH on the 15th day of gestation (Poulton and Reece, 1957). This was followed by a marked increase of both substances on the 21st day of pregnancy and a sharp drop at parturition. The authors concluded that a gradual increase occurs in the secretory activity of the adrenal cortex which reaches a peak on the 15th day of pregnancy in the rat. Thereafter the activity decreased until parturition when a marked increase was observed. The initial decrease in pituitary ACTH potency followed by an increase after day 15 is interpreted as an initial increase in ACTH release followed by a decreased release. The decrease in pituitary ACTH potency at parturition is compatible with the marked increase in adrenocortical activity at this time if the decreased pituitary ACTH activity is interpreted as indicative of ACTH release.

Maintenance of pregnancy in rats hyl')ophysectomized early in pregnancy was obtained with prolactin by Cutuly (1942), although Lyons, Simpson and Evans (1943) reported negative results with a purified prolactin. However, a partial maintenance of pregnancy was obtained with purified prolactin and estrone.


F. Placenta

The placenta is not only involved in the synthesis of hormones during pregnancy but also in the transfer of substances between mother and fetus. It is obvious that the transfer of substances is limited and the l^lacenta does offer a barrier. This problem bears not onlv on the matter of fetal nutrition, but also on the fetal environment and as such is important in the sexual development of the fetus (see chapter by Burns) .


The presence of estriol in the urine of newborn male infants has led to the conclusion that estrogens can pass through the placenta because of their low molecular weight (Diczfalusy, Tillinger and Westman, 1957). Studies on the transfer of estrogens across the placental barrier in the guinea pig with C^'^-labeled estradiol revealed an extremely rapid disappearance of radioactivity from the maternal blood following intravenous injection of the hormone into the mother, and the appearance of large amounts of water-so.luble radioactivity in the fetal plasma (Dancis, Money, Condon and Levitz, 1958). However, no estradiol was found in the fetal plasma. Replacement of fetal circulation with a perfusion system indicated that estradiol did not j^ass the placenta although estriol was readily transferred in both directions. These authors reported that the placenta was relatively impermeable to the water-soluble estrogens found in the urine, wliich are essentially glucuronides.

The discovery in 1927 of large amounts of estrogens and gonadotrophins in the blood and urine of pregnant w^omen led to the cjuestion as to whether the placenta is a gland of internal secretion. This can be answered with an uneciuivocal yes. Nevertheless, several questions are still unanswered: (1) the number of hormones produced by the placenta, (2) the quantities, and (3) the secretory activity of the placenta in different species.

Data on the presence of gonadotrophins in the placenta have already been discussed. At least three different types of gonadotrophins have been extracted from the placentas of the human being, mare, and rat. These have been defined physiologically and appear to be different in the three species. Cole and his co-workers have identified the endometrial cups as the source of PJVIS in the mare, whereas the elegant experiments of Stewart, Sano and Montgomery (1948) indicate that HCG in the human being is secreted by the Langhans cells. These investigators grew human placental cells in tissue culture and obtained ^ gonadotrophin in the culture. They also noted a direct correlation between the growth of the Langhans cells and the production of gonadotrophic hormone (see also the discussion of this subject in the chapter by Wislocki and Padykula).

The initial discovery of a progressive rise in the secretion of adrenal corticoids in pregnancy (Venning, 1946) has been confirmed by numerous investigators. Gemzell ( 1953) attributed the steady rise to a stimulation of the adrenal glands by excessive amounts of estrogen present during pregnancy and to hyperactivity of the fetal adrenals. The hypertrophy of the fetal adrenal cortex in the rat following adrenalectomy of the pregnant mother was first reported by Ingle and Fisher in 1938 and confirmed by Walaas and Walaas (1944), and Knobil and Briggs (1955). However, the 17-ketosteroid and corticoid level of fetal urine is very low (Day, 1948; Jailer and Knowlton, 1950) as are the 17-hydroxycorticosteroids in the blood of the newborn infant (Klein, Fortunato and Papados, 1953). ACTH-like activity has been found in extracts of the placenta (Jailer and Knowlton, 1950; Tarantino, 1951; Opsahl and Long, 1951) and corticoid activity has been found in the placenta of horses and human beings, as demonstrated by the glycogen deposition and growth-survival test in adrenalectomized rats (Johnson and Haines, 1952). Berliner, Jones and Salhanick (1956) isolated 17a-hydroxy corticoids from the human placenta.

Pincus (1956) reported that ACTH can stimulate steroidocorticogenesis in the perfused placenta. Using the ascorbic acid depletion test, Assali and Hamermesz (1954) assayed the blood in the intervillous space and the chorionic villous tissue for ACTH. Good activity was observed in the blood from the intervillous spaces and in the tissue of the chorionic villi. Corticotrophic activity was also obtained by Lundin and Holmdahl (1957) from placentas obtained at full term, but the activity was small compared with that obtained from the pituitary gland.

The possible role of the fetal pituitary was investigated by Knobil and Briggs (1955) who noted that hypophysectomy of the mother prevented the fetal adrenal weight increase observed following adrenalectomy of the pregnant mother. However, complete atrophy of the adrenal gland was not observed in the pregnant mother if the conceptus was present. It was concluded that ACTH can cross the placental barrier and that the fetus or placenta or both produce a sufficient amount of ACTH, to influence the maternal adrenal gland in the absence of the maternal hypophysis. It is still questionable, however, whether these sources, i.e., placenta and fetal pituitary, are of sufficient magnitude to account for the increased release of adrenal corticoids. Hofmann, Knobil and Caton (1954) showed that the ability of the hypophysectomized nonpregnant rat to secrete a water load is not greater than that of the hypophysectomized pregnant rat. Hence the contribution of the fetal pituitary or j^lacenta to the corticoid pool is not of sufficient magnitude to influence water balance.


As with the gonadotrophins, the increased amounts of estrogen ancl pregnanediol during pregnancy were thought to be derived from the placenta. In 1933, Selye, Collip and Thompson presented evidence to indicate that the placentas of rats jiroduce both estrogen and gestagen. Many physiologic data have been accumulated to prove this point, but completely convincing evidence was obtained only when these hormones were identified in placental extracts and in fluid perfused through the placenta. Diczfalusy and Lindkvist (1956) identified estradiol in the placenta and the presence of progesterone was described by Salhanick, Noall, Zarrow and Samuels (1952) and by Pearlman and Cerceo (1952).

Perfusion experiments on human placentas have revealed that this organ secretes a number of steroids (Pincus, 1956). These include progesterone, desoxycorticosterone Cortisol, and a number of unidentified steroids. Addition of ACTH to the perfusate had no effect on the concentration of Cortisol, but it did increase the concentration of the reduced corticosteroids, namely, the tetrahydro derivatives of cortisone and Cortisol. This was interpreted as a stimulation of the placenta by ACTH resulting in an increased release of the corticoid as demonstrated by the increase in the degradation products.

The identification of the placenta as a source of both sex steroids and certain gonadotrophins clarifies the manner by which jiregnancy can be maintained in certain species in the absence of the pituitary gland or ovary (see sections above on ovary and pituitary gland). Newton and Beck (1939) and others showed the hypophysectomy of the pregnant mouse does not precipitate abortion. Studies of the ovary reveal that, if the placentas are retained, the corpora lutea remain normal but removal of the placentas causes immediate degeneration of the corpora lutea (Deanesly and Newton, 1940). A comparable situation appears to exist in the rabbit and rat ; it is assumed, therefore, that the placenta takes over control of the corpus luteum in pregnancy in those species that require the ovary for successful gestation. In other species, such as man, sheep, cattle, and guinea pig, it seems that the placenta can supplant the ovary after pregnancy has progressed to a certain stage.


G. Pelvic Adaptation

The discovery that pelvic changes are under hormonal control in certain species was the result of extensive studies on pelvic adaptations associated with parturition (see reviews by Allen, Hisaw and Gardner, 1939; Hisaw and Zarrow, 1951). It has been argued that, in general, a narrow pelvis is present in mammals living in burrows. This would have the advantage of permitting an animal to turn within narrow confines, but a narrow pelvis would also interfere with the delivery of the young at parturition. As Hisaw pointed out in his extensive studies, this problem has been met by special adaptations on the part of different species. This has varied from a resorption of the cartilaginous pubic arch in the male and female mole iScalopiis aquaticus machrinus, Raf.) which is independent of the endocrine system (Hisaw and Zilley, 1927) to elongation of the pubic ligament which is directly under hormonal control (Hisaw and Zarrow, 1951).

The symphysis pubis of the pocket gopher, Geomys bursarius (Shaw), behaves as a female secondary sexual character so that a sex dimorphism exists in this species. The pubic cartilages ossify in both sexes and unite to form a complete pelvis with a rigid symphysis pubis. At this stage, the pelvis is too small for the passage of the young, but with the first estrus in the female, the pubic bones are gradually resorbed, leaving the pelvis open ventrally. The pelvis in the male remains intact (Hisaw, 1925). Treatment with estrogen alone can readily bring about the resorption of the pubic bones.

A third type of adaptive mechanism has been described in great detail in the guinea pig and led to the discovery of the hormone, relaxin. A sex dimorphism of the pelvis exists in the guinea pig, as in the pocket gopher, but in addition parturition is further facilitated by marked relaxation of the pubic ligaments and of the sacroiliac joint. Thus far extensive pelvic relaxation has been described in the guinea pig (Hisaw, 1926, 1929 », mouse (Gardner, 1936; Newton and Lits, 1938; Hall and Newton, 1946a), women (see review by Hisaw and Zarrow, 1951), and rhesus monkey (Straus, 1932; Hartman and Straus, 1939). No relaxation of the pubic symphysis has been reported in the ewe but a relaxation of the sacroiliac joint and an elongation of the sacrosciatic ligament was noted the 2nd to 3rd month of gestation. These changes increased as pregnancy progressed (Bassett and Phillips, 1955). Treatment with stilbestrol alone caused a marked loosening of the sacroiliac joint and the sacrosciatic ligament. The addition of relaxin to the treatment was without effect (Bassett and Phillips, 1954).

The role of relaxin in the relaxation of the pubic symphysis has been studied most extensively in the guinea pig and mouse. The work before 1950 was reviewed by Hisaw and Zarrow in 1951. The controversies (de Fremery, Kober and Tausk, 1931 ; Haterius and Fugo, 1939) as to whether such a hormone exists need not be discussed here, in detail, except to point out that the evidence supporting this opinion is more than adeciuate. Zarrow ( 1946, 1948) showed that pubic relaxation could be induced by estradiol alone, by a combination of estradiol and progesterone, or by relaxin in an estrogen primed animal (Table 16.13). The difference in the time required to induce relaxation, i.e., 23 days for estrogen alone, 13 days for estrogen and progesterone, and 6 hours for relaxin, and data indicating that progesterone caused the presence of relaxin in the blood of guinea pig only if a uterus was present led to the concept that pubic relaxation may be produced independ


TABLE 16.13

Relaxation of the symphysis pubis and relaxin content of blood, urine, and uteri of castrated and castrated,

hysterectomized guinea pigs after treatment with moderate doses of estradiol and progesterone

(From M. X. Zarrow, Endocrinology, 42, 129, 1948.)



Treatment, Daily


Average Relaxation Time


Relaxin Content


No. of Guinea Pigs


Estradiol


Progesterone


Total


After progesterone treatment


Blood serum


Urine


Uterus



MS

mg.



days


days


G.P.U./ml.


G.P.U./ml.


G.P.U./gm.


Castrated 9« 


10


1 from day


11


13.5

(13-14)


3.5


0.5


0.3


10


10


10


2 from day


11


13.0


3


0.5


0.5


10


10


10




(12-14)

23.7 (16.31)



Negative at 4 ml.


Negative at 5 ml.


Negative


Castrated, hys



terectomized 11


10


1 from day


11


23.7 (17-30)


13.7


Negative at 4 ml.


Negative at 8 ml.



10


10




25.6 (18-32)



Negative at 4 ml.


Negative at 4 ml.



One guinea pig not included in the table refiuired 22 days of treatment for pubic relaxation.


ently by estradiol (prolonged treatment) or relaxin (single injection). It is also possible to conclude that the action of progesterone is indirect and due to the formation of relaxin in the uterus (Zarrow, 1948; Hisaw, Zarrow, Money, Talmage and Abramovitz, 1944) . In the mouse, however, progesterone inhibits the action of relaxin on the pubic symphysis (Hall, 1949).

Further evidence that two hormones are involved in pubic relaxation was provided by histologic examination of the pubic ligament. Symphyseal relaxation following estrogen appeared to be due to a resorption of bone and a proliferation of loose fibrous connective tissue with an increase in mucoid alkaline phosphatase and water content (Talmage, 1947a, 1947b, 1950; Heringa and van der Meer, 1948). Relaxin produced a breakdown and splitting of the collagenous fibers into thin threads and a similar change was noted with progesterone (Talmage, 1947a, 1950).

Histochemical and biocliemical studies of the pubic symphysis have recently been reviewed (Frieden and Hisaw, 1933) and tend to show that relaxin produces specific changes. These include loss of metachromasia (Heringa and van der Meer, 1948) , accumulation of Evans blue m vivo, and increased solubility of the glycoproteins in the McManus-Hotchkiss reaction, all of wiiich indicate that a depolymerization of the ground substance and basement membrane glycoproteins had occurred (Perl and Catchpole, 1950) . Frieden and Hisaw (1951) found an increase in water content of the symphyseal tissue, but failed to find a decrease in the water-soluble hexose and hexoseamine following a single injection of relaxin. On the basis of a depolymerization of ground substance, a decrease should have occurred. However, repeated injections of relaxin led to a decrease in the insoluble hexoses and hexoseamines. In addition, consistent decreases in collagen content and trypsin-resistant protein content were noted. No hyaluronidase was found, but ^-glucuronidase was increased during relaxation. Gersh and Catchpole (1949) reported the presence of a collagenase from histochemical studies, but no confirmation has been forthcoming. Relaxin also has a protein


anabolic effect which occurs in the absence of pubic relaxation (Frieden, 1956). This action was demonstrated by the increased up-take of labeled glycine by the connective tissue proteins of the pubic symphysis. Recent experiments indicate that relaxin not only acts in conjunction with the female sex steroids but can also act alone (Brennan and Zarrow, 1959). However, it is apparent that the available data are still inadequate for a clear understanding of the mechanism of action of relaxin.

Relaxation of the pubic symphysis of the mouse has been studied in great detail by Hall. In a series of reports she showed that pubic relaxation occurs in the mouse during pregnancy and following treatment with estradiol and relaxin (Hall and Newton, 1946a, b). This was later confirmed by Kliman, Salhanick and Zarrow (1953). Contrary to the results reported following work on the guinea pig, progesterone not only failed to influence the effect of estrone on the pubic symphysis of the mouse, but progesterone also inhibited the action of relaxin. It was suggested that this inhibition is the result of an antagonism by progesterone on the action of relaxin and that a true species difference exists (Hall, 1949, 1955). Histologic studies revealed that changes in the pubic symphysis during pregnancy and after treatment with relaxin and estradiol are similar (Hall, 1947) . These changes consist of proliferation of articular hyaline cartilage, resorption of the medial ends of the pubes, lengthening of the pubic ligament by formation of new cartilage, and reversion of the cartilage to collagenous connective tissue. Hall (1956) suggested that estradiol causes a depolymerization of the mucopolysaccharides through enzymatic action resulting in a matrix sufficiently pliable to respond to the tensions set up by relaxin. Evidence presented in support of this concept was the loss of metachromasia and the increase in water. In addition, a two-step effect was seen with relaxin: (1) complete degradation of the matrix, and (2) the appearance of a gap in the cranial part of the cartilage produced by stretching of the symphyseal cleft. Some data in support of the latter part of this concept were presented by van der Meer (1954) who showed that inhihition of pelvic muscle tension inhibited relaxation in the guinea pig. In a similar type of experiment Crelin (1954) tied together the innominate bones of a mouse before pregnancy and obtained some dorsoventral displacement of the pubic symphysis but normal relaxation was inhibited.


H. Dilation of the Uterine Cervix

Dilation or softening of the uterine cervix in the pregnant woman at the time of labor has been known for a long time. This reaction has been used to determine whether delivery can be anticipated. Within recent years this phenomenon has been described in a number of animals and some analysis of the hormonal control of the reaction has been attempted.

Relaxation of the uterine cervix of the rat during pregnancy was first reported by de Vaal in 1946 and confirmed by Uyldert and de Vaal in 1947. Relaxation was measured by the insertion of a gauging pin into a cervix that had been removed and the diameter determined at the point where resistance is first felt. The measurements revealed a marked rise from approximately 3.5 mm. on the 17th day of pregnancy to 10 mm. at parturition. Recently, both Harkness and Harkness (1956) and Yochim and ZaiTow (1959) have taken in vitro measurements of the relaxation of the uterine cervix of the rat and observed marked relaxation during the latter part of gestation and at parturition. Yochim and Zarrow (1959) removed the cervix, suspended it from a rod and measured the stretch due to weights added at fixed intervals until the cervix broke. The amount of relaxation of the cervix was determined by the amount of stretch obtained with a weight of 50 gm. Under these conditions, the curve for relaxation of the cervix showed two sloi^es as pregnancy progressed (Fig. 16.35). The initial slope between day 12 and day 20 showed a rise of approximately 4 mm., with an extremely abrupt rise of 14 mm. on day 21. By 24 hours after parturition the degree of dilation had fallen to 3 mm. It is of interest that the curve for the tensile strength of the cervix (expressed in grams force necessary to tear 1 mg. cervical tissue in a rat weighing 100 gm.) was the opposite to that seen for cervical dilation. The tensile strength fell from approximately 50 gm. force to a low of 3 gm. at parturition and then rose during the postpartum period. The drop in tensile strength preceded the changes in the dilation of the cervix and was essentially completed 5 to 6 days before parturition or when the abrupt increase in dilatability of the cervix occurred.

Similar changes have been described in the dilatability of the cervix of the mouse (Steinetz, Beach and Kroc, 1957) with increased dilatability progressed beyond the 15th day (Fig. 16.36). The diameter of the cervix increased from a])proximately 2 mm. to about 5 mm. at delivery. It is apparent that the rate of the reaction, i.e., dilation, is much more rapid in the rat, although it is possible that the method of measurement is responsible for the differences.

The induction of cervical dilation by relaxin was reported by Graham and Dracy (1953) in the cow, and by Zarrow, Sikes and Neher (1954) in the sow and the heifer. Treatment with stilbestrol followed by relaxin caused a dilation of the uterine cervix of the gilt from % or % inch to 1 inch (Zarrow, Neher, Sikes, Brennan and Bullard, 1956). Measurements were made by the passage of aluminum rods, and, although the technique is not too exact, the differences are significant. Stilbestrol given alone or in combination with progesterone had no effect on the cervical dilation. On the other hand. Smith and Nalbandov (1958) have recently reported that estrogen treatment constricted the uterine cervix of the sow and that relaxin was without effect. A cue with respect to the mechanism of action of relaxin is given by the similarity of the action of relaxin on the pubic symphyseal ligament and the uterine cervix. In both instances, an increase in water content and a marked dei)olymerizatioii occurs.


Fig. 16.35. Dilation and ten.sile strength of the uterine cervLx of the rat during estrus, pregnancy, and 2 days postpartum. The dihition of the cervix in mm. of stretch per 50 gm. of added weight. The tensile strength is expressed in grams force necessary to tear 1 mg. cervical tissue in a rat weighing 100 gm. E = estrus; P = parturition. (From J. Yochim and M. X. Zarrow, Fed. Proc, 18, 174, 1959.)


Cullen and Harkness ( 1958) observed relaxation of the uterine cervix of the rat with estradiol alone, or with estradiol and progesterone, or with estradiol and relaxin, but maximal dilation was obtained only with a combination of estradiol, progesterone, and relaxin. In general Kroc, Steinetz and Beach (1959b) obtained comparable results in the rat. Estrogen alone caused some in crease in dilatability when 5 fxg. estradiol cyclopentylpropionate were given, and a decrease when 50 /Ag. were given. Progesterone had no consistent effect either alone or in estrogen-primed animals. Relaxin alone caused some softening of the cervix, but gave a maximal effect only when given with progesterone in estrogen-primed animals. Normal cervical dilation was also obtained in pregnant rats castrated the 15th day of gestation and maintained with progesterone, estradiol, and relaxin (Kroc, Steinetz and Beach, 1959; Yochim and Zarrow, 1959). Data on dilation of the uterine cervix of the mouse are rather sparse; nevertheless, softening of the cervix with relaxin has been reported (Kroc, Steinetz and Beach, 1959). It is not the purpose of this review to evaluate the data on cervical softening in the human female. The nature of the action of relaxin in the human female is controversial. Nevertheless, softening of the cervix following treatment with relaxin has been reported (Eichner, Waltner, Goodman and Post, 1956; Stone, Sedlis and Zuckerman, 1958) although McGaughey, Corey and Thornton (1958) reported no effect on the cervix following relaxin.



Fig. 16.36. Increased length of the pubic ligament, inciea.sed cervical dilatability, and increased responsiveness to oxytocin with the length of pregnanc.y in the mouse. L = lactating; NL = not lactating. (From B. G. Steinetz, V. L. Beach and R. L. Kroc, Endocrinology, 61, 271, 1957.)


XII. Uterine Myometrial Activity

The classical and well known description of uterine muscular activity has been more than adequately reviewed by Reynolds (1949). Since then Csapo and his colleagues have reported a series of elegant experiments involving the action of estrogen and progesterone on the uterine myometrium and have evolved the concept of "i)rogesterone block" in the control of uterine activity (1956a, 1956b). It has been shown that the ovarian steroid hormones regulate myometrial activity and that the uterine contractions are dependent on the relative amounts of the two hormones. Contractility is dependent basically on the concentration of the high energy phosphates which are maintained by estrogen w^iich in turn is involved in the synthesis of these substances (Csapo, 1950; Menkes and Csapo, 1952). Discovery of the staircase phenomenon in the uterine myometrium similar to that exhibited by cardiac muscle led to a marked difference between the action of estrogen and progesterone (Csapo and Corner, 1952 ) .


With decreasing freciuency of electrical stimidation in an isometric arrangement, tension decreased if the uterus was dominated by estrogen and increased if it was dominated by progesterone. Uteri from castrated rabbits were insensitive to the frequency of electrical stimulation. Thus estrogen induced a "positive staircase" response and progesterone a "negative staircase" response, although in the latter instance some estrogen is also present. These staircase responses have been used successfully as a measure of hormone domination and have been shown to be a function of the Na+ and K+ gradients across the myometrial cell membrane.

Uterine motility during estrus, the diestrum, and pregnancy has been described by many investigators in great detail (for a review see Reynolds, 1949). The diestrous uterus shows extremely slow, feeble, uncoordinated movements. The contractions may arise in any part of the uterus and extend in any direction. At estrus, the uterine contractions become rhythmic and sweep over


Fig. 16.37. Change from a positive to a negative staircase as the hormone dominance of the myometrium moves from the estrus to the progestational state after mating. X and O indicate the two strains of rabbits used. (From B. M. Schofield, J. Physiol., 138, 1, 1957.)


the uterine horn in a wave starting at the tubal end. Both amplitude and rate are increased. During pregnancy the uterus becomes relatively quiescent. In general this pattern of myometrial activity has been reproduced with both hormones, estradiol and progesterone.

Recently Schofield 11957), using the Csapo technique, has studied, in vivo, myometrial activity in the rabbit. In a series of experiments she was able to show in several strains of rabbits that, when mating occurs during estrus, the uterine myometrium is dominated by estrogen. Within 20 to 28


STAIRCASE Negative

Transient Positive


o


o o


o o


8x

OX


o







ox


X X X








o o o


ox

X



26 27 28 29 30 31 Day of pregnancy


32


Fig. 16.38. Change from negative through transient to positive staircase as the hormone dominance reverses at the end of pregnane}', indicating estrogen dominance. X and O indicate the two strains of rabbits used. (From B. M. Schofield, J. Physiol. 138, 1, 1957.)


hours after mating, the positive staircase effect passes through a transient effect to a negative effect indicating the development of progesterone dominance (Fig. 16.37j. This condition remained in effect throughout pregnancy until 24 hours before parturition when a reversion to estrogen domination was indicated by the positive staircase response ( Fig. 16.38) . Thus the progesteronedominated uterus is maintained throughout pregnancy and the uterus is nonreactive to oxytocin. Csapo (1956a) and others have shown that labor cannot be induced by oxytocin in the rabbit before day 30 of gestation, but 24 hours later, on removal of the progesterone block, 96 per cent of the rabbits delivered following treatment with oxytocin. He believes that the specific action of progesterone involves a blocking of the excitation-contraction coupling which is a consequence of the disturbed ionic balance in the myometrial cell. Thus a block is set up to the propagation of the contraction wave which can be removed only by a decrease in the level of progesterone.

The role of the water-soluble extract, relaxin in myometrial activity, is still uncertain. That an inhibition of estrogen-induced uterine contractions is obtained in certain species, such as the rat, mouse, and guinea pig, with relaxin preparations is un(luestionable. However, we still have not answered the questions as to w'hether this hormone plays a role in uterine contractions under normal physiologic conditions and whether the uterine contraction-inhibiting substance is relaxin or a contaminant of the relaxin extract.

Krantz, Bryant and Carr (1950) reported than an aqueous extract of the corpus luteum would produce an inhibition or decrease of uterine activity in the guinea pig and rabbit previously primed with estrone. This has been amply confirmed with both in vivo and in vitro preparations involving spontaneous contractions measured isometrically in the guinea pig (Felton, Frieden and Bryant, 1953; Wada and Yuhara, 1956; JMiller, Kisley and Murray, 1957) , rat (Sawyer, Frieden and Martin, 1953; Wada and Yuhara, 1956; Bloom, Paul and Wiqvist, 1958), and mouse (Kroc, Steinetz and Beach, 1959). However, Miller, Kisley and Murray (1957) failed to show any action of relaxin on uterine motility in the rabbit and the human being in vitro. Thus, the information on the rabbit is contradictory and a similar situation exists with regard to the human female for whom both positive and negative results have been reported following treatment with relaxin for threatened abortion (McGaughey, Corey and Thornton, 1958; Stone, Sedlis and Zuckerman, 1958; Eichner, Herman, Kritzer, Platock and Rubinstein, 1959). In briefly summarizing the action of relaxin on the uterine myometrium it should be pointed out that ( 1 ) relaxin inhibits uterine motility in an estrogen-primed animal, (2) the action may be species-limited, and (3) relaxin treatment docs not interfere with the action of pitocin.


XIII. Parturition

A. Progesterone

A number of theories have been suggested to explain the hormonal control of parturition. The most popular is that parturition is due to a decrease in the level of progesterone which allows oxytocin to exert its effect on the uterus. Evidence has already been presented indicating that pregnancy can be maintained in the castrated rabbit by an extract of corpora lutea, or progesterone, and even prolonged in rats (Nelson, Pfiffner and Haterius, 1930; Miklos, 1930), mice (Mandelstamm and Tschaikowsky, 1931), and rabbits (Zarrow, 1947a). Snyder (1934) and Koff and Davis (1937) prolonged gestation in rabbits by inducing the formation of new corpora lutea during the last trimester of pregnancy.

Knaus (1930) originally noted a marked antagonism between posterior pituitary extract and the corpus luteum hormone and Koff and Davis (1937) reported that in prolonged gestation induced by progesterone, posterior pituitary extract was ineffective until two days after the last injection. Csapo (1956a) performed a series of elegant experiments and concluded that progesterone blocks the uterine contractions, and that premature labor could not be induced with oxytocin before the 30th day of gestation in the rabbit except for a very small percentage of animals. This has been confirmed by Fuchs and Fuchs (1958).

Zarrow and Neher (1955) found the serum gestagen levels in the pregnant rabbit fell only after parturition was under way. Hence the problem arose as to how parturition could begin while a high blood concentration of gestagen was present. A partial answer was obtained in experiments by Csapo (1956b) and Schofield (1957) who showed that the progesterone-dominated uterus of the pregnant rabbit becomes estrogen-dominated and responsive to oxytocin 24 hours before parturition. Hence the concentration of progesterone in the serum is meaningless by itself and it could be theorized that the significant point is the ratio of estrogen to progesterone. Csapo (1956a), however, offered an alternative solution. He observed a local effect of placental progesterone on the myometrium so that the myometrium closest to the placenta is under a greater progesterone-dominance than that portion of the myometrium lying more distant. Hence the local level of progesterone would be the significant factor in the onset of parturition and not the systemic level.


B. Oxytocin

It is now generally believed that parturition is the result of the action of the posterior pituitary hormone on the myometrium of the uterus sensitized by estrogen. The development of this hypothesis followed from the well known fact that oxytocin produces uterine contractions and induces labor and delivery of the young. It is apparent, however, that a mass of contradictory data exist and the hypothesis is still in need of better evidence before it can be fully accepted (for review of early literature see Reynolds, 1949) .

Some of the evidence supporting the above hypothesis is the fact of the presence, to a limited degree, of a deficiency syndrome in parturition following removal of the posterior pituitary gland. The data, however, are still equivocal. Labor is apparently prolonged in the monkey (Smith, 1946) and guinea pig (Dey, Fisher and Ranson, 1941 ) after total hypophysectomy. Nevertheless, parturition will occur normally after removal of the pituitary gland in the rabbit (Robson, 1936), cat (Allen and Wiles, 1932), mouse (Gardner and Allen, 1942), and rat (Smith, 1932). Even where there is some indication of interference with labor, delivery occurs. However, the lack of consistent results and species differences may be due to the recent finding that the posterior pituitary hormones are synthesized in the hypothalamus and that removal of the posterior pituitary is only effective under limited conditions because the source of the hormone is still present. These experiments have also been criticized on the ground that the anterior pituitary was also removed and hence interference with many other hormones occurred.

Additional evidence in favor of a role for the neurohypophysis in the delivery of the young is the increase in uterine motility following stimuli that bring about release of the posterior pituitary hormones, and the lack of an effect on the uterus when release of the hormone is blocked.

Positive evidence for the release of oxytocin at the time of parturition is still lacking as are measurements of the concentration in the blood. Fitzpatrick (1957) takes the view that oxytocin is liberated as an essential part of normal parturition and cites the following evidence. (1) A superficial similarity exists between spontaneous labor and that induced by oxytocin. Harris (1955) also stresses the similarity in the uterine response to oxytocin and to electrical stimulation of the supraoptic hypophyseal nucleus. (2) Mechanical dilation of the uterus or cervix evokes an increase in uterine contractions presumably by way of a nervous reflex release of oxytocin (Ferguson, 1941). (3) Oxytocin is decreased in the posterior pituitary gland of the rat and the dog after labor (Dicker and Tyler, 1953).

Evidence from the attempts to measure the concentration of oxytocin in body fluids at the time of parturition is inadequate. The early reports of higher concentrations in the urine (Cockrill, Miller and Kurzrok, 1934) and blood (Bell and Morris, 1934; Bell and Robson, 1935) during parturition are questioned because of the inadequate methods of extraction and lack of specificity in the assay. Recently, Hawker and Robertson (1957, 1958) reinvestigated the problem and concluded that two oxytocic substances are present in the blood and hypothalamus of cats, cows, and rats and blood of women. However, they found that the concentration of oxytocin in the blood fell during labor from a high during pregnancy. It is apparent that this presents a paradoxical situation in view of the fact that the concentration of oxytocin is low at the time of parturition; a time when the hormone is supposedly exerting its greatest effect. The situation is further complicated by the presence of two oxytocic factors and the presence of an oxytocinase in the blood and l)lacenta (von Fekete, 1930; Page, 1946; Woodbury, Ahlquist, Abreu, Torpin and Watson, 1946; Hawker, 1956). Although more work is required on this problem and esi)ecially with regard to the specificity and concentration of the oxytocinase, there is some indication of a fall in enzyme level before parturition. Tyler (1955) reported a decrease in the blood level of the enzyme towards the end of pregnancy and Sawyer (1954) reported a decrease in oxytocinase activity in rat tissues at the end of pregnancy.

C. Relaxin

Recently, the discovery of the action of relaxin on the pubic symphysis, uterine cervix, and uterine motility has raised the question of the role of this hormone in parturition. Certainly in the species that normally show pubic relaxation, relaxin would appear to play a significant role. However, this phenomenon is a special adaptation and the question of cervical dilatability becomes more important because it seems to occur in all species examined thus far. It would seem that relaxin can induce cervical dilatability in conjunction with the sex steroids and that cervical dilation is a necessary event in parturition, but whether relaxin controls this event under physiologic conditions is still unknown and direct evidence is unavailable. It is also apparent in some species that relaxin can inhibit uterine contractions w'ithout interfering with the action of oxytocin. Kroc, Steinetz and Beach (1959) reported that relaxin actually restored responsiveness to oxytocin in mice treated with progesterone. Again the question is raised as to whether this is merely a good experiment or a part of the normal physiologic events.


In a general way the events leading to labor may be summarized as follows. As pregnancy approaches term, the uterus becomes subject to increasing pressure from within, due to a differential change in the growth rates of the fetus and the uterus (Woodbury, Hamilton and Torpin, 1938). Concurrently, a reversal from progesterone to estrogen domination occurs, which also contributes to an increase in uterine tonus. As intra-uterine tension increases, spontaneous contractions acquire a greater efficiency and forcefulness. Because the radius of curvature in the human uterus is greater at the fundus than at the cervix, and because the myometrium is thicker at the upper pole (by a factor of 2) the contractile force is stronger at the fundus than at the cervical end. This contractile gradient i^roduces a thrust toward the cervix.

Utilization of a type of strain gauge, the tokodynamometer, has afforded information on the rate and strength of contraction of the various parts of the parturient uterus simultaneously (Reynolds, Heard, Bruns and Hellman, 1948). These measurements have indicated that, during the first stage of labor, the fundus exerts strong contractions of rather long duration. The corpus exhibits less intense contractions, usually of shorter duration, which frequently diminish in force as labor advances. The lower uterine segment is almost inactive throughout the first stage of parturition. According to Reynolds (1949), both the fundus and the midportion contract at the same time, but the fundus remains contracted for a longer period of time than the corpus beneath, thus building up a force downward. If cervical dilation has not occurred, the three areas of the uterus will continue to contract. As cervical dilation begins, the contractions in the midportion of the uterus decrease in intensity and the contractions in the lower segment disappear. Cervical dilation has been observed only when there is a preponderance of rhythmic activity of the fundus over the rest of the uterus.

When amniotic fluid is lost after the rupture of the membranes, the absolute tension within the wall of the uterus is reduced so that the ratio of force between fundus and cervix is increased. Thus rupture of the membranes decreases the tension in the cervix more than the fundus and the net effect is an increased force from the fundus. This change tends to precipitate the parturition more rapidly.

As pregnancy nears term, both increased tonus of the myometrium and rapid growth of the fetus cause a rise in intra-uterine pressure. This rise results in a decrease of effective arterial blood pressure in the placenta. During this period also, thrombosis is observed in many of the venous sinuses of the placenta and many of the blood vessels become more or less obstructed by giant cells. During parturition, the systemic blood pressure of the mother rises with each contraction, but, due to the increased intrauterine pressure produced by the contractions, the effective maternal arterial blood pressure in the placenta decreases to zero. Thus maternal circulation is cut off from the fetus.

Measurements of intra-uterine pressure at term show that the human uterus contracts with a pressure wave which varies from 25 to 95 mm. Hg (Woodbury, Hamilton and Torpin, 1938). The uterine wall is subjected to an average tension of 500 gm. per cm.- and, during delivery of the head, may, with the aid of abdominal musculature, develop as much as 15 kg. force.

In animals giving birth to multiple young (rat and mouse) evacuation of the horn proceeds in an orderlv fashion beginning at the cervical end. As evacuation of the lowest implantation site starts, changes occur in the periods of contractions of segments of uterine artery near its entrance into the uterine wall (Knisely, 1934; Keiffer, 1919). Gradually the constriction phase becomes proportionately longer than the dilation phase until the arterial lumen is obliterated. The myometrium in the area of the constricting segments becofes more active and, after long intense local contractions of the uterine muscle, the fetuses and the placentas separate and are discharged through the dilated cervix. After evacuation, a relaxation of the contracted segment of uterus occurs and the process is repeated at the next implantation site.

Recently, Cross (1958) re-examined the problem of labor in the rabbit. He concluded that (1) oxytocin in physiologic amounts can induce labor that is comparable to the events normally seen, (2) oxytocin is released during a normal labor, and (3) oxytocin can induce delivery without supplementary mechanisms. He noted that straining movements involving reflex abdominal contractions initiated by distention of the vagina and cervix aided in expulsion of the fetus. It is also possible that this might cause reflexly an increased secretion of oxytocin. Other reflex mechanisms have been suggested, but evidence is inadequate. Cross cites a report by Kurdinowski published in 1904 in which the entire process of labor and delivery in an isolated full-term rabbit uterus perfused with Locke's solution is described. In these experiments orderly delivery of the viable fetuses was affected by the contractile efforts of the uterus and vagina in absence of any hormonal or nervous stimuli.


XIV. Conclusion

Although we have garnered much information, no major conclusions can be drawn at this time concerning gestation in the mammal. This is probably true because of the vastness of the subject and the lack of sufficient data, especially that of a comparative nature. It is probably fitting to close this chapter with the final statement written by Newton in the second edition of Sex and Internal Secretion, "It seems rather that the investigation of endocrine relationships during pregnancy is still in the exploratory stage and that the time is not ripe for systematization."

It is true that many data have been accumulated in the last two decades since the publication of the second edition of this book. It is also probably true that some systematization can now be started. But above all we need more data on different species in order to systematize fully the role of the various hormones and glands in pregnancy and to evaluate the metabolic and other changes that occur at this time.

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Aclcleiitluin

Several reports on an exteroceptive block to pregnancy in mice appeared since this manuscript was completed. In a series of three articles, Bruce (Nature, London, 184, 105, 1959; Science, 131, 1526, 1960; and J. Reprod. Fertil, 1, 96, 1960) has shown that exposure of newly mated, female mice to strange males caused an inhibition of pregnancy that ran as high as 80 per cent. Prior removal of the olfactory bulbs abolished the reaction. The pregnancy block in tliis instance consisted in a failure of the blastocysts to implant.

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