Book - Sex and internal secretions (1961) 1: Difference between revisions

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reciprocal cross the value was 31. Aside  
 
 
BIOLOGIC BASIS OF SEX
 
 
 
by frequent gynandromorphs, XO males,
and dominant lethals among the rod and
ring zygotes. It has been suggested that the
instability is due to heterochromatic elements. Hinton (1959) has observed the
chromosome behavior of these types in
Feulgen mounts of whole eggs that were in
cleavages 3 to 8. He found strikingly abnormal chromosome behavior in these
cleaving nuclei. For some cell divisions
chromosome reproduction was interpreted
as being through chromatid-type breakage
fusion bridge cycles. As a result of this behavior mosaics are formed which are intermediate between those of the half gynandromorphs and those which occur much
later because of somatic crossing over. In
terms of volume of cells included, the abnormal types may include only a few cells
of the total organism, a fair proportion of
the cells, or a full half of the whole body.
These unstable ring chromosome mosaics
may be a part of the secondary reproductive system or for that matter any other
region of the body. When the mosaic cells
are incorporated in the region of sex organ
differentiation male or female type organs
or parts of organs may develop as governed
by the cell nuclei being X, XX or some fraction thereof.
 
Gynandromor|)lis appear sporadically and
rarely in many species but in some instances genes which activate mechanisms
for their formation are known. In the presence of recessive homozygous claret in the
eggs of D. siniulans, gynandromorphs constitute a noticeable percentage of the
emerging adults. The gene nearly always
operates on the X received from the mother
causing it to be eliminated from the cell.
The resulting gynandromorphs are similar
to those of D. melanogaster. The fact that
the claret gene should affect the X and a
particular X chromosome is suggestive of
the manner in which given chromosomes arc
eliminated in Sciara. Other types of sex
mosaics will be found in the descriptions of
other species, i)articularly in the Hymenoptci'a.
 
C. PARTHENO(iENESIS IN DROSOPHILA
 
Parthenogenesis is of interest as it
changes the sex ratios in families and brings
to light new sex types and novel methods
 
 
 
for their development (Stalker, 1954), A
survey of 28 species of Drosophila showed a
low rate of parthenogenesis in 23 species.
Adult progeny were obtained for only 3
species. For D. 'parthenogenetica the original rate was 8 in 10,000 whereas that for D.
polymorpha was 1 in 19,000. These rates
could be increased by selection of higher
rate parents: 151 and 70 per 10,000 unfertilized eggs of the first and second species
respectively.
 
D. parthenogenetica diploid virgins produced diploid and triploid daughters as well
as rare XO sterile diploid males. Triploid
virgins produced diploid and triploid females and large numbers, 40 per cent, of
sterile XO diploid males. Diploid virgins
heterozygous for sex-linked recessive garnet
produced homozygous and heterozygous
diploid females as well as +/+/g and
+ /'g/g triploid females. No homozygous
wild-type or homozygous garnet triploid females or garnet mosaics were found. Diploid females crossed to fertile diploid males
produced few if any polyploid progeny or
jjrimary X chromosome exceptional types.
Of the unfertilized eggs from diploid virgins
which started development, 80 per cent died
in late embryonic or early larval stages.
The i)arthenogenesis in diploid females depended on two normal meiotic divisions followed by fusion of two of the derived haploid nuclei to form diploid progeny, or the
fusion of three such nuclei to form triploitl
progeny. In the triploid virgins similar fusions of the maturation nuclei may produce
diploid and triploid females but the large
number of dii)loid XO sterile males were
picsunicd to be the result of cleavage without prior nuclear fusion. Such cleavages
without fusion in eggs of dijiloid virgins
would lead to the production of haploid
embryos. They were presumed i-esponsible
for the large early larval and embryonic
<h'atlis. These obser\ali()ns have been confirmed by the study of S|)rackling (1960)
in\-olving some 2200 eggs at various stages
of cleavage. Evidence from XXY diploid
virgins indicated that biiuiclcar fusion in
unfertilized eggs involved two terminal
haploid nuclei or two central nuclei. The
fact that tetraploids were not observed as
progeny of ti'iploid vii'gins was considered
indicative of relative inviability of this
 
 
 
FOUNDATIONS FOR SEX
 
 
 
23
 
 
 
type. Successful parthenogenesis was under
partial control of the inheritance as 80 generations of selection increased the rate
about 20-fold. Similarly outcrossing to bisexually reproducing males and reselection
resulted in both pronounced increases in
and survival of the parthenogenetic types.
 
Carson, Wheeler and Heed (1957) and
Murdy and Carson (1959) have established
a strain of Drosophila mangabeirai with
only thelytokous reproduction. Males have
been captured in nature but are rare. Fecundity of the virgin females is low but the
egg hatch is 60 per cent and 80 per cent
survive to adult stage. The progeny of the
virgins are always diploid. In meiotic spindle formation D. mangabeirai differs from
other Drosophila species in that its orientation increases the probability for fusion of
two haploid nuclei into structurally heterozygous diploid females. The study of
Feulgen whole mounts of freshly laid eggs
indicated automictic behavior with two
meiotic divisions followed by a fusion of
two of the four haploid meiotic products.
The absence of adult structural homozygotes in wild populations is probabl}^ explained by death during early development
and by possible fusion of second division
meiotic products derived from different secondary oocytes. Stalker (1956a) postulated
such selective fusion in order to account for
the heterozygous condition in females of
Lonchoptera dubia, an automictic, parthenogenetic fly in which males are rare or unknown.
 
A case in which jihenotypically rudimentary females, supposed homozygous for r/r
give rare and unexpected type progeny, has
suggested that polar body fusion may also
take place in D. melanogaster (Goldschmiclt, 1957). The rudimentary mothers
producing the peculiar type are interpreted
as formed by a most unusual series of
events: a fertilization nucleus derived from
the fusion of an r containing egg fertilized
by an r containing sperm and a polar copulation nucleus derived from the fusion of a
polar body containing an r genome and one
having wild type. Both cell types become
incorporated into the ovary. The progeny
which come from these supposed rudimentary mothers are presumed to be derived
from the maturation into eggs of the cells
 
 
 
derived from the heterozygous i)olar copulation nuclei. If these progeny-producing
rudimentary mothers arise in the presumed
manner they give the basis for a parthenogenetic mode of reproduction and the sex
types which have been described in other
Drosophila species by Stalker and Carson.
There are similar, as well as other forms
of parthenogenesis which affect sex (Smith,
1955) or assist in maintaining trijiloid conditions (Smith-White, 1955). A number of
these types have been reviewed by Suomalainen (1950, 1954). The reader may be referred to this material for other cases and
chromosome behaviors.
 
D. SEX INFLUENCE OF THE Y CHROMOSOME
 
The first function discovered for the Y
chromosome in D. melanogaster was that it
was necessary to male fertility (Bridges,
1916). Two and possibly more Y chromosome-borne, genetic factors were involved
(Stern, 1929). Gamete maturation when
these factors were lacking ceased just short
of the sperm's becoming motile (Shen,
1932). The motility conferred on the sperm
by the presence of the Y chromosome factors was fixed for the testes at an early
stage of development as transplantation experiments, sterile testes to fertile larvae
and fertile testes to sterile larvae, showed
motility to be a property determined by
early localized somatic influences on the
developing gametes or predetermined in the
diploid phase (Stern and Hadorn, 1938).
This Y chromosome function was sex limited, because females without a Y were the
normal fertile females and those with an
extra Y also were fertile.
 
Neuhaus (1939) followed by Cooper
(1952, 1959) and Brosseau (1960) further
analyzed the Y chromosome for fertility
loci. The latter showed at least two fertility
loci on the short arm and five on the long
arm of the Y chromosome. Data are compatible with a linear order of the genes. An
additional fertility factor common to the X
and Y was suggested. The Y chromosome
fertility factors consequently fall in line
with Bridges' concept of multiple gene loci
distributed in a more or less random manner which may affect sex.
 
The Y chromosome has other attributes
which help to explain its significance to sec
 
 
24
 
 
 
BIOLOGIC BASIS OF SEX
 
 
 
ondary if not primary sex characteristics.
As with many other species it has loci for
genes which are also found in the X chromosome, i.e., bobbed, as well as a limited
number of bands in the salivary gland chromosome. Nucleolus organizers and at least
two specialized pairing organelles similar
to those in the X chromosome are found
within the Y chromosome (Cooper, 1952,
1959). One of the most significant properties is the effect of extra Y chromosomes on
the variegation observed for various gene
phenotypes either in the normal chromosome pattern or in that accompanying
translocation. In variegation one Y chromosome as extra to the normal complex is
sufficient to eliminate or more rarely to
much reduce the variegated expression
(Gowen and Gay, 1933). When the Y chromosomes are 2 above the normal complement, the phenotypes gain two new features
(Cooper, 1956). Both males and females
become variegated in the expression of their
eye characteristics. The males become sterile. These effects are unexpected for they
are counter to any previous trends in the Y
effects on these characteristics. They have
reversed the direction of the effects as established by the two previous chromosome
types. The variegation of the XX2Y + 2A
females and X3Y + 2A males resembles
that of the XX + 2A females and XY +
2A males but is more extreme, whereas the
XXY + 2A and X2Y + 2A are largely
nonvariegated. The fertility relations are
equally aberrant: the X3Y + 2A males
have the same type sterility through loss of
sperm motility as that of the XO + 2A
males. Bundles of sperm are formed but
they do not become motile. Full-sized Y
chromosomes are not required to bring
about these effects, because females having
a whole Y plus a piece of a second, or males
with 2Y plus a piece of a third, will show
the effects.
 
The fractional Y chromosomes furnish
opportunities to test for the partial independence of the variegation and sterility effects. The two Y hyperploid males differ in
their degree of fertility according to the
fraction of the Y chromosome which may
be present whereas the effects on variegation may be constant among groups. This is
in accord with the sterility l)eing in part
 
 
 
independent of the factors causing the variegations. Similarly, the variegations may
be shown to be partially free of the action
of some elements that are not themselves
members of the two sets of factors influencing fertility in the normal male.
 
Other phenotypic irregularities appear;
eye facets may be roughened, legs shortened, and wing membranes become abnormal. On the negative side two extra Y chromosomes in females homozygous for the
transformer gene, tra, do not increase in
maleness or function.
 
The variegations of the so-called V-type
position effects with translocations are suppressed, as in the normal type described,
by one extra Y but are nonetheless variegated when two extra Y chromosomes are
present. That these effects are caused by
there being two supernumerary Y's is indicated by the fact that XX2Y females are
fertile and X3Y males sometimes lose a Y
chromosome in their germinal tracts and
become fertile. A number of mechanisms
have been suggested to account for these
results but most have proven unsatisfactory. A balance interpretation for the X, Y
and autosomes like that for sex, as suggested by Cooper (1956) is compatible with
the somatic cell variegations for euchromatic loci transferred to the heterochromatic regions and the sterility-fertility
relations expressed by the different chromosomal types.
 
The variegation effects of the Y chromosome take on further significance. Baker
and Spofford (1959) have shown that 15
different fragments of the Y chromosome
when studied for their contributions to variegation differ in their effects with the differences often not related to the size of the
fragment, thus indicating that the Y chromosome has linearly differentiated factors
capable of modifying the variegated phenotype.
 
Other indications of genetic activity of
the Y chromosome were given by Aronson
(1959) in her study of the segregation observed in 3rd chromosome translocations. A
deficiency for the region of the 3rd chromosome centromere is lethal when homozygous. Both males and females are fully
\'iable when this deficiency is heterozygous.
XO or haplo IV deficient males die. How
 
 
FOUNDATIONS FOR SEX
 
 
 
25
 
 
 
ever, in the presence of a Y chromosome the
deficient haplo IV males become viable.
The lability of this last class indicates that
the Y chromosome is genetically active, can
compensate for the autosomal deficiency,
and thus alter the progeny sex ratio.
 
In D. virilis the situation is somewhat
different from that observed by Cooper in
1956 in D. melanogaster. Baker (1956) has
shown that, in a translocation, males having
two Y chromosomes plus a Y marked with
a 5th chromosome peach are fertile. These
results seem to indicate a species difference
in the effect of the extra Y on fertility or
the Y with the inserted peach locus is not
a complete Y and in consequence the true
composition of these males is X + 2Y plus
a fragment of the Y. The X chromosomal
associations in the multichromosomal types
are shown to be by trivalents or by tetravalents. The segregation data indicate that
the pattern of disjunction of trivalents is a
function of the particular Y chromosome
involved. In X2Y males with normal Y's or
with one normal and one marked Y, the Y's
disjoin almost twice as frequently as they
do from trivalents with two identical Y's.
Tetravalent segregation is almost entirely
two by two, with no preference for any of
the three types of disjunction.
 
An odd situation was reported by Tokunaga (1958) in substrains of Aphiochaeta
.vanthina Speiser. When a male of the substrain was crossed to individuals bearing
3rd chromosome genes of the original
strains, the mutant genes for brown, and so
on, behaved as if they were partially sexlinked in the following generations. On the
other hand, when a female carrying the partially sex-linked genes on the X and Y
chromosomes. Abrupt or Occhi chiari, was
crossed to the male of the substrain the
characters segregated as though they were
autosomal. As a working hypothesis it was
suggested that in this species the Y chromosome had the major male determining
factors. The "special" male arose as a translocation of these factors to the third chromosome with the consequent change in linkage
relations. Data on the role of the X chromosome in sex determination in this species
have not yet been obtained but if they support the interpretation they indicate real
differences between this species and that of
 
 
 
Drosopliila in the location of the sex genes.
The results are reminiscent of those obtained by Winge in Lebistes as well as those
in Melandrium and other forms in which
the Y or W chromosomes may contain
strong sex genes for either sex. They would
further support the thesis that sex genes
may be distributed to almost any loci within
the inheritance complex.
 
E. M.\TERNAL INFLUENCES ON SEX RATIO
 
Aside from chance and specific genetic
factors, sex ratio is subject to effects from
agents intrinsic in the cells of the mothers
(Buzzati-Traverso, 1941; Magni, 1952).
Strains of Drosophila bifasciata (Magni,
1952, 1953, 1957), D. prosaltans (Cavalcanti and Falcao, 1954), D. willistoni and
D. paulistorum Spassky, 1956 have been
isolated which were nearly all of the female
sex even though the mothers were outbred
to other strains having normal ratio bisexual
progeny. Study of these strains by the above
workers and Malogolowkin (1958) IVIalogolowkin and Poulson (1957), and Malogolowkin, Poulson and Wright (1959), as
well as by Carson (1956) have shown inheritance strictly through the mother regardless of the genetic nature of the males
to which they were bred. The unbalanced
ratio was retained even when the original
chromosomes had been replaced by homologous genomes from lines giving normal male
and female progenies. Transmission of this
unbalanced sex ratio was through the cytoplasm. Eggs fertilized by Y-bearing sperm
died early in the course of development.
Malogolowkin, Poulson and Wright (19591
have shown that the high female ratio and
embryonic male deaths may be transferred
from affected females to those which do not
normally show the condition through injection of ooplasm from infected females.
Ooplasm of this same type is, w^hen injected
into males, suflficient to cause death to occur within 3 days. In the females a latent
period of 10 to 14 days after ooplasm injection was apparently necessary to establish
egg sensitization. Once established the condition could be transmitted through the
female line for several generations. The
ooplasm injections were not as efficient in
establishing these lines as females found
naturally infected. Unisexual broods may
 
 
 
26
 
 
 
BIOLOGIC BASIS OF SEX
 
 
 
fail to appear, in some instances fail to
transmit the condition or produce intermediate progeny ratios, as well as in some
instances to skip a generation. The infectious agent was found to vary in different
species. Magni (1953, 1954) for D. bifasciata found that temperature above normal tended to remove the cytoplasmic agent
making it ineffective. This result has a parallel to the action of temperature on certain
viruses, as for instance that involved in one
of the peach tree diseases. On the other
hand, Malogolowkin (1958) ior D . willistoni
found no temperature effect. Malogolowkin
further found that the cytoplasmic factor
was not independent of chromosomal genes
since some wildtype mutant strains induced
reversions to normal sex ratio. Recently
Poulson has established the complete correlation of the high female progeny characteristic with the presence of a Trepomena in
the fly's lymph. As with mouse typhoid variations, lethality was genotype dependent.
 
These cases have interest from more than
the sex ratio viewpoint. Cytoplasmically
inherited susceptibility of some strains of
D. 7nelanogaster to poisoning by carbon dioxide as studied by L'Heritier (1951, 1955)
depended on the presence of some cytoplasmic entity which passed through the egg
cytoplasm and also, but less efficiently, by
means of the male sex cells to the progeny.
The carbon dioxide susceptibility was transmitted through injections of hemolymph or
transplantation of organs of susceptible
strains. The transmissible substance had a
further property of heat susceptibility. The
COo susceptibility differed from that of "sex
ratio" in being partially male transmitted.
It agrees with ''sex ratio" D. bifasciata in
being heat susceptible but differs from D.
willistoni "sex ratio" in this respect.
 
D. bifasciata may behave differently than
D. willistoni in that Rasmussen (1957) and
Moriwaki and Kitagawa (1957) both conducted transplantation experiments with
negative results. However, it is jiossible that
these experiments may be affected by a
different incubation period for the injected
material as contrasted with that for D.
willistoni. A range of possibilities evidently
exist for extrinsic effects in sex ratio.
 
Carson (1956) found a female producing
strain of D. borealis Patterson, which car
 
 
ried on for a period of 8 generations, produced 1327 females with no males. The
strain showed no chromosomal abnormalities. It had 3 inversions. The females would
not produce young unless mated to males
from other strains having biparental inheritance. This requirement, together with
gene evidence, showing that the females
were of biparental origin, is against the female progenies being derived by thelytokous
reproduction.
 
F. MALE-INFLUENCED TYPE OF
FEMALE SEX RATIO
 
Sturtevant in 1925 described exj^eriments
with a stock of D. affinis in which a great
deficiency of sons was obtained from certain
males, regardless of the source of females
to which such males were mated. The few
males obtained from such matings were normal in behavior but some of the sons of
females from such anomalous cultures again
gave very few sons (]Morgan, Bridges and
Sturtevant, 1925).
 
Gershenson (1928) in a sampling of 19
females caught in nature found two that
were heterozygous for a factor causing
strong deviations toward females (96 per
cent to 4 per cent males) whereas the normal D. pseudoobscura ratio was nearly 1 to
1. The factor was localized in the X chromosome and was transmitted like an ordinary
sex-linked gene. Its effect was sex limited
as it was not manifest in either heterozygous or homozygous females. It had no
effect on the development of zygotes already formed but strongly influenced the
mechanism of sex determination through the
almost total removal of the spermatozoa
with the Y chromosome from the fertilization process. Egg counts showed the divergent ratio toward females was not caused
by death of the male zygotes inasmuch as
there was no greater mortality from such
cultures than from controls giving 1 to 1 sex
ratios.
 
Sturtevant and Dobzhansky (1936)
showed that an identical or nearly identical
phenotypc to that obscM'ved in Drosophila
obscura was present in D. pseudoobscura.
The D. pseudoobscura carrying the factor
were scattered over rather wide geographical areas. Comparable types also were found
in two other species D. athabasca and D.
 
 
 
FOUNDATIONS FOR SEX
 
 
 
27
 
 
 
azteca. This genie "sex ratio"' sr lies in the
right limb of the X of races A and B of
D. pseudoobscura. Like the other cases
analyzed, males carrying sr have mostly
daughters and few sons regardless of the
genotypes of their mates. Structurally the
female sex ratio came through modification
of the development of the sex cells of the
male to give a majority of X-bearing sperm.
Cytologic study showed that in "sex ratio"
males the X underwent equational division
at each meiotic division, whereas the autosomes behaved normally. The Y chromosome lagged on the first spindle, remained
much condensed, and its spindle attachment
end was not attenuated. The Y chromosome
was not included in either telophase group
of the first meiotic division but was left behind. It was sometimes noted in one of the
daughter cells where it formed a small nucleus. Among 64 spermatocytes examined,
all had an X chromosome and none a Y
chromosome in their main nuclei. The micronuclei containing Y played no further
part in the division, became smaller and
exceedingly contracted. The final fate of the
micronuclci was uncertain, but there was no
indication that any spermatids died or were
abnormal.
 
G. HIGH MALE SEX RATIO OF GENETIC ORIGIN
 
In 1920, Thompson described a recessive
mutant in D. melanogaster which killed all
homozygous females but changed the male
phenotypes only slightly, the wings standing erect above the back. The locus of the
mutant was 38 in the X chromosome. This
mutant was a leader for a class in which the
genes affect only one sex but are innocuous
to the other.
 
Bobbed-lethal, a sex-linked gene found
i)y Bridges, is a gene of this class but one in
which the mechanism of protection to the
other sex is known. It kills homozygous females but does not kill the males because of
the wild type allele which the males have in
their Y chromosomes. The presence of this
bobbed-lethal in a population consequently
leads to male ratios higher than wild type.
 
A recessive gene in chromosome II, discovered by Redfield (1926), caused the
early death of the majority of female zygotes and led to a sex ratio of about 1 female to 5.5 males. The effect was trans
 
 
mitted througli both males and females. The
missing females may have died largely in
the egg stage although disproportionate
losses also occurred in the larvae and pupae.
The maternal effect was attributed to an
influence exerted by the chromosome constitution of the mother on the eggs before
they left the mother's body. Because of this
maternal lethal, the families from these
mothers have the normal number of sons
but few or no daughters.
 
A culture was observed by Gowen and
Nelson (1942) which yielded only male
progeny, 136 in all. Some of the male progeny were able to transmit the male-producing characteristics to half of their daughters
without regard to the characteristics of the
mates to which they were bred. The inheritance was without phenotypic effect on
the males. When present in the heterozygous condition in females, the female's
own phenotype gave no indication of the
gene's presence. The inheritance was sex
limited in that it affected only the eggs laid
by the mothers carrying it. In these eggs it
acted as a dominant lethal for the XX zygotes. The gene was found located in the 3rd
chromosome between the marker genes for
hairy and for Dicheate at approximately 31.
The X eggs carrying the gene, Ne, died in the
egg stage at between 10 and 15 hours under
25°C. temperature. The gene was as effective in triploids as in diploids. One dose
of this gene in triploids caused them to have
only male progeny and male type intersexes.
The presence of this gene caused the elimination of any embryos with chromosomal
capacities for initiating and developing the
primary or secondary female sexual systems. Since the gene itself was heterozygous
in the female the meiotic divisions of the
eggs would cause the gene to pass into the
polar bodies as often as to remain in the
fertilization nucleus yet the lethal effects
are found in all eggs. The antagonism was
between the egg cytoplasm and the XX
fusion products. Supernumerary sperm of
the Y type were not sufficient to overcome
the lethal effects. An XY fertilization nucleus was necessary for survival.
 
This case has parallel features with that
observed by Sturtevant (1956) for a 3rd
chromosome gene that destroys individuals
bearing the first chromosome recessive gene
 
 
 
28
 
 
 
BIOLOGIC BASIS OF SEX
 
 
 
for prune. The gene responsible was found
to be a dominant, prune killer, K-pn, which
was located in the end of the 3rd chromosome at 104.5. Prune killer was without
phenotypic effect so far as could be observed except that it acted as a killer for
flies containing any known allele of prune.
It was equally effective in either males or
females, hemizygous or homozygous, for
prune. The larvae died in the 2nd instar
but no gross abnormalities were detected
in the dying larvae. This case has interest
for the Ne gene in that the killer gene occupies a locus in the 3rd chromosome although removed by about 70 units from Ne,
and acts on a specific genotype, the prune
genotype, whereas Ne acts on the specific
XX genotype. The known base for action
of the prune killer is genetically much narrower than that for the female killer, Ne,
in that prune killer acts on an allele within
a specific locus in the X chromosome, and
Ne acts on a type coming as a product of
the action of two whole sex chromosomes. It
is, of course, conceivable that when ultimately traced each action may be dependent
upon specific changes of particular chemical syntheses. The action of these genes is
also of interest from another viewpoint.
 
In mice there is a phenotype caused by
the homozygous condition of a recessive
gene (Hollander and Gowen, 1959) which
acts on its own specific dominant allelic
type in its progeny so as to cause an increased number of deaths between birth
and two weeks of age, as well as causing the
long bones to break and the joints to show
large swellings. The lethal nature of this
interaction is not a product of the mother's
milk nor does it show humoral effects such
as those observed with erythroblastosis in
the human. The interallelic interaction is
sex limited in that it is confined to the
mother and is without effect when the male
has the same genotype.
 
H. FEMALE-MALE SEX RATIO INTERACTIONS
 
A case in D. affinis involving the interaction of a "female sex ratio" factor and an
autosomal "male sex ratio" factor has been
studied by Novitski (1947). Starting from
stock which had a genetic constitution for
the "sex ratio" X chromosome which ordinarily causes males carrying it to produce
 
 
 
only daughters, he was able to establish a
recessive gene in chromosome B in whose
presence only male offspring resulted. The
genetic constitution of the male was alone
important. High sterility accompanied the
"male sex ratio" males breeding performance. The "male sex ratio" parents yielded
95 fertile cultures having an average of 25
individuals per culture. Females appeared
in 10 of these cultures with an average of 3
per culture for those producing females. The
total sex ratio was 77 males per female. The
males were morphologically normal. They
carried an X chromosome of their mothers.
Cytologic observation of spermatogonial
metaphases of 3 progeny showed that the
Fi males may or may not have carried a Y
of their fathers. This agreed with the tendency for sterility in these "male sex ratio"
males. The ventral receptacles of the females from 4 such sterile cultures when examined proved devoid of sperm. The occasional female offspring of the "male sex
ratio" males had one X chromosome from
each parent. Females mated to "male sex
ratio" males show large numbers of sperm
in the ventral receptacles (7 out of 8 cases) ,
although such females had usually produced
no or very few offspring. The sperm, if
capable of fertilization, must have had a
lethal effect on the zygotes. The recessive
nature of the factor in chromosome B indicated that its potential lethal effect originated during spermatogenesis rather than
at the time of fertilization. This lethal period corresponds to that when the "female
sex ratio" factor in the X chromosome is
active.
 
The research on sex ratio in Drosophila
reviewed shows that through the interplay
of the sex chromosome-located and autosomal-located factors all types of sex ratios
from only females in the family to only
males in the family may be generated.
 
V. Sex Determination in Other Insects
 
 
 
Sciara, a fungus gnat, offers sex-determining mechanisms quite different from any
yet offered in Drosophila. Sciara is unique,
yet in its uniqueness, it illustrates basic
facts that were rediscovered in other species only through the study of abnormal
 
 
 
FOUNDATIONS FOR SEX
 
 
 
29
 
 
 
forms some of which were created as induced developmental, chromosomal, or hormonal abnormalities. The complexities responsible for the remarkable facts were
analyzed by Metz and his collaborators.
The following brief review is based upon
Metz's (1938) summarization of the chromosome behavior problem to which the
reader is referred for further information.
Of Sciara species studied 12 out of 14 have
their basic chromosome groups composed of
three types of chromosomes: autosomes,
sex chromosomes, and "limited" chromosomes. Two species, S. ocellaris Comstock
and S. reynoldsi Metz, lack the "limited"
chromosomes. Two sets of autosomes and
three sex chromosomes are found in the
zygotes of all Sciara species at the completion of fertilization. The behavior of these
chromosomal types will be considered in
sequence.
 
The autosomes behave normally in somatic mitosis and in oogenesis. There is
cytologic and genetic evidence for synapsis,
crossing over, random segregation, and regular distribution of chromosomes and genes
in the female.
 
In spermatogenesis the story is quite different. The first maturation division is unipolar. Genetic evidence shows a complete,
selective segregation of the maternally derived autosomes and sex chromosomes from
those of paternal origin. The paternal homologues move away from the pole, are
extruded, and degenerate. The second
spermatocyte division is likewise unequal
and one of the products, a bud, degenerates.
Thus a spermatogonial cell passing through
meiosis gives rise to only one sperm. At the
second spermatocyte division all of the
chromosomes (maternal homologues) except
the sex chromosome undergo an equational
division but both halves of the sex chromosome enter into the same nucleus. This nucleus becomes the sperm nucleus. The chromosomes at the opposite pole form a bud
and degenerate. Fertilization of the Sciara
egg is normally monospermic not polyspermic.
 
The sex chromosomes XXX of the fertilization nucleus are derived, two from the
sperm and one from the oocyte. Their destiny depends on whether the egg they are
 
 
 
in develops into a male or a female imago.
In male early development, those nuclei
which are to become male soma lose the two
sex chromosomes XX, contributed by the
father's sperm, to give X + 2A soma. These
chromosomes fail to complete mitosis at
the 7th or 8th cleavage division and are left
to degenerate in the general cytoplasm when
there are no true cells in the soma region
and no membranes surround the nuclei.
Those embryos which are to become female
soma at the same cleavage cycle eliminate
but one paternal X chromosome. On a chromosome basis the soma cells respectively
become X, plus two sets of autosomes, give
rise to males on differentiation, or become
XX + 2A and develop female organs (Du
Bois, 1932). The germ line nuclei for each
sex, on the other hand, remain unrestricted
in their development. They retain their
XXX constitutions until the first day of
larval life, or about 6 hours before the formation of the left and right gonads (Berry,
1941), when they eliminate a single paternally derived X. The X which is rejected or
makes its own exit is always one of two
sister chromosomes contributed by the
father. The process of loss is strikingly different from that noted in the soma-building
nuclei. Both cell walls and nuclear membranes are present. The path of the X chromosome is through the nuclear membrane
into the cytoplasm where degeneration
eventually takes place. The loss occurs at
a time when there is no mitotic activity and
the chromosomes are separated from the
cytoplasm by an intact nuclear membrane.
Some Sciara species are characterized by
females which produce only unisexual families — practically all females or practically
all males. Genetically, the sex of progeny is
accounted for by sex chromosomes, X' and
X, which are so designated because of their
physiologic properties. Mothers having only
female progeny are characterized by always
having the X' chromosome in all their cells,
X'X + 2A, whereas mothers whose progeny
are all males are XX + 2A (Moses and
Metz, 1928). The all female and all male
broods are observed in about equal numbers. The male has no influence on sex ratio.
Normally the progeny in all female broods
will be X'X + 2A or XX + 2A in soma
 
 
 
30
 
 
 
BIOLOGIC BASIS OF SEX
 
 
 
and germ lines, whereas the all male progeny broods will be only X + 2A in the soma
and XX + 2A in the germ line.
 
Studies of Grouse (1943, 1960a, b) show
nondisjunction may occur and eggs entirely
lacking sex chromosomes or having double
the normal number may be produced. When
the nondisjunctional eggs lacking X chromosomes are from X'X females and are fertilized by sperm contributing two sister X
chromosomes, one X (as expected of X'X
mothers, not two as expected of XX cells) is
eliminated at the 7th or 8th cleavage to give
cells which develop into "exceptional" males
with XO + 2A soma and XX + 2A germ
line, all X chromosomes being of paternal
origin. The casting out of the single X chromosome is consequently controlled by the
characteristics of the egg cytoplasm derived
from the X'X mother rather than by the
kind of chromosomes present in the nuclei
at the 7th or 8th cleavage stage of the eml)ryo.
 
Similarly when nondisjunctional eggs
having both XX chromosomes of the XX
mothers are fertilized by the normal spermbearing XX chromosomes, the zygotes are
XXXX. At the 7th or 8th cleavage both
paternal X chromosomes are eliminated and
the resulting soma develops into an ''exceptional" female, XX + 2A, often with 3 X's
in her germ line. These observations confirm the earlier interpretations that the X'X
or XX constitution of the mother conditions
her eggs respectively to cast out 1 or 2
parental X chromosomes at the 7th or 8th
cleavage according to the cytoplasmic constitution of the egg. Somatic sex development is visually determined only after this
stage when the chromosomes of the somatic
cells take on the familiar aspect of either
XX + 2A for the females or X + 2A for the
males.
 
Although at present there are few examples in other species, this cytoplasmic conditioning of early embryologic development
by the mother's genotyj)e may be a common
rather than a rare occurrence of development. The expression of the events to which
this control may lead may be quite different in different cases. The spiral of snail
shells was an early recognized case (Sturtcvant, 1923) . In Drosophila, a gene acting
through the mother's genome allows onlv
 
 
 
male embryos to survive (Gowen and Nelson, 1942), suggesting that this gene acts
through her cytoplasm in a manner comparable to that of the X' and X chromosomes of Sciara. In either case the genotype would not be considered a primary sex
factor, profound as the effect on sex may
ultimately be.
 
Sciara also has strains or species where
the progeny of single matings are of both
sexes, bisexual, yet the chromosomal elimination mechanisms remain as described
above save for the fact that they now operate within broods rather than between
broods. Sex ratios for the families within
these bisexual strains are extremely variable, 1:0 or 0:1 ratios not being infrequent
and 1 : 1 ratios being the exception. An instance of a bisexual strain arising from a
unisexual line has been analyzed by Reynolds (1938). The females were found to be
3X in their germ line and produced 2X eggs
which developed into daughters and X eggs
which became sons. Loss of the extra X
from this line resulted in the line's return to
unisexual reproduction.
 
In general, bisexuality as contrasted with
unisexuality seems to be caused by differences in the X'X-XX mechanisms, either
specific for the mechanism, or induced by
modifying genes which cause different embryos within the same progeny to cast out
sometimes one or sometimes two X chromosomes from all their somatic nuclei. This
casting out occurs uniformly for all nuclei
of a given embryo as is shown by the fact
that sex mosaics or gynandromorphs are as
seldom observed in bisexual as unisexual
strains.
 
Some species of Sciara have large chromosomes which are in essence "limited" to
the germ line since they are lost from the
somatic nuclei at the 5th or 6th cleavage
division. Other species lack these chromosomes, yet agree with the rest in the behavior of their remaining chromosomes and
in sex determination. So far as is now
known the "limited" chromosomes seem
empty of inheritance factors influencing either sex or unisexual vs. bisexual broods or
for that matter other characteristics (their
persistence seems to make this latter unlikely I. Their presence does lead to a question of the efficacv of desoxvribonucleic acid
 
 
 
FOUNDATIONS FOR SEX
 
 
 
31
 
 
 
(DNA) as the all inclusive agent in inheritance for they are clearly DNA-positive.
 
These cytogenetic observations clear up
most of the events leading to sex differentiation and the delayed time in embryologic
development when it takes place, but, as
INIetz points out, other puzzling questions
are raised. Observed chromosome extrusions
from cell nuclei are rarities today even
though discovered for Ascaris 60 years ago.
Why should this mechanism be so well defined in Sciara? Why should the mode of
elimination be so accurately timed and yet
be different in method and time for the
soma and germ cell lines? Two of the three
X chromosomes in the fertilized egg are
sisters from the father and should be identical. What is special in the 7th or 8th cleavage that causes elimination in the soma
plasm but not in the germ plasm? Why
should the casting out of one of these same
paternal X's in the germ plasm of both
sexes be reserved for a much later stage in
cleavage and by a different procedure?
 
The observations of Grouse (1943, 1960a),
obtained from translocations and species
crosses, are critical in showing that the
chromosome first moA'ing in its entirety to
the first differentiating pole of the second
spermatocyte division is the X and is the
one taking part in the later postfertilization
sex chromosome elimination of the 7th or
8th cleavage. The X heterochromatic end of
the chromosome and not the centromere region seems to be responsible for the unique
behavior of this chromosome (Grouse,
1960b). Induced nondisjunctional eggs of
X'X (female-producing) mothers having as
a consequence no sex chromosomes, when
fertilized by the normal sperm bringing in
XX chromosomes, develop into embryos
eliminating one of these X's at the 7th or
8th cleavage, as expected of the eggs of
X'X mothers, and become "exceptional"
males with X + 2A soma and XX paternal
germ line. Imagoes of these embryos become
functional but partially sterile males transmitting the paternal X, a reversal of the
normal condition. Similarly the nondisjunctional eggs retaining both X's, XX
from male-producing mothers, and having
4 X's on fertilization eliminate the two paternal X's, as occurs normally in XXX eml)ryos, to become "exceptional" females with
 
 
 
2X or sometimes in species hybrids 3X
soma.
 
Phenotypic sex in Sciara on this evidence
depends on the adjustment of the X chromosome numbers and their contained genes
with this adjustment regularly taking place,
not at fertilization as in most forms like
Drosophila, but at the 7th or 8th cell cleavage of embryologic development. The X' vs.
X chromosomes of the mother predispose
her haploid egg cytoplasm to time specific
chromosome elimination. The germ line cells
on the other hand may regularly have other
chromosome numbers than the soma or exceptionally tolerate other numbers as extra
X's (Grouse, 1943), or extra sets (Metz,
1959).
 
Triploids of 3X and 3A soma have not
been found in pure Sciara species. Salivary
gland cells of a triploid hybrid S. ocellaris x
aS. reynoldsi have two sets of S. ocellaris and
one of S. reynoldsi chromosomes (Metz,
1959). Ghromosome banding in 2X -I- 3A,
3X + 3A larval glands showed the »S. ocellaris homologues were heterozygous. The
3X + 3A type chromosomes were of typical
female appearance. The 2X:3A-type had
X chromosomes of typical male type, very
thick, pale and diffuse. The mosaic salivaries were a mixture of the two types of
cells. These results suggest that the 3X:3A
would be a typical female. The intersexes
2X -I- 3A conceivably have a range in male
and female organ development depending
on how the S. ocellaris and S. reynoldsi
chromosomes act. If the chromosomes are
equivalent, the gene expression would be
expected to be that of a 2X to 3A or a
phenotypic intersex. If they act separately
the S. ocellaris genes would be in balance for
female determination, whereas the S. reynoldsi autosomal genes would have no X S.
reynoldsi genes to balance them and presumably would be overwhelmingly male in
effect. The phenotypic outcome would be
in doubt.
 
The maternally governed cleavage and
chromosomal sequence occurring before the
8th embryonic cell division, although related to the subsequent events leading to the
particular sex, is unique to Sciara and its
relatives. Phenotypic sex expression apparently starts with the somatic nuclei having
either X + 2A or XX + 2A as their chro
 
 
32
 
 
 
BIOLOGIC BASIS OF SEX
 
 
 
mosomal constitutions. Sciara is like Drosophila and many other genera in its basic
sex-determining mechanism. There is a sexindifferent period when the maternal genotype may influence development through
cytoplasmic substances. This is followed by
active differentiation when the sex phenotypes may be influenced by various agencies
but when passed becomes fixed for phenotypic sex. The indifferent period may represent a fairly large number of early cell generations as in amphibia or fish. Somatic
and germ cells are labile but are normally
susceptible to control only by forces developed within the organism as present
knowledge indicates for Sciara. In Sciara,
only the somatic cells conform in chromosome number to their expected phenotypes.
The germ cells show not only a regulated instability of their chromosome number but
the number is different from that of the
supporting somatic cells. The fact that sex
mosaics in Sciara may develop both an
ovary and testis in the same animal (Metz,
1938; Grouse, 1960a, b) shows localized
phenotypic sex determination occurring
fairly late in development rather than the
generalized determination that would occur if sex were established at fertilization.
 
The germ line chromosomal differentiation from that of the somatic tissue has
parallels in a number of fairly widely dispersed species. The significance of these
types to soma line determination and to the
reversibility or irreversibility of differential gene activation in differentiated cells
subsequent to embryogenesis has been considered by Beermann (1956). Although
these types are yet in the fringe areas of
our knowledge surrounding the general
paths taken in the evolution of sex determining mechanisms, they promise to have
more generality as clarification of gene action becomes more exact.
 
B. APIS AND HABROBRACON
 
Hymenopteran interi:)retations of sex determination have largely turned on studies
of the honey bee. Apis mellijera Linnaeus
and Habrohracon juglandis Ashmead. Dzierzon early established that the drones of
honey bees come from unfertilized eggs,
whereas the females, queens, and workers
come from fertilized eggs so that sex differ
 
 
entiation is marked by an N set of chromosomes for the male and 2N for the female.
This mechanism was satisfactory until it
was realized that on a balanced theory the
doubling of a set of chromosomes, if truly
identical, should not change the gene balance and consequently not the sex. Further
doubt was cast on the N-2N hypothesis for
sex determination by the discovery of diploid males by Whiting and "Whiting (1925).
These difficulties were resolved by Whiting
(1933a, b) with the suggestion that the two
genomes in the female were not truly alike
but actually contained a sex locus linked
with the gene for fused which was occupied
by different sex alleles as Xa and Xb . In
this A'iew the heterozygous condition would
lead to the production of females, whereas in
the haploid or homozygous condition either
of these genes would react to produce males.
Difficulties with this hypothesis became evident in that the ratios of males to females in
certain crosses were significantly divergent
from those which were expected. Evidence
was collected and trials made to interpret
these difficulties (Whiting, 1943a, b) by assuming that on fertilization by Xa-bearing
sperm the polar body spindle would turn
so as to cast out the Xa chromosome and retain the complementary Xb in the majority
of cases instead of in a random half. Snell
(1935) offered the hypothesis that there
could be several loci for sex genes, such that
the X locus might be a master locus, but
when this locus was in homozygous condition the sex would then be controlled by
genes in other loci in the genome. As pointed
out by Bridges (1939) this hypothesis would
satisfy that of sex gene balances postulated
in Drosophila and of change in emphasis on
loci for sex genes as observed by Winge
(1934) in Lebistes. However, the work of
Bostian (1939) called both hypotheses into
question. Consideration of these further results led to the multiple allele hypothesis of
complementary sex determination for
Habrobracon (Whiting, 1943a) taking a
similar form to that of the sterility relations
observed for a single locus in the white
clover of INIelilotus. Under this system the
sex locus would be occupied by multii)le alleles, any one of which or any combination
of identical alleles would be male-producing,
but any combination of two different al
 
 
FOUNDATIONS FOR SEX
 
 
 
83
 
 
 
Iclcs would l»e female determining. By having a sufficient number of these genes the
jiroduction of diploid males would be curtailed to a point at which their various frequencies could be explained. In support of
this liyi)othesis, multiple alleles to the number of at least 9 were found to exist for
thi.> single sex locus.
 
In principle at least, the honey bee could
have the Habrobracon scheme of sex determination. Rothenbuhler (1958) has recently collected the researches which test
this possibility. Tests of the multiple allele
hypothesis as applied to the honey bee were
made by Mackensen (1951, 1955) who interpreted evidence for inviable progeny produced by mating of closely related individuals as proof that this species as well as
Habrobracon juglandis follows the multiple
allelic system. The discovery of male tissue
of bii)arental origin in mosaic bees from related i)arents was considered as further evidence for the multiple allelic theory of sex
determination (Rothenbuhler, 1957).
 
Most recent cytologic evidence supports
the concept that there are 16 chromosomes
in the gonadal cells of the male and 32 in
those of the female (Sanderson and Hall,
1948, 1951; Ris and Kerr, 1952; Hachinohe
and Onishi, 1952; Wolf, 1960». Hachinohe
and Onishi (1952) found 16 chromosomes
as characteristic of the meiosis in the drone.
Wolf observed a nucleus in both bud and
spermatocyte of the only maturation (equational) division.
 
The greatest progress has been made in
understanding the mechanisms of sex mosaicism in the Hymenoptera species. These
mosaics, although ordinarily of rather rare
occurrence, have a direct bearing on sex
determination and development. In Apis,
iiolyspermy furnishes the customary basis
for their formation. One sperm fertilizes
the haploid egg nucleus and another sperm,
which has entered the egg instead of degenerating as it ordinarily does, enters into
mitotic cleavage and eventually forms islands of haploid cells of paternal origin
among the diploid cells derived from the
fertilized egg (Rothenbuhler, Gowen and
Park, 1952). Evidence showing that genetic
influences affect the sperm nucleus toward
stimulating its independent cleavage is
found to exist in Apis material (Rothen
 
 
buhler, 1955, 1958). Tliousands of gynandromorphs have been observed in Apis, all
but a small number of which have been produced in this manner. This method of initiating sex mosaics also exists for Habrobracon (Whiting, 1943b) but is rather rare.
In Habrobracon the frequent mode has a
different origin. The gynandromorph is
formed from the cleavage products of the
normal fertilization of the egg nucleus combined with those of a remaining nuclear
product of oogonial meiosis. Under these
conditions the female tissue is 2N of biparental origin and the male tissue is N of
maternal origin (Whiting, 1935, 1943b).
This type is less frequent in Apis but one
specimen has been described by Mackensen
(1951).
 
A number of other ways in which sex
mosaics may occur are occasionally expressed in these species. Three different
kinds of male tissue have been observed in
individual honey bee mosaics produced by
doubly mated queens — haploid male tissue
from one father, haploid male tissue from
the other genetically different father, and
diploid male tissue of maternal-paternal
origin. In other cases, the diploid, biparental
tissue was female and associated with two
kinds of male tissue (Rothenbuhler, 1957,
1958) . Cases where the haploid portions of
the sex mosaics are of two different origins,
one paternal and the other maternal, while
the female portion is representative of the
fertilized egg, are known in Habrobracon
(Whiting, 1943b). Similarly, Taber (1955)
observed females which were mosaics for
two genetically different tissues and which
he accounted for as the result of binucleate
eggs fertilized by two sperm. Mosaic drones
of yet another type were observed by
Tucker (1958) as progeny of unmated
queens. They were interpreted as the cleavage products from two of the separate nuclei formed in meiosis. These cases represent
a number of the possible types that arise
through meiotic or cleavage disfunctions
under particular environmental or hereditary conditions.
 
Tucker (1958) studied the method by
which impaternate workers were formed
from the eggs of unmated queens. For this
purpose he used genetic markers, red, chartreuse, ivory, and cordovan. Observations
 
 
 
34
 
 
 
BIOLOGIC BASIS OF SEX
 
 
 
Avore nuult' i)ii 237 workers from hotcrozygous mothers. For the chartreuse loeus. 12 to
20 per cent were homozygous, for x\w i\ory
locus 1.8 per cent, and (he cordovan locus
on lesser numbers per t-ent. An egg which
is destined to become an automictic worker,
a gynandromorph with somatic male tissue
or a mosaic male, is retained within the
queen for an unusually long time during
which meiosis is suspended in anaphase 1.
Normal reorientation of first division
spindle is i)ossibly inhibited by this aging
so that after the egg is laid meiosis II occurs
with two second division spindles on sejiarate axes as Goldschmidt conjectured for
rarely fertile rudimentary Drosophila
(19")7i. Two polar l)odies and two egg
prontich^i are formed. The polar bodies take
no fiu'ther part in develoinnent. In most of
the unusual eggs the two egg pronuclei
unite to form a diploid cleavage nucleus
which develops into a female. Rarely the
two egg pronuclei develop separately as two
haploid cleavage nuclei to form a mosaic
male. Two unlike haploid cleavage nuclei,
one descending from each of the two secondary oocytes after at least one cleavage
di\ision. unite to form a dijiloid cleavage
nucleus which develops together with the
remaintler of the haploid cleavage nuclei
to produce a gynandromorph with mosaic
male tissues. The male and female tissues
within these unusual gynandromor[ihs or
female types were identical with normal
drone or normal female tissues so were
probably haploid and diploid resiiectively.
Genetic segregation observed within the
mothers of automictic workers allows the
estimation of the distance between the locus
of the gene and its centromere. "With random
recombination and "central union" Tucker
estimates this distance for the chartreuse
locus to centromere as 28.8 units and for the
ivory to its centromere 3.6 units. Four lines
of bees of diverse origin all showed a low
percentage of automictic or gynandromorphic types produced from queens in each
line. Various chance environmental conditions apparently influence the rate of production of these types. However, there were
some females in two of the lines with higher
frequencies inciicating that innate factors
may have significant eft'ects on their frequencies. Observations on Drosophila spe
 
 
cies — I), porthenogeuetica (Stalker, 1956b),
I). ))ia)njabeirai (INIurdy and Carson, 1959)
and D. nielanogaster (Goldschmidt, 1957) —
strongly suppt)i-t this A-iew.
 
The [irohleni of si'x determination in Hal)rol)i'acon presently stands as a function of
multii)le alleles in one locus, the heterozygotes being female and the azygotes and
homozygotes male. The occasionally diploid
males are regularly produced from fertilized
eggs in two allele crosses after inbreeding.
These diploid males are of low viability and
are nearly sterile. Their few daughters are
triploids, their sperm being dijiloid. Apis
probably follows the same scheme, as a
few cases of mosaics with diploid male tissue are known and close inbreeding results
in a sufficient number of deaths in the egg
to account for diploid males which might be
formed. ^lormoniella (Whiting, 1958), however, shows that this scheme for sex determination does not hold for all Hymenoptera.
In this form diploid males may occur
through some form of mutation. They may
then develop from unfertilized eggs laid by
triploid females. In contrast to Habrobracon the dijiloid males are highly viable and
fertile. Their sperm are diploid and their
numerous daughters triploid. Virgin triploid
females produce 6 kinds of males, 3 haploid
and 3 dij^loid. Similarly ]\Ielittobia has still
a different and as yet unexplained form of
sex determination. Haploid eggs develop
into males. After mating many eggs are laid
which develop into nearly 97+ per cent females. The method of reproduction is close
inbreeding but no dijiloid males or "bad"
eggs are formed. The prol)lem of the sex
determining mechanism remains open
(Schmieder and Whitiuii, 1947).
 
 
 
The silkworm, Bomby.r mori, differs from
Drosophila, Lymantria and the species thus
far discussed in having a single region in the
^^' chromosome (Hasimoto, 1933; Tazima,
1941, 1952) occupied by a factor or factors
of high female potency. The strong female
potency has thus far been connnon to all
races. The chromosome patterns of the
sexes are like those of Abraxas and Lymantria: males ZZ + 2A and females ZwV 2A.
The diploid chromosome number is 56 in
both sexes. Extensive, well executed studies
 
 
 
FOUNDATIONS FOR SEX
 
 
 
35
 
 
 
liave revealed no W chruniosome loci for
genes expressed as morphologic traits. From
radiation-treated material it has been possible to pick up a translocation of chromosome II to the W chromosome as well as a
cross-over from chromosome Z. This chromosome together with tests of hypoploids
and hyperploids have materially aided in
understanding how the normal chromosome
complexes determine sex. The sex types resulting from different chromosome arrangements have been summarized by Yokoyama
1 1959) and are presented in Table 1.3.
 
Whenever the W chromosome was absent a male resulted. Extra Z or A chromosomes did not influence the result. Similarly
with a W chromosome in the fertilized egg
a female developed. Again extra Z or A
chromosomes did not influence the result.
 
A full Z chromosome was essential to survival. Hypoploids deficient for different
amounts of the Z chromosome in the presence of a normal W chromosome all died
without regard to the portion deleted. Hyperploids for the Z chromosome, on the other
hand, when accompanied with a W chromosome all lived and showed no abnormal sexual cliaracteristics. Parthenogenesis led to
the pioduction of both sexes, although the
males were more numerous than the females. Diploidy was necessary for the eml)ryo to go beyond the blastoderm stage.
Triploid and tetraploid cells were often
found. High temperature treatments led to
merogony (Hasimoto, 1929, 1934). The exceptional males were homozygous for a sexlinked recessive gene and were explained
by assuming that the egg nuclei were inactivated by the high temperature and
the exceptional males developed from the
union of two sperm nuclei. This conclusion
was supported by cytologically observed
l)olyspermy (Kawaguchi, 1928) and by
cytologic observation of the union of two
sperm nuclei by Sato ( 1942 ) . Binucleate eggs
were also believed to occur, which when
fertilized by different sperm may each construct half of the future body. This type of
mosaicism was influenced by heredity
iGoldschmidt and Katsuki, i927, 1928,
1931 ). Polar body fertilization was also believed to occur, one side of the embryo originating from the ordinary fertilized egg nucleus and the other side from the union of
 
 
 
TABLE 1.:^
Sex in Bombyx iitori
(Summarized by T. Yokoyama, 1959.)
 
 
 
Sex
 
 
Chromosome Types and Numbers
 
 
 
 
W
 
 
z
 
 
A
 
 
Male
 
 
W
II.W.ZL
 
w
w
 
WW
WW
 
 
zz
 
zz
 
zzz
 
z
 
z
 
zz
zzz
zz
zz
 
 
AA
 
 
Male
 
Male
 
Female
 
Female
 
Female
 
Female
 
Female
 
Female
 
 
AAA
AAA
 
AA
 
AA
 
AAA
 
AAAA
 
AAA
 
AAAA
 
 
 
nuclei of two of the polar bodies. Similarly,
dispermic merogony was noted following
the formation of one part of the body from
the fertilization nucleus, the other part from
the union of two sperm nuclei, the result
being a gynandromorph or mosaic.
 
VI. Sex Determination in Dioecious
Plants
 
\. MELAXDRUM t LYCHNIS]
 
Over the last 20 years studies on several
species of dioecious plants have made notable advances in unclerstanding the mechanisms by which sex is determined.
 
Melandrium album has been shown to
have the same chromosome arrangement as
Drosophila. The male has an X and Y plus
22 autosomes, whereas the female has XX
plus 22 autosomes. Sex-linked inheritance
is known for genes borne in the X chromosomes as well as for genes born in the Y
chromosome. The X and Y chromosomes
are larger than any of the autosomes with
the Y chromosome about 1.6 times that of
the X in the materials studied by Warmke
( 1946) . Separate male and female plants are
characteristic of the species. By use of
colchicine and other methods, Warmke and
Blakeslee (1939), Warmke (1946), and
Westergaard (1940) have made various
l^olyploid types from which they could derive other new X, Y and A chromosome
combinations from which information was
obtained on the location of the sex determining elements. The Y chromosome carries
the male determining elements, the X chro
 
 
36
 
 
 
BIOLOGIC BASIS OF SEX
 
 
 
mosome the female determining elements.
The guiding force of the elements in the Y
chromosome during development is sufficient to override the female tendencies of
several X chromosomes. Data derived by
each of these investigators are shown in
Table 1.4.
 
From these data Warmke (1946) concluded that the balances between the X and
the Y chromosomes essentially determined
sex with the autosomes of relatively little
importance. Where no Y chromosomes were
present but the numbers of X chromosomes
ranged from 1 to 5, only females were observed, even though the autosomes varied in
number from two to four sets. When a Y
chromosome was present the individual was
of the male type unless the Y was balanced
by at least 3 X chromosomes when an occasional hermaphroditic blossom was formed.
 
TABLE L4
 
Numhers of X, Y, chromosomes and A, autosome sets
 
and the sex of the various Melandrium plants
 
(Data from H. E. Warmke, 1946; and
 
M. Westergaard, 1953.)
 
 
 
 
 
 
 
Ratio
 
 
 
 
Chromosome
 
 
Warmlce
 
 
 
 
Westergaard
 
 
Constitution
 
 
 
 
 
 
 
 
 
 
X/A
 
 
 
 
4A 5X
 
 
Female
 
 
1.3
 
 
 
 
4A 4X
 
 
Female
 
 
1.0
 
 
Female
 
 
4A 3X
 
 
Female
 
 
0.8
 
 
 
 
4A 2X
 
 
Female
 
 
0.5
 
 
 
 
3A 3X
 
 
Female
 
 
1.0
 
 
Female
 
 
3A 2X
 
 
Female
 
 
0.7
 
 
Female
 
 
2A 3X
 
 
Female
 
 
1.5
 
 
 
 
2A 2X
 
 
Female
 
 
1.0
 
 
Female
 
 
 
 
Bisexual*
 
 
X/Y
 
 
 
 
4A 4X Y
 
 
4.0
 
 
Male
 
 
4A 3X Y
 
 
Malet
 
 
3.0
 
 
Male
 
 
4A 2X Y
 
 
Malet
 
 
2.0
 
 
Male
 
 
4A X Y
 
 
Male
 
 
1.0
 
 
 
 
3A 3X Y
 
 
Malet
 
 
3.0
 
 
 
 
3A 2X Y
 
 
Malet
 
 
2.0
 
 
Male
 
 
3A X Y
 
 
Male
 
 
1.0
 
 
 
 
2A 2X Y
 
 
Malet
 
 
2.0
 
 
 
 
2A X Y
 
 
Male
 
 
1.0
 
 
Male
 
 
4A 4X YY
 
 
Malet
 
 
2.0
 
 
 
 
4A 3X YY
 
 
Male
 
 
1.5
 
 
 
 
4A 2X YY
 
 
Male
 
 
1.0
 
 
Male
 
 
2A X YY
 
 
Male
 
 
0.5
 
 
 
 
 
* Occasional staminate but never carpellate
blossom.
 
t Occasional licrina])hr(i(lit ic blossom.
 
 
 
When 4 X chromosomes were present together with a Y, the plants were hermaphroditic but occasionally had a male blossom.
Two Y chromosomes almost doubled the
male effect. Two Y chromosomes balanced
4 X chromosomes to give a majority of male
plants. Only an occasional plant showed an
hermaphroditic blossom. Autosomal sex
effects, if present, were only observed when
plants had 4 sets and 3 or 4 X chromosomes
balanced by a Y chromosome. Warmke used
the ratio of the numbers of X to Y chromosomes as a scale against which to measure
clianges from complete male to hermaphroditic types. No mention is made of quantitative measures of the sex character changes
with increasing X chromosome dosages.
This is of interest since in many forms
changes in chromosome balance are accompanied by changes of phenotype which are
unrelated to sex. That such phenotypic
changes do accompany changes in autosomal
balance in Melandrium are proven, however, by further observations of Warmke in
4 trisomic types coming from crosses of
triploids by diploids. Of 36 such trisomies
analyzed, 5 or 6 of them were of different
growth habits and morphologic types. These
differences did not affect the sex patterns
since all were females. Warmke and Blakeslee in 1940 observed an almost complete
array of chromosome types from 25 to 48 in
progeny derived from crosses of 3N x 3N,
4N X 3N, and 3N x 4N. Out of about 200
plants studied, only 4 were found to show
indications of hermaphroditism. These types
were 2XY and 3XY. As noted from the
table, even the euploid plants would occasionally be expected to have an hermaphroditic blossom. Of the 200 plants, all with a
Y (XY, 2XY, 3XY) were males and all
plants without the Y (2X, 3X, 4X) were
females. In an 8-year period up to 1946.
Warmke was able to observe only one male
trisomic. From these facts he concluded
that the autosomes are unimportant in the
sex determining mechanism utilized by this
species. In their crosses they were unsuccessful in getting a 5XY plant, the point at
which the female factor influence of the X
chromosomes might be expected to nearly
equal or slightly surpass that of the single
Y. From the j^hysiologic side the obscrva
 
 
FOUNDATIONS FOR SEX
 
 
 
37
 
 
 
tion of Strassburger in 1900, as quoted by
both Warmke and Westergaard, that the
fungus Ustilago violacea when it infects
Melandrium will cause diseased plants to
produce mature blossoms with well developed stamens (filled with fungus spores) as
well as fertile pistils, shows that these females have the potentialities of both male
and female development. The case suggests
that sex hormone-like substances may be
produced by the fungus which acting on the
developing Melandrium sex structures
cause sex reversions. Should this be true,
Melandrium cells would have a parallel
with those of fish where sex hormones incor|)orated in the developing organism in sufficient quantities can cause the soma to
develop a phenotype opposite to that expected of their chromosomal type. For other
aspects see Burn's chapter and Young's
chapter on hormones.
 
Westergaard's studies (1940) with European strains of Melandrium were in progress at the same time as those of Warmke
and Blakeslee. In their broad aspects both
sets of data are concordant in showing the
l^rimary role of elements found within the
Y chromosome in determining the male sex
and of elements in the X chromosomes for
the female sex. Examination of Table 1.4
shows that the strain used by Westergaard
has a Y chromosome containing elements of
greater male sex potentialities than the
strain used by Warmke.
 
A similar difference appeared in the sex
potencies of the autosomes of European
strains. Instead of obtaining essentially
only male and female plants in crosses involving aneuploid types, Westergaard obtained from 3N females (3A + 3X) x 3N
males (3A + 2XY) 10 plants which were
more or less hermaphroditic, 21 females, and
15 males. Studies of the offspring of these
hermaphrodites through several generations
showed that their sex expression required
effects by both the X chromosomes and certain autosome combinations which under
special conditions counterbalanced the female suppressor in the Y chromosome. Increasing the X chromosomes from 1 to 4
increased the hermaphrodites from to 100
per cent in the presence of a Y chromosome.
However, in euploids these types would be
 
 
 
all males. The significance of the autosomes
is further shown by the fact that among 205
aneuploid 3XY plants, 72 were males and
133 were hermaphrodites.
 
As pointed out for Drosophila, quantitative studies on the effects of sex chromosomes and autosomes in Melandrium are
handicapped by not having a suitable scale
for the evaluation of the different sex types.
The data presented by Westergaard and by
Warmke make this difficulty become particularly evident. In the interest of quantizing
the X, Y, and A chromosome on sex the
author has assigned a value of 1 for the
male type, 3 for the female type, and 2
when the types are said to be hermaphroditic. When the types are mixed, as for example, in the data of Warmke where he says
a particular type is male with a few blossoms, the type is assigned a value of 1.05 or
1.10, depending on the numbers of these
blossoms. His bisexual type which comes
as a consequence of Y, 4X and 4A chromosome arrangement is given a value of 2, although possibly the value should be somewhat higher as it may well be that the fully
bisexuals are further along in the scale toward female development than the hermaphroditic types. The data are treated on the
additive scale both as between chromosomal
types and within chromosomal type. This is
apparently unfair if we examine the work
of Westergaard in which it looks as if particular autosomes rather than autosomes in
general make a contribution to sex determination. The results, when these methods are
used, are as follows:
 
Westergaard in Tables 1 to 5 of his 1948
paper gives information on sex types with
a determination of the numbers of their
different kinds of chromosomes. Analysis
of these data by least square methods shows
that the sex type may be predicted from
the equation
 
Sex type = - 1.37 Y + 0.10 X
 
+ 0.01 A + 2.34
 
This equation fits the data fairly well considering that the correlation between the
variables and the sex type is 0.87. This
analysis again shows that the Y chromosome has a strong effect toward maleness.
The X chromosomes are next in importance
 
 
 
38
 
 
 
BIOLOGIC BASIS OF SEX
 
 
 
with an effect of each X only about 1/13
that of the Y and in the direction of femaleness, the autosomes have one tenth the effect
of the X chromosomes but they too have
a composite effect toward femaleness. It is
to be remembered that the Y chromosome
variation is limited to 2 chromosomes
whereas the X chromosomes may total 4,
and the autosomes may range from 22 up
to 42, so that the total effect of the autosomes is definitely more than their single
effects. These data are for aneuploids. Examining Westergaard's data for 1953 for the
euploids and assigning the value of 1.5 for
the type observed when there was one Y
chromosome, four X, and four sets of autosomes, we have the following equation:
 
Sex value = -1.29 Y + 0.10 X - 0.01
 
(autosome sets) + 2.53
 
In these data, as distinct from those above,
the autosomes are treated as sets of autosomes since they are direct multiples of each
other so the value of the individual autosome is but 1/11 that given in the equation.
 
This equation shows no pronounced difference from that when the aneuploids were
utilized. The Y chromosomes have slightly
less effect toward the male side. The X
chromosomes have practically identical effects but there has been a shift in direction
of the autosomal effects on sex, although
the value is small. The constants are subject to fairly large variations arising
through chance.
 
In Table 10 of Westergaard's 1948 paper
he presented data on the chromosome constitution and sex in the aneuploids which
carried a Y chromosome. These data are of
particular interest as the plants are counted
for the i)roi)ortions of those which are male
to those which are hermaphroditic. The
plants with a Y chromosome plus an X are
all males. Those which have either one, two
or three Y chi'omosomes balanced by two
X chromosomes have 89 per cent males.
The plants with three X chromosomes and
one oi- two Y's have 36 p(T cent males and
those which have one or two ^■ chroiiiosoincs
and four X chromosomes have no males.
The woik iiiv()l\-cd in getting these data is,
of course, large indeed and is definitely
handicapped by the diiiicuHies in obtaining
 
 
 
certain types. Thus the XXYYY and the
4X + 2Y types depend only on one plant.
There are eight observations but the fitting
of the data for the X and Y constitutions
eliminates three degrees of freedom from
that number so that statistically the observations are few. The data do have the advantage that the sex differences can be
measured on an independent quantitative
scale. The equation coming from the results
is:
 
Percentage of males = 13.2 Y - 36.4 X
 
+ 134.4
 
These results show that the Y chromosome
increases the proportion of males and the
X chromosome increases the proportion of
intersexes. The data are not comparable
with those analyzed earlier as these data
are describing simply the ratio between the
males and intersexes, instead of the relations betw'een the males, intersexes, and females. The equation fits the observations
rather well, as indicated by the fact that
the correlation between the X's and Y's
and the percentages of males is 0.98, but
there are large uncertainties.
 
As a contrast to these data we have those
presented by Warmke (1946) in his Tables
2 and 3. These data give the numbers of X
and Y chromosomes found within the plants
but not the numbers of autosomes, the autosomes being considered as 2, 3, and 4 genomes. Analyzing these data in the same
manner as those of Westergaard's Table 1,
we find that the
 
Sex type = -1.05 Y + 0.22 X -0.04 A
 
-f2.25
 
As indicated for Westergaard's data, the A
effect is now in terms of the diploid type
e(iualing 2, the trijiloid 3, and the tetral)loid 4.
 
The Y chromosome has a i)ronounccd effect toward maleness, the effect l)eing someW'hat less in Warmke's data than that of
Westergaard's. The X chromosomes on the
other hand, have nearly twice the female
infhience in Warmke's data that they do in
the phiiits grown by Westergaartl. A difference in sign exists for the effect of the A
rhi'oinosnnie genom(\s as well as a difference
in [\\v (|uantitati\'e effect. Th(^ values for
 
 
 
FOUNDATIONS FOR SEX
 
 
 
39
 
 
 
both sets of data are small and toward the
male side. As Westergaard points out, the
strains used by these investigators are of
different geographic origins. The evolutionary history of the two strains may have a
bearing on the lesser Y and greater X effects on the sex of the American types.
Chromosome changes seem to have occurred
in the strains before the studies of Warmke
and Westergaard and will be discussed.
 
The location of the sex determiners has
been studied by both investigators utilizing
techniques by which the Y chromosome becomes broken at different places. These
breakages may occur naturally and at fairly
high rates in individuals which are Y + 2X
+ 2A. These facts suggest that the breakage of the Y chromosome occurs in meiosis
since the breakage comes in selfed individuals of highly inbred stocks where heterozygosity is not to be expected, in the Y
chromosome which has no homologue thus
does not synapse, and in the second meiotic
<li vision. Plants showing these initial breakages seem to have the same constitution in
all of the somatic cells of different organs as
well as in the germ cells, again pointing to
meiosis as the time of breakage.
 
In AVarmke, Davidson and LeClerc's
(1945) material, the normal offspring which
resulted from selfing 2A, 2XY plants, were
2A, 2XY (male hermaphrodites), 2A, 2X
(female), 2A, XY (male), and 2A, X2Y
(suiiermales) , and in addition two abnormal
hermaphroditic classes. These were: (1) a
type in which the female structures were
liighly developed, essentially as well developed as in 2A, 2X females and with normal stamens; and (2) a type in which there
was a complete failure of stamen development shortly after meiosis. Cytologic examination showed these types to be associated with the Y chromosome breaks.
The first type occurred when the homologous (synaptic) arm of the Y was deficient. The deficiencies ranged in size
from a short terminal loss to one which
seemed to include the entire, or nearly entire, homologous arm. It was of importance
that the degree of abnormality was not prol)ortional to the length of the deficiency.
Once a small terminal segment was lost the
change in sex type occurred and larger
 
 
 
losses seemed to have no more pronounced
effect. Chromosomes of this type showed
complete asynapsis of the Y chromosome,
indicating that its synaptic element comparable with the X was lost. Loss of as much as
one-fifth to one-fourth of the arm prevented
synapsis. These results seemed to indicate
that the Y chromosomes had lost elements
which acted as suppressors to the female development.
 
The second type observed by Warmke
was associated with a break in the differential arm of the Y chromosome. A
small terminal loss of this differential
arm was sufficient to cause male development to be arrested and sterility to result. The plants that had lost as little as
one-fourth the differential arm were therefore male sterile and were also indistinguishable from plants that had lost both
arms. The altered X chromosomes retained
their centromeres and were carried through
mitotic growth divisions to every cell of the
])lant. Only in rare cases, and with very
small fragments, was there evidence that
somatic loss may have occurred. From these
results Warmke concluded that the Y chromosome contained at least three gene complexes which operated in the development
of maleness. First there was one near the
centromere and present in the smallest fragment of the Y chromosome which initiated
male development. The stamens developed
but only just past meiosis. The second factor was found near the end of the differential arm of the Y chromosome and infiuenced complete male development. When
the entire differential arm of the Y chromosome was present, full male development
resulted. The third element appeared on the
terminal fourth of the homologous arm of
the Y chromosome and suppressed female
development. Whether this was in the pairing segment or close to it was uncertain. In
individuals with entire Y chromosomes,
plus two X chromosomes, the female structures were underdeveloped with only a
small percentage of the blossoms capable
of setting capsules with seed. When the
homologous arm was deficient the female
development was complete and every blossom produced seed-filled capsules, again
supporting the conclusion that this part of
 
 
 
40
 
 
 
BIOLOGIC BASIS OF SEX
 
 
 
the Y chromosome acted as a positive suppressor of female determining regions in the
X chromosomes. That these regions were
strictly located and the effect not due to the
quantities of the Y chromosome which may
have been present or absent was indicated
by crosses of the different types of fragments. In these crosses the resulting total Y
chromatin may have been considerably
greater in size than a normal single Y chromosome, yet the observed changes in sex
characteristics of the specific regions lost
were present. The results indicated that the
sex elements located in the Y chromosomes
were qualitatively distinct from one another
in their action and cannot be substituted
for another in quantitati^'e fashion. The
causes of the differential changes in each
case could rest on single gene differences or
possibly a closely associated nest of such
genes.
 
Westergaard's studies showed that his
plants behaved differently in some particulars from those of Warmke. His search for
the male determining elements in the Y
chromosome of his Danish plants emphasized these differences. He was able (1946a,
b) to divide the Y chromosome into four
different regions, a region corresponding to
the X chromosome in which there was
synapsis and three regions containing various sex initiating elements. When these elements were compared with those of
Warmke's it was found that they were comparable in action but differed in their order
within the Y chromosomes. The Danish
l)lants had the female suppressor region in
the end opposite the pairing region at the
extremity of the differential region. The elements initiating anther development were
found near the centromere, but toward the
pairing region. The element which coml)leted development was found near the
l)airing region or homologous section. When
compared with Warmke's results the i^osition of the different elements in the Westergaard material was the reverse of that in
the American material. Westergaard explained this difference on the assumption of
a centric inversion in the Y chromosome resulting in the change of positions. As he
suggested, it would certainly be interesting
to know the geographic distribution of
 
 
 
these two types and what would result in
progeny of crosses between them.
 
Westergaard (1948) summarized his
views on the sex-determining mechanism in
Alelandrium as follows. A trigger mechanism is built up by an absolute linkage between the female suppressor region and at
least two blocks of essential male genes in
the Y chromosome. This trigger mechanism
operates with the X chromosomes and autosomes in which the X chromosomes have female potencies and certainly the autosomes
contribute to them. The action of these two
types can only be demonstrated through
the breaking of the normal balance by polyploidy or aneuploidy. As yet, the female
])otentials of the X chromosome and certain
of the autosomes have not been analyzed to
the extent of showing whether they contain
major female sex genes or flocks of modifying genes. W^estergaard favors the hypothesis of modifying genes.
 
B. RUMEX
 
Rumex studies on sex determination took
their origin as with most dioecious plants
in chromosome examinations of the different
members of this genus (Kihara and Ono,
1923; Kihara, 1925; Ono, 1930, 1935;
Kihara and Yamamoto, 1935). These
studies showed that the species Rumex acetosa had the normal diploid female complement of 14 chromosomes consisting of a pair
of X chromosomes and 6 pairs of autosomes
and the male had one X chromosome opposed by 2 Y chromosomes with 6 pairs of
autosomes. Occasionally intersexes found in
nature had 2 X chromosomes, 2 Y chromosomes, and 3 sets of autosomes. Sex determination in this earlier data, as summarized
in Tables 3 and 4 of Yamamoto's excellent
1938 paper, when analyzed by us utilizing
the metliods of least squares, showed tiiat
X chromosomes had large female effects in
both euploids and aneuploids whereas the
net effects of the Y chromosomes and autosome sets were but one-sixth to one-tenth
as great and in the male direction. Since
that time great advances have been made
through the studies of Yamamoto (1938),
Love (1944), Smith (1955), Love and
Sarkar ( 1956) , and Love (1957). As Bridges
foi'ctold in 1939, "It may now be suggested
 
 
 
FOUNDATIONS FOR SEX
 
 
 
41
 
 
 
from genetic studies that the occurrence of
translocations is responsible for (a) the
production of multiple elements from originally single elements, for (b) the frequent
change in type of sex chromosome configuration in closely related forms, such as in the
various species of Rumex and Humulus, and
for (c) the associations and non-random
segregation of compound elements." The
]n-edictions have been borne out in the coml)lex chromosomal and genetic systems observed in some of the more recent studies.
 
Polyploids and trisomies of R. acetosa
were studied, particularly by Ono (1935)
and Yamamoto (1938). Yamamoto identified each chromosome found in each sex
type. He showed that the 6 pairs of autosomes were not ecjually balanced toward the
promoting of the male sex. The chromosomes called ai , a4 , and ae , had net effects
toward the males, whereas chromosome
pairs denoted by ao and as had net effects
toward female determination. Different balances of the different chromosome types and
pairs lead to the production of types named
after those of Bridges, supermales, males,
intersexes, females, triploids, and superfemales.
 
In his studies of euploid types, Yamamoto
set up ratios similar to those used by
Bridges in Drosophila, except that he gave
the X chromosome a weight of 100 and
each set of autosomes a weight of 60. Like
Bridges he considered Y chromosome empty
of sex genes. By using these weights he was
able to arrange the sex types in a consistent
series in which the so-called supermales had
an index of 0.56, the males 0.83, the intersexes 1.11 to 1.43, females 1.67, and superfemales 2.50. As in Drosophila the assigning
of these different values was handicapped by
the lack of any really quantitative measure
of the sex evaluations.
 
If Yamamoto's carefully tabulated data
are assigned 1 for male, 3 for female, 2 for
intersex, 3.5 for superfemale, and 0.5 for
supermale and then analyzed by least
squares for the effects of the different chromosomes on sex, the resulting equation is
 
Sex value = 1.96 + 1.09 X - 0.1 8Yi
 
- O.27Y2 - 0.28ai , + 0.06ao - O.OSa.,
 
- 0.23a4 + 0.12a.5 - 0.23a6 .
 
 
 
The X chromosomes contribute a strong
female influence and each Y a less effective
male influence. The autosomes ai , Sn and
ae are somewhat more potent toward the
male type than the Y chromosomes. Chromosomes &2 , Siz , and as have their sex genes
almost in balance. As may be noted, this
form of quantitative analysis leads to conclusions in agreement with those of Yamamoto.
 
In another section of the genus, Rumex
paucifolms, Love and Sarkar (1956) have
analyzed a tetraploid type with 28 chromosomes. The sex chromosomes were suggested
as of the XXXX and XXXY types, the
male being heterogametic. The X chromosomes were the longest whereas the Y was
the smallest chromosome in the complement. They concluded that the mechanism
of sex determination in this species is dependent on the Y chromosome's having
strongly epistatic male determinants. This
conclusion was based on the fact that the
species is dioecious and the belief that the
plants are polyploids so that the sex mechanism must be based on strong male determinants in the Y chromosomes. The strength
of these male determinants is suggested to
be less than to allow the production of
dioecious hexaploids, inasmuch as the tetraploid included not only true females and
males, but also a low frequency of androgynous individuals (Love, 1957).
 
In another group of species classified by
Love (1957) in the subgenus Acetosella
there were 5 species: 2 diploid, 1 tetraploid,
1 hexaploid, and 1 octoploid. The diploid
species have the XX and XY arrangement.
The natural tetraploid, R. tenuijolius, shows
about the same degree of pairing at meiosis
as do hybrids between it and experimentally
produced panautotetraploids of R. angiocarpus. The natural tetraploids show 4 X's
or 3X + Y for the females and males. Hexaploids derived by alloploidy from the diploid
R. angiocarpus and the tetraploid R. tenuifolius have 6 X chromosomes for the female
and 5X + Y for the male. Similarly the
octoploid R. graminifolius is an autotetraploid oi R. tenuifolius with 8 X chromosomes
in the female and 7X + Y in the male. Only
in this stage do slightly intersexual individuals occur as 2N = 57 or 58 chromosomes
 
 
 
42
 
 
 
BIOLOGIC BASIS OF SEX
 
 
 
instead of 56. At least one of the extra
chromosomes is an X. From these observations Love (1957) concluded "detailed studies and comparisons of the sex chromosomes
and their pairing in natural and experimentally produced polyploids lead to the
conclusion that the sex mechanism in this
group must be based on the evolution of a
strong male determinant in the Y chromosome, of much the same kind as in Melandrium, but stronger."
 
Within this one genus, Rumex, species are
present which seem to have the male determining elements (a) located in the autosomes as in Drosophila, and (b) in the Y
chromosomes as in man. When contrasted
with the female-determining elements of the
X chromosome these male elements seem
to vary in their sex-determining capacity
in the different species.
 
C. SPINACIA
 
Spinach is dioecious l)ut the X and Y
•chromosomes are cytologically indistinguishable from each other in at least some
species. Spinacia oleracea has 6 chromosome pairs. Recent work on sex-determining
mechanisms for this species has been conducted by Bemis and Wilson ( 1953) , Janick
and Stevenson (1955a, b), Dressier (1958),
and Janick, Mahoney and Pfahler (1959).
Dressier has indicated that each pair of the
6 chromosomes can be identified by different
morphology although most other investigators have been unable to make these
separations within their own material. He
assigns the role of sex differentiation to
the chromosome pair having the largest
size. The Y chromosome bears a satellite,
whereas the X chromosome does not. Janick
and Ellis (1959) located the sex chromosome pair through the use of the six primary
trisomies each of which is differentiated
morphologically. These trisomies have been
obtained as progeny from triploid pistillate
XXX bred to diploid staminate XY. Five of
the crosses between staminate plants of the
six trisomies mated with pistillate diploids
gave the one male to one female sex ratio
indicative of independence of the sex complex from the particular trisomies. The sixth
cross utilizing the reflex trisomic gave a one
male to two female ratio indicative of the
sex complex being within the chromosome
 
 
 
pair which in triploid condition showed the
reflex type. Each of the morphologic trisomies was associated with one of the six
chromosomes. The chromosome associated
with reflex trisomic, the sex chromosome, is
the longest chromosome and is characterized
by submedian centromere. In the somatic
cells Janick and Ellis were unable to observe obvious heteromorphism in this
chromosome pair. These results, although
not agreeing in detail with those of Zoschke
(1956) and of Dressier (1958) confirmed
the existence of races which differ with respect to morphology of the chromosomes
containing the XY factors. Janick and
Stevenson (1955b) considered that the
monoecious character did not depend on
unaltered balance between the X and Y
factors but seemed to be caused by an allele
as well as by other modifying genes. They
found that in polyploidy the sex expression
in spinach indicated that a single Y factor
was male-determining even when opposed
by three doses of the X. In their results only
the XX, XXX, and XXXX formed pistillate flowers, whereas the XY, YY, XXY,
XXXY, and XXYY were of the staminate
type. Extra doses of the Y may have further effects as illustrated by the fact that
YY plants do not produce seed, whereas
sometimes the XY staminate progenies obtained from selfing staminate plants do,
indicating that staminate plants come to
their fullest expression with the YY genotype. Chromosome recovery in the progeny
from crosses of 2N females mated to 3N
(XYY and XXY) males revealed that the
functional gametes from staminate triploids
were not confined to N and N 4- 1 types
(Janick, IVIahoney and Pfahler, 1959). The
progeny produced contained 49 per cent
diploids, 18 per cent trisomies, 0.5 per cent
14 chromosomes, 1.2 per cent 16 chromosomes, 11 per cent 17 chromosomes, 19 per
cent triploids, and 0.8 per cent 19 chromosomes. The reciprocal cross in which the
female was of the 3N type and the male
2N gave distinctly higher ratios of ancuploids. Of the progeny, 28 per cent were 12
chromosomes, 36 per cent were 13, 7 per
cent wTre 14, 0.9 per cent were 15, 2.8 per
cent were 16, 13 per cent were 17, 13 per
cent were 18, or 60 per cent of the total
progeny were aneuploids, whereas in the reciprocal cross the value was 31. Aside  
from some indication of preferential X-YY  
from some indication of preferential X-YY  
segregation in the triploid staminate parent  
segregation in the triploid staminate parent  

Revision as of 13:51, 10 June 2020


22


BIOLOGIC BASIS OF SEX


by frequent gynandromorphs, XO males, and dominant lethals among the rod and ring zygotes. It has been suggested that the instability is due to heterochromatic elements. Hinton (1959) has observed the chromosome behavior of these types in Feulgen mounts of whole eggs that were in cleavages 3 to 8. He found strikingly abnormal chromosome behavior in these cleaving nuclei. For some cell divisions chromosome reproduction was interpreted as being through chromatid-type breakage fusion bridge cycles. As a result of this behavior mosaics are formed which are intermediate between those of the half gynandromorphs and those which occur much later because of somatic crossing over. In terms of volume of cells included, the abnormal types may include only a few cells of the total organism, a fair proportion of the cells, or a full half of the whole body. These unstable ring chromosome mosaics may be a part of the secondary reproductive system or for that matter any other region of the body. When the mosaic cells are incorporated in the region of sex organ differentiation male or female type organs or parts of organs may develop as governed by the cell nuclei being X, XX or some fraction thereof.

Gynandromor|)lis appear sporadically and rarely in many species but in some instances genes which activate mechanisms for their formation are known. In the presence of recessive homozygous claret in the eggs of D. siniulans, gynandromorphs constitute a noticeable percentage of the emerging adults. The gene nearly always operates on the X received from the mother causing it to be eliminated from the cell. The resulting gynandromorphs are similar to those of D. melanogaster. The fact that the claret gene should affect the X and a particular X chromosome is suggestive of the manner in which given chromosomes arc eliminated in Sciara. Other types of sex mosaics will be found in the descriptions of other species, i)articularly in the Hymenoptci'a.

C. PARTHENO(iENESIS IN DROSOPHILA

Parthenogenesis is of interest as it changes the sex ratios in families and brings to light new sex types and novel methods


for their development (Stalker, 1954), A survey of 28 species of Drosophila showed a low rate of parthenogenesis in 23 species. Adult progeny were obtained for only 3 species. For D. 'parthenogenetica the original rate was 8 in 10,000 whereas that for D. polymorpha was 1 in 19,000. These rates could be increased by selection of higher rate parents: 151 and 70 per 10,000 unfertilized eggs of the first and second species respectively.

D. parthenogenetica diploid virgins produced diploid and triploid daughters as well as rare XO sterile diploid males. Triploid virgins produced diploid and triploid females and large numbers, 40 per cent, of sterile XO diploid males. Diploid virgins heterozygous for sex-linked recessive garnet produced homozygous and heterozygous diploid females as well as +/+/g and + /'g/g triploid females. No homozygous wild-type or homozygous garnet triploid females or garnet mosaics were found. Diploid females crossed to fertile diploid males produced few if any polyploid progeny or jjrimary X chromosome exceptional types. Of the unfertilized eggs from diploid virgins which started development, 80 per cent died in late embryonic or early larval stages. The i)arthenogenesis in diploid females depended on two normal meiotic divisions followed by fusion of two of the derived haploid nuclei to form diploid progeny, or the fusion of three such nuclei to form triploitl progeny. In the triploid virgins similar fusions of the maturation nuclei may produce diploid and triploid females but the large number of dii)loid XO sterile males were picsunicd to be the result of cleavage without prior nuclear fusion. Such cleavages without fusion in eggs of dijiloid virgins would lead to the production of haploid embryos. They were presumed i-esponsible for the large early larval and embryonic <h'atlis. These obser\ali()ns have been confirmed by the study of S|)rackling (1960) in\-olving some 2200 eggs at various stages of cleavage. Evidence from XXY diploid virgins indicated that biiuiclcar fusion in unfertilized eggs involved two terminal haploid nuclei or two central nuclei. The fact that tetraploids were not observed as progeny of ti'iploid vii'gins was considered indicative of relative inviability of this


FOUNDATIONS FOR SEX


23


type. Successful parthenogenesis was under partial control of the inheritance as 80 generations of selection increased the rate about 20-fold. Similarly outcrossing to bisexually reproducing males and reselection resulted in both pronounced increases in and survival of the parthenogenetic types.

Carson, Wheeler and Heed (1957) and Murdy and Carson (1959) have established a strain of Drosophila mangabeirai with only thelytokous reproduction. Males have been captured in nature but are rare. Fecundity of the virgin females is low but the egg hatch is 60 per cent and 80 per cent survive to adult stage. The progeny of the virgins are always diploid. In meiotic spindle formation D. mangabeirai differs from other Drosophila species in that its orientation increases the probability for fusion of two haploid nuclei into structurally heterozygous diploid females. The study of Feulgen whole mounts of freshly laid eggs indicated automictic behavior with two meiotic divisions followed by a fusion of two of the four haploid meiotic products. The absence of adult structural homozygotes in wild populations is probabl}^ explained by death during early development and by possible fusion of second division meiotic products derived from different secondary oocytes. Stalker (1956a) postulated such selective fusion in order to account for the heterozygous condition in females of Lonchoptera dubia, an automictic, parthenogenetic fly in which males are rare or unknown.

A case in which jihenotypically rudimentary females, supposed homozygous for r/r give rare and unexpected type progeny, has suggested that polar body fusion may also take place in D. melanogaster (Goldschmiclt, 1957). The rudimentary mothers producing the peculiar type are interpreted as formed by a most unusual series of events: a fertilization nucleus derived from the fusion of an r containing egg fertilized by an r containing sperm and a polar copulation nucleus derived from the fusion of a polar body containing an r genome and one having wild type. Both cell types become incorporated into the ovary. The progeny which come from these supposed rudimentary mothers are presumed to be derived from the maturation into eggs of the cells


derived from the heterozygous i)olar copulation nuclei. If these progeny-producing rudimentary mothers arise in the presumed manner they give the basis for a parthenogenetic mode of reproduction and the sex types which have been described in other Drosophila species by Stalker and Carson. There are similar, as well as other forms of parthenogenesis which affect sex (Smith, 1955) or assist in maintaining trijiloid conditions (Smith-White, 1955). A number of these types have been reviewed by Suomalainen (1950, 1954). The reader may be referred to this material for other cases and chromosome behaviors.

D. SEX INFLUENCE OF THE Y CHROMOSOME

The first function discovered for the Y chromosome in D. melanogaster was that it was necessary to male fertility (Bridges, 1916). Two and possibly more Y chromosome-borne, genetic factors were involved (Stern, 1929). Gamete maturation when these factors were lacking ceased just short of the sperm's becoming motile (Shen, 1932). The motility conferred on the sperm by the presence of the Y chromosome factors was fixed for the testes at an early stage of development as transplantation experiments, sterile testes to fertile larvae and fertile testes to sterile larvae, showed motility to be a property determined by early localized somatic influences on the developing gametes or predetermined in the diploid phase (Stern and Hadorn, 1938). This Y chromosome function was sex limited, because females without a Y were the normal fertile females and those with an extra Y also were fertile.

Neuhaus (1939) followed by Cooper (1952, 1959) and Brosseau (1960) further analyzed the Y chromosome for fertility loci. The latter showed at least two fertility loci on the short arm and five on the long arm of the Y chromosome. Data are compatible with a linear order of the genes. An additional fertility factor common to the X and Y was suggested. The Y chromosome fertility factors consequently fall in line with Bridges' concept of multiple gene loci distributed in a more or less random manner which may affect sex.

The Y chromosome has other attributes which help to explain its significance to sec


24


BIOLOGIC BASIS OF SEX


ondary if not primary sex characteristics. As with many other species it has loci for genes which are also found in the X chromosome, i.e., bobbed, as well as a limited number of bands in the salivary gland chromosome. Nucleolus organizers and at least two specialized pairing organelles similar to those in the X chromosome are found within the Y chromosome (Cooper, 1952, 1959). One of the most significant properties is the effect of extra Y chromosomes on the variegation observed for various gene phenotypes either in the normal chromosome pattern or in that accompanying translocation. In variegation one Y chromosome as extra to the normal complex is sufficient to eliminate or more rarely to much reduce the variegated expression (Gowen and Gay, 1933). When the Y chromosomes are 2 above the normal complement, the phenotypes gain two new features (Cooper, 1956). Both males and females become variegated in the expression of their eye characteristics. The males become sterile. These effects are unexpected for they are counter to any previous trends in the Y effects on these characteristics. They have reversed the direction of the effects as established by the two previous chromosome types. The variegation of the XX2Y + 2A females and X3Y + 2A males resembles that of the XX + 2A females and XY + 2A males but is more extreme, whereas the XXY + 2A and X2Y + 2A are largely nonvariegated. The fertility relations are equally aberrant: the X3Y + 2A males have the same type sterility through loss of sperm motility as that of the XO + 2A males. Bundles of sperm are formed but they do not become motile. Full-sized Y chromosomes are not required to bring about these effects, because females having a whole Y plus a piece of a second, or males with 2Y plus a piece of a third, will show the effects.

The fractional Y chromosomes furnish opportunities to test for the partial independence of the variegation and sterility effects. The two Y hyperploid males differ in their degree of fertility according to the fraction of the Y chromosome which may be present whereas the effects on variegation may be constant among groups. This is in accord with the sterility l)eing in part


independent of the factors causing the variegations. Similarly, the variegations may be shown to be partially free of the action of some elements that are not themselves members of the two sets of factors influencing fertility in the normal male.

Other phenotypic irregularities appear; eye facets may be roughened, legs shortened, and wing membranes become abnormal. On the negative side two extra Y chromosomes in females homozygous for the transformer gene, tra, do not increase in maleness or function.

The variegations of the so-called V-type position effects with translocations are suppressed, as in the normal type described, by one extra Y but are nonetheless variegated when two extra Y chromosomes are present. That these effects are caused by there being two supernumerary Y's is indicated by the fact that XX2Y females are fertile and X3Y males sometimes lose a Y chromosome in their germinal tracts and become fertile. A number of mechanisms have been suggested to account for these results but most have proven unsatisfactory. A balance interpretation for the X, Y and autosomes like that for sex, as suggested by Cooper (1956) is compatible with the somatic cell variegations for euchromatic loci transferred to the heterochromatic regions and the sterility-fertility relations expressed by the different chromosomal types.

The variegation effects of the Y chromosome take on further significance. Baker and Spofford (1959) have shown that 15 different fragments of the Y chromosome when studied for their contributions to variegation differ in their effects with the differences often not related to the size of the fragment, thus indicating that the Y chromosome has linearly differentiated factors capable of modifying the variegated phenotype.

Other indications of genetic activity of the Y chromosome were given by Aronson (1959) in her study of the segregation observed in 3rd chromosome translocations. A deficiency for the region of the 3rd chromosome centromere is lethal when homozygous. Both males and females are fully \'iable when this deficiency is heterozygous. XO or haplo IV deficient males die. How


FOUNDATIONS FOR SEX


25


ever, in the presence of a Y chromosome the deficient haplo IV males become viable. The lability of this last class indicates that the Y chromosome is genetically active, can compensate for the autosomal deficiency, and thus alter the progeny sex ratio.

In D. virilis the situation is somewhat different from that observed by Cooper in 1956 in D. melanogaster. Baker (1956) has shown that, in a translocation, males having two Y chromosomes plus a Y marked with a 5th chromosome peach are fertile. These results seem to indicate a species difference in the effect of the extra Y on fertility or the Y with the inserted peach locus is not a complete Y and in consequence the true composition of these males is X + 2Y plus a fragment of the Y. The X chromosomal associations in the multichromosomal types are shown to be by trivalents or by tetravalents. The segregation data indicate that the pattern of disjunction of trivalents is a function of the particular Y chromosome involved. In X2Y males with normal Y's or with one normal and one marked Y, the Y's disjoin almost twice as frequently as they do from trivalents with two identical Y's. Tetravalent segregation is almost entirely two by two, with no preference for any of the three types of disjunction.

An odd situation was reported by Tokunaga (1958) in substrains of Aphiochaeta .vanthina Speiser. When a male of the substrain was crossed to individuals bearing 3rd chromosome genes of the original strains, the mutant genes for brown, and so on, behaved as if they were partially sexlinked in the following generations. On the other hand, when a female carrying the partially sex-linked genes on the X and Y chromosomes. Abrupt or Occhi chiari, was crossed to the male of the substrain the characters segregated as though they were autosomal. As a working hypothesis it was suggested that in this species the Y chromosome had the major male determining factors. The "special" male arose as a translocation of these factors to the third chromosome with the consequent change in linkage relations. Data on the role of the X chromosome in sex determination in this species have not yet been obtained but if they support the interpretation they indicate real differences between this species and that of


Drosopliila in the location of the sex genes. The results are reminiscent of those obtained by Winge in Lebistes as well as those in Melandrium and other forms in which the Y or W chromosomes may contain strong sex genes for either sex. They would further support the thesis that sex genes may be distributed to almost any loci within the inheritance complex.

E. M.\TERNAL INFLUENCES ON SEX RATIO

Aside from chance and specific genetic factors, sex ratio is subject to effects from agents intrinsic in the cells of the mothers (Buzzati-Traverso, 1941; Magni, 1952). Strains of Drosophila bifasciata (Magni, 1952, 1953, 1957), D. prosaltans (Cavalcanti and Falcao, 1954), D. willistoni and D. paulistorum Spassky, 1956 have been isolated which were nearly all of the female sex even though the mothers were outbred to other strains having normal ratio bisexual progeny. Study of these strains by the above workers and Malogolowkin (1958) IVIalogolowkin and Poulson (1957), and Malogolowkin, Poulson and Wright (1959), as well as by Carson (1956) have shown inheritance strictly through the mother regardless of the genetic nature of the males to which they were bred. The unbalanced ratio was retained even when the original chromosomes had been replaced by homologous genomes from lines giving normal male and female progenies. Transmission of this unbalanced sex ratio was through the cytoplasm. Eggs fertilized by Y-bearing sperm died early in the course of development. Malogolowkin, Poulson and Wright (19591 have shown that the high female ratio and embryonic male deaths may be transferred from affected females to those which do not normally show the condition through injection of ooplasm from infected females. Ooplasm of this same type is, w^hen injected into males, suflficient to cause death to occur within 3 days. In the females a latent period of 10 to 14 days after ooplasm injection was apparently necessary to establish egg sensitization. Once established the condition could be transmitted through the female line for several generations. The ooplasm injections were not as efficient in establishing these lines as females found naturally infected. Unisexual broods may


26


BIOLOGIC BASIS OF SEX


fail to appear, in some instances fail to transmit the condition or produce intermediate progeny ratios, as well as in some instances to skip a generation. The infectious agent was found to vary in different species. Magni (1953, 1954) for D. bifasciata found that temperature above normal tended to remove the cytoplasmic agent making it ineffective. This result has a parallel to the action of temperature on certain viruses, as for instance that involved in one of the peach tree diseases. On the other hand, Malogolowkin (1958) ior D . willistoni found no temperature effect. Malogolowkin further found that the cytoplasmic factor was not independent of chromosomal genes since some wildtype mutant strains induced reversions to normal sex ratio. Recently Poulson has established the complete correlation of the high female progeny characteristic with the presence of a Trepomena in the fly's lymph. As with mouse typhoid variations, lethality was genotype dependent.

These cases have interest from more than the sex ratio viewpoint. Cytoplasmically inherited susceptibility of some strains of D. 7nelanogaster to poisoning by carbon dioxide as studied by L'Heritier (1951, 1955) depended on the presence of some cytoplasmic entity which passed through the egg cytoplasm and also, but less efficiently, by means of the male sex cells to the progeny. The carbon dioxide susceptibility was transmitted through injections of hemolymph or transplantation of organs of susceptible strains. The transmissible substance had a further property of heat susceptibility. The COo susceptibility differed from that of "sex ratio" in being partially male transmitted. It agrees with sex ratio" D. bifasciata in being heat susceptible but differs from D. willistoni "sex ratio" in this respect.

D. bifasciata may behave differently than D. willistoni in that Rasmussen (1957) and Moriwaki and Kitagawa (1957) both conducted transplantation experiments with negative results. However, it is jiossible that these experiments may be affected by a different incubation period for the injected material as contrasted with that for D. willistoni. A range of possibilities evidently exist for extrinsic effects in sex ratio.

Carson (1956) found a female producing strain of D. borealis Patterson, which car


ried on for a period of 8 generations, produced 1327 females with no males. The strain showed no chromosomal abnormalities. It had 3 inversions. The females would not produce young unless mated to males from other strains having biparental inheritance. This requirement, together with gene evidence, showing that the females were of biparental origin, is against the female progenies being derived by thelytokous reproduction.

F. MALE-INFLUENCED TYPE OF FEMALE SEX RATIO

Sturtevant in 1925 described exj^eriments with a stock of D. affinis in which a great deficiency of sons was obtained from certain males, regardless of the source of females to which such males were mated. The few males obtained from such matings were normal in behavior but some of the sons of females from such anomalous cultures again gave very few sons (]Morgan, Bridges and Sturtevant, 1925).

Gershenson (1928) in a sampling of 19 females caught in nature found two that were heterozygous for a factor causing strong deviations toward females (96 per cent to 4 per cent males) whereas the normal D. pseudoobscura ratio was nearly 1 to 1. The factor was localized in the X chromosome and was transmitted like an ordinary sex-linked gene. Its effect was sex limited as it was not manifest in either heterozygous or homozygous females. It had no effect on the development of zygotes already formed but strongly influenced the mechanism of sex determination through the almost total removal of the spermatozoa with the Y chromosome from the fertilization process. Egg counts showed the divergent ratio toward females was not caused by death of the male zygotes inasmuch as there was no greater mortality from such cultures than from controls giving 1 to 1 sex ratios.

Sturtevant and Dobzhansky (1936) showed that an identical or nearly identical phenotypc to that obscM'ved in Drosophila obscura was present in D. pseudoobscura. The D. pseudoobscura carrying the factor were scattered over rather wide geographical areas. Comparable types also were found in two other species D. athabasca and D.


FOUNDATIONS FOR SEX


27


azteca. This genie "sex ratio"' sr lies in the right limb of the X of races A and B of D. pseudoobscura. Like the other cases analyzed, males carrying sr have mostly daughters and few sons regardless of the genotypes of their mates. Structurally the female sex ratio came through modification of the development of the sex cells of the male to give a majority of X-bearing sperm. Cytologic study showed that in "sex ratio" males the X underwent equational division at each meiotic division, whereas the autosomes behaved normally. The Y chromosome lagged on the first spindle, remained much condensed, and its spindle attachment end was not attenuated. The Y chromosome was not included in either telophase group of the first meiotic division but was left behind. It was sometimes noted in one of the daughter cells where it formed a small nucleus. Among 64 spermatocytes examined, all had an X chromosome and none a Y chromosome in their main nuclei. The micronuclei containing Y played no further part in the division, became smaller and exceedingly contracted. The final fate of the micronuclci was uncertain, but there was no indication that any spermatids died or were abnormal.

G. HIGH MALE SEX RATIO OF GENETIC ORIGIN

In 1920, Thompson described a recessive mutant in D. melanogaster which killed all homozygous females but changed the male phenotypes only slightly, the wings standing erect above the back. The locus of the mutant was 38 in the X chromosome. This mutant was a leader for a class in which the genes affect only one sex but are innocuous to the other.

Bobbed-lethal, a sex-linked gene found i)y Bridges, is a gene of this class but one in which the mechanism of protection to the other sex is known. It kills homozygous females but does not kill the males because of the wild type allele which the males have in their Y chromosomes. The presence of this bobbed-lethal in a population consequently leads to male ratios higher than wild type.

A recessive gene in chromosome II, discovered by Redfield (1926), caused the early death of the majority of female zygotes and led to a sex ratio of about 1 female to 5.5 males. The effect was trans


mitted througli both males and females. The missing females may have died largely in the egg stage although disproportionate losses also occurred in the larvae and pupae. The maternal effect was attributed to an influence exerted by the chromosome constitution of the mother on the eggs before they left the mother's body. Because of this maternal lethal, the families from these mothers have the normal number of sons but few or no daughters.

A culture was observed by Gowen and Nelson (1942) which yielded only male progeny, 136 in all. Some of the male progeny were able to transmit the male-producing characteristics to half of their daughters without regard to the characteristics of the mates to which they were bred. The inheritance was without phenotypic effect on the males. When present in the heterozygous condition in females, the female's own phenotype gave no indication of the gene's presence. The inheritance was sex limited in that it affected only the eggs laid by the mothers carrying it. In these eggs it acted as a dominant lethal for the XX zygotes. The gene was found located in the 3rd chromosome between the marker genes for hairy and for Dicheate at approximately 31. The X eggs carrying the gene, Ne, died in the egg stage at between 10 and 15 hours under 25°C. temperature. The gene was as effective in triploids as in diploids. One dose of this gene in triploids caused them to have only male progeny and male type intersexes. The presence of this gene caused the elimination of any embryos with chromosomal capacities for initiating and developing the primary or secondary female sexual systems. Since the gene itself was heterozygous in the female the meiotic divisions of the eggs would cause the gene to pass into the polar bodies as often as to remain in the fertilization nucleus yet the lethal effects are found in all eggs. The antagonism was between the egg cytoplasm and the XX fusion products. Supernumerary sperm of the Y type were not sufficient to overcome the lethal effects. An XY fertilization nucleus was necessary for survival.

This case has parallel features with that observed by Sturtevant (1956) for a 3rd chromosome gene that destroys individuals bearing the first chromosome recessive gene


28


BIOLOGIC BASIS OF SEX


for prune. The gene responsible was found to be a dominant, prune killer, K-pn, which was located in the end of the 3rd chromosome at 104.5. Prune killer was without phenotypic effect so far as could be observed except that it acted as a killer for flies containing any known allele of prune. It was equally effective in either males or females, hemizygous or homozygous, for prune. The larvae died in the 2nd instar but no gross abnormalities were detected in the dying larvae. This case has interest for the Ne gene in that the killer gene occupies a locus in the 3rd chromosome although removed by about 70 units from Ne, and acts on a specific genotype, the prune genotype, whereas Ne acts on the specific XX genotype. The known base for action of the prune killer is genetically much narrower than that for the female killer, Ne, in that prune killer acts on an allele within a specific locus in the X chromosome, and Ne acts on a type coming as a product of the action of two whole sex chromosomes. It is, of course, conceivable that when ultimately traced each action may be dependent upon specific changes of particular chemical syntheses. The action of these genes is also of interest from another viewpoint.

In mice there is a phenotype caused by the homozygous condition of a recessive gene (Hollander and Gowen, 1959) which acts on its own specific dominant allelic type in its progeny so as to cause an increased number of deaths between birth and two weeks of age, as well as causing the long bones to break and the joints to show large swellings. The lethal nature of this interaction is not a product of the mother's milk nor does it show humoral effects such as those observed with erythroblastosis in the human. The interallelic interaction is sex limited in that it is confined to the mother and is without effect when the male has the same genotype.

H. FEMALE-MALE SEX RATIO INTERACTIONS

A case in D. affinis involving the interaction of a "female sex ratio" factor and an autosomal "male sex ratio" factor has been studied by Novitski (1947). Starting from stock which had a genetic constitution for the "sex ratio" X chromosome which ordinarily causes males carrying it to produce


only daughters, he was able to establish a recessive gene in chromosome B in whose presence only male offspring resulted. The genetic constitution of the male was alone important. High sterility accompanied the "male sex ratio" males breeding performance. The "male sex ratio" parents yielded 95 fertile cultures having an average of 25 individuals per culture. Females appeared in 10 of these cultures with an average of 3 per culture for those producing females. The total sex ratio was 77 males per female. The males were morphologically normal. They carried an X chromosome of their mothers. Cytologic observation of spermatogonial metaphases of 3 progeny showed that the Fi males may or may not have carried a Y of their fathers. This agreed with the tendency for sterility in these "male sex ratio" males. The ventral receptacles of the females from 4 such sterile cultures when examined proved devoid of sperm. The occasional female offspring of the "male sex ratio" males had one X chromosome from each parent. Females mated to "male sex ratio" males show large numbers of sperm in the ventral receptacles (7 out of 8 cases) , although such females had usually produced no or very few offspring. The sperm, if capable of fertilization, must have had a lethal effect on the zygotes. The recessive nature of the factor in chromosome B indicated that its potential lethal effect originated during spermatogenesis rather than at the time of fertilization. This lethal period corresponds to that when the "female sex ratio" factor in the X chromosome is active.

The research on sex ratio in Drosophila reviewed shows that through the interplay of the sex chromosome-located and autosomal-located factors all types of sex ratios from only females in the family to only males in the family may be generated.

V. Sex Determination in Other Insects


Sciara, a fungus gnat, offers sex-determining mechanisms quite different from any yet offered in Drosophila. Sciara is unique, yet in its uniqueness, it illustrates basic facts that were rediscovered in other species only through the study of abnormal


FOUNDATIONS FOR SEX


29


forms some of which were created as induced developmental, chromosomal, or hormonal abnormalities. The complexities responsible for the remarkable facts were analyzed by Metz and his collaborators. The following brief review is based upon Metz's (1938) summarization of the chromosome behavior problem to which the reader is referred for further information. Of Sciara species studied 12 out of 14 have their basic chromosome groups composed of three types of chromosomes: autosomes, sex chromosomes, and "limited" chromosomes. Two species, S. ocellaris Comstock and S. reynoldsi Metz, lack the "limited" chromosomes. Two sets of autosomes and three sex chromosomes are found in the zygotes of all Sciara species at the completion of fertilization. The behavior of these chromosomal types will be considered in sequence.

The autosomes behave normally in somatic mitosis and in oogenesis. There is cytologic and genetic evidence for synapsis, crossing over, random segregation, and regular distribution of chromosomes and genes in the female.

In spermatogenesis the story is quite different. The first maturation division is unipolar. Genetic evidence shows a complete, selective segregation of the maternally derived autosomes and sex chromosomes from those of paternal origin. The paternal homologues move away from the pole, are extruded, and degenerate. The second spermatocyte division is likewise unequal and one of the products, a bud, degenerates. Thus a spermatogonial cell passing through meiosis gives rise to only one sperm. At the second spermatocyte division all of the chromosomes (maternal homologues) except the sex chromosome undergo an equational division but both halves of the sex chromosome enter into the same nucleus. This nucleus becomes the sperm nucleus. The chromosomes at the opposite pole form a bud and degenerate. Fertilization of the Sciara egg is normally monospermic not polyspermic.

The sex chromosomes XXX of the fertilization nucleus are derived, two from the sperm and one from the oocyte. Their destiny depends on whether the egg they are


in develops into a male or a female imago. In male early development, those nuclei which are to become male soma lose the two sex chromosomes XX, contributed by the father's sperm, to give X + 2A soma. These chromosomes fail to complete mitosis at the 7th or 8th cleavage division and are left to degenerate in the general cytoplasm when there are no true cells in the soma region and no membranes surround the nuclei. Those embryos which are to become female soma at the same cleavage cycle eliminate but one paternal X chromosome. On a chromosome basis the soma cells respectively become X, plus two sets of autosomes, give rise to males on differentiation, or become XX + 2A and develop female organs (Du Bois, 1932). The germ line nuclei for each sex, on the other hand, remain unrestricted in their development. They retain their XXX constitutions until the first day of larval life, or about 6 hours before the formation of the left and right gonads (Berry, 1941), when they eliminate a single paternally derived X. The X which is rejected or makes its own exit is always one of two sister chromosomes contributed by the father. The process of loss is strikingly different from that noted in the soma-building nuclei. Both cell walls and nuclear membranes are present. The path of the X chromosome is through the nuclear membrane into the cytoplasm where degeneration eventually takes place. The loss occurs at a time when there is no mitotic activity and the chromosomes are separated from the cytoplasm by an intact nuclear membrane. Some Sciara species are characterized by females which produce only unisexual families — practically all females or practically all males. Genetically, the sex of progeny is accounted for by sex chromosomes, X' and X, which are so designated because of their physiologic properties. Mothers having only female progeny are characterized by always having the X' chromosome in all their cells, X'X + 2A, whereas mothers whose progeny are all males are XX + 2A (Moses and Metz, 1928). The all female and all male broods are observed in about equal numbers. The male has no influence on sex ratio. Normally the progeny in all female broods will be X'X + 2A or XX + 2A in soma


30


BIOLOGIC BASIS OF SEX


and germ lines, whereas the all male progeny broods will be only X + 2A in the soma and XX + 2A in the germ line.

Studies of Grouse (1943, 1960a, b) show nondisjunction may occur and eggs entirely lacking sex chromosomes or having double the normal number may be produced. When the nondisjunctional eggs lacking X chromosomes are from X'X females and are fertilized by sperm contributing two sister X chromosomes, one X (as expected of X'X mothers, not two as expected of XX cells) is eliminated at the 7th or 8th cleavage to give cells which develop into "exceptional" males with XO + 2A soma and XX + 2A germ line, all X chromosomes being of paternal origin. The casting out of the single X chromosome is consequently controlled by the characteristics of the egg cytoplasm derived from the X'X mother rather than by the kind of chromosomes present in the nuclei at the 7th or 8th cleavage stage of the eml)ryo.

Similarly when nondisjunctional eggs having both XX chromosomes of the XX mothers are fertilized by the normal spermbearing XX chromosomes, the zygotes are XXXX. At the 7th or 8th cleavage both paternal X chromosomes are eliminated and the resulting soma develops into an exceptional" female, XX + 2A, often with 3 X's in her germ line. These observations confirm the earlier interpretations that the X'X or XX constitution of the mother conditions her eggs respectively to cast out 1 or 2 parental X chromosomes at the 7th or 8th cleavage according to the cytoplasmic constitution of the egg. Somatic sex development is visually determined only after this stage when the chromosomes of the somatic cells take on the familiar aspect of either XX + 2A for the females or X + 2A for the males.

Although at present there are few examples in other species, this cytoplasmic conditioning of early embryologic development by the mother's genotyj)e may be a common rather than a rare occurrence of development. The expression of the events to which this control may lead may be quite different in different cases. The spiral of snail shells was an early recognized case (Sturtcvant, 1923) . In Drosophila, a gene acting through the mother's genome allows onlv


male embryos to survive (Gowen and Nelson, 1942), suggesting that this gene acts through her cytoplasm in a manner comparable to that of the X' and X chromosomes of Sciara. In either case the genotype would not be considered a primary sex factor, profound as the effect on sex may ultimately be.

Sciara also has strains or species where the progeny of single matings are of both sexes, bisexual, yet the chromosomal elimination mechanisms remain as described above save for the fact that they now operate within broods rather than between broods. Sex ratios for the families within these bisexual strains are extremely variable, 1:0 or 0:1 ratios not being infrequent and 1 : 1 ratios being the exception. An instance of a bisexual strain arising from a unisexual line has been analyzed by Reynolds (1938). The females were found to be 3X in their germ line and produced 2X eggs which developed into daughters and X eggs which became sons. Loss of the extra X from this line resulted in the line's return to unisexual reproduction.

In general, bisexuality as contrasted with unisexuality seems to be caused by differences in the X'X-XX mechanisms, either specific for the mechanism, or induced by modifying genes which cause different embryos within the same progeny to cast out sometimes one or sometimes two X chromosomes from all their somatic nuclei. This casting out occurs uniformly for all nuclei of a given embryo as is shown by the fact that sex mosaics or gynandromorphs are as seldom observed in bisexual as unisexual strains.

Some species of Sciara have large chromosomes which are in essence "limited" to the germ line since they are lost from the somatic nuclei at the 5th or 6th cleavage division. Other species lack these chromosomes, yet agree with the rest in the behavior of their remaining chromosomes and in sex determination. So far as is now known the "limited" chromosomes seem empty of inheritance factors influencing either sex or unisexual vs. bisexual broods or for that matter other characteristics (their persistence seems to make this latter unlikely I. Their presence does lead to a question of the efficacv of desoxvribonucleic acid


FOUNDATIONS FOR SEX


31


(DNA) as the all inclusive agent in inheritance for they are clearly DNA-positive.

These cytogenetic observations clear up most of the events leading to sex differentiation and the delayed time in embryologic development when it takes place, but, as INIetz points out, other puzzling questions are raised. Observed chromosome extrusions from cell nuclei are rarities today even though discovered for Ascaris 60 years ago. Why should this mechanism be so well defined in Sciara? Why should the mode of elimination be so accurately timed and yet be different in method and time for the soma and germ cell lines? Two of the three X chromosomes in the fertilized egg are sisters from the father and should be identical. What is special in the 7th or 8th cleavage that causes elimination in the soma plasm but not in the germ plasm? Why should the casting out of one of these same paternal X's in the germ plasm of both sexes be reserved for a much later stage in cleavage and by a different procedure?

The observations of Grouse (1943, 1960a), obtained from translocations and species crosses, are critical in showing that the chromosome first moA'ing in its entirety to the first differentiating pole of the second spermatocyte division is the X and is the one taking part in the later postfertilization sex chromosome elimination of the 7th or 8th cleavage. The X heterochromatic end of the chromosome and not the centromere region seems to be responsible for the unique behavior of this chromosome (Grouse, 1960b). Induced nondisjunctional eggs of X'X (female-producing) mothers having as a consequence no sex chromosomes, when fertilized by the normal sperm bringing in XX chromosomes, develop into embryos eliminating one of these X's at the 7th or 8th cleavage, as expected of the eggs of X'X mothers, and become "exceptional" males with X + 2A soma and XX paternal germ line. Imagoes of these embryos become functional but partially sterile males transmitting the paternal X, a reversal of the normal condition. Similarly the nondisjunctional eggs retaining both X's, XX from male-producing mothers, and having 4 X's on fertilization eliminate the two paternal X's, as occurs normally in XXX eml)ryos, to become "exceptional" females with


2X or sometimes in species hybrids 3X soma.

Phenotypic sex in Sciara on this evidence depends on the adjustment of the X chromosome numbers and their contained genes with this adjustment regularly taking place, not at fertilization as in most forms like Drosophila, but at the 7th or 8th cell cleavage of embryologic development. The X' vs. X chromosomes of the mother predispose her haploid egg cytoplasm to time specific chromosome elimination. The germ line cells on the other hand may regularly have other chromosome numbers than the soma or exceptionally tolerate other numbers as extra X's (Grouse, 1943), or extra sets (Metz, 1959).

Triploids of 3X and 3A soma have not been found in pure Sciara species. Salivary gland cells of a triploid hybrid S. ocellaris x aS. reynoldsi have two sets of S. ocellaris and one of S. reynoldsi chromosomes (Metz, 1959). Ghromosome banding in 2X -I- 3A, 3X + 3A larval glands showed the »S. ocellaris homologues were heterozygous. The 3X + 3A type chromosomes were of typical female appearance. The 2X:3A-type had X chromosomes of typical male type, very thick, pale and diffuse. The mosaic salivaries were a mixture of the two types of cells. These results suggest that the 3X:3A would be a typical female. The intersexes 2X -I- 3A conceivably have a range in male and female organ development depending on how the S. ocellaris and S. reynoldsi chromosomes act. If the chromosomes are equivalent, the gene expression would be expected to be that of a 2X to 3A or a phenotypic intersex. If they act separately the S. ocellaris genes would be in balance for female determination, whereas the S. reynoldsi autosomal genes would have no X S. reynoldsi genes to balance them and presumably would be overwhelmingly male in effect. The phenotypic outcome would be in doubt.

The maternally governed cleavage and chromosomal sequence occurring before the 8th embryonic cell division, although related to the subsequent events leading to the particular sex, is unique to Sciara and its relatives. Phenotypic sex expression apparently starts with the somatic nuclei having either X + 2A or XX + 2A as their chro


32


BIOLOGIC BASIS OF SEX


mosomal constitutions. Sciara is like Drosophila and many other genera in its basic sex-determining mechanism. There is a sexindifferent period when the maternal genotype may influence development through cytoplasmic substances. This is followed by active differentiation when the sex phenotypes may be influenced by various agencies but when passed becomes fixed for phenotypic sex. The indifferent period may represent a fairly large number of early cell generations as in amphibia or fish. Somatic and germ cells are labile but are normally susceptible to control only by forces developed within the organism as present knowledge indicates for Sciara. In Sciara, only the somatic cells conform in chromosome number to their expected phenotypes. The germ cells show not only a regulated instability of their chromosome number but the number is different from that of the supporting somatic cells. The fact that sex mosaics in Sciara may develop both an ovary and testis in the same animal (Metz, 1938; Grouse, 1960a, b) shows localized phenotypic sex determination occurring fairly late in development rather than the generalized determination that would occur if sex were established at fertilization.

The germ line chromosomal differentiation from that of the somatic tissue has parallels in a number of fairly widely dispersed species. The significance of these types to soma line determination and to the reversibility or irreversibility of differential gene activation in differentiated cells subsequent to embryogenesis has been considered by Beermann (1956). Although these types are yet in the fringe areas of our knowledge surrounding the general paths taken in the evolution of sex determining mechanisms, they promise to have more generality as clarification of gene action becomes more exact.

B. APIS AND HABROBRACON

Hymenopteran interi:)retations of sex determination have largely turned on studies of the honey bee. Apis mellijera Linnaeus and Habrohracon juglandis Ashmead. Dzierzon early established that the drones of honey bees come from unfertilized eggs, whereas the females, queens, and workers come from fertilized eggs so that sex differ


entiation is marked by an N set of chromosomes for the male and 2N for the female. This mechanism was satisfactory until it was realized that on a balanced theory the doubling of a set of chromosomes, if truly identical, should not change the gene balance and consequently not the sex. Further doubt was cast on the N-2N hypothesis for sex determination by the discovery of diploid males by Whiting and "Whiting (1925). These difficulties were resolved by Whiting (1933a, b) with the suggestion that the two genomes in the female were not truly alike but actually contained a sex locus linked with the gene for fused which was occupied by different sex alleles as Xa and Xb . In this A'iew the heterozygous condition would lead to the production of females, whereas in the haploid or homozygous condition either of these genes would react to produce males. Difficulties with this hypothesis became evident in that the ratios of males to females in certain crosses were significantly divergent from those which were expected. Evidence was collected and trials made to interpret these difficulties (Whiting, 1943a, b) by assuming that on fertilization by Xa-bearing sperm the polar body spindle would turn so as to cast out the Xa chromosome and retain the complementary Xb in the majority of cases instead of in a random half. Snell (1935) offered the hypothesis that there could be several loci for sex genes, such that the X locus might be a master locus, but when this locus was in homozygous condition the sex would then be controlled by genes in other loci in the genome. As pointed out by Bridges (1939) this hypothesis would satisfy that of sex gene balances postulated in Drosophila and of change in emphasis on loci for sex genes as observed by Winge (1934) in Lebistes. However, the work of Bostian (1939) called both hypotheses into question. Consideration of these further results led to the multiple allele hypothesis of complementary sex determination for Habrobracon (Whiting, 1943a) taking a similar form to that of the sterility relations observed for a single locus in the white clover of INIelilotus. Under this system the sex locus would be occupied by multii)le alleles, any one of which or any combination of identical alleles would be male-producing, but any combination of two different al


FOUNDATIONS FOR SEX


83


Iclcs would l»e female determining. By having a sufficient number of these genes the jiroduction of diploid males would be curtailed to a point at which their various frequencies could be explained. In support of this liyi)othesis, multiple alleles to the number of at least 9 were found to exist for thi.> single sex locus.

In principle at least, the honey bee could have the Habrobracon scheme of sex determination. Rothenbuhler (1958) has recently collected the researches which test this possibility. Tests of the multiple allele hypothesis as applied to the honey bee were made by Mackensen (1951, 1955) who interpreted evidence for inviable progeny produced by mating of closely related individuals as proof that this species as well as Habrobracon juglandis follows the multiple allelic system. The discovery of male tissue of bii)arental origin in mosaic bees from related i)arents was considered as further evidence for the multiple allelic theory of sex determination (Rothenbuhler, 1957).

Most recent cytologic evidence supports the concept that there are 16 chromosomes in the gonadal cells of the male and 32 in those of the female (Sanderson and Hall, 1948, 1951; Ris and Kerr, 1952; Hachinohe and Onishi, 1952; Wolf, 1960». Hachinohe and Onishi (1952) found 16 chromosomes as characteristic of the meiosis in the drone. Wolf observed a nucleus in both bud and spermatocyte of the only maturation (equational) division.

The greatest progress has been made in understanding the mechanisms of sex mosaicism in the Hymenoptera species. These mosaics, although ordinarily of rather rare occurrence, have a direct bearing on sex determination and development. In Apis, iiolyspermy furnishes the customary basis for their formation. One sperm fertilizes the haploid egg nucleus and another sperm, which has entered the egg instead of degenerating as it ordinarily does, enters into mitotic cleavage and eventually forms islands of haploid cells of paternal origin among the diploid cells derived from the fertilized egg (Rothenbuhler, Gowen and Park, 1952). Evidence showing that genetic influences affect the sperm nucleus toward stimulating its independent cleavage is found to exist in Apis material (Rothen


buhler, 1955, 1958). Tliousands of gynandromorphs have been observed in Apis, all but a small number of which have been produced in this manner. This method of initiating sex mosaics also exists for Habrobracon (Whiting, 1943b) but is rather rare. In Habrobracon the frequent mode has a different origin. The gynandromorph is formed from the cleavage products of the normal fertilization of the egg nucleus combined with those of a remaining nuclear product of oogonial meiosis. Under these conditions the female tissue is 2N of biparental origin and the male tissue is N of maternal origin (Whiting, 1935, 1943b). This type is less frequent in Apis but one specimen has been described by Mackensen (1951).

A number of other ways in which sex mosaics may occur are occasionally expressed in these species. Three different kinds of male tissue have been observed in individual honey bee mosaics produced by doubly mated queens — haploid male tissue from one father, haploid male tissue from the other genetically different father, and diploid male tissue of maternal-paternal origin. In other cases, the diploid, biparental tissue was female and associated with two kinds of male tissue (Rothenbuhler, 1957, 1958) . Cases where the haploid portions of the sex mosaics are of two different origins, one paternal and the other maternal, while the female portion is representative of the fertilized egg, are known in Habrobracon (Whiting, 1943b). Similarly, Taber (1955) observed females which were mosaics for two genetically different tissues and which he accounted for as the result of binucleate eggs fertilized by two sperm. Mosaic drones of yet another type were observed by Tucker (1958) as progeny of unmated queens. They were interpreted as the cleavage products from two of the separate nuclei formed in meiosis. These cases represent a number of the possible types that arise through meiotic or cleavage disfunctions under particular environmental or hereditary conditions.

Tucker (1958) studied the method by which impaternate workers were formed from the eggs of unmated queens. For this purpose he used genetic markers, red, chartreuse, ivory, and cordovan. Observations


34


BIOLOGIC BASIS OF SEX


Avore nuult' i)ii 237 workers from hotcrozygous mothers. For the chartreuse loeus. 12 to 20 per cent were homozygous, for x\w i\ory locus 1.8 per cent, and (he cordovan locus on lesser numbers per t-ent. An egg which is destined to become an automictic worker, a gynandromorph with somatic male tissue or a mosaic male, is retained within the queen for an unusually long time during which meiosis is suspended in anaphase 1. Normal reorientation of first division spindle is i)ossibly inhibited by this aging so that after the egg is laid meiosis II occurs with two second division spindles on sejiarate axes as Goldschmidt conjectured for rarely fertile rudimentary Drosophila (19")7i. Two polar l)odies and two egg prontich^i are formed. The polar bodies take no fiu'ther part in develoinnent. In most of the unusual eggs the two egg pronuclei unite to form a diploid cleavage nucleus which develops into a female. Rarely the two egg pronuclei develop separately as two haploid cleavage nuclei to form a mosaic male. Two unlike haploid cleavage nuclei, one descending from each of the two secondary oocytes after at least one cleavage di\ision. unite to form a dijiloid cleavage nucleus which develops together with the remaintler of the haploid cleavage nuclei to produce a gynandromorph with mosaic male tissues. The male and female tissues within these unusual gynandromor[ihs or female types were identical with normal drone or normal female tissues so were probably haploid and diploid resiiectively. Genetic segregation observed within the mothers of automictic workers allows the estimation of the distance between the locus of the gene and its centromere. "With random recombination and "central union" Tucker estimates this distance for the chartreuse locus to centromere as 28.8 units and for the ivory to its centromere 3.6 units. Four lines of bees of diverse origin all showed a low percentage of automictic or gynandromorphic types produced from queens in each line. Various chance environmental conditions apparently influence the rate of production of these types. However, there were some females in two of the lines with higher frequencies inciicating that innate factors may have significant eft'ects on their frequencies. Observations on Drosophila spe


cies — I), porthenogeuetica (Stalker, 1956b), I). ))ia)njabeirai (INIurdy and Carson, 1959) and D. nielanogaster (Goldschmidt, 1957) — strongly suppt)i-t this A-iew.

The [irohleni of si'x determination in Hal)rol)i'acon presently stands as a function of multii)le alleles in one locus, the heterozygotes being female and the azygotes and homozygotes male. The occasionally diploid males are regularly produced from fertilized eggs in two allele crosses after inbreeding. These diploid males are of low viability and are nearly sterile. Their few daughters are triploids, their sperm being dijiloid. Apis probably follows the same scheme, as a few cases of mosaics with diploid male tissue are known and close inbreeding results in a sufficient number of deaths in the egg to account for diploid males which might be formed. ^lormoniella (Whiting, 1958), however, shows that this scheme for sex determination does not hold for all Hymenoptera. In this form diploid males may occur through some form of mutation. They may then develop from unfertilized eggs laid by triploid females. In contrast to Habrobracon the dijiloid males are highly viable and fertile. Their sperm are diploid and their numerous daughters triploid. Virgin triploid females produce 6 kinds of males, 3 haploid and 3 dij^loid. Similarly ]\Ielittobia has still a different and as yet unexplained form of sex determination. Haploid eggs develop into males. After mating many eggs are laid which develop into nearly 97+ per cent females. The method of reproduction is close inbreeding but no dijiloid males or "bad" eggs are formed. The prol)lem of the sex determining mechanism remains open (Schmieder and Whitiuii, 1947).


The silkworm, Bomby.r mori, differs from Drosophila, Lymantria and the species thus far discussed in having a single region in the ^^' chromosome (Hasimoto, 1933; Tazima, 1941, 1952) occupied by a factor or factors of high female potency. The strong female potency has thus far been connnon to all races. The chromosome patterns of the sexes are like those of Abraxas and Lymantria: males ZZ + 2A and females ZwV 2A. The diploid chromosome number is 56 in both sexes. Extensive, well executed studies


FOUNDATIONS FOR SEX


35


liave revealed no W chruniosome loci for genes expressed as morphologic traits. From radiation-treated material it has been possible to pick up a translocation of chromosome II to the W chromosome as well as a cross-over from chromosome Z. This chromosome together with tests of hypoploids and hyperploids have materially aided in understanding how the normal chromosome complexes determine sex. The sex types resulting from different chromosome arrangements have been summarized by Yokoyama 1 1959) and are presented in Table 1.3.

Whenever the W chromosome was absent a male resulted. Extra Z or A chromosomes did not influence the result. Similarly with a W chromosome in the fertilized egg a female developed. Again extra Z or A chromosomes did not influence the result.

A full Z chromosome was essential to survival. Hypoploids deficient for different amounts of the Z chromosome in the presence of a normal W chromosome all died without regard to the portion deleted. Hyperploids for the Z chromosome, on the other hand, when accompanied with a W chromosome all lived and showed no abnormal sexual cliaracteristics. Parthenogenesis led to the pioduction of both sexes, although the males were more numerous than the females. Diploidy was necessary for the eml)ryo to go beyond the blastoderm stage. Triploid and tetraploid cells were often found. High temperature treatments led to merogony (Hasimoto, 1929, 1934). The exceptional males were homozygous for a sexlinked recessive gene and were explained by assuming that the egg nuclei were inactivated by the high temperature and the exceptional males developed from the union of two sperm nuclei. This conclusion was supported by cytologically observed l)olyspermy (Kawaguchi, 1928) and by cytologic observation of the union of two sperm nuclei by Sato ( 1942 ) . Binucleate eggs were also believed to occur, which when fertilized by different sperm may each construct half of the future body. This type of mosaicism was influenced by heredity iGoldschmidt and Katsuki, i927, 1928, 1931 ). Polar body fertilization was also believed to occur, one side of the embryo originating from the ordinary fertilized egg nucleus and the other side from the union of


TABLE 1.:^ Sex in Bombyx iitori (Summarized by T. Yokoyama, 1959.)


Sex


Chromosome Types and Numbers



W


z


A


Male


W II.W.ZL

w w

WW WW


zz

zz

zzz

z

z

zz zzz zz zz


AA


Male

Male

Female

Female

Female

Female

Female

Female


AAA AAA

AA

AA

AAA

AAAA

AAA

AAAA


nuclei of two of the polar bodies. Similarly, dispermic merogony was noted following the formation of one part of the body from the fertilization nucleus, the other part from the union of two sperm nuclei, the result being a gynandromorph or mosaic.

VI. Sex Determination in Dioecious Plants

\. MELAXDRUM t LYCHNIS]

Over the last 20 years studies on several species of dioecious plants have made notable advances in unclerstanding the mechanisms by which sex is determined.

Melandrium album has been shown to have the same chromosome arrangement as Drosophila. The male has an X and Y plus 22 autosomes, whereas the female has XX plus 22 autosomes. Sex-linked inheritance is known for genes borne in the X chromosomes as well as for genes born in the Y chromosome. The X and Y chromosomes are larger than any of the autosomes with the Y chromosome about 1.6 times that of the X in the materials studied by Warmke ( 1946) . Separate male and female plants are characteristic of the species. By use of colchicine and other methods, Warmke and Blakeslee (1939), Warmke (1946), and Westergaard (1940) have made various l^olyploid types from which they could derive other new X, Y and A chromosome combinations from which information was obtained on the location of the sex determining elements. The Y chromosome carries the male determining elements, the X chro


36


BIOLOGIC BASIS OF SEX


mosome the female determining elements. The guiding force of the elements in the Y chromosome during development is sufficient to override the female tendencies of several X chromosomes. Data derived by each of these investigators are shown in Table 1.4.

From these data Warmke (1946) concluded that the balances between the X and the Y chromosomes essentially determined sex with the autosomes of relatively little importance. Where no Y chromosomes were present but the numbers of X chromosomes ranged from 1 to 5, only females were observed, even though the autosomes varied in number from two to four sets. When a Y chromosome was present the individual was of the male type unless the Y was balanced by at least 3 X chromosomes when an occasional hermaphroditic blossom was formed.

TABLE L4

Numhers of X, Y, chromosomes and A, autosome sets

and the sex of the various Melandrium plants

(Data from H. E. Warmke, 1946; and

M. Westergaard, 1953.)




Ratio



Chromosome


Warmlce



Westergaard


Constitution






X/A



4A 5X


Female


1.3



4A 4X


Female


1.0


Female


4A 3X


Female


0.8



4A 2X


Female


0.5



3A 3X


Female


1.0


Female


3A 2X


Female


0.7


Female


2A 3X


Female


1.5



2A 2X


Female


1.0


Female



Bisexual*


X/Y



4A 4X Y


4.0


Male


4A 3X Y


Malet


3.0


Male


4A 2X Y


Malet


2.0


Male


4A X Y


Male


1.0



3A 3X Y


Malet


3.0



3A 2X Y


Malet


2.0


Male


3A X Y


Male


1.0



2A 2X Y


Malet


2.0



2A X Y


Male


1.0


Male


4A 4X YY


Malet


2.0



4A 3X YY


Male


1.5



4A 2X YY


Male


1.0


Male


2A X YY


Male


0.5



  • Occasional staminate but never carpellate

blossom.

t Occasional licrina])hr(i(lit ic blossom.


When 4 X chromosomes were present together with a Y, the plants were hermaphroditic but occasionally had a male blossom. Two Y chromosomes almost doubled the male effect. Two Y chromosomes balanced 4 X chromosomes to give a majority of male plants. Only an occasional plant showed an hermaphroditic blossom. Autosomal sex effects, if present, were only observed when plants had 4 sets and 3 or 4 X chromosomes balanced by a Y chromosome. Warmke used the ratio of the numbers of X to Y chromosomes as a scale against which to measure clianges from complete male to hermaphroditic types. No mention is made of quantitative measures of the sex character changes with increasing X chromosome dosages. This is of interest since in many forms changes in chromosome balance are accompanied by changes of phenotype which are unrelated to sex. That such phenotypic changes do accompany changes in autosomal balance in Melandrium are proven, however, by further observations of Warmke in 4 trisomic types coming from crosses of triploids by diploids. Of 36 such trisomies analyzed, 5 or 6 of them were of different growth habits and morphologic types. These differences did not affect the sex patterns since all were females. Warmke and Blakeslee in 1940 observed an almost complete array of chromosome types from 25 to 48 in progeny derived from crosses of 3N x 3N, 4N X 3N, and 3N x 4N. Out of about 200 plants studied, only 4 were found to show indications of hermaphroditism. These types were 2XY and 3XY. As noted from the table, even the euploid plants would occasionally be expected to have an hermaphroditic blossom. Of the 200 plants, all with a Y (XY, 2XY, 3XY) were males and all plants without the Y (2X, 3X, 4X) were females. In an 8-year period up to 1946. Warmke was able to observe only one male trisomic. From these facts he concluded that the autosomes are unimportant in the sex determining mechanism utilized by this species. In their crosses they were unsuccessful in getting a 5XY plant, the point at which the female factor influence of the X chromosomes might be expected to nearly equal or slightly surpass that of the single Y. From the j^hysiologic side the obscrva


FOUNDATIONS FOR SEX


37


tion of Strassburger in 1900, as quoted by both Warmke and Westergaard, that the fungus Ustilago violacea when it infects Melandrium will cause diseased plants to produce mature blossoms with well developed stamens (filled with fungus spores) as well as fertile pistils, shows that these females have the potentialities of both male and female development. The case suggests that sex hormone-like substances may be produced by the fungus which acting on the developing Melandrium sex structures cause sex reversions. Should this be true, Melandrium cells would have a parallel with those of fish where sex hormones incor|)orated in the developing organism in sufficient quantities can cause the soma to develop a phenotype opposite to that expected of their chromosomal type. For other aspects see Burn's chapter and Young's chapter on hormones.

Westergaard's studies (1940) with European strains of Melandrium were in progress at the same time as those of Warmke and Blakeslee. In their broad aspects both sets of data are concordant in showing the l^rimary role of elements found within the Y chromosome in determining the male sex and of elements in the X chromosomes for the female sex. Examination of Table 1.4 shows that the strain used by Westergaard has a Y chromosome containing elements of greater male sex potentialities than the strain used by Warmke.

A similar difference appeared in the sex potencies of the autosomes of European strains. Instead of obtaining essentially only male and female plants in crosses involving aneuploid types, Westergaard obtained from 3N females (3A + 3X) x 3N males (3A + 2XY) 10 plants which were more or less hermaphroditic, 21 females, and 15 males. Studies of the offspring of these hermaphrodites through several generations showed that their sex expression required effects by both the X chromosomes and certain autosome combinations which under special conditions counterbalanced the female suppressor in the Y chromosome. Increasing the X chromosomes from 1 to 4 increased the hermaphrodites from to 100 per cent in the presence of a Y chromosome. However, in euploids these types would be


all males. The significance of the autosomes is further shown by the fact that among 205 aneuploid 3XY plants, 72 were males and 133 were hermaphrodites.

As pointed out for Drosophila, quantitative studies on the effects of sex chromosomes and autosomes in Melandrium are handicapped by not having a suitable scale for the evaluation of the different sex types. The data presented by Westergaard and by Warmke make this difficulty become particularly evident. In the interest of quantizing the X, Y, and A chromosome on sex the author has assigned a value of 1 for the male type, 3 for the female type, and 2 when the types are said to be hermaphroditic. When the types are mixed, as for example, in the data of Warmke where he says a particular type is male with a few blossoms, the type is assigned a value of 1.05 or 1.10, depending on the numbers of these blossoms. His bisexual type which comes as a consequence of Y, 4X and 4A chromosome arrangement is given a value of 2, although possibly the value should be somewhat higher as it may well be that the fully bisexuals are further along in the scale toward female development than the hermaphroditic types. The data are treated on the additive scale both as between chromosomal types and within chromosomal type. This is apparently unfair if we examine the work of Westergaard in which it looks as if particular autosomes rather than autosomes in general make a contribution to sex determination. The results, when these methods are used, are as follows:

Westergaard in Tables 1 to 5 of his 1948 paper gives information on sex types with a determination of the numbers of their different kinds of chromosomes. Analysis of these data by least square methods shows that the sex type may be predicted from the equation

Sex type = - 1.37 Y + 0.10 X

+ 0.01 A + 2.34

This equation fits the data fairly well considering that the correlation between the variables and the sex type is 0.87. This analysis again shows that the Y chromosome has a strong effect toward maleness. The X chromosomes are next in importance


38


BIOLOGIC BASIS OF SEX


with an effect of each X only about 1/13 that of the Y and in the direction of femaleness, the autosomes have one tenth the effect of the X chromosomes but they too have a composite effect toward femaleness. It is to be remembered that the Y chromosome variation is limited to 2 chromosomes whereas the X chromosomes may total 4, and the autosomes may range from 22 up to 42, so that the total effect of the autosomes is definitely more than their single effects. These data are for aneuploids. Examining Westergaard's data for 1953 for the euploids and assigning the value of 1.5 for the type observed when there was one Y chromosome, four X, and four sets of autosomes, we have the following equation:

Sex value = -1.29 Y + 0.10 X - 0.01

(autosome sets) + 2.53

In these data, as distinct from those above, the autosomes are treated as sets of autosomes since they are direct multiples of each other so the value of the individual autosome is but 1/11 that given in the equation.

This equation shows no pronounced difference from that when the aneuploids were utilized. The Y chromosomes have slightly less effect toward the male side. The X chromosomes have practically identical effects but there has been a shift in direction of the autosomal effects on sex, although the value is small. The constants are subject to fairly large variations arising through chance.

In Table 10 of Westergaard's 1948 paper he presented data on the chromosome constitution and sex in the aneuploids which carried a Y chromosome. These data are of particular interest as the plants are counted for the i)roi)ortions of those which are male to those which are hermaphroditic. The plants with a Y chromosome plus an X are all males. Those which have either one, two or three Y chi'omosomes balanced by two X chromosomes have 89 per cent males. The plants with three X chromosomes and one oi- two Y's have 36 p(T cent males and those which have one or two ^■ chroiiiosoincs and four X chromosomes have no males. The woik iiiv()l\-cd in getting these data is, of course, large indeed and is definitely handicapped by the diiiicuHies in obtaining


certain types. Thus the XXYYY and the 4X + 2Y types depend only on one plant. There are eight observations but the fitting of the data for the X and Y constitutions eliminates three degrees of freedom from that number so that statistically the observations are few. The data do have the advantage that the sex differences can be measured on an independent quantitative scale. The equation coming from the results is:

Percentage of males = 13.2 Y - 36.4 X

+ 134.4

These results show that the Y chromosome increases the proportion of males and the X chromosome increases the proportion of intersexes. The data are not comparable with those analyzed earlier as these data are describing simply the ratio between the males and intersexes, instead of the relations betw'een the males, intersexes, and females. The equation fits the observations rather well, as indicated by the fact that the correlation between the X's and Y's and the percentages of males is 0.98, but there are large uncertainties.

As a contrast to these data we have those presented by Warmke (1946) in his Tables 2 and 3. These data give the numbers of X and Y chromosomes found within the plants but not the numbers of autosomes, the autosomes being considered as 2, 3, and 4 genomes. Analyzing these data in the same manner as those of Westergaard's Table 1, we find that the

Sex type = -1.05 Y + 0.22 X -0.04 A

-f2.25

As indicated for Westergaard's data, the A effect is now in terms of the diploid type e(iualing 2, the trijiloid 3, and the tetral)loid 4.

The Y chromosome has a i)ronounccd effect toward maleness, the effect l)eing someW'hat less in Warmke's data than that of Westergaard's. The X chromosomes on the other hand, have nearly twice the female infhience in Warmke's data that they do in the phiiits grown by Westergaartl. A difference in sign exists for the effect of the A rhi'oinosnnie genom(\s as well as a difference in [\\v (|uantitati\'e effect. Th(^ values for


FOUNDATIONS FOR SEX


39


both sets of data are small and toward the male side. As Westergaard points out, the strains used by these investigators are of different geographic origins. The evolutionary history of the two strains may have a bearing on the lesser Y and greater X effects on the sex of the American types. Chromosome changes seem to have occurred in the strains before the studies of Warmke and Westergaard and will be discussed.

The location of the sex determiners has been studied by both investigators utilizing techniques by which the Y chromosome becomes broken at different places. These breakages may occur naturally and at fairly high rates in individuals which are Y + 2X + 2A. These facts suggest that the breakage of the Y chromosome occurs in meiosis since the breakage comes in selfed individuals of highly inbred stocks where heterozygosity is not to be expected, in the Y chromosome which has no homologue thus does not synapse, and in the second meiotic

  • eriments with fowl pox it has been shown that the number of eggs developing parthenogenetically increases considerably following vaccination. The factors leading to parthenogenesis are considered to be the genetic characteristics of the strain of birds and the presence of an activating agent or agents in the blood stream of the hens. The parthenogenetic forms are of particular interest to the problem of sex determination. The females should l)e producing two types of oocytes Z -I- A and W -h A of which presumably the Z + A alone survive since the embryos capable of being sexed are all males. The embryos are also diploids. The 2Z -I- 2A could be derived from a fusion of the Z -H A polav body nuclei as noted earlier or possibly chromosome doubling coming later in the early cleavage. A genetic element seems partially to control the parthenogenetic process. Chromosome doubling would lead to cells with identical pairs of chromosomes. The gene would be homozygous. Inbreeding of poultry leads to a continning and rai^id loss in the vialiility of FOUNDATIONS FOR SEX 49 most strains of chickens. A greater loss would be expected for truly homozygous chickens or poults as birds are known for the large numbers of sublethal genes they carry. In fact, it is surprising that any survive to the adult stage. The doubling of the W and A type would result in individuals lacking the Z chromosome. From what was observed in Amphibia and fish the WW + 2A, individual if it survived, would be expected to be female. Since this type has not as yet been detected it may be inferred that it is inviable because of loss of certain essential genes in the Z chromosome. X. Sex Determination in Mammals A. GOAT HERMAPHRODITES Goat hermaphroditism as reported by Asdell (1936), Eaton (1943, 1945) and Kondo (1952, 1955a, b) is of particular interest when comjjared with human hermaphroditism as observed by Overzier (1955) and of testicular feminization as reported by Jacobs, Baikie, Court Brown, Forrest, Roy, Stewart and Lennox (1959) and others. In each species the phenotypic range in sexual development extended from nearly perfect female to nearly perfect male, with the most frequent class as an intermediate. External appearance of each was partially correlated with internal structure. When internal female structures as the INIiillerian ducts were present, the external appearance was more female-like. When the male structures AVolffian ducts were developed, the external api^earance was more male-like. The presence of the dual systems within certain of these hermaphroditic types indicates, as in Drosophila, that there is independence of development of each system without a socalled turning point calling for differentiation of the female sex followed by that of the male sex or vice versa. In goats the hermaphroditic types were traced to the action of a recessive autosomal gene (Eaton, 1945; Kondo, 1952, 1955a, b). This gene apparently acts only on the female zygote. In homozygous condition the eml)ryos bearing them develop simultaneously toward the male as well as toward the female types. This development resembles closely that of the Hr gene in Drosophila, because, although Hr is dominant and the one in goats is recessive, they both operate only on the female type and both tend to develop jointly both male and female systems in sexual development. One jarring note comes in relating the cytologic basis for sex determination in goats with that for the intersexes. The sex ratios for the different crosses clearly place the hermaphrodites as genetic females expected to have the XX chromosome constitution. The XX constitution would then also agree with that found for human hermaphrodites as discussed later in this paper. Makino (1950) has shown for one case of the intersexual goat that its sex chromosomes were of the male type. Makino's excellent studies with other species made this observation of particular significance as it was contrary to the other morphologic and genetic evidence on these hermaphrodites. The implications were fully realized by Makino when the cytologic observations were made so that as far as possible the observations should be critical on this point. However, there are several sources of cell variation that suggest the desirability of further checks. The chromosome number of the goat is large, normal mitoses rarely appear in the gonads of the intersexes, and the chromosomes of the goat's spermatogenesis are so small as to make difficult details of structure or identification. Some of the difficulties possibly could be avoided by making tissue cultures and determining the somatic chromosome numbers of their cells. Kondo (1955b) has shown that under the breeding conditions of Japan when the sire was heterozygous, the percentage of intersexes actually approached the expected value 7.3 per cent. When the sires were homozygous recessive individual matings showed 14.6 per cent hermaphrodites as was expected. Continued mating of homozygotes should show 25 per cent of the total kids hermaphrodites, or the equivalent of 50 per cent of the female progeny. Hermaphroditism in goats has a further advantage in that the locus is apparently linked closely to the horned or polled condition. The horned condition, in consequence, becomes a valuable indicator marking the presence of the hermaphroditic factor in the 50 BIOLOGIC BASIS OF SEX otherwise indistinguishable male types. With these characteristics the goat types have remarkable advantages over other species for the solution of problems of hermaphroditism. The gene for goat hermaphroditism has even more interest when it is contrasted with that of another gene, tra, discovered by Sturtevant (1945). Tra is recessive wdth no distinguishable heterozygous effect. In the homozygous state it converts the zygotic female into a form with completely male genitalia and internal reproductive tract with no evidence of the female sexual reproductive system. The gene effects in Drosophila are more extreme than those in goats but are concordant in showing that there are loci in the autosomes which may be occupied by recessive genes having direct effects on phenotypic development of the genotypic female. This evidence indicates the significance of these genes rather than the happenstance of their being in the autosome, X or Y chromosome. B. SEX IN THE MOUSE The mouse has the XY + 38 A chromosomal arrangement for the males and XX + 38 A for the females. Similar karyotype patterns have been reviewed for some Amphibia and fish. Other Amphibia and fish may have their karyotypes reversed as both forms are found in nature or observed in breeding studies. Similar reversals may be made experimentally in the phenotypes even though the genotypes remain unaltered. Birds show the sex differentiating arrangement of ZW for the females and ZZ for the males. Parthenogenesis seems to lead to males of ZZ type in domestic fowl and turkeys. In an evolutionary sense the mammals could have originated from and perpetuated either of the major karyotype sex arrangements. Mice and men are alike in that the X has female-determining properties and the Y male potencies. How much part the genes in the autosomes have in sex develojoment is not yet clear. Welshons and Russell (1959) have shown that mice of the presumed X() constitution are females and arc fertile. They have 39 as the modal number of chromosomes found in their bone marrow cells, wiiereas the genetically proven XX types have 40 cln'omosomcs. X ('hroinosoinc linked genes' behavior substantiate the chromosomal constitutions of XO and XX as females and XY as males. These results are further supported by the breeding behavior of the X-linked recessive gene, scurfy (Russell, Russell and Gower,. 1959). This gene is lethal to the hemizygous males before breeding. The genetics of the scurfy females have been analyzed by transplanting the ovaries to normal recipient females and obtaining offspring from them. In the scurfy stock the XO type occurred as 0.9 per cent of the progeny. The YO progeny w^ere not identified and probably die prematurely. Nondisjunction of the X and Y chromosomes in the males could result in sperm carrying neither X nor Y chromosomes. These sperm on fertilization of the X egg would give an XO + 2A type individual. Because the result is a female, this would support the Y chromosome as of male potency. The mouse arrangement may then be expected to be like Melandrium in which a well worked out series of types is known. Sex ratio in mice is strain dependent over what has thus far proven to be a 10 to 15 per cent range. Weir (1958) has shown that for two strains of mice established by selecting for low and high pH, the sex ratio figures were 33 and 53 per cent for artificially inseminated mice and 41 and 52 per cent for natural matings of these respective strains. The differential pH values for the bloods of the low line were 7.498 ± 0.006 and for the high line 7.557 ± 0.007 as of the sixth generation of selection. The parents with the more alkaline bloods tended to have greater percentages of males in their progenies. These results direct attention to the genotype dependent phenotypic factor which may be of some importance for variations in sex ratios. C. SEX AND STERILITY IN THE CAT The tortoiseshell male cat has long interested geneticists because it has seemed that by theory it should not be. However, nature has wonderful ways of circumventing best laid hypotheses, sometimes when they are fals(\ sometimes when they have not been probed dee])ly enough. The yellow gene for coat color in cats is sex-linked. This gene operates on an autosomal background of ^(■lu's for black oi' tabby. Tlu^ females may FOUNDATIONS FOR SEX 51 be phenotypically orange as the double dose, 0/0, covers up the effects of the other coat color genes; or tortoiseshell, 0/+; or black or tabby, +/+. The males may be orange, '0/, black or tabby, +/, and the type unexpected tortoise. The tortoiseshell males are timid, keep away from other males, and are generally sterile. Testes are of much reduced size and solid consistency. Exceptionally, tortoiseshell males may mate and offspring presumed from the matings may be born. Active study of these males commenced as early as 1904. Komai (1952) has offered a unified hypothesis for their origin. Komai and Ishihara (1956) have contributed added information and a review of the literature to which the reader is referred. The cat has 38 chromosomes including an X-Y pair for the males. The tortoise males agree in having this arrangement (Ishihara, 1956) , the X being 3 or 4 times the length of the Y in all cytologic preparations from Japanese cats. Komai (1952) visualizes the cat X chromosomes as composed of a pairing segment containing the kinetochore and gene loci among which is that for the orange gene and a differential segment, not found in the Y chromosome, containing the factor-complex for femaleness. The Y chromosome is visualized as having a segment containing the kinetochore and capable of pairing with the X chromosome. This segment may cross over with the X so that it may acquire the locus for orange or its wild type. The Y chromosome is viewed as containing two differential segments. The one carrying the factor complex for maleness is located to correspond with the X differential segment carrying the female sex factor. The second Y differential segment is at the other end of the chromosome and contains the male fertility complex. The tortoiseshell sterile males are interpreted as caused by a Y chromosome crossing over with the X chromosome to incorporate the male segment and the gene in the resulting Y chromosome but with the loss of the male fertility segment. The gamete carrying this modified Y fertilizing an egg with a normal X chromosome containing the wild type instead of the gene develops into the sterile tortoiseshell male. The data show that the probability of these events occurring is small. Komai records as reliable ■65 tortoiseshell male cats where the inci dence of the O gene in the whole population of Japanese cats is 25 to 40 per cent. Of the 65, 3 were apparently fertile. These cases and the few others found in the literature are regarded as caused by those rare occasions when the Y chromosome incorporates the gene but retains the male fertility complex as might occur in double crossing over. The hypothesized factor locations and crossing over arrangements also may explain the unexpected black females which are known to occur in some matings. Although not mentioned, black males and orange males showing the same sterility features as the sterile tortoiseshell males should also be found in the cat poi)ulation. If found they would further strengthen the hypotheses. It is difficult to understand why, even with its low initial frequency, the fertile tortoiseshell male would not establish itself in the Japanese cat population, inasmuch as they are so admired and sought after by all the people if any tortoiseshell males became as fertile as the tortoiseshell male "lucifer" (Bamber and Herdman, 1932) known to have sired 56 kittens. Ishihara's work (1956) seems to close the door on another attractive hypothesis to explain the origin of these unexpected cat types. Tortoiseshell male reproductive organs include small, firm testes showing reduced spermatogonial development. Together with the interaction of the gene with the wild type allele they suggest the human types XXY + 2A which may arise from nondisjunction. However, the chromosome type is shown to be XY -f- 2A = 38 which is fatal to this hypothesis. It is of interest that Komai in 1952 postulated the male complex and fertility factors in the Y chromosome of a mammal. The case has a further parallel in the plant Melandrium in that the work of both Westergaard (1946) and Warmke (1946) indicated the Y chromosomes of this plant to contain such factor complexes although in differing arrangements. D. DEVIATE SEX TYPES IN CATTLE AND SWINE As a caution in the mushrooming of cytologic interjiretations of sex development, attention may be directed to the freemartin types known particularly from the work of 52 BIOLOGIC BASIS OF SEX Keller and Tandler (1916), Lillie (1917), and the researches stimulated by their observations on cattle twins. The freemartin in cattle develops in the same uterus with its twin male. The blood circulations anastomose so that blood and the products it contains are common to both fetuses during development. The development of the female twin is intersexual, presumably because of substances contributed by the male twin to the common blood during uterine growth. The freemartin intersexuality may be graded into perfectly functioning fertile females to types with external female genitalia and typically male sex cords except germ cells are absent, vasa efferentia, and elements of the vasa deferentia. The conditions are similar to those discussed for amphibia, fish, and rabbits in which early sex development passes through neutral stages during which it may be directed toward one sex or the other by the right environmental stimuli. Intersexes in swine have been interpreted as owing to similar causes (Hughes, 1929; Andersson, 1956) although the resulting phenotypes may not be quite as extreme. The resulting intersexes for both cattle and swine presumably are not caused by chromosomal misbehavior but to the right environmental stimuli operating on suitable gene backgrounds. The observations of Johnston, Zeller and Cantwell (1958) on 25 intersexual pigs all from one breeding group of Yorkshires suggest significant inheritance effects. The intersexes were of two types, "male pseudohermaphrodites" and "true hermaphrodites," but there was some intergrading of their phenotypes suggesting that they may be the products of like causes. Common organs between the two groups included uteri, vulvae, vaginae, testes, epididymis, and penis or enlarged clitori. The "true hermaphrodites" were separated on the basis of no prostates, bulbo-urethral glands, or seminal vesicles as well as having testes or ovotestes with ovaries. A similar case was described by Hammond (1912) but, as in one of the above cases, the supposed ovaries when sectioned seemed to be lymphatic tissue. Favorable nerve tissue^ from 6 of the Yorkshire pigs was examined foi- nuclear chromatin. The cases were found chromatin positive. Phenotypically these cases also have parall(>ls in mice and man. E. .SEX-iN man: chromosomal basis A surprise even to its discoverers, Tjio and Levan (1956), came with the observation that the somatic number of chromosomes in cultures of human tissue was 46 rather than the previously supposed 48. Search for the true number has been going on for more than half a century. In early investigations the numbers reported varied widely. Difficulties of proper fixation and spreading of the chromosomes of human cells accounted for most of this variation and the numerous erroneous interpretations. Among the observations that of de Winiwarter (1912) was of particular interest in showing the chromosome number as 46 autosomes plus one sex chromosome with the Y being absent. This number was also found later by de Winiwarter and Oguma (1926). Observations by Painter (1921, 1923) showed 46 chromosomes plus an X and a Y, a total of 48. This number was subsequently reported by a series of able investigators, Evans and Swezy (1929), Minouchi and Ohta (1934), Shiwago and Andres (1932), Andres and Navashin (1936), Roller (1937), Hsu (1952), Mittwoch (1952), and Darlington and Haque (1955). As Tjio and Levan indicated, the acceptance of 48 as the correct number, with X and Y as the sex chromosome arrangement, was so general that when Drs. Eva Hanson-Melander and S. Kullander had earlier found 46 chromosomes in the liver cells of the material they were studying they temporarily gave up the study. In the few years since 1956, the acceptance of 46 chromosomes as the normal complement of man has become nearly universal. There are 22 paired autosomes plus the X and Y sex chromosomes. The reasons which have warranted this change of viewpoint are no doubt many, but three improvements in technique are certainly significant. The first came as a consequence of simplifying the culture of human somatic cells. The second followed Hsu's (1952) recognition that pretreatment of these cells before fixation with hypotonic solutions tended to better spreads of the chromosomes on the division plates when subsefiuently stained by the squash techniciuo. Pretreatment of the cultures with FOUNDATIONS FOR SEX 53 colchicine made the studies more attractive by increasing the numbers of usable cells that were in the metaphase of cell division. Ford and Hamerton (1956) in an independent investigation, closely following that of Tjio and Levan, observed that the human cell complement contained 46 chromosomes. They, too, agreed with Painter and others that followed him that the male was XY and the female XX in composition. A flood of confirming evidence soon followed: Hsu, Pomerat and Moorhead (1957), Bender (1957), Syverton (1957), Ford, Jacobs and Lajtha (1958), Tjio and Puck (1958), Puck (1958), Chu and Giles (1959), and a number of others. In most instances the results of the different investigators were surprisingly consistent in showing that the individual cell chromosome counts nearly always totaled 46. This was no doubt due in part to the desirability of single layers of somatic cells for identifying and separating the different chromosomes into distinct units. Chu and Giles' results illustrate this consistency. For 34 normal human subjects, including 29 American whites and 4 American Negroes, and one of unknown race, and regardless of sex, age, or tissue, the diploid chromosome number of the somatic cells was overwhelmingly 46. In only five individuals were other numbers observed in isolated cells. Out of 620 counts, 611 had 46 chromosomes; two individuals, whose majority of cells showed 46, had 3 cells with 45 chromosomes; three other individuals, the majority of whose cells showed 46, had 6 cells with 47 chromosomes. Average cell plates counted per individual was nearly 20. The only recent observations at variance with these results were those of Kodani (1958) who studied spermatogonial and first meiotic metaphases in the testes from 15 Japanese and 8 whites. In these studies at least several good spermatogonial metaphases in which the chromosomes could be counted accurately, and secondly at least 15 spermatocyte metaphases in which the structure of individual chromosomes could be observed clearly, were made on each specimen. The numbers of cells studied in metaphase were generally above these numbers, one reaching 60 metaphases. Some variation was noted within individuals. Among individuals, numbers of 46, 47, and 48 were observed. Among 15 Japanese, 9 had 46, 1 had 47, and 5 had 48 chromosomes, whereas among the whites 7 had 46, and 1 had 48. Sixteen of the 23 individuals had 46 chromosomes. Karyotype analyses indicated that the numerical variation was caused by a small supernumerary chromosome. On the basis of these observations it would appear that individuals within races may vary in chromosome number and yet be of normal phenotype. However, in view of the extensive observations by others, it seems unlikely that the variation between individuals is as large as that indicated. It will require much further study to establish any other number than 46 as the normal karyotype of man. This is particularly true in view of the work of Makino and Sasaki (1959) and Alakino and Sasaki cited by Ford (1960), in which they studied the human cell cultures of 39 Japanese and found without exception 46 chromosomes, and the earlier work of Ford and Hamerton (1956) on spermatogonial material where they, too, found 46 chromosomes in that tissue. The best features of these human chromosome studies will come in the identification of the individual chromosomes making up the human group. The chromosome pairs may be ordered according to their lengths. The longest chromosome is about 8 times the length of the smallest. The chromosomes may be classified according to their centromere positions. The chromosomes are said by most observers to be fairly easily separated into 7 groups. Separation of the individual chromosome pairs from each other and designation of the pairs so that they can be identified by trained investigators in all good chromosome preparations is not possible according to some ciualified cytologists and admitted difficult by all students. However, standardized reporting in the rapidly growing advances in human cell studies should refine observations, reduce errors, and encourage better techniques. With this in mind, 17 investigators working in this field met in Denver in 1959 in what has come to be called the "Denver conference" (Editorial, 1960). From an examination of the available evidence on chromosome morphologies an idiogram was set up as a standard for the somatic chromosome 54 BIOLOGIC BASIS OF SEX CD LO 00 CM ^ m CM CM 'li m ^ CM i| m m 8 1^ 9m c £ II CM C30 £ Sg ^
    • o
    1 «. C^ ►5 '^ ^ 9^
    -f = O ^ •^ CD in ^ i <7> ^D 4M .£ i -^ CD «  «  >t J 2 ?: C^ -• «  CO cording to not easily ] and Thood( € < CM CM )ered ac ich are H. Tjio C < S s numl tes \vh I of J. n «  CM Ill • CD ^ m-m %J7
    1. c #• *—
    iO CM in \^ 00 2 >e #c i£ CM s s S c £ £ ^-^i a> d -e LO S J^ £ PI O q; i CO o ^ct v£ Fig. 1.1. Id r number, th chromosom r^ 00 FOUNDATIONS FOR SEX 55 complement of the normal human genome. A reproduction of this standard is presented in Figure 1.1, as kindly loaned by Dr. Theodore T. Puck for this purpose. The autosomes were first ordered in relation to their size and such attributes as would help in their positive identification. Numbers were given to each chromosome as a means of permanent identification. Basically, identification is assisted by the ratio of the length of the long arm to that of the short arm; the centromeric index calculated from the ratio of the length of the shorter arm to the whole length of the chromosome ; and the presence or absence of satellites. Classification is assisted by dividing the chromosome pairs into seven groups. Groups 1-3. Large chromosomes with approximatel}^ median centromeres. The three chromosomes are readily distinguished from each other by size and centromere position. Group 4-6. Large chromosomes with submedian centromeres. The two chromosomes are difficult to distinguish, but chromosome 4 is slightly longer. Group 6-12. Medium sized chromosomes with submedian centromeres. The X chromosome resembles the longer chromosomes in this group, especially chromosome 6, from which it is difficult to distinguish. This large group is the one which presents major difficulty in identification of individual chromosomes. Group 13-15. INledium sized chromosomes with nearly terminal centromeres ("acrocentric" chromosomes). Chromosome 13 has a prominent satellite on the short arm. Chromosome 14 has a small satellite on the short arm. No satellite has been detected on chromosome 15. Group 16-18. Rather short chromosomes with approximately median (in chromosome 16) or sul>median centromeres. Group 19-20. Short chromosomes with approximately median centromeres. Group 21-22. Very short, acrocentric chromosomes. Chromosome 21 has a satellite on its short arm. The Y chromosome belongs to this group. Separations of the human chromosome pairs into the seven groups is not as difficult as designating the pairs within groups (Patau, 1960). The svstem is a notable ad vance in summarizing visually the current information in the hope that availability of such a standard will promote further refinements, lessen misclassification, and contribute to a better understanding of the problems by cytologists and other workers in the field. 1. Xuclear Chromatin, Sex Chromatin Sexual dimorphism in nuclei of man (Barr, 1949-59) and certain other mammals may be detected by the observable presence of nuclear chromatin adherent to the inner surfaces of the nuclear membrane. The material is about 1 /x in diameter. It frequently can be resolved into two components of equal size. It has an affinity for basic dyes and is Feulgen and methyl green positive. Nuclear chromatin can be recognized in 60 to 80 per cent of the somatic nuclei of females and not more than 10 per cent of males. It is known to be identifiable in the females of man, monkey, cat, dog, mink, marten, ferret, raccoon, skunk, coyote, wolf, bear, fox, goat, deer, swine, cattle, and opossum, but is not easily usable for sex differentiation in rabbit and rodents because these forms have multiple large particles of chromatin in their nuclei. The tests can be made quickly and easily on skin biopsy material or oral smears. Extensive utilization of the presence or absence of nuclear chromatin in cell samples of man has been made for assigning the presumed genetic sex to individuals who are phenotypically deviates from normal sex types. (See also chapters by Hampson and Hampson, and by Money.) Numerous studies on normal individuals seem to support the test's high accuracy. However, in certain cases involving sexual modification, questions have arisen which are only now being resolved. In male pseudohermaphroditism, sex, determined by nuclear chromatin, is male, thus agreeing with the major aspects of the phenotype. For female pseudohermaphroditism, individuals with adrenal hyperplasia or those without adrenal hyperplasia give the female nuclear chromatin test. For cases listed as true hermaphrodites Grumbach and Barr (1958) list 6 of the male type and 19 of the female type. For the syndrome of gonadal dysgenesis they list 90 as male and 12 as female among the proved cases and 15 more as female among those that are 56 BIOLOGIC BASIS OF SEX suspected. In the syndrome of seminiferoustubule dysgenesis where there is tubular fibrosis, 9 are listed as male and 18 female. Where there is germinal aplasia, 15 are listed as male and 1 as female. The seeming difficulties in assigning a sex constitution to some of these types are now being dissipated through the study of the full chromosome complements which are responsible for these different disease conditions. As observations on different chromosome types have been extended, evidence has accumulated to show that the numbers of sex nuclear chromatins, for at least some of the nuclei making up the organism, often equals (n — 1) times the number of X chromosomes. The majority of male XY nuclei are chromatin negative as are most of the Turner XO type. Female nuclei XX have a single chromatin positive element as do the XXY and XXYY types. The XXX and XXXY have 14 and 40 per cent respectively with two Barr bodies in cases for which quantitative data are available. However, a child with 49 chromosomes, but whose cultured cell chromosomes appear as single heteropycnotic masses making identification of the individual chromosomes difficult, showed 50 per cent of the cell nuclei with three Barr elements (Fraccaro and Lindsten, 1960) . The chromosome constitution of these nuclei was interpreted as trisomic for 8, 11, and sex chromosomes. Sandberg, Crosswhite and Gordy (1960) report the case of a woman 21 years old having various somatic changes which does not fit this sequence. The chromosome number was 47 and the nuclei were considered trisomic for the sixth largest chromosome. Two chromatin positive bodies were ])rosent in the nuclei. 2. Chrotnosome Complement und Phenotijpe in Man Experience of the past 50 years has emphasized that genes and trisomies or other types of aneuploid chromosome complexes may lead to the development of abnormal phenotypes expressing a variety of characteristics. Drosophila led the way in illustrating how the different gene or chromosome arrangements may affect sex expression. Investigations of human abnormal types, particularly those with altered sex differentiation, have reccntlv .^liown that man follow.-^ other species in this regard. The Y carries highly potent male influencing factors. Gene differences often lead to characteristic phenotypes of unique form. 3. Testicular Feminization The testicular feminization syndrome illustrates one of these types. As described by Jacobs, Baikie, Court Brown, Forrest, Roy, Stewart and Lennox (1959), "In complete expression of this syndrome the external genitalia are female, pubic and axillary hair are absent or scanty, the habitus at puberty is typically female, and there is primary amenorrhoea. The testes can be found either within the abdomen, or in the inguinal canals, or in the labia majora, and as a rule the vagina is incompletely developed. An epididymis and vas deferens are commonly present on both sides, and there may be a rudimentary uterus and Fallopian tubes. The condition is familial and is transmitted through the maternal line." A sex-linked recessive, a sex-limited dominant, and chromosome irregularities of the affected persons have been postulated as mechanisms causing the apparent inheritance of this condition. Chromosome examinations of the cells of affected persons have shown 46 as the total number and X and Y as the sex complement. The karyotype analysis agrees with the Barr nuclear chromatin test in that the cells are chromatin-negative but both are at variance with the sex phenotypes in the sense that aside from suppressed testes the patients are so completely female. Genetically, Stewart (1959) has described two color-blind patients with the testicular feminization syndrome in the first five patients he reported. The limited data from these cases suggest that the genie basis for this condition is either independent or but loosely linked with color blindness. This evidence does not exclude sex-linkage but does make it less probable. The third hypothesis of autosomal inheritance may take one of several forms. A recessive gene which affects only the male phenotypes when in homozygous condition is apparently untenable because the matings from which these individuals come are of the outbreeding type and the ratios apparently do not differ from the one-to-one ratio expected of a heterozygous dominant instead of that re FOUNDATIONS FOR SEX 57 quired for an autosomal recessive. The hypothesis advanced by Witschi, Nelson and Segal (1957), that the presence of an autosomal gene in the mother converts all her male offspring into phenotypes of more or less female constitution, in a manner comparable to that of the Ne gene in Drosophila (Gowen and Nelson, 1942) which causes the elimination of all the female type zygotes, is also made unlikely by the ratios of normal to testicular feminization phenotypes observed in the progenies of these affected mothers. The evidence favors a simple autosomal dominant, acting only in the male zygotes and perhaps balanced by some genes of the X chromosome, which have sufficient influence on the developing male zygote to guide it toward an intermediate to nearly female phenotype. The observations of Puck, Robinson and Tjio ( 1960) indicate that the action of a gene for this condition may not be entirely absent in the female, because in heterozygous condition in an XX individual it seemed to delay menarche as much as 8 years. If this delay be diagnostic for the heterozygote, it will further assist in the genetic analysis of this problem. Evidence on this point should be a part of the genetic studies. Cases closely similar to those described by Jacobs, Baikie, Court Brown, Forrest, Roy, Stewart and Lennox (1959) are presented by Sternberg and Kloepfer (1960). The patients show no trace of masculinity. They are remarkably uniform in anatomic expression. Except for failure to menstruate due to lack of uteri they undergo normal female puberty. Cryptorchid testes, usually intra-abdominal, if removed precipitate menopause symptoms. Four unrelated cases were found in this one study with 7 additional cases traced through pedigree information. A total of 11 affected individuals was found in 6 sibships having 26 siblings of whom 5 were normal males. In each kindred the inheritance was compatible with that of a sex-linkecl recessive gene. A chromosomal study of a thyroid tissue culture from one case revealed 46 chromosomes with normal XY male configuration. The individuals observed were designated as simulant females." 4- Superfemale The human superfemale has been recognized by Jacobs, Baikie, Court Brown, MacGregor, Maclean and Harnden (1959) in a girl of medium height and weight, breasts underdeveloped, genitalia infantile, vagina small, and uterocervical canal 6 cm. in length. Ovaries appeared postmenopausal with normal stroma, and as indicated by a biopsy specimen, deficient in follicle formation. Menstruation was thought to have begun at age 14, but was irregular, occurring every 3 to 4 months and lasting 3 days. The last spontaneous menstruation was at 19. Estrogen therapy caused some development of the breasts and external genitalia, vagina, and uterus with slight uterine bleeding. The patient's parents were above 40 years of age, mother 41, at time of her daughter's birth. Examination of sternal marrow cultures showed 47 chromosomes in over 80 per cent of the cells examined. The extra chromosome was the X, the chromosomal type being XXX plus 22 pairs of autosomes. Buccal smears showed 47 per cent of nuclei contained a single chromatin body and 14 per cent contained 2 chromatin bodies as expected of a multiple XX or XXX genotype. In comparison, 25 smears from 20 normal women had 36 to 51 per cent chromatin positive cells but none of these contained 2 chromatin bodies. Two chromatin bodies were seen in some cells of the ovarian stromal tissue. The patient showed a lack of vigor, mentally was subnormal, was underdeveloped rather than overly developed in the phenotypic sexual characteristics. Examination of the patient's mother showed her to be XX plus 22 pairs of autosomes, the normal 46 chromosomes. Other cases show that types with XXX plus 22 pairs of autosomes are of female l)henotype but may vary in fertility and development of the secondary sexual characteristics from nonfunctional to functional females bearing children ( Stewart and Sanderson, 1960; Eraser, Campbell, MacGillivray, Boyd and Lennox, 1960). The triplo X condition in man has a greater range of development and fertility than in Drosophila. In man ovaries may develop spontaneously. In Drosophila they require transplantation 58 BIOLOGIC BASIS OF SEX to a diploid female host where they may attach to the oviducts and release eggs for fertilization (Beadle and Ephrussi, 1937). These cases present confirmation of two facts already mentioned for Drosophila. They show that when the X chromosome has primarily sex determining genes, the organism generally becomes unbalanced when 3 of these X chromosomes are matched against two sets of autosomes. The resulting phenotypes are female but relatively undeveloped rather than overdeveloped. The second is that the connotations evoked by the prefix "super" are by no means applicable to this human type or to the Drosophila type. The characteristics of the patient also suggest that the autosomes may be carrying sex genes opposing those of female tendencies as observed in both Drosophila and Rumex genie imbalance. 5. Klinefelter Syndrome In the Klinefelter syndrome there is male differentiation of the reproductive tracts with small firm descended testes. Meiotic or mitotic divisions are rare, sperm are ordinarily not found in the semen. The type is eunuchoid in appearance with gynecomastia, high-pitched voice, and sparse facial hair growth. Seminiferous tubules showing an increased number of interstitial cells are atrophic and hyalinized. Urinary excretion of pituitary gonadotrophins is generally increased, whereas the level of 17-ketosteroids may be decreased. The nuclear chromatin is typically female. Of the dozen or more cases studied (Jacobs and Strong, 1959; Ford, Jones, Miller, Mittwoch, Penrose, Ridler and Sha])iro, 1959; Bergman and Reitalu quoted by Ford, 1960), only one, having but 5 metaphase figures, had less than 47 chromosomes in the somatic cells and XXY sex chromosomes. That case was thought to have typical female chromosomes XX + 22 AA. Two other cases were of particular interest as indicating further chromosome aberration. Ford, Jones, Miller, Mittwoch, Penrose, Ridler and Shapiro (1959) studied one patient who displayed both the Klinefelter and Mongoloid syndromes. The chromosome number was 48, the sex chromosomes being XXY and the 48tli chromosoinc being small acrocentric. This individual had evidently developed from an egg carrying 2 chromosomal aberrations, one for the sex chromosomes and the second for one of the autosomes. The other case, Bergman and Reitalu as cited by Ford (1960), had 30 per cent of its cells with an additional acrocentric chromosome which had no close counterpart in the normal set. Data where the Klinefelter syndrome occurs in families showing color blindness (Polani, Bishop, Ferguson-Smith, Lennox, Stewart and Prader, 1958; Nowakowski, Lenz and Parada, 1959; and Stern, 1959a) further test the XXY relationship and give information on the possible position of the color blindness locus with reference to the kinetochore. Polani, Bishop, FergusonSmith, Lennox, Stewart and Prader (1958) tested 72 sex chromatin-positive Klinefelter patients for their color vision and found that none was affected by red-green color blindness. Nowakowski, Lenz and Parada ( 1959) tested 34 cases and detected 3 affected persons, 2 of whom were deuteranomalous and one protanopic. Stern (1959a) l^oints out that these cases and their ratios are compatible with the interpretation of the Klinefelter syndrome as XXY. One of the deuteranomalous cases had a deuteranomalous mother and a father with normal color vision. This case could have originated from a nondisjunctional egg carrying 2 maternal X chromosomes fertilized by a sperm carrying a Y chromosome. The other two cases had normal fathers with heterozygous mothers. There are several explanations by which the color-blind Klinefelter progenies could be obtained. The heterozygotes might manifest the color-blind condition. The second hypothesis, which is favored, is that of crossing over between the kinetochore and the color-blind locus at the first meiotic division to form eggs each carrying 2 X chromosomes, one homozygous for color blindness, and the other for normal vision. An equational nondisjunction would form eggs homozygous for color blindness which on fertilization by the Y chromosomes of the male would give the necessary XXY constitution for the color-blind male which is Klinefelter in phenotype. A third possibiHty is that these exceptions may arise without crossing over as the result of FOUNDATIONS FOR SEX 59 nondisjunction at the second meiotic division. If the hypothesis of crossing over is accepted, the color-blind locus separates freely from its kinetochore and would suggest that the position of the locus is at some distance from the kinetochore of the X chromosome. A disturbed balance between the X and the Y chromosomes alters the sexual type. A single Y chromosome, contributing factors important to male development, is able to alter the effects of two sets of female influencing X chromosomes. Yet two Y chromosomes in a complex of XXYY plus 44 autosomes seem to have little or no more influence than one Y (Muldal and Ockey, 1960). The locations of the sex-influencing genes in man are thus more like those of the plant Melandrium than of Drosophila in which the male-determining factors occur in the autosomes. The relative potencies of the male sex factors compared with those of the female, however, are much less than those in Melandrium. 6. Turner Syndrome Turner's syndrome or ovarian agenesis further substantiates the female influence of the X chromosomes. The cases occur as the developmental expression of accidents in the meiotic or mitotic divisions of the chromosomes. These accidents lead to adults unbalanced for the female tendencies of the X chromosome. The gonads consist of connective tissue. The rest of the reproductive tract is female. Growth stimuli of puberty are lacking, resulting in greatly reduced female secondary sexual development. Patients are noticeably short and may be abnormal in bone growth. In its more extreme form, designated as Turner's syndrome, the individuals may show skin folds over the neck, congenital heart disease, and subnormal intellect, as well as other metabolic conditions. Earlier work (Barr, 1959; Ford, Jones, Polani, de Almeida and Briggs, 1959) shows that 80 per cent of the nuclear chromatin patterns are of the male type. Evidence from families having both this condition and color blindness suggested that at least some of the Turner cases would be found to have 45 chromosomes, the sex chromosome being a lone X (Polani, Lessof and Bishop, 1956). Work of Ford, Jones, Polani, de Almeida and Briggs, (1959) has confirmed this hypothesis and added the fact that some of these individuals are also mosaics of cells having 45 and 46 chromosomes. The 45 chromosome cells had but one X, whereas the 46 had two X's. This finding may explain the female-chromatin cell type observed in about 20 per cent of the cases having the Turner syndrome. Such mosaics of different chromosome cell types could also be significant in reducing the severity of the Turner syndrome and in increasing the range of symptoms which characterize this chromosome-caused disease as contrasted with those characterizing Turner's disease. Further cases observed in other investigations, Fraccaro, Kaijser and Lindsten (1959), Tjio, Puck and Robinson (1959), Harnden, and Jacobs and Stewart cited by Ford (1960) have all shown 45 chromosome cells and a single X chromosome. As with the XXX plus 44 autosome super females, the Turner type, X plus 44 autosomes, also shows a rather wide range in development from sterility with extensive detrimental secondary effects to nearly normal in all respects. Bahner, Schwarz, Harnden, Jacobs, Hienz and Walter (1960) report a case which gave birth to a normal boy. Other cases have been described (Hoffenberg, Jackson and jVIuller, 1957; Stewart, 19601 in which menstruation was established over a period of years. The XO type in man and Melandrium is morphologically female. In Drosophila on the other hand, the XO type is phenotypically nearly a perfect male. It is further to be noted that the X chromosome of Drosophila appears to have a less pronounced female bias than that of man when balanced against its associated autosomes, inasmuch as the XO + 2A type in Drosophila is male as contrasted with the XO + 2A type in man which is female. At the same time it seems that the autosomes in the human may be influential in that the female gonadal development is suppressed instead of going to completion as it does in the XX type. 7. Hermaphrodites Hermaphroditic phenotypes in man, to the number of at least 74 (Overzier, 1955), have been observed and recorded since 1900. Types with a urogenital sinus pre 60 BIOLOGIC BASIS OF SEX dominated. The uteri were absent in some cases, even when complete external female genitalia were present. Ovotestes were found on the right side of the body in over half the cases; separate left ovaries or testes were about equally frequent ; in three cases separate testis and ovary were indicated. The left side of the body showed a different distribution of gonad types; about onefourth had ovotestes, another fourth ovaries, and one-twelfth testes. Unilateral distribution of gonad types was most frequent. The presence or absence of the prostate seemed to have significance because it is sometimes absent in purely female types. In recent literature similar cases have been called true hermaphrodites. This is an exaggeration in terms of long established practice in plants and animals where true hermaphroditism includes fully functioning gametes of each sex. Hungerford, Donnelly, Nowell and Beck (1959» have reported on a case of a Negro in which the culture cells had the chromosome complement of a normal female 46, with XX sex chromosomes. Unfortunately, the possibility that this case may be a chromosome mosaic was not tested by karyotype samples from several parts of the body. Harnden and Armstrong (1959) established separate skin cultures from both sides of the body of another hermaphroditic type. The majority of the cells were apparently of XX constitution with a total of 46 chromosomes. However, in one of the 4 cultures established, some 7 per cent of cells had an abnormal chromosome present, suggesting that the case might involve a reciprocal translocation between chromosomes 3 and 4 when the chromosomes were ordered according to size. All the other cell nuclei were normal. The fact that the majority of the cells in these two cases were XX and with 46 chromosomes seems to predicate against the view that either changes in chromosome number or structure of the fertilized egg are necessary for the initiation of hermaplu'odites. Ferguson-Smith (1960) describes two cases of gynandromorphic type in which the reproductive organs on the left side were female and on the right side were male. The recognizable organs were Fallopian tube, ovary with primordial follicles only, imma ture uterus in one case, none in the other, rudimentary prostate, small testis and epididymis, vas deferens, bifid scrotum, phallus, perineal urethra, pubic and axillary hair, breasts enlarging at 14 years. Testicular development with hyperplasia of Leydig cells, germinal aplasia, and hyalinization of the tubules was suggestive of the Klinefelter syndrome. Nuclear-chromatin was positive in both cases. Modal chromosome number was 46. The sex chromosomes were interpreted as XX. The 119 cell counts on one patient showed a rather wide range; 7 per cent had 44 chromosomes, 13 per cent had 45, 62 per cent had 46, and 18 per cent had 47 chromosomes. The extra chromosome within the cells containing 47 chromosomes was of medium size with submedian kinetochore as generally observed for chromosomes of group 3. It is surprising that the male differentiation in these four and other hermaphroditic cases (Table 1.5) is as complete as it is. Other observations show that the Y chromosome contains factors of strong male potency, yet in its absence the hermaphrodites develop an easily recognized male system. It is not complete but the degree of gonadal differentiation is as great as that observed in the XXY 4- 2A Klinefelter types. The bilateral sex differentiation in hermaphrodites would seem to require other conditions than those heretofore considered. Another case of hermaphroditism is that presented by Hirschhorn, Decker and Cooper (1960). The patient's j:)henotype was intersexual with phallus, hypospadias, vagina, uterus. Fallopian tubes, two slightly differentiated gonads in the position of ovaries. The child was 4 months old. Culture of bone marrow cells showed that the individual was a mosaic of two types. About 60 per cent of the cells had 45 chromosomes of XO karyotype, and 40 per cent had 46 chromosomes with a karyotA'^pe XY. The Y chromosome when present was larger than Y chromosomes of normal individuals. The change in size may be related to the association of the XO and XY cells and be similar etiologically to the case discussed by yivtz (1959) in Sciara triploids. There are mosaics in Drosophila formed from the loss by the female in some cells of one of lioi- X chromosomes, as for instance FOUNDATIONS FOR SEX 61 in ring chromosome types, which may display primary and secondary hermaphroditic development. For this to happen the altered nuclei apparently find their way into the region of the egg cytoplasm which is to differentiate into the reproductive tract. As seen in the adults, organ tissue of one chromosome type is cell for cell sharply differentiated from that of the other chromosome type with regard to sex. These observations indicate that for these mosaics the basic chromosome structure of the cell itself determines its development. In fact most mosaics of this species show this cell-restricted differentiation. Several problems arise when these well tested observations are considered in comparison with those now arising in the chromosome mosaics of the sex types in man. It would seem unlikely that the bone marrow cells or for that matter any somatic cells not a part of the reproductive tract would operate to modify the adult sex or a part thereof. Rather the developmental secjuence should start from cell differences within the early developing reproductive tract. Circulatory cells or substances would be of dubious direct significance from another viewpoint. All cells of the body would ultimately be about equally affected by any cells or elements circulating in the blood. With strong male elements and strong female elements the result expected would be a reduction in sex development of either sex instead of the sharply differentiated organ systems which are observed. This raises the question, are the chromosomally differentiated cell sex mosaics primary to or secondarily derived from the tissues of the ultimate hermaphrodites? Study of the cell structure of the sex organs themselves as well as much other information will be necessary to clear up this problem. There are, however, other types of controlled sex development, as by various genes, which lead to the presence of both male and female sexual systems. Genes for these phenotypes are relatively rare, but once found are transmitted as commonly expected. The inherited hermaphroditic cases in Drosophila are certainly relevant to the testicular feminization syndrome in man. Are they equally pertinent to the highly sporadic hermaphroditic forms just considered for man? If so, they indicate a genie basis for these types which would probably be beyond the range of the microscope to detect. The low frequency of true hermaphrodites in the human, together with lack of information on possible inheritance mitigates against the genie explanation ; although genie predisposition acting in conjunction with rare environmental events as occurs in our Balb/Gw mice (Hollander, Go wen and Stadler, 1956 ) could explain the rare hermaphrodites observed in that particular line of mice and a limited number of its descendants. 8. XX XY + U Autosome Type The XXXY -I- 44 autosome type in the human has been studied by Ferguson-Smith, Johnson and Handmaker (1960) and Ferguson-Smith, Johnston and Weinberg (1960). The two cases described were characterized by primary amentia, microorchidism and by two sex chromatin bodies in intermitotic nuclei. The patients were similar in having disproportionately long legs; facial, axillary, and abdominal hair scant; pubic hair present; penes and scrota medium to well developed ; small testes and prostates; vasa deferentia and epididymides normally developed on both sides of the body and no abnormally developed Miillerian derivatives. Testes findings were like those in Klinefelter cases with chromatinpositive nuclei, small testes with nearly complete atrophy, and hyalinization of seminiferous tubules and islands of abnormal and pigmented Leydig cells in the hyalinized areas. The few seminiferous tubules present were lined with Sertoli cells but were without germinal cells. Nuclear chromatin was of female type. About twofifths of the nuclei had double and twofifths single sex chromatin. The modal chromosome count for bone marrow cells was 48, 75 per cent of the cells having this number. Chromosome counts spread from 45 to 49. This type, XXXY plus 44 autosomes, may be looked upon as a superfemale plus a Y or a Klinefelter plus an X chromosome. In either case the male potency of the genes in the Y chromosome is able to dominate the female tendencies of XXX to develop nearly complete male phenotypes. Both cases had severe mental defects but TABLE 1.5 Chromosome kinds and numbers for different recognized sex types in man External Type Male Female Female (rare hemophilic) Eunuchoid female Female Female Female Female Female Female Male . Male. Male. Male. Male. Male . . . Female. Female . Female . Female . Male. Male. Hermaphrodite Hermaphrodite. Intersex Numbers of Chromosomes chromosoil rearrange 1 + X fragment 1 2 2 2 2 Interpreted a.s XX trisomic for Sand 11 or as XXXX 4X + Yl 3X+ Yj 1 3 3 2l /mosaic Missing or Extra Chromosomes ? Chr mosome 3 orT(X;A) Small autosome ? Large chromosome or T(X;A) X or Y (X or Y) +21 46,47,48 cliromosomes X, Y X or Y -fA set Sex Chromatm Negative Positive Negative Negative Positive Negative Positive Negative Negative Negative Negative Negative ± Negative Positive Positive Triple positi Negative Double posit Double posit Negative Double posit Negative Negative Negative Designating Term Normal male Normal female Pure gonadal dysgenesis CJonadal dysgenesis Testicular feiuinizati Turner Turner type female Tinner? gave birth to boy Turner Turner Klinefelter Klinefelter mongoloid Klinefelter Klinefelter Klinefelter Klinefelter Sujierfemale Superfemale gave birtli to children Testicular deficiency Precocious puberty Triploid Hermaphrodite or intersex dei)cnding on definition Investigators* 2 3, 4, 5, 5, 12, 13, 14, 40, 41 41 16, 17, 18, 40, 41 19 40 23, 40 24, 25 41 26 29, 30, 31,. 32, 33 35, 39, 40 34 41 Tjio and Levan, 1956. Nilsson, Bergman, Reitalu and Waldenstrom, 1959. Harnden and Stewart, 1959. Stewart, 1960b. Stewart, 1960a. Elliott, Sandler and Rabinowitz, 1959. Jacobs, Baikie, Court Brown, Forrest, Roy, Stewart and Lennox, 1959. Stewart, 1959 Lubs, Vilar and Bergenstal, 1959. Sternberg and Kloepfer, 1960. Puck, Robinson and Tjio, 1960. Ford, Jones, Polani, de Almeida and Briggs, 1959. Fraccaro, Kaijser and Lindsten, 1959. Fraccaro, Kaijser and Lindsten, 1960a. 15. Bahner, Schwarz, Harnden, Jacobs, Hienz and Walter, 1960. 16. Jacobs and Strong, 1959. 17. Bergman, Reitalu, Nowakowski and Lenz, 1960. 18. Nelson, Ferrari and Bottura, 1960. 19. Ford, Jones, Miller, Mittwoch, Penrose, Hidlor and Shapiro, 1959. 20. Ford, Polani, Briggs and Bishor), 1959. 21. Crooke and Hayward, 1960. 22. Muldal and Ockey, 1960. 23. Jacobs, Baikie, Court Broun, MacCJregor, Maclean and Harnden, 1959. 24. Eraser, Campbell, .MacCiillivray, Boyd and Lennox, 1960. 25. Stewart and Sanderson, 1960. 26. Jacobs, Harnden, Court Brown, Cohl.stein, Close, Mac Gregor, Maclean and Strong, 1960. 62 FOUNDATIONS FOR SEX 63 27. Ferguson-Smith, Johnston and Handiiiaker, 196( 28. Book and Santesson, 1960. 29. Harnden and Armstrong, 1959. 30. Hungerford, Donnelly, Nowell and Beck, 1959. 31. Ferguson-Smith, Johnston and Weinberg, 1960. 32. deAssis, Epps, Bottura and Ferrari, 1960. 33. Gordon, O'Gorman, Dewhurst and Blank, 1960 34. Hirschhorn, Decker and Cooper, 1960. 35. Sasaki and Makino, 1960. 36. Bloise, Bottura, deAssis, and Ferrari, 1960, 37. Fraccaro, Kaijser and Lindsten, 1960c. 37a. Fraccaro and Lindsten, 1960. 38. Fraccaro, Ikkos, Lindsten, Luft and Kaijser, 1960. 39. Harnden, 1960. 40. Ferguson-Smith and Johnston, 1960. 41. Sandberg, Koepf, Crosswhite and Hauschka, 1960. 42. Hayward, 1960. it should be remembered that they were sought in institutions for which this is a criterion of admittance. Their mental ability was distinctly less than that of Klinefelter XXY cases which have come under study. The pattern of the XXXY effects on the reproductive tract, however, was comparable with that observed in the XXY genotypes. The effects of one Y chromosome were balanced by either two or three X chromosomes to give nearly equal phenotypic effects. 9. XXY + 66 Autosome Type XXY + 66 autosome type was established by Book and Santesson (1960) for an infant boy having several somatic anomalies which may or may not be relevant to the sex type. Externally the genitalia were normal for a male of his age, penis and scrotum with testes present in the scrotum. Again the Y chromosome demonstrates its male potencies over two X's even in the presence of three sets of autosomes. The case is of particular significance since further development may indicate what male potencies an extra set of autosomes may possess. 10. Summary of Types Other types of sex modifying chromosomal combinations and their contained genes have been observed particularly as mosaics or as chromosomal fragments added or substracted from the normal genomes. No doubt other types will be discovered during the mushroom growth of this period. Time can only test the soundness of the observations for the field of human chromosomal genetics and cytology is difficult at best requiring special aptitudes and experience. Mistakes, no doubt, will be made. The status of the subject is summarized in Table 1.5. 11. Types Unrelated to Sex Other cases not related to sex or only secondarily so were scrutinized during the course of these studies. The information gained from them is valuable as it strengthens our respect for the mechanisms involved. The sex types which are dependent on loss or gain of the X and/or Y chromosomes belong to the larger category of monosomies or trisomies. Numbers of autosomal monosomic and trisomic syndromes have also been identified in the course of these investigations. Similarly, not all cases that have been studied have turned out to be associated with chromosomal changes. This in itself is important since it lends confidence in those that have, as well as redirects research effort toward the search for other causes than chromosomal misbehavior. The first trisomic in man was identified through the study of Mongolism. The condition affects a number of primary characteristics but not those of sex, for males and females occur in about equal numbers. The broad spectrum of these effects points to a loss of balance for an equally extensive group of genes in the two sexes. The common association of characteristics making up these Mongoloids, together with their sporadic appearance and their change in frequency with maternal age, all suggest the findings which Lejeune, Gautier and Turpin (1959a, b) and Lejeune, Turi)in and Gautier (1959a, b) were able to demonstrate so successfully. They established that the tissue culture cells of Mongoloid imbeciles had 47 chromosomes and that the extra chromosome was in the small acrocentric group. Lejeune, Gautier and Turpin (1959a, b) have now confirmed these observations on not less than nine cases. Jacobs, Baikie, Court Brown and Strong (1959), Book, Fraccaro and Lindsten (1959) and Fraccaro (cited by Ford, 1960) as well as later observers have substantiated the results on more than ten other cases. The well known maternal age effect, whereby women over 40 have a chance of having IMongoloid offspring 10 to 40 times as frequently as those of the younger ages, would seem to point to non 64 BIOLOGIC BASIS OF SEX disjunction in oogenesis as the most important cause of this condition. Some women who have had previous JMongoloid progeny have an increased risk of having others. This is an important consideration in that genetic factors may materially assist in bringing about nondisjunction in man as they are known to do in Drosophila (Gowen and Gowen, 1922; Gowen, 1928). The products of the nondisjunctions approach those expected on random distribution of the chromosomes (Gowen, 1933) so that occasionally more than one type of chromosome disjunction will appear in a given individual. Such a case is that illustrated by Ford, Jones, Miller, Mittwoch, Penrose, R idler and Shapiro (1959) in which the nondisjunctional type included not only that for the chromosome important to Mongolism but also the sex chromosomes significant in determining the Klinefelter condition. This individual showed 48 chromosomes, 22 pairs of normal autosomes, 3 sex chromosomes XXY, and a small acrocentric chromosome matching a pair of chromosomes, the 21st, within the smallest chromosomes of the human idiogram. The analysis of Mongolism showed the way for the separation of the various human sex types through chromosome analyses. Chromosome translocations furnish another means of establishing an anomaly that may then continue on an hereditary basis as either the male or female may transmit the rearranged chromosomes. Polani, Briggs, Ford, Clarke and Berg (1960), Fraccaro, Kaijser and Lindsten (1960b), Penrose, Ellis and Delhanty (1960) and Carter, Hamerton, Polani, Gunalp and Weller (1960) have studied Mongoloid cases which they interpreted in this manner. In some cases the rearranged chromosomes have been transmitted for three generations. Several of the translocations were considered to include chromosomes 15 and 21. Another trisomic autosomal type was rc])ortcd by Patau, Smith, Therman, Inhorn and Wagner (1960). The patient was female and had 47 chromosomes. The extra chromosome was a medium-sized acrocentric autosome belonging to the D group. Despite extensive malformations affecting several organs the patient lived more than a year. Another female iiortraying the same syndrome has since been found, so other cases may be expected. Among the characteristics are mental retardation, minor motor seizures, deafness, apparent micro or anophthalmia, horizontal palmar creases, trigger thumbs, Polydactyly, cleft i)alate, and hemangiomata. The third trisomic type was also reported by Patau, Smith, Therman, Inhorn and Wagner (1960). Six individuals have been observed. The characters affected are mental retardation, hypertonicity (5 patients), small mandible, malformed ears, flexion of fingers, index finger overlaps third, big toe dorsiflexed (at least 4), hernia and/or diaphragm eventration, heart anomaly (at least 4), and renal anomaly (3). The sexes were two males and four females. The extra chromosome was in the E group and was diagnosed as number 18. Edwards, Harnden, Cameron, Crosse and Wolff (1960) have described a similar case but they consider the trisomic to be number 17. Ultimate comparisons of these types no doubt will decide if this is a 4th trisomic or if all the cases belong in the same group. The Sturge-Weber syndrome apparently is caused by another trisomic. Locomotor and mental abilities are retarded. Hayward and Bower (1960) interpret the 3 chromosomes responsible as the smallest autosomes, number 22, of the human group. Trisomic frequencies should be matched by equal numbers of monosomies. Turpin, Lejeune, Lafourcade and Gautier (1959) have reported polydysspondylism in a child with low intelligence, dwarfing, and multil)le malformations of spine and sella turcica. The somatic cell chromosome count was only 45 but one of the smallest acrocentric chromosomes appeared to have been translocated, the greatest part of this chromosome being observed on the short arm of one of the 3 longer acrocentric chromosomes. Th(> condition appears to be unique and not likely to be found in other unrelated famiVws. However, the phenotyj^ic effects were so severe that all members of the proband's family would seemingly be worthy of careful sur\'('y for their chromosome characteristics. The comi)lex pattern of multiple anomalies renders each syndrome distinct from the othei's. Chromosome losses or gains from FOUNDATIONS FOR SEX Go the normal diploid would be expected to lead to the complex changes. Mongolism is influenced by age of the mother and probably to some extent by her inheritance. It is to be expected that the other trisomies may show parallel relations. Other trisomies may be expected although, as the chromosomes increase in size, a group of them will have less opportunity to survive because of loss of balance with the rest of the diploid set. Thus far most of these conditions affect the sex phenotypes. This is in accord with the results in Drosophila. Changes in the balance of the X chromosomes are less often lethal than the gain or loss of an autosome. Other animals show like effects. In plants, loss or gain of a chromosome, although generally detrimental, often causes less severe restrictions on life. Harmful effects are observed but do not cause early deaths. This may be because many aneuploids are within what are presumably polyploid plant species. Ford (1960) has collected the data on 13 different phenotypes that could come under suspicion of chromosomal etiology as examined by a number of workers. Careful cytologic examination of patients suffering from one or another of these diseases has shown that the idiograms were normal in both number and structure of the chromosomes. The disease conditions were: acrocephalosyndactyly, arachnodactyly (Marfan's syndrome), chondrodystrophy, Crouzon's disease, epiloia, gargoylism, Gaucher's disease, hypopituitary dwarfism, juvenile amaurotic idiocy, Laurence-MoonBiedl syndrome. Little's disease, osteogenesis imperfecta, phenylketonuria, and anencephalic types. To this list Sandberg, Koepf , Crosswhite and Hauschka (1960) have now been added neurofibromatosis, Lowe's syndrome, and pseudohypoparathyroidism. F. SEX RATIO IN MAN Sex ratio studies on human and other animal populations have always been large in volume. The period since 1938 is no exception. Geissler's (1889) data on family sex ratios, containing more than four million births, have been reviewed and questions raised by several later analysts. Edwards (1958) has reanalyzed the clata from this population and considered these points and reviewed the problems in the light of the following questions: (1) Does the sex ratio vary between families of the same size? (2) Do parents capable of producing only unisexual families exist? (3) Can the residual deviations in the data be satisfactorily explained? Probability analyses were based on Skellam's modified binomial distribution, a special case of the hypergeometrical. The following conclusions were drawn. The probability of a birth being male varies between families of the same size among a complete cross-section of this 19th century German population. There is no evidence for the existence of parents capable of producing only unisexual families. With the assumption that proportions of males vary within families, the apparent anomalies in the data appear to be explicable. These studies have a bearing on the variances observed in further work dealing with family differences such as that of Cohen and Glass ( 1959) on the relation of ABO blood groups to the sex ratio and that of Novitski and Kimball (1958) on birth order, parental age, and sex of offspring. Novitski and Kimball's data are of basic significance, for the interpretations are based on a large volume of material covering a one-year period in which improved statistical techniques were utilized in the data collection, in showing that within these data sex ratio variation showed relatively little dependence on age of mother, whereas it did show dependence on age of father, birth order, and interactions between them. These observations have direct bearing on the larger geographic differences observed in sex ratios as discussed by Russell (1936) and have recently been brought to the fore through the studies of Kang and Cho (1959a, b). If these data stand the tests for biases, they are of significance in showing Korea to have one of the highest secondary sex ratios of any region, 113.5 males to 100 females, as contrasted with the American ratio of about 106 males to 100 females. Of similar interest is the lower rate of twin births, 0.7 per cent in Korea vs. about 1 per cent in Caucasian populations and the fact that nearly twothirds of these tW'in births in Korean peoples are identical, whereas those in the Caucasian groups are only about half that number. The reasons for these differences must 66 BIOLOGIC BASIS OF SEX lie in the relations of the human X and Y chromosomes and autosomes and the balance of their contained genes. Little or nothing is known about how these factors operate in the given situations. Acknowledgment. In formulating and organizing the material on which this paper is based I have been fortunate in the helpful discussions and analytical advice contributed so generously by Doctors H. L. Cai'son, K. W. Cooper, H. V. Grouse, C. W. Metz, S. B. Pipkin, W. C. Rothenbuhler, and H. D. Stalker, and others having primary research interests in this field. To them, and particularly to my research associates Doctors S. T. C. Fung and Janice Stadler, our secretary Gladys M. Dicke and to my wife, ]\Iarie S. Gowen, I tender grateful acknowledgment for their contributions to the manuscript. Direct reference is made to some 300 investigations in the body of the paper but the actual number, many of which could equally well have been incorporated, that furnished background to these advances is much larger, more than 1600. 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