Difference between revisions of "Book - The Development of the Albino Rat 9"

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==Late Stages in Egg-Cylinder Differentiation and the Anlage of the Mesoderm==
  
 +
In the rat series there are found 24 egg cjdinders showing the stages of development considered in this section; certain of them are cut longitudinally and others cross-wise.
 +
 +
 +
For the special consideration of egg-C3'linder formation just
 +
prior to the anlage of the mesoderm, I present two egg-cylinders
 +
obtained during the latter half of the ninth day after insemination ;
 +
one of these was cut longitudinally, the other in favorable crosssection. The egg-cylinder shown in figure 29, rat No. 40, 8
 +
days, 17 hours after insemination, seems unusually well fixed,
 +
as evidenced by its symmetrical outline, and is cut in a very
 +
favorable plane. The sections are from a series cut at right angles to the long axis of the uterine horn. The decidual crypts
 +
lodging the egg-cylinders of this stage are by this time nearly
 +
completely separated from the lumen of the uterus, and are
 +
surrounded by a well-developed decidua. Extra vasated maternal l)lood nearly surrounds such egg-cylinders.
 +
 +
 +
Fig. 29 Longitudinal, sagittal section of egg-cylinder of the albino rat showing the final mesoderm-free stage. X 200. Rat No. 40, 8 days, 17 hours, after insemination; ect.pl., ectoplacental cone or Trager; p.ect., parietal or transitory ectoderm; pr.emh.ect., primary embryonic ectoderm; ex.ect., extraembryonic ectoderm; pr.c, proamniotic cavity; r.ent., visceral entoderm, absorptive for maternal hemoglobin, cells showing the three zones described by Sobotta; pr.emh.ent., primary embryonic entoderm.
 +
 +
 +
The egg-cylinder shown in figure 29 presents a total length
 +
of 1.15 mm., a width of approximately 0.18 mm. The ectoplacental cone presents a length of 0.4 mm. and of the proamniotic
 +
cavity, 0.5 mm., of which 0.2 mm. falls to the antimesometrial
 +
portion lined by primary embryonic ectoderm. This egg-cylinder differs only in shape and size from that shown in C of figure
 +
27, obtained 8 days after insemination. The primary embryonic
 +
and extraembryonic ectoderm lining or enclosing the proamniotic cavity are readily differentiated. The primary embryonic
 +
ectoderm, derived from the ectodermal node, constitutes a pseudostratified epithelium, composed of relatively long colunmar
 +
cells, with nuclei radially placed with reference to the lumen of
 +
the proamniotic cavity, and shows active cell division, no less
 +
than 12 mitotic figures occurring in the section figured. The
 +
protoplasm of its cells stains distinctly deeper than does that
 +
of the cells of the extraembryonic ectoderm. The cells of the
 +
latter are of cubic, short columnar, or polyhedral shape, arranged in a single or double layer, with no definite arrangement
 +
of the long axes of its nuclei. It is, therefore, possible readily
 +
to distinguish — by reason of shape and size of cells, relative position of nuclei, reaction to stain of protoplasm — between the
 +
cells of the primary embryonic and extraembryonic ectoderm,
 +
and to determine the sharp hne of junction at which the two
 +
types of cells form a continuous layer, a fact which will receive
 +
further consideration in dealing with the anlage of the mesoderm
 +
as observed in slightly more advanced stages. At the mesometriai end of the i)roanmiotic cavity, the cells of the extraembryonic ectoderm become continuous with the cells at the base
 +
of the ectoplacental cone; in the region of this junction, active
 +
mitosis are often to be observed. In this egg-cylinder the visceral
 +
entoderm may readily be differentiated into two portions. The
 +
portion which surrounds the primary embryonic ectoderm to
 +
nearly the region of its junction with the extraembryonic ectoderm, consists of a single layer of broad, flattened cells which
 +
assume a cubic or short columnar shape as the mesometrial
 +
border of the primary embryonic ectoderm is approached.
 +
This portion of the visceral entoderm we have designated as the primary oiiibrvonic CMit()<l(M-ni. 'I'ho portion of tlic Nisccral
 +
ont()d(M'iH surrouiidiiiji; tlic sides of tlic cjiir-cvliiidcr in the region
 +
of the oxtiacnihiyonic ectoderm, to ncai- the base of the cctoplacentul cone, consists of a single layer of eolunniar cells, regularly arranged and presenting the three zones described by Sobotta. In this stage of egg-cylinder development of the al})ino
 +
rat, the absorption of hemoglobin granules derived from maternal
 +
blood cells, first shown for the mouse by Sobotta and Kolstei',
 +
may be readily made out. In preparations stained in hematoxylin
 +
and Congo red, in and on the outer zone of the visceral entodennal
 +
cells there may be observed granules staining deeph' in the Congo
 +
red, presenting the color reaction of hemoglobin. In the middle zone of these cells the protoplasm is distinctly \acuolated,
 +
while the inner zone, containing the nuclei, presents a denser
 +
protoplasm. The transitory' or parietal ectoderm consists of a
 +
homogeneous membrane, closely adherent to the maternal decidua, especially along the sides of the egg-cylinder. This layer
 +
presents scattered nucleated protoplasmic masses of spindle or
 +
dome shape on its inner surface, the relations and distribution
 +
of which may be clearly seen in the figure. Attention needs
 +
yet be drawn to the ectoplacental cone of the egg-cylinder. Its
 +
relation to the maternal decidua is very intimate, so that in
 +
places, owing to blood extravasations, it is difficult to differentiate
 +
between embryonic and maternal tissue. Many of the cells
 +
of the ectoplacental cone present a vacuolated protoplasm, the
 +
\'acuoles enclosing maternal blood cells. Therefore, they are
 +
distinctly' phagocytic. Sobotta has also observed and described
 +
this for the mouse. Referring to a slightly older stage after the
 +
anlage of the mesoderm, his own words read as follows:
 +
 +
:Weitcrhin sehen wir iin Stadium der Fig. 5 audi eine starke Verlangerung und Vorgnissorung des Ectoplacontarconus, an dcm im mesonietralcn Tcile jctzt ^'a(•uole^ auftreten, die in spateren Stadien regelmassig gefunden worden und zwar crfiillt niit miitterlichcn Blutextravasaten. Die Ehrniihrung dcs Embryo mit miitterlichem Hiimoglobin * * * * jg^ jetzt im vollen (Jang.
 +
 +
 +
Absorption of maternal hemoglobin by the cells of the ectoplacental cone appears to be established at a relatively earlier
 +
period in the rat than in the mouse.
 +
 +
 +
The egg-cylinder presented in figure 29 constitutes the final
 +
mesoderm-free stage, the final stage in which no distinct bilaterality may be determined. I assume that the egg-cylinder presented in the figure is cut in the sagittal plane. This assumption
 +
is based on the fact that the primary embryonic ectoderm extends slightly farther toward the mesometrial pole on the one
 +
side than on the other. In good frontal sections one side of the
 +
egg-cylinder in this stage of development should present a mirror picture of the other side. The side on which the primary
 +
embryonic ectoderm extends farther toward the mesometrial
 +
pole, the left in the figure, is regarded as containing the caudal
 +
end of the future embrj^o. In the primary embryonic ectoderm
 +
of this region, it is believed, will develop the primitive streak and
 +
groove, and thus the anlage of the mesoderm. Not in all the
 +
egg-cylinders of this stage of development found in my series can
 +
the caudal end of the future embryonic area be postulated prior
 +
to the anlage of the mesoderm, and in cross-sections no such
 +
differentiation can be made. The proamniotic cavity of the
 +
egg-cylinder shown in figure 29 presents a regular and nearly
 +
smooth contour, not divisible into regions such as described
 +
for a similar stage for the mouse by Selenka, MeUssinos, and
 +
Sobotta. A very slight constriction is to be observed onh^ in
 +
the region where the primary embryonic and extraembryonic
 +
ectoderm are joined in a continuous layer. I am thus wholly
 +
in accord with Widakowich, who in describing a similar stage in
 +
one of his preparations, states: "Das war die einzige konstante,
 +
bald starker, bald schwiicher ausgepragte Einschniirung der
 +
Proamnionhohle," as previously quoted.
 +
 +
 +
A series of figures of critical regions taken from a series of cross-sections of an egg-cylinder of a stage nearly identical with that shown in figure 29, though of a shghtly smaller egg-cjdinder,
 +
is given in figure 30, rat No. 42, 8 days, 16 hours, after insemination. The sections chosen for the several drawings, A to D, are from the following regions, as may be ascertained by comparison with figure 29; A, through about the middle of the ectoplacental cone; B, through the proamniotic cavity just below its mesometrial end; C, through the proamniotic cavity just above the region of the junction of the primary embryonic and extraeml)ryonic ectoderm; 1), a Httle above the middle of the antimesometrial j)oi-ti()ii of tiie i)i'oamiii()tic cavity. The levels of the several sections drawn in figure 30 is approximately indicated by the several crosses found to the left of the egg cylinder drawn in figure 29.
 +
 +
 +
 +
 +
Fig. 30 Four figures from a series of cross sections of an egg-cylinder of the albino rat in the stage of development shown in figure 29. X 200. Rat No. 42, 8 days, 16 hours after insemination.
 +
 +
The levels at which the several sections drawn were taken is ai)i)roximately
 +
indicated by the several crosses found to the left of figure 29. A, middle of
 +
cctoplacental cone; B, ectoplacental end of the proamniotic cavity; C, just above
 +
level of junction of the primary embryonic and extraembryonic ectoderm; a
 +
little above the middle of primary embryonic ectoderm. The want of any definite
 +
bilateral sjinmetry of albino rat egg-cylinders of this stage of development is
 +
shown by this series of sections; p.ect., parietal or transitorj^ ectoderm; cx.ect.,
 +
extraembryonic ectoderm, surrounding mesometrial portion of proamniotic cavity; p?-.em6.ec<., primary embrj-onic ectoderm; r.e»/., visceral entoderm; p.emb.
 +
ent., primary embryonic entoderm; pr.c, proamniotic cavity.
 +
 +
 +
In A of figure 30, there may be observed a vacuolization of the
 +
protoplasm of the more peripherally placed cells of the ectoplacental cone, the vacuoles enclosing maternal blood cells. The
 +
more centrally placed cells of this ectoplacental cone show a
 +
tendency to concentric arrangement. Figures B and C present
 +
structural appearances nearly identical. The egg-cylinder is
 +
bounded by the thin layer of parietal or transitory ectoderm having
 +
scattered masses of nucleated protoplasm on its inner surface.
 +
This membrane of apparently homogeneous structure stains
 +
sharply in well fixed preparations and may be readily discerned.
 +
The cells of the \dsceral entoderm, somewhat taller in the section
 +
taken nearer the antimesometrial pole (C), present clearly the
 +
three zones to which attention has been drawn. The cells of
 +
the extraembryonic ectoderm bounding the mesometrial portion
 +
of the proamniotic cavity, are of cubic, short columnar, or polyhedral form disposed in single or double layer, presenting relatively lightly staining protoplasm. In D of figure 30, the cells forming the primary embryonic ectoderm are of distinct columnar
 +
shape, with relatively deeply staining protoplasm and nuclei
 +
arranged nearl}^ in a single layer except for such as show mitotic
 +
phases. The cells of the primary embryonic entoderm are of
 +
a broad, pavement type for a greater part of the circumference,
 +
and may be contrasted with the cells of the visceral entoderm
 +
shown in B and C of the figure; the latter are absorptive cells, the
 +
former not. This series of figures, more especially B, C, and D,
 +
show clearlj' the absence of bilaterality in the egg-cylinders of
 +
the albino rat at this stage of development. The slight compression observed in this egg-cylinder, as shown in the fi gures,
 +
I regard as not of moment.
 +
 +
 +
Fig. 31 Longitudinal sagittal section of egg-cylinder of the albino rat showing anlage of the mesoderm. X 200. Rat Xo. 34, 8 days, 18 hours, after insemination; eel. pi., ectoplacental cone or Trager; p.ect., parietal or transitorjectoderm; pr.emb.ect., primary embryonic ectoderm; ex.ect., extraembryonic
 +
ectoderm; pr.emfc.eni., primary embryonic entoderm; wcs., mesoderm in anlage;
 +
pr.c, proamniotic cavity; v.oit., visceral entoderm.
 +
 +
 +
 +
Grosser has figured in his figures 68 and 114, an egg-cyUnder
 +
of the albino rat which measures nearly 2 nini. in length. The
 +
age of this is given as 8| days. So far as may be determined from
 +
his figures, the preparation is not described in his text, the age,
 +
size, form, and structure of the egg cylinder shown in figure 29
 +
and Grosser's figures 68 and 114, are very similar. In Grosser's
 +
figures, I see no evidence of his having differentiated between
 +
primary embryonic and extraembryonic ectoderm, while the
 +
reference letters for ectoderm and entoderm are reversed. Selenka's figure 31, plate 45, may be of a similar stage. This figure is,
 +
however, too diagrammatic to admit of close study. No difference is shown in the shape and structure of the cells bounding
 +
the two parts of the proamniotic cavity. Christiani's figure
 +
39 may be of the same stage, but is too schematically drawn.
 +
Figure 4 of the article of AYidakowich is of a slightly older stage
 +
and presents only a part of the egg-cylinder; it is recorded as
 +
about 6f days old. The stage under consideration is not figured
 +
by Widakowich, although his text description corresponds closely
 +
with what has been here presented.
 +
 +
 +
The next stage and the one with which this communication
 +
is to be completed is one of importance since it is characterized
 +
by the anlage of the mesoderm. My own observations may be
 +
introduced with the consideration of an egg-cylinder, a section
 +
of which is presented in figure 31, rat No. 34, 8 days, 17 hours,
 +
after insemination. This was cut in the sagittal plane and
 +
measures 1.1 mm. by 0.2 mm., of which 0.4 mm. fall to the
 +
ectoplacental cone. This egg-cylinder is almost an exact duplicate, both in size and form, of that figured in figure 29 of the
 +
same age. In the egg-cylinder shown in figure 31, however,
 +
there may be observed, to one side, in the region of the junction
 +
of the primary embryonic and extraembryonic ectoderm, and
 +
between primary embryonic ectoderm and entoderm, a small
 +
group of cells which lie in close relation to the ectoderm and
 +
constitute early mesodermal cells. The sections of this series
 +
pass not exactly parallel to the mid- sagittal plane throughout the
 +
whole extent of the egg-cylinder; especially is this true of its antimesometrial portion, in the region of the primary embryonic ecloclcnn. This portion in the section iij^ured, passes a httle
 +
to one side of the mid-sagittal plane. The two sections preceding the one figured enclose the mid-sagittal plane, and in them,
 +
the groii]:) of cells found between primary embryonic ectoderm
 +
and (Miloderm are in closer relation to the ectodermal hiyer and
 +
al all points (Hstinctly separated fi'om the entoderm. They
 +
are regarded as having wandered from the primary embryonic
 +
ectoderm to the place they occupy, a fact which is more easily
 +
ascertained in cross sections of a similar stage, as will appear
 +
from further discussion. From a study of very slightly older
 +
stages it can be determined that this region constitutes the
 +
primitive streak region of the future embryonic area. It is not
 +
my purpose at this time and in this communication to give especial consideration to the much discussed ciuestion of the
 +
origin of the mesoderm in Mammalia. In the rat, this cjuestion
 +
is complicated by the question of the anlage of the anmiotic
 +
fold, which separates the proamniotic cavity into amniotic
 +
cavity proper and the ectoplacental cavity, the (hnelopment
 +
of which will be considered in a projected contribution. In
 +
anticipation of this second publication, however, the following
 +
facts may here receive consideration. Widakowich presents
 +
in his figure 4, giving only the antimesometrial end of an eggcylinder obtained the latter part of the 7th day, the anlage of the
 +
mesoderm as observed by him. This figure and my own figure
 +
31 present almost identical relations, his figure showing only
 +
thi'ee mesodermal cells between primary embryonic ectoderm
 +
and entoderm. His own words concerning the anlage of the
 +
mesoderm in the albino rat, with which I find myself in full
 +
accord, except as to the age of the egg-cylinder, read as follows:
 +
 +
:Das erstc auftreten des Mesoderms beobachtete ich an Keimen vom Ende des 7 Tages. Die ersten Mesodermzollen licgen im Bereiche der vom mesometralen Ende des stiirkcr far])baren priniaren cmbryonalen Ectoderm gebildeten Faltc. Es koninit hier einc ganz bestimmte Stelle in Betracht, die dort liegt, wo sich spater das hintere Ende des Primitivstreifens liefindet.
 +
 +
 +
There is, however, wide divergence of the views of authors as concerns the anlage of the mesoderm in the rat and mouse.
 +
 +
 +
Selenka, it would seem, in part at least, interpreted correctly
 +
the development of the mesoderm in the rat, although a stage
 +
showing its anlage was not observed. Duval believes that the
 +
mesoderm has origin from a thickened part of the entoderm,
 +
probably in the region of the anterior portion of the future embryonic area; the primitive streak was not recognized. Christiani's figures 45 and 47, transverse sections of the egg-cylinder
 +
from the eighth day, give correctly the relative position of the
 +
mesoderm with reference to the primitive streak; however, they
 +
show stages some little time after the anlage of the mesoderm.
 +
According to Robinson, in the early part of the eighth day the
 +
cavities of the epiblast (primary embryonic ectoderm) and of
 +
the trophoblast (extraembryonic ectoderm) meet and fuse to
 +
form a hollow cylinder, the proamniotic cavity. He states
 +
that "For a time the united cavities of the epiblast and trophoblast increase in size, together with the general growth of the
 +
ovum, and this increase continues until in the latter part of the
 +
eighth day the mesoblast appears around the margin of the
 +
epiblast where it is in apposition with the trophoblast." Robinson was able to differentiate between the primary embryonic
 +
ectoderm (epiblast) and the extraembryonic ectoderm (trophoblast) and his figure 14 (plate 23-24), though schematic, shows
 +
that he recognized the positions of the anlage of the mesoderm
 +
correctly, as also its derivation from the primary embryonic
 +
ectoderm. The observations of Melissinos, bearing on the anlage of the mesoderm have been critically reviewed by both
 +
Widakowdch and Sobotta, and I am wholly in accord with their
 +
views when they state that no credence can be given these observations since it is clear that IVIelissinos has confused sagittal
 +
and frontal sections in such a way as to make his observations
 +
of no value. According to Melissinos, the mesoderm arises
 +
from the outer surface of the middle fold of the egg cylinder,
 +
in the region of its union with the antimesometrial ectodermal
 +
fold; it is certain that it does not arise from the part of the eggcylinder that has differentiated from the primary embryonic
 +
ectoderm; but, if I interpret him correctly, from the extraembryonic portion of the ectoderm. That Melissinos did not have before him the stages showing the aiilage of tlic mesoderm
 +
seems clear. S()))otta's ('11) observations, mouse material,
 +
deserve fuller consideration. In interpreting his results, I am
 +
mindful of the fact that he was unable to locate the line of union
 +
between primary embryonic and extraembryonic ectoderm, as
 +
can readily be done in suitable rat material, as has previously
 +
been shown by Robinson and Widakowich, and to which attention has constantly been drawn in this communication. I am
 +
unable to state from personal observation whether in the white
 +
mouse these two types of ectoderm which form the lining of the
 +
proamniotic cavity, can be differentiated on ascertaining the
 +
right technical method. Sobotta's material seems well fixed.
 +
If not, it would seem to me difficult to determine definitely
 +
the exact place of origin of the mesodermal cells, whether extraembryonic or embryonic. Sobotta recognized the anlage of
 +
the mesoderm in the mouse during the last hours of the seventh
 +
day or first hours of the eighth day. This is said to appear at
 +
the caudal end of the future eml:)ryo as a group of loosely arranged cells lying between the inner and outer layers of the
 +
egg-cylinder. At the place where the mesodermal cells arise
 +
from the inner layer of the egg-cylinder, there is developed a
 +
fold, recognized as the caudal amniotic fold (''Schwanzfalte
 +
des Amnios"). After discussing these observations at length,
 +
Sobotta concludes as follows:
 +
 +
:Was die Deutung dieser friihen Stadien der Mosodcnnbikhing in der Keiinl)laso der Alaus anlangt, so haiuielt es sich hier niclit uni die Bildung des einbryonalen Mesoderms, die erst mit der eigentlichen Gastrulation spater einsetzt, sonderii uni Entsteliung ausserembryonalen ^Mesoderms, besonder des Teils des mittleren Keimblattes, dass bei der Bildung der primaren Eihiiute, Amnios und Chorion in Betraeht kommt und des den ausserembryonalen Teil der Leibeshrihle, das Exocoelom auskliedet, der Hohle, die eben Amnios und Chorion voneinander trennt. Es erfolgt also, um einen kurzen Ausdruck zu gebrauchen, die Bildung des Amniosmesodcrms.
 +
 +
 +
An embryonic anlage is said not to exist at this stage; this is
 +
recognized only after the development of the primitive streak.
 +
It is not my purpose to enter fully into a discussion of this important question in this communication. This would involve consideration of older stages, and the making of a number of
 +
reconstructions, ^yhich it is not contemplated to consider now.
 +
It must suffice to state at this time that in the albino rat, as
 +
shown by Widakowich and here shown by me, it is possible to
 +
delineate clearly the primary embryonic ectoderm and to show
 +
that the first evidence of the mesoderm is found antimesometrial
 +
to the future amniotic fold and in the region of the future primitive streak; therefore is mesoderm which I would regard as
 +
peristomal mesoderm in the sense of C. Rabl, reference to which
 +
is made bj^ Sobotta in his discussion of this question. It may
 +
be that the rat offers more suitable material for the elucidation
 +
of this question than is to be found in the mouse. In the albino
 +
rat, the anlage of the mesoderm is from the sagittal portion of
 +
the caudal region of the primary embryonic ectoderm, the caudal
 +
part of the future primitive streak and antimesometrial to the
 +
amniotic fold. Sobotta gives very favorable consideration to
 +
the observations of Widakowich, touching this question, which
 +
he regards as ''Bei weitem die beste Darstellung des Gegenstandes." My own observations fully confirm those of Widakowich.
 +
These questions will receive fuller consideration in a later publication dealing with the embryology of the albino rat, carrying
 +
the development from the time of the anlage of the amniotic fold
 +
to the stage of embryo form, the material for which is at hand.
 +
 +
 +
In figure 32 are shown cross-sections of the antimesometrial
 +
portion of three egg-cylinders in the region of the developing
 +
mesoderm. Sections drawn in A and B, were taken respectively
 +
from egg-cylinders obtained from the same uterus as was the
 +
one shown in sagittal section in figure 31, rat No. 34, 8 days, 17
 +
hours, after insemination; C, from rat No. 41, 8 daj^s, 16 hours,
 +
after insemination. It is very probable that the series from
 +
which A of this figure was drawn, is net cut in exactly the cross
 +
plane. A stud}^ of the series shows, however, that the deviation
 +
from this plane is not marked. The sections from which this
 +
figure was drawn pass a little below (antimesometrial) to the
 +
region of junction of the primary embryonic and extraembryonic ectoderm. To one side, the lower in the figure, the primary
 +
embryonic ectoderm shows a slight thickening and evidence of
 +
 +
 +
 +
Fig. 32 Three cross sections from egg-cj'lindcrs of the albino rat, showing early stages in the development of the mesoderm. X 200. A and B, rat
 +
No. 34, 8 days, 17 hours; C, rat No. 41, 8 days, 16 hours, after insemination.
 +
 +
These sections taken from three egg-cylinders are through the primary embr}'onic ectoderm, near its junction with the extraembryonic ectoderm, thus through
 +
the antimesometrial portion of the proamniotic cavity A, early stage, in
 +
anlage of the mesoderm; B, anlage of the primitive streak and groove; C, well
 +
developed primitive streak and groove, with lateral wings of mesoderm; pr.e?rt6.
 +
ect., primary embrj'onic ectoderm; pr.emb.ent., primarj^ embryonic entoderm;
 +
mes., mesoderm; pr.sir., primitive streak; pr.gr., primitive groove; p. ect., parietal or transitory ectoderm; pr.c, proanmiotic cavity.
 +
 +
 +
 +
cell proliferation. The cells of this region have not the form
 +
of tall columnar cells, such as seen in the greater part of the
 +
remaining primary embryonic ectoderm, but are of polyhedral form and are continuous, in the mid sagittal plane, with cells
 +
that have wandered between the primary embryonic ectoderm
 +
and entoderm, cells regarded as constituting the mesoderm.
 +
In all of the sections of this series, so far as the mesoderm extends, this is distinctly separable from the entoderm, and is
 +
continuous with the primary embryonic ectoderm only along a
 +
narrow region of thickened primary embryonic ectoderm, situated in the mid-sagittal plane, and which may in this series be
 +
regarded as the anlage of the primitive streak. From the sides
 +
of this region of slightly thickened primary embryonic ectoderm,
 +
the extent of which is evidenced by the absence of an external
 +
limiting membrane, cells wander laterally to form the mesoderm.
 +
B, of figure 32, presents essentially the same appearance, although
 +
representing a slightly older stage. The sections of this series
 +
I regard as cut fairh' well in a plane at right angles to the long
 +
axis of the respective egg-cylinder. The section taken for the
 +
sketch is situated a very little further away from the line of
 +
junction of the primary embryonic and extraembrj^onic ectoderm, than is the section the drawing of which is shown in A
 +
of this figure, as may be judged from the more uniformly pavement tj'pe of the entodermal cells. The triangular form of the
 +
proamniotic cavit}' is regarded as normal, and as indicating an
 +
early stage in the anlage of the primitive groove. In this figure,
 +
in its lower portion, the region of the primitive streak is readily
 +
discernible by reason of the fact that there is wanting here an
 +
external limiting membrane, and further by reason of the form
 +
of the cells and the form and relative position of their nuclei;
 +
certain of these cells indicating, both by their form and their
 +
position, the source and the direction of the wandering of the
 +
cells which constitute the anlage of the mesoderm. The wandering of the mesodermal cells between the primary embryonic
 +
ectoderm and entoderm, to form the lateral mesodermal wings,
 +
is clearly shown in this figure, especially to the left. The antimesometrial ends of the egg-cylinders, sections of which are
 +
shown in A and B of this figure, are as yet free from the invading
 +
mesoderm, as is also the part of the egg-cylinders lying opposite
 +
the region of the primitive streak, the upper portions of the
 +
respective figures, these forming the region of the future anterior
 +
ends of the respective embryos. In C of figure 32 is shown a
 +
drawing of one of the sections of a series of cross-sections of an
 +
egg-cylinder taken from rat No. 41, 8 days, 16 hours, after insemination, presenting a stage in which the primitive groove
 +
may be definitely made out. This figure is not unhke figure (>
 +
of the article of Widakowich, obtained from an egg-cylinder
 +
secured on the eighth day. Concerning this figure he states:
 +
"Das Ectoderm steht in direktem Zusammenhange mit zwei
 +
Mesodermzungen die gegen die der Primitivrinne gegeniiberhegende Seite zu auswachsen." The section drawn in C of this
 +
figure is taken from the region very near the junction of the
 +
primary embryonic and the extraembryonic ectoderm, as may
 +
be observed from the character of the entodermal cells, in the
 +
lower part of the figure. The increase in the thickness of the
 +
mesodermal wings, the result, in part at least, of proliferation
 +
of mesodermal cells, as evidenced by the presence of mitotic
 +
figures, is clearly shown in this figure. The mesoderm is distinctly separable from the entoderm as also from the primary
 +
embryonic ectoderm except in the region of the primitive streak
 +
and groove. The growth of the mesoderm after its anlage has
 +
been correctly shown for the albino rat by Selenka, Robinson,
 +
and Widakowich; the latter especially giving excellent figures.
 +
His figure 5 is especially instructive. In this, he represents the
 +
appearances shown by two views of an isolated egg-cylinder, with
 +
the primiti^'e groove in anlage, showing the lateral extensions
 +
of the mesoderm. Sobotta ('11) has given the best and most
 +
comprehensive account of the anlage and growth of the mesoderm in the mouse. An excellent cross-section of a mouse eggcylinder in the primitive streak stage is presented in his figure
 +
6, which presents very similar appearances to my C of figure 32.
 +
None of the figures of cross-sections of egg-cylinders included
 +
by me show the very beginning of the anlage of the mesoderm,
 +
though A of figure 32 approaches this very closely, as does also
 +
figure 31, presenting a sagittal section. The evidence at hand
 +
warrants the conclusion that in the albino rat, the mesoderm
 +
has its anlage in the caudal region of the primary embryonic ectoderm, from a narrow zone of cells situated in the region of
 +
the future primitive streak. From this region there is an outwandering of cells which invade the potential cleft between primary embryonic ectoderm and entoderm, spreading laterally
 +
in wing-like sheets. This I would regard as prostomial mesoderm
 +
in the sense of C. Rabl. The anlage of the mesoderm in the
 +
albino rat, and the early stages of its lateral extension, with the
 +
anlage of the primitive streak and groove, falls to the latter
 +
part of the ninth day after insemination.
 +
 +
 +
Beginning with the pronuclear stage, found at the end of the first day, 8 days are required for the completion of the process of segmentation, blastodermic vesicle formation and the formation of the primary germ layers — ectoderm, mesoderm, and entoderm — in all, 9 days out of a possible 21 to 23 days, the normal gestation period of the albino rat.
 +
 +
==Conclusions==
 +
 +
Early stages of mammalian development may readily be
 +
obtained from the albino rat (Mus norvegicus albinus). When
 +
care is exercised, mating may be observed and the age of the
 +
embryo, reckoned from the time of mating (insemination),
 +
determined with a fair degree of accuracy. Ovulations occur
 +
about the time of parturition and again 29 to 30 days post partem.
 +
This latter period is more favorable for obtaining insemination
 +
and semination, thus fertilized ova. The process of fertilization
 +
probably takes place during the latter half of the first day after
 +
insemination.
 +
 +
 +
The pronuclear stage, a stage which extends through a period
 +
of perhaps 12 to 15 hours, in the middle phase, is observed at
 +
the end of the first day after insemination; the fertilized ova
 +
having wandered about one-fourth of the length of the oviduct
 +
by that time. Of the two pronuclei, the female pronucleus is
 +
slightly the larger. The two pronuclei lie near the center of
 +
the ovum, are distinctly membraned, and do not fuse prior
 +
to the formation of the first segmentation spindle.
 +
 +
 +
The formation of the first segmentation spindle and the first segmentation occur during the early part of the second day after insemination. The resulting 2-c(41 stage extends for a period of about 24 hours and is found in about the middle of the oviduct. The first two blast omeres are equivalent cells. One of these segments before the other, resulting in a 3-cell stage, present for each ovum for only a relatively short period.
 +
 +
 +
The 4-cell stage is observed at the end of the third day after insemination. The ova have by this time traversed about ninetenths of the length of the oviduct.
 +
 +
 +
The 8-cell stage is observed the latter half of the fourth day after insemination and at the end of the fourth day the ova pass from the oviduct to the uterus in the 12-ccll to 16-cell stage. The oolemma is lost usually in the 4-cell stage, the segmenting ova conforming in shape to the general form of that portion of the oviduct in which they are found.
 +
 +
 +
Three successive segmentation stages, spaced at intervals of about 18 hours, resulting in 2-, 4-, and 8-cell stages occur during transit through the oviduct. During the foiu-th segmentation the ova pass from the oviducts to the uterine horns, at the end of the fourth day.
 +
 +
 +
The mass increase of the ova during the first three segmentations is approximately from 0.15 c.mm. in the pronuclear stage
 +
to 0.18 c.mm. in the 8-cell stage. The slow rate of segmentation
 +
and the relatively small mass increase may be attributed to the
 +
relative scarcity of the embryotroph during transit through
 +
the oviducts.
 +
 +
 +
During the early hours of the fifth day after insemination, all of the segmenting ova are found lying free in the lumen of the uterus, spaced about as in the later stages of development, the fifth series of segmentations having been completed by this time, the resulting morula masses having ovoid form, measuring approximately 80 /x by 50 /i and consisting of from 24 to 32 cells. The mechanism operative in spacing the ova in the uterine horns has not been determined.
 +
 +
 +
The early stages of blastodermic vesicle formation are observed during the middle and latter half of the fifth day. The segmentation cavity begins as a single, irregularly crescentic space, eccentric in position, and arising between the cells of the morula.
 +
 +
 +
By the end of the fifth day after insemination, all fertilized,
 +
normal ova are found in the blastodermic vesicle stage. One
 +
pole of each vesicle, its floor, consists of a relatively thick mass
 +
of cells, in which there is no differentiation in layers and no
 +
evidence of ectodermal and entodermal cells. The other pole
 +
of each vesicle, its roof, consists of a single laj^er of flattened
 +
cells, bordering the segmentation cavity.
 +
 +
 +
During the sixth day, the blastodermic vesicles which still lie free in the lumen of the uterus, increase in size, partly as a result of extension of the roof cells, partly owing to rearrangement and flattening of the cells of the floor. This portion of the vesicle now presents the form of a concavo-convex disc, forming about one-sixth of the vesicle wall and consisting, as a rule, of three layers of cells, the inner of which is now differentiated to form the yolk entoderm.
 +
 +
 +
During the seventh day after insemination the blastodermic
 +
vesicles become definitely oriented in a decidual crypt, the
 +
thicker portion, its floor, being directed toward the mesometrial
 +
border. The phenomenon of the ''inversion of the germ layers"
 +
or "entypy of the germ layers" is initiated, the result of cell
 +
rearrangement and cell enlargement in the germinal disc, manifested as an outgrowth to form the ectoplacental cone or Trager
 +
and an ingrowth into the vesicle, the anlage of the egg-plug or
 +
egg-cylinder. In the egg-plug there is recognized a circumscribed, compact mass of cells, staining more deeply than surrounding cells, which constitute the ectodermal node, the anlage
 +
of the primary embryonic ectoderm of the future embryo. This
 +
ectodermal node, so far as it extends into the cavity of the
 +
blastodermic vesicle, is surrounded by yolk entoderm.
 +
 +
 +
During the eighth day after insemination, the egg-cylinder
 +
comes in definite relation with the maternal decidua and receives as embryotroph maternal hemoglobin, partly through
 +
phagocytic action of the cells of the ectoplacental cone, partly
 +
through absorption of maternal hemoglobin by the cells of the
 +
entoderm, initiating a period of very active growth as evidenced
 +
by active mitosis. The egg-cjdinder increases in length, and
 +
entypy is completed. A cavity develops in the ectodermal node, the antinipsonu'trial portion of llic i)n)ainiii()tic cavity.
 +
A little later a second cavity develops in the extraembryonic
 +
ectoderm, the mesometrial portion of the proamniotic cavity,
 +
the two cavities fusing by the end of the eighth da}" to form a
 +
single proamniotic cavity, lined in its antimesometrial portion
 +
by primary embryonic ectoderm, and in its mesometrial portion
 +
by extraembryonic ectoderm, the two types of ectoderm forming
 +
a continuous layer with the line of junction readily distinguisliable. No evidence of bilateral synnnetry is at this stage observed in the egg-cylinder.
 +
 +
 +
During the ninth day after insemination there is observed the anlage and the early developmental stage of the mesoderm and the anlage of the primitive streak and groove. The mesoderm has its anlage in the caudal portion of the i^rimary embryonic ectoderm in the sagittal region and is of the nature of prostomial mesoderm, extending laterally in wing-like extensions between the ectoderm and entoderm.
  
  
  
 
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Huber GC. The Development of the Albino Rat (Mus norvegicus albinus). (1915) J Morphol. 26(2).

Normal: Introduction | Materials and Methods | Ovulation, Maturation and Fertilization | Pronuclear Stage | Segmentation Stages | 2-ceIl stage | 4-ceIl stage | 12 to 16-ceIl stages | Summary of segmentation stages | Completion of segmentation and blastodermic vesicle formation | Blastodermic vesicle | Late stages blastodermic vesicle | Egg-cylinder formation | Late stages in egg-cylinder | Conclusions | Literature cited | Figures
Abnormal: Introduction | Half Embryos in Mammalia | Degeneration of ova at the end of segmentation | Incomplete or retarded segmentation | Abnormal segmentation cavity formation | Degeneration of ova as a result of pathologic mucosa | Imperfect development of ectodermal vesicle | Two egg-cylinders in one decidual crypt | Conclusions | Literature cited
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Pages where the terms "Historic" (textbooks, papers, people, recommendations) appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms, interpretations and recommendations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)

Late Stages in Egg-Cylinder Differentiation and the Anlage of the Mesoderm

In the rat series there are found 24 egg cjdinders showing the stages of development considered in this section; certain of them are cut longitudinally and others cross-wise.


For the special consideration of egg-C3'linder formation just prior to the anlage of the mesoderm, I present two egg-cylinders obtained during the latter half of the ninth day after insemination ; one of these was cut longitudinally, the other in favorable crosssection. The egg-cylinder shown in figure 29, rat No. 40, 8 days, 17 hours after insemination, seems unusually well fixed, as evidenced by its symmetrical outline, and is cut in a very favorable plane. The sections are from a series cut at right angles to the long axis of the uterine horn. The decidual crypts lodging the egg-cylinders of this stage are by this time nearly completely separated from the lumen of the uterus, and are surrounded by a well-developed decidua. Extra vasated maternal l)lood nearly surrounds such egg-cylinders.


Fig. 29 Longitudinal, sagittal section of egg-cylinder of the albino rat showing the final mesoderm-free stage. X 200. Rat No. 40, 8 days, 17 hours, after insemination; ect.pl., ectoplacental cone or Trager; p.ect., parietal or transitory ectoderm; pr.emh.ect., primary embryonic ectoderm; ex.ect., extraembryonic ectoderm; pr.c, proamniotic cavity; r.ent., visceral entoderm, absorptive for maternal hemoglobin, cells showing the three zones described by Sobotta; pr.emh.ent., primary embryonic entoderm.


The egg-cylinder shown in figure 29 presents a total length of 1.15 mm., a width of approximately 0.18 mm. The ectoplacental cone presents a length of 0.4 mm. and of the proamniotic cavity, 0.5 mm., of which 0.2 mm. falls to the antimesometrial portion lined by primary embryonic ectoderm. This egg-cylinder differs only in shape and size from that shown in C of figure 27, obtained 8 days after insemination. The primary embryonic and extraembryonic ectoderm lining or enclosing the proamniotic cavity are readily differentiated. The primary embryonic ectoderm, derived from the ectodermal node, constitutes a pseudostratified epithelium, composed of relatively long colunmar cells, with nuclei radially placed with reference to the lumen of the proamniotic cavity, and shows active cell division, no less than 12 mitotic figures occurring in the section figured. The protoplasm of its cells stains distinctly deeper than does that of the cells of the extraembryonic ectoderm. The cells of the latter are of cubic, short columnar, or polyhedral shape, arranged in a single or double layer, with no definite arrangement of the long axes of its nuclei. It is, therefore, possible readily to distinguish — by reason of shape and size of cells, relative position of nuclei, reaction to stain of protoplasm — between the cells of the primary embryonic and extraembryonic ectoderm, and to determine the sharp hne of junction at which the two types of cells form a continuous layer, a fact which will receive further consideration in dealing with the anlage of the mesoderm as observed in slightly more advanced stages. At the mesometriai end of the i)roanmiotic cavity, the cells of the extraembryonic ectoderm become continuous with the cells at the base of the ectoplacental cone; in the region of this junction, active mitosis are often to be observed. In this egg-cylinder the visceral entoderm may readily be differentiated into two portions. The portion which surrounds the primary embryonic ectoderm to nearly the region of its junction with the extraembryonic ectoderm, consists of a single layer of broad, flattened cells which assume a cubic or short columnar shape as the mesometrial border of the primary embryonic ectoderm is approached. This portion of the visceral entoderm we have designated as the primary oiiibrvonic CMit()<l(M-ni. 'I'ho portion of tlic Nisccral ont()d(M'iH surrouiidiiiji; tlic sides of tlic cjiir-cvliiidcr in the region of the oxtiacnihiyonic ectoderm, to ncai- the base of the cctoplacentul cone, consists of a single layer of eolunniar cells, regularly arranged and presenting the three zones described by Sobotta. In this stage of egg-cylinder development of the al})ino rat, the absorption of hemoglobin granules derived from maternal blood cells, first shown for the mouse by Sobotta and Kolstei', may be readily made out. In preparations stained in hematoxylin and Congo red, in and on the outer zone of the visceral entodennal cells there may be observed granules staining deeph' in the Congo red, presenting the color reaction of hemoglobin. In the middle zone of these cells the protoplasm is distinctly \acuolated, while the inner zone, containing the nuclei, presents a denser protoplasm. The transitory' or parietal ectoderm consists of a homogeneous membrane, closely adherent to the maternal decidua, especially along the sides of the egg-cylinder. This layer presents scattered nucleated protoplasmic masses of spindle or dome shape on its inner surface, the relations and distribution of which may be clearly seen in the figure. Attention needs yet be drawn to the ectoplacental cone of the egg-cylinder. Its relation to the maternal decidua is very intimate, so that in places, owing to blood extravasations, it is difficult to differentiate between embryonic and maternal tissue. Many of the cells of the ectoplacental cone present a vacuolated protoplasm, the \'acuoles enclosing maternal blood cells. Therefore, they are distinctly' phagocytic. Sobotta has also observed and described this for the mouse. Referring to a slightly older stage after the anlage of the mesoderm, his own words read as follows:

Weitcrhin sehen wir iin Stadium der Fig. 5 audi eine starke Verlangerung und Vorgnissorung des Ectoplacontarconus, an dcm im mesonietralcn Tcile jctzt ^'a(•uole^ auftreten, die in spateren Stadien regelmassig gefunden worden und zwar crfiillt niit miitterlichcn Blutextravasaten. Die Ehrniihrung dcs Embryo mit miitterlichem Hiimoglobin * * * * jg^ jetzt im vollen (Jang.


Absorption of maternal hemoglobin by the cells of the ectoplacental cone appears to be established at a relatively earlier period in the rat than in the mouse.


The egg-cylinder presented in figure 29 constitutes the final mesoderm-free stage, the final stage in which no distinct bilaterality may be determined. I assume that the egg-cylinder presented in the figure is cut in the sagittal plane. This assumption is based on the fact that the primary embryonic ectoderm extends slightly farther toward the mesometrial pole on the one side than on the other. In good frontal sections one side of the egg-cylinder in this stage of development should present a mirror picture of the other side. The side on which the primary embryonic ectoderm extends farther toward the mesometrial pole, the left in the figure, is regarded as containing the caudal end of the future embrj^o. In the primary embryonic ectoderm of this region, it is believed, will develop the primitive streak and groove, and thus the anlage of the mesoderm. Not in all the egg-cylinders of this stage of development found in my series can the caudal end of the future embryonic area be postulated prior to the anlage of the mesoderm, and in cross-sections no such differentiation can be made. The proamniotic cavity of the egg-cylinder shown in figure 29 presents a regular and nearly smooth contour, not divisible into regions such as described for a similar stage for the mouse by Selenka, MeUssinos, and Sobotta. A very slight constriction is to be observed onh^ in the region where the primary embryonic and extraembryonic ectoderm are joined in a continuous layer. I am thus wholly in accord with Widakowich, who in describing a similar stage in one of his preparations, states: "Das war die einzige konstante, bald starker, bald schwiicher ausgepragte Einschniirung der Proamnionhohle," as previously quoted.


A series of figures of critical regions taken from a series of cross-sections of an egg-cylinder of a stage nearly identical with that shown in figure 29, though of a shghtly smaller egg-cjdinder, is given in figure 30, rat No. 42, 8 days, 16 hours, after insemination. The sections chosen for the several drawings, A to D, are from the following regions, as may be ascertained by comparison with figure 29; A, through about the middle of the ectoplacental cone; B, through the proamniotic cavity just below its mesometrial end; C, through the proamniotic cavity just above the region of the junction of the primary embryonic and extraeml)ryonic ectoderm; 1), a Httle above the middle of the antimesometrial j)oi-ti()ii of tiie i)i'oamiii()tic cavity. The levels of the several sections drawn in figure 30 is approximately indicated by the several crosses found to the left of the egg cylinder drawn in figure 29.



Fig. 30 Four figures from a series of cross sections of an egg-cylinder of the albino rat in the stage of development shown in figure 29. X 200. Rat No. 42, 8 days, 16 hours after insemination.

The levels at which the several sections drawn were taken is ai)i)roximately indicated by the several crosses found to the left of figure 29. A, middle of cctoplacental cone; B, ectoplacental end of the proamniotic cavity; C, just above level of junction of the primary embryonic and extraembryonic ectoderm; a little above the middle of primary embryonic ectoderm. The want of any definite bilateral sjinmetry of albino rat egg-cylinders of this stage of development is shown by this series of sections; p.ect., parietal or transitorj^ ectoderm; cx.ect., extraembryonic ectoderm, surrounding mesometrial portion of proamniotic cavity; p?-.em6.ec<., primary embrj-onic ectoderm; r.e»/., visceral entoderm; p.emb. ent., primary embryonic entoderm; pr.c, proamniotic cavity.


In A of figure 30, there may be observed a vacuolization of the protoplasm of the more peripherally placed cells of the ectoplacental cone, the vacuoles enclosing maternal blood cells. The more centrally placed cells of this ectoplacental cone show a tendency to concentric arrangement. Figures B and C present structural appearances nearly identical. The egg-cylinder is bounded by the thin layer of parietal or transitory ectoderm having scattered masses of nucleated protoplasm on its inner surface. This membrane of apparently homogeneous structure stains sharply in well fixed preparations and may be readily discerned. The cells of the \dsceral entoderm, somewhat taller in the section taken nearer the antimesometrial pole (C), present clearly the three zones to which attention has been drawn. The cells of the extraembryonic ectoderm bounding the mesometrial portion of the proamniotic cavity, are of cubic, short columnar, or polyhedral form disposed in single or double layer, presenting relatively lightly staining protoplasm. In D of figure 30, the cells forming the primary embryonic ectoderm are of distinct columnar shape, with relatively deeply staining protoplasm and nuclei arranged nearl}^ in a single layer except for such as show mitotic phases. The cells of the primary embryonic entoderm are of a broad, pavement type for a greater part of the circumference, and may be contrasted with the cells of the visceral entoderm shown in B and C of the figure; the latter are absorptive cells, the former not. This series of figures, more especially B, C, and D, show clearlj' the absence of bilaterality in the egg-cylinders of the albino rat at this stage of development. The slight compression observed in this egg-cylinder, as shown in the fi gures, I regard as not of moment.


Fig. 31 Longitudinal sagittal section of egg-cylinder of the albino rat showing anlage of the mesoderm. X 200. Rat Xo. 34, 8 days, 18 hours, after insemination; eel. pi., ectoplacental cone or Trager; p.ect., parietal or transitorjectoderm; pr.emb.ect., primary embryonic ectoderm; ex.ect., extraembryonic ectoderm; pr.emfc.eni., primary embryonic entoderm; wcs., mesoderm in anlage; pr.c, proamniotic cavity; v.oit., visceral entoderm.


Grosser has figured in his figures 68 and 114, an egg-cyUnder of the albino rat which measures nearly 2 nini. in length. The age of this is given as 8| days. So far as may be determined from his figures, the preparation is not described in his text, the age, size, form, and structure of the egg cylinder shown in figure 29 and Grosser's figures 68 and 114, are very similar. In Grosser's figures, I see no evidence of his having differentiated between primary embryonic and extraembryonic ectoderm, while the reference letters for ectoderm and entoderm are reversed. Selenka's figure 31, plate 45, may be of a similar stage. This figure is, however, too diagrammatic to admit of close study. No difference is shown in the shape and structure of the cells bounding the two parts of the proamniotic cavity. Christiani's figure 39 may be of the same stage, but is too schematically drawn. Figure 4 of the article of AYidakowich is of a slightly older stage and presents only a part of the egg-cylinder; it is recorded as about 6f days old. The stage under consideration is not figured by Widakowich, although his text description corresponds closely with what has been here presented.


The next stage and the one with which this communication is to be completed is one of importance since it is characterized by the anlage of the mesoderm. My own observations may be introduced with the consideration of an egg-cylinder, a section of which is presented in figure 31, rat No. 34, 8 days, 17 hours, after insemination. This was cut in the sagittal plane and measures 1.1 mm. by 0.2 mm., of which 0.4 mm. fall to the ectoplacental cone. This egg-cylinder is almost an exact duplicate, both in size and form, of that figured in figure 29 of the same age. In the egg-cylinder shown in figure 31, however, there may be observed, to one side, in the region of the junction of the primary embryonic and extraembryonic ectoderm, and between primary embryonic ectoderm and entoderm, a small group of cells which lie in close relation to the ectoderm and constitute early mesodermal cells. The sections of this series pass not exactly parallel to the mid- sagittal plane throughout the whole extent of the egg-cylinder; especially is this true of its antimesometrial portion, in the region of the primary embryonic ecloclcnn. This portion in the section iij^ured, passes a httle to one side of the mid-sagittal plane. The two sections preceding the one figured enclose the mid-sagittal plane, and in them, the groii]:) of cells found between primary embryonic ectoderm and (Miloderm are in closer relation to the ectodermal hiyer and al all points (Hstinctly separated fi'om the entoderm. They are regarded as having wandered from the primary embryonic ectoderm to the place they occupy, a fact which is more easily ascertained in cross sections of a similar stage, as will appear from further discussion. From a study of very slightly older stages it can be determined that this region constitutes the primitive streak region of the future embryonic area. It is not my purpose at this time and in this communication to give especial consideration to the much discussed ciuestion of the origin of the mesoderm in Mammalia. In the rat, this cjuestion is complicated by the question of the anlage of the anmiotic fold, which separates the proamniotic cavity into amniotic cavity proper and the ectoplacental cavity, the (hnelopment of which will be considered in a projected contribution. In anticipation of this second publication, however, the following facts may here receive consideration. Widakowich presents in his figure 4, giving only the antimesometrial end of an eggcylinder obtained the latter part of the 7th day, the anlage of the mesoderm as observed by him. This figure and my own figure 31 present almost identical relations, his figure showing only thi'ee mesodermal cells between primary embryonic ectoderm and entoderm. His own words concerning the anlage of the mesoderm in the albino rat, with which I find myself in full accord, except as to the age of the egg-cylinder, read as follows:

Das erstc auftreten des Mesoderms beobachtete ich an Keimen vom Ende des 7 Tages. Die ersten Mesodermzollen licgen im Bereiche der vom mesometralen Ende des stiirkcr far])baren priniaren cmbryonalen Ectoderm gebildeten Faltc. Es koninit hier einc ganz bestimmte Stelle in Betracht, die dort liegt, wo sich spater das hintere Ende des Primitivstreifens liefindet.


There is, however, wide divergence of the views of authors as concerns the anlage of the mesoderm in the rat and mouse.


Selenka, it would seem, in part at least, interpreted correctly the development of the mesoderm in the rat, although a stage showing its anlage was not observed. Duval believes that the mesoderm has origin from a thickened part of the entoderm, probably in the region of the anterior portion of the future embryonic area; the primitive streak was not recognized. Christiani's figures 45 and 47, transverse sections of the egg-cylinder from the eighth day, give correctly the relative position of the mesoderm with reference to the primitive streak; however, they show stages some little time after the anlage of the mesoderm. According to Robinson, in the early part of the eighth day the cavities of the epiblast (primary embryonic ectoderm) and of the trophoblast (extraembryonic ectoderm) meet and fuse to form a hollow cylinder, the proamniotic cavity. He states that "For a time the united cavities of the epiblast and trophoblast increase in size, together with the general growth of the ovum, and this increase continues until in the latter part of the eighth day the mesoblast appears around the margin of the epiblast where it is in apposition with the trophoblast." Robinson was able to differentiate between the primary embryonic ectoderm (epiblast) and the extraembryonic ectoderm (trophoblast) and his figure 14 (plate 23-24), though schematic, shows that he recognized the positions of the anlage of the mesoderm correctly, as also its derivation from the primary embryonic ectoderm. The observations of Melissinos, bearing on the anlage of the mesoderm have been critically reviewed by both Widakowdch and Sobotta, and I am wholly in accord with their views when they state that no credence can be given these observations since it is clear that IVIelissinos has confused sagittal and frontal sections in such a way as to make his observations of no value. According to Melissinos, the mesoderm arises from the outer surface of the middle fold of the egg cylinder, in the region of its union with the antimesometrial ectodermal fold; it is certain that it does not arise from the part of the eggcylinder that has differentiated from the primary embryonic ectoderm; but, if I interpret him correctly, from the extraembryonic portion of the ectoderm. That Melissinos did not have before him the stages showing the aiilage of tlic mesoderm seems clear. S()))otta's ('11) observations, mouse material, deserve fuller consideration. In interpreting his results, I am mindful of the fact that he was unable to locate the line of union between primary embryonic and extraembryonic ectoderm, as can readily be done in suitable rat material, as has previously been shown by Robinson and Widakowich, and to which attention has constantly been drawn in this communication. I am unable to state from personal observation whether in the white mouse these two types of ectoderm which form the lining of the proamniotic cavity, can be differentiated on ascertaining the right technical method. Sobotta's material seems well fixed. If not, it would seem to me difficult to determine definitely the exact place of origin of the mesodermal cells, whether extraembryonic or embryonic. Sobotta recognized the anlage of the mesoderm in the mouse during the last hours of the seventh day or first hours of the eighth day. This is said to appear at the caudal end of the future eml:)ryo as a group of loosely arranged cells lying between the inner and outer layers of the egg-cylinder. At the place where the mesodermal cells arise from the inner layer of the egg-cylinder, there is developed a fold, recognized as the caudal amniotic fold (Schwanzfalte des Amnios"). After discussing these observations at length, Sobotta concludes as follows:

Was die Deutung dieser friihen Stadien der Mosodcnnbikhing in der Keiinl)laso der Alaus anlangt, so haiuielt es sich hier niclit uni die Bildung des einbryonalen Mesoderms, die erst mit der eigentlichen Gastrulation spater einsetzt, sonderii uni Entsteliung ausserembryonalen ^Mesoderms, besonder des Teils des mittleren Keimblattes, dass bei der Bildung der primaren Eihiiute, Amnios und Chorion in Betraeht kommt und des den ausserembryonalen Teil der Leibeshrihle, das Exocoelom auskliedet, der Hohle, die eben Amnios und Chorion voneinander trennt. Es erfolgt also, um einen kurzen Ausdruck zu gebrauchen, die Bildung des Amniosmesodcrms.


An embryonic anlage is said not to exist at this stage; this is recognized only after the development of the primitive streak. It is not my purpose to enter fully into a discussion of this important question in this communication. This would involve consideration of older stages, and the making of a number of reconstructions, ^yhich it is not contemplated to consider now. It must suffice to state at this time that in the albino rat, as shown by Widakowich and here shown by me, it is possible to delineate clearly the primary embryonic ectoderm and to show that the first evidence of the mesoderm is found antimesometrial to the future amniotic fold and in the region of the future primitive streak; therefore is mesoderm which I would regard as peristomal mesoderm in the sense of C. Rabl, reference to which is made bj^ Sobotta in his discussion of this question. It may be that the rat offers more suitable material for the elucidation of this question than is to be found in the mouse. In the albino rat, the anlage of the mesoderm is from the sagittal portion of the caudal region of the primary embryonic ectoderm, the caudal part of the future primitive streak and antimesometrial to the amniotic fold. Sobotta gives very favorable consideration to the observations of Widakowich, touching this question, which he regards as Bei weitem die beste Darstellung des Gegenstandes." My own observations fully confirm those of Widakowich. These questions will receive fuller consideration in a later publication dealing with the embryology of the albino rat, carrying the development from the time of the anlage of the amniotic fold to the stage of embryo form, the material for which is at hand.


In figure 32 are shown cross-sections of the antimesometrial portion of three egg-cylinders in the region of the developing mesoderm. Sections drawn in A and B, were taken respectively from egg-cylinders obtained from the same uterus as was the one shown in sagittal section in figure 31, rat No. 34, 8 days, 17 hours, after insemination; C, from rat No. 41, 8 daj^s, 16 hours, after insemination. It is very probable that the series from which A of this figure was drawn, is net cut in exactly the cross plane. A stud}^ of the series shows, however, that the deviation from this plane is not marked. The sections from which this figure was drawn pass a little below (antimesometrial) to the region of junction of the primary embryonic and extraembryonic ectoderm. To one side, the lower in the figure, the primary embryonic ectoderm shows a slight thickening and evidence of


Fig. 32 Three cross sections from egg-cj'lindcrs of the albino rat, showing early stages in the development of the mesoderm. X 200. A and B, rat No. 34, 8 days, 17 hours; C, rat No. 41, 8 days, 16 hours, after insemination.

These sections taken from three egg-cylinders are through the primary embr}'onic ectoderm, near its junction with the extraembryonic ectoderm, thus through the antimesometrial portion of the proamniotic cavity A, early stage, in anlage of the mesoderm; B, anlage of the primitive streak and groove; C, well developed primitive streak and groove, with lateral wings of mesoderm; pr.e?rt6. ect., primary embrj'onic ectoderm; pr.emb.ent., primarj^ embryonic entoderm; mes., mesoderm; pr.sir., primitive streak; pr.gr., primitive groove; p. ect., parietal or transitory ectoderm; pr.c, proanmiotic cavity.


cell proliferation. The cells of this region have not the form of tall columnar cells, such as seen in the greater part of the remaining primary embryonic ectoderm, but are of polyhedral form and are continuous, in the mid sagittal plane, with cells that have wandered between the primary embryonic ectoderm and entoderm, cells regarded as constituting the mesoderm. In all of the sections of this series, so far as the mesoderm extends, this is distinctly separable from the entoderm, and is continuous with the primary embryonic ectoderm only along a narrow region of thickened primary embryonic ectoderm, situated in the mid-sagittal plane, and which may in this series be regarded as the anlage of the primitive streak. From the sides of this region of slightly thickened primary embryonic ectoderm, the extent of which is evidenced by the absence of an external limiting membrane, cells wander laterally to form the mesoderm. B, of figure 32, presents essentially the same appearance, although representing a slightly older stage. The sections of this series I regard as cut fairh' well in a plane at right angles to the long axis of the respective egg-cylinder. The section taken for the sketch is situated a very little further away from the line of junction of the primary embryonic and extraembrj^onic ectoderm, than is the section the drawing of which is shown in A of this figure, as may be judged from the more uniformly pavement tj'pe of the entodermal cells. The triangular form of the proamniotic cavit}' is regarded as normal, and as indicating an early stage in the anlage of the primitive groove. In this figure, in its lower portion, the region of the primitive streak is readily discernible by reason of the fact that there is wanting here an external limiting membrane, and further by reason of the form of the cells and the form and relative position of their nuclei; certain of these cells indicating, both by their form and their position, the source and the direction of the wandering of the cells which constitute the anlage of the mesoderm. The wandering of the mesodermal cells between the primary embryonic ectoderm and entoderm, to form the lateral mesodermal wings, is clearly shown in this figure, especially to the left. The antimesometrial ends of the egg-cylinders, sections of which are shown in A and B of this figure, are as yet free from the invading mesoderm, as is also the part of the egg-cylinders lying opposite the region of the primitive streak, the upper portions of the respective figures, these forming the region of the future anterior ends of the respective embryos. In C of figure 32 is shown a drawing of one of the sections of a series of cross-sections of an egg-cylinder taken from rat No. 41, 8 days, 16 hours, after insemination, presenting a stage in which the primitive groove may be definitely made out. This figure is not unhke figure (> of the article of Widakowich, obtained from an egg-cylinder secured on the eighth day. Concerning this figure he states: "Das Ectoderm steht in direktem Zusammenhange mit zwei Mesodermzungen die gegen die der Primitivrinne gegeniiberhegende Seite zu auswachsen." The section drawn in C of this figure is taken from the region very near the junction of the primary embryonic and the extraembryonic ectoderm, as may be observed from the character of the entodermal cells, in the lower part of the figure. The increase in the thickness of the mesodermal wings, the result, in part at least, of proliferation of mesodermal cells, as evidenced by the presence of mitotic figures, is clearly shown in this figure. The mesoderm is distinctly separable from the entoderm as also from the primary embryonic ectoderm except in the region of the primitive streak and groove. The growth of the mesoderm after its anlage has been correctly shown for the albino rat by Selenka, Robinson, and Widakowich; the latter especially giving excellent figures. His figure 5 is especially instructive. In this, he represents the appearances shown by two views of an isolated egg-cylinder, with the primiti^'e groove in anlage, showing the lateral extensions of the mesoderm. Sobotta ('11) has given the best and most comprehensive account of the anlage and growth of the mesoderm in the mouse. An excellent cross-section of a mouse eggcylinder in the primitive streak stage is presented in his figure 6, which presents very similar appearances to my C of figure 32. None of the figures of cross-sections of egg-cylinders included by me show the very beginning of the anlage of the mesoderm, though A of figure 32 approaches this very closely, as does also figure 31, presenting a sagittal section. The evidence at hand warrants the conclusion that in the albino rat, the mesoderm has its anlage in the caudal region of the primary embryonic ectoderm, from a narrow zone of cells situated in the region of the future primitive streak. From this region there is an outwandering of cells which invade the potential cleft between primary embryonic ectoderm and entoderm, spreading laterally in wing-like sheets. This I would regard as prostomial mesoderm in the sense of C. Rabl. The anlage of the mesoderm in the albino rat, and the early stages of its lateral extension, with the anlage of the primitive streak and groove, falls to the latter part of the ninth day after insemination.


Beginning with the pronuclear stage, found at the end of the first day, 8 days are required for the completion of the process of segmentation, blastodermic vesicle formation and the formation of the primary germ layers — ectoderm, mesoderm, and entoderm — in all, 9 days out of a possible 21 to 23 days, the normal gestation period of the albino rat.

Conclusions

Early stages of mammalian development may readily be obtained from the albino rat (Mus norvegicus albinus). When care is exercised, mating may be observed and the age of the embryo, reckoned from the time of mating (insemination), determined with a fair degree of accuracy. Ovulations occur about the time of parturition and again 29 to 30 days post partem. This latter period is more favorable for obtaining insemination and semination, thus fertilized ova. The process of fertilization probably takes place during the latter half of the first day after insemination.


The pronuclear stage, a stage which extends through a period of perhaps 12 to 15 hours, in the middle phase, is observed at the end of the first day after insemination; the fertilized ova having wandered about one-fourth of the length of the oviduct by that time. Of the two pronuclei, the female pronucleus is slightly the larger. The two pronuclei lie near the center of the ovum, are distinctly membraned, and do not fuse prior to the formation of the first segmentation spindle.


The formation of the first segmentation spindle and the first segmentation occur during the early part of the second day after insemination. The resulting 2-c(41 stage extends for a period of about 24 hours and is found in about the middle of the oviduct. The first two blast omeres are equivalent cells. One of these segments before the other, resulting in a 3-cell stage, present for each ovum for only a relatively short period.


The 4-cell stage is observed at the end of the third day after insemination. The ova have by this time traversed about ninetenths of the length of the oviduct.


The 8-cell stage is observed the latter half of the fourth day after insemination and at the end of the fourth day the ova pass from the oviduct to the uterus in the 12-ccll to 16-cell stage. The oolemma is lost usually in the 4-cell stage, the segmenting ova conforming in shape to the general form of that portion of the oviduct in which they are found.


Three successive segmentation stages, spaced at intervals of about 18 hours, resulting in 2-, 4-, and 8-cell stages occur during transit through the oviduct. During the foiu-th segmentation the ova pass from the oviducts to the uterine horns, at the end of the fourth day.


The mass increase of the ova during the first three segmentations is approximately from 0.15 c.mm. in the pronuclear stage to 0.18 c.mm. in the 8-cell stage. The slow rate of segmentation and the relatively small mass increase may be attributed to the relative scarcity of the embryotroph during transit through the oviducts.


During the early hours of the fifth day after insemination, all of the segmenting ova are found lying free in the lumen of the uterus, spaced about as in the later stages of development, the fifth series of segmentations having been completed by this time, the resulting morula masses having ovoid form, measuring approximately 80 /x by 50 /i and consisting of from 24 to 32 cells. The mechanism operative in spacing the ova in the uterine horns has not been determined.


The early stages of blastodermic vesicle formation are observed during the middle and latter half of the fifth day. The segmentation cavity begins as a single, irregularly crescentic space, eccentric in position, and arising between the cells of the morula.


By the end of the fifth day after insemination, all fertilized, normal ova are found in the blastodermic vesicle stage. One pole of each vesicle, its floor, consists of a relatively thick mass of cells, in which there is no differentiation in layers and no evidence of ectodermal and entodermal cells. The other pole of each vesicle, its roof, consists of a single laj^er of flattened cells, bordering the segmentation cavity.


During the sixth day, the blastodermic vesicles which still lie free in the lumen of the uterus, increase in size, partly as a result of extension of the roof cells, partly owing to rearrangement and flattening of the cells of the floor. This portion of the vesicle now presents the form of a concavo-convex disc, forming about one-sixth of the vesicle wall and consisting, as a rule, of three layers of cells, the inner of which is now differentiated to form the yolk entoderm.


During the seventh day after insemination the blastodermic vesicles become definitely oriented in a decidual crypt, the thicker portion, its floor, being directed toward the mesometrial border. The phenomenon of the inversion of the germ layers" or "entypy of the germ layers" is initiated, the result of cell rearrangement and cell enlargement in the germinal disc, manifested as an outgrowth to form the ectoplacental cone or Trager and an ingrowth into the vesicle, the anlage of the egg-plug or egg-cylinder. In the egg-plug there is recognized a circumscribed, compact mass of cells, staining more deeply than surrounding cells, which constitute the ectodermal node, the anlage of the primary embryonic ectoderm of the future embryo. This ectodermal node, so far as it extends into the cavity of the blastodermic vesicle, is surrounded by yolk entoderm.


During the eighth day after insemination, the egg-cylinder comes in definite relation with the maternal decidua and receives as embryotroph maternal hemoglobin, partly through phagocytic action of the cells of the ectoplacental cone, partly through absorption of maternal hemoglobin by the cells of the entoderm, initiating a period of very active growth as evidenced by active mitosis. The egg-cjdinder increases in length, and entypy is completed. A cavity develops in the ectodermal node, the antinipsonu'trial portion of llic i)n)ainiii()tic cavity. A little later a second cavity develops in the extraembryonic ectoderm, the mesometrial portion of the proamniotic cavity, the two cavities fusing by the end of the eighth da}" to form a single proamniotic cavity, lined in its antimesometrial portion by primary embryonic ectoderm, and in its mesometrial portion by extraembryonic ectoderm, the two types of ectoderm forming a continuous layer with the line of junction readily distinguisliable. No evidence of bilateral synnnetry is at this stage observed in the egg-cylinder.


During the ninth day after insemination there is observed the anlage and the early developmental stage of the mesoderm and the anlage of the primitive streak and groove. The mesoderm has its anlage in the caudal portion of the i^rimary embryonic ectoderm in the sagittal region and is of the nature of prostomial mesoderm, extending laterally in wing-like extensions between the ectoderm and entoderm.



Normal: Introduction | Materials and Methods | Ovulation, Maturation and Fertilization | Pronuclear Stage | Segmentation Stages | 2-ceIl stage | 4-ceIl stage | 12 to 16-ceIl stages | Summary of segmentation stages | Completion of segmentation and blastodermic vesicle formation | Blastodermic vesicle | Late stages blastodermic vesicle | Egg-cylinder formation | Late stages in egg-cylinder | Conclusions | Literature cited | Figures
Abnormal: Introduction | Half Embryos in Mammalia | Degeneration of ova at the end of segmentation | Incomplete or retarded segmentation | Abnormal segmentation cavity formation | Degeneration of ova as a result of pathologic mucosa | Imperfect development of ectodermal vesicle | Two egg-cylinders in one decidual crypt | Conclusions | Literature cited
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