Difference between revisions of "Paper - Contributions to the embryology of the marsupialia 4-2"

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==Chapter II.  -  The Ovum of Dasyueus==
 
==Chapter II.  -  The Ovum of Dasyueus==
  
1. Structure of the Ovarian Ovum.
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===1. Structure of the Ovarian Ovum===
  
The full-grown ovarian ovum of Dasyurus (PI. 1, fig. 1)  
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The full-grown ovarian ovum of Dasyurus (PI. 1, fig. 1) appears as a rounded, or more usually, ovalish cell, the diameter of which varies in section in ten eggs measured from '28 X ‘126 mm. to '27 x '26 mm. (average, '24 mm.), and is therefore large relatively to the ova of Eutheria. It is enclosed by a tliin, but very definite I'efractive membrane or zona (vitelline membrane of Caldwell) of an approximate thickness of '002 mm. (fig. 1, z-p-), on which the cells of the discus proligerus (fig. I, d.p.) directly abut, a differentiated corona radiata and syncytial layer being absent. It appears to be identical in its relations and optical characters with the membrane investing the monotreme ovum, and never shows in section any trace of radial striations (though I believe I have detected an extremely faint appearance of such in the fresh zona), or of the extension into it of protoplasmic processes from the adjacent cells of the discus ‘proligerus, such as Caldwell figures in the case of the ovum of Phascolarctus (cf. his PI. 29, fig. 5). Within the zona the peripheral cytoplasm of the ovum is differentiated to form an exceedingly thin but distinct bounding layer or egg-membrane (vitelline membrane, sensu stricto).
appears as a rounded, or more usually, ovalish cell, the  
 
diameter of which varies in section in ten eggs measured  
 
from '28 X ‘126 mm. to '27 x '26 mm. (average, '24 mm.),  
 
and is therefore large relatively to the ova of Eutheria. It is enclosed by a tliin, but very definite I'efractive membrane  
 
or zona (vitelline membrane of Caldwell) of an approximate  
 
thickness of '002 mm. (fig. 1, z-p-), on which the cells of the  
 
discus proligerus (fig. I, d.p.) directly abut, a differentiated  
 
corona radiata and syncytial layer being absent. It appears  
 
to be identical in its relations and optical characters with the  
 
membrane investing the monotreme ovum, and never shows  
 
in section any trace of radial striations (though I believe I  
 
have detected an extremely faint appearance of such in the  
 
fresh zona), or of the extension into it of protoplasmic  
 
processes from the adjacent cells of the discus ‘proligerus,  
 
such as Caldwell figures in the case of the ovum of Phascolarctus (cf. his PI. 29, fig. 5). Within the zona the  
 
peripheral cytoplasm of the ovum is differentiated to form an  
 
exceedingly thin but distinct bounding layer or egg-membrane  
 
(vitelline membrane, sensu stricto).  
 
  
  
The cytoplasmic body of the ovum exhibits a very obvious  
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The cytoplasmic body of the ovum exhibits a very obvious and striking differentiation into two regions in correspondence with the presence in it of two definitely localised varieties of deutoplasmic material, respectively granular and fluid. Peripherally it consists of a relatively narrow cytoplasmic zone of practically uniform width, dense and finely granular in appearance owing to the presence in it of numei'ous particles of deutoplasmic nature. This we may distinguish as the foi'mative zone (fig. 1, f.z.). In it lies embedded the large vesicular nucleus (about ‘06 x ‘03 mm. in diam.). Centrally and forming the main bulk of the ovum is a mass of greatly vacuolated cytoplasm presenting the appearance of a clear wide-meshed reticulum. Its framework is coarser peripherally where it passes over without definite limit into the formative zone, with which it is structurallv identical, but much finer and wider-meshed centrally, so fine, indeed, that it almost invariably breaks down under the action of fixatives, and appears in sections as an irregular space, perhaps crossed by a few fine interlacing strands (fig. 1, d.z.). The meshes of this reticulum are occupied by a clear fluid which must be held to constitute the central deutoplasm of the egg. We may accordingly designate this central reticular area as the deutoplasmic zone.
and striking differentiation into two regions in correspondence  
 
with the presence in it of two definitely localised varieties of  
 
deutoplasmic material, respectively granular and fluid. Peripherally it consists of a relatively narrow cytoplasmic zone of  
 
practically uniform width, dense and finely granular in  
 
appearance owing to the presence in it of numei'ous particles  
 
of deutoplasmic nature. This we may distinguish as the  
 
foi'mative zone (fig. 1, f.z.). In it lies embedded the large  
 
vesicular nucleus (about ‘06 x ‘03 mm. in diam.). Centrally  
 
and forming the main bulk of the ovum is a mass of greatly  
 
vacuolated cytoplasm presenting the appearance of a clear  
 
wide-meshed reticulum. Its framework is coarser peripherally  
 
where it passes over without definite limit into the formative  
 
zone, with which it is structurallv identical, but much finer  
 
and wider-meshed centrally, so fine, indeed, that it almost  
 
invariably breaks down under the action of fixatives, and  
 
appears in sections as an irregular space, perhaps crossed by  
 
a few fine interlacing strands (fig. 1, d.z.). The meshes of  
 
this reticulum are occupied by a clear fluid which must be  
 
held to constitute the central deutoplasm of the egg. We may accordingly designate this central reticular area as the  
 
deutoplasmic zone.  
 
  
  
If we pass now from the full-grown to the ripe ovarian  
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If we pass now from the full-grown to the ripe ovarian ovum (PI. 1, figs. 2 and 3), i. e. an ovum in which either thefirst polar spindle has appeared or the first polar body hasalready been separated off, it at once becomes evident that important changes have occurred in the disposition and relative proportions of the two constituent regions of the eggcytoplasm. The full-grown ovum is of the centrolecithal type, the central deutoplasmic zone forming its main bulk and being completely surrounded by the thin formative zone. The ripe ovum, on the other hand, exhibits an obvious and unmistakable polarity, and is of the telolecithal type, as the following facts show. The cytoplasmic body evidently consists of the same two regions as form that of the full-gi-own ovum, but here the dense formative region now forms its main bulk, and no longer surrounds the clear deutoplasmic region as a uniform periphei*al layer. It has not only increased considerably in amount as compared with that of the full-grown egg, and at the expense apparently of the moreperipheral coarser portion of the deutoplasmic zone, but it has undergone polar segregation, with the result that it now occupies rather more than one hemisphere of the egg as a dense finely granular mass, with vacuoles of varying sizesparsely scattered through it (figs. 2 and 3, /.z.). It accordingly defines one of the ovular poles. The opposite pole is just as markedly chai'acterised by the presence immediately below it of a more or less rounded clear mass, eccentrically situated, and composed of an extremely finecytoplasmic reticulum with wide fluid-filled meshes. It is completely surrounded by formative cytoplasm (though over the polar region the enclosing layer is so extremely thin that it here almost reaches the sui'face), and its cytoplasmic framework is perfectly continuous with the same, the line of junction of the two being abrupt and well defined. So delicate, however, is this framewoi'k that it breaks down more or less completely under the action of fixatives of such excellence even as the fluids of Flemming and Hermann, and thus in sections usually all that represent it ai'e a few irregular cytoplasmic strands crossing a large, sharply defined clear space (figs. 2 and 3, d.z.). The mass in question has thus all the characters of the deutoplasmic zone of the fullgrown ovum, and it must undoubtedly be held to represent the central portion of that which has not been utilised in the upbuilding of the formative cytoplasm, and which has been forced to take up an excentric position immediately below the polar region of one hemisphere, owing to the increase of the formative cytoplasm and its segregation in the other hemisphere.
ovum (PI. 1, figs. 2 and 3), i. e. an ovum in which either thefirst polar spindle has appeared or the first polar body hasalready been separated off, it at once becomes evident that  
 
important changes have occurred in the disposition and  
 
relative proportions of the two constituent regions of the eggcytoplasm. The full-grown ovum is of the centrolecithal  
 
type, the central deutoplasmic zone forming its main bulk  
 
and being completely surrounded by the thin formative zone.  
 
The ripe ovum, on the other hand, exhibits an obvious and  
 
unmistakable polarity, and is of the telolecithal type, as the  
 
following facts show. The cytoplasmic body evidently consists of the same two regions as form that of the full-gi-own  
 
ovum, but here the dense formative region now forms its  
 
main bulk, and no longer surrounds the clear deutoplasmic  
 
region as a uniform periphei*al layer. It has not only  
 
increased considerably in amount as compared with that of  
 
the full-grown egg, and at the expense apparently of the moreperipheral coarser portion of the deutoplasmic zone, but it  
 
has undergone polar segregation, with the result that it now  
 
occupies rather more than one hemisphere of the egg as a  
 
dense finely granular mass, with vacuoles of varying sizesparsely scattered through it (figs. 2 and 3, /.z.). It  
 
accordingly defines one of the ovular poles. The opposite  
 
pole is just as markedly chai'acterised by the presence immediately below it of a more or less rounded clear mass,  
 
eccentrically situated, and composed of an extremely finecytoplasmic reticulum with wide fluid-filled meshes. It is  
 
completely surrounded by formative cytoplasm (though over  
 
the polar region the enclosing layer is so extremely thin that  
 
it here almost reaches the sui'face), and its cytoplasmic framework is perfectly continuous with the same, the line of  
 
junction of the two being abrupt and well defined. So  
 
delicate, however, is this framewoi'k that it breaks down  
 
more or less completely under the action of fixatives of such excellence even as the fluids of Flemming and Hermann,  
 
and thus in sections usually all that represent it ai'e a few  
 
irregular cytoplasmic strands crossing a large, sharply defined  
 
clear space (figs. 2 and 3, d.z.). The mass in question has  
 
thus all the characters of the deutoplasmic zone of the fullgrown ovum, and it must undoubtedly be held to represent  
 
the central portion of that which has not been utilised in the  
 
upbuilding of the formative cytoplasm, and which has been  
 
forced to take up an excentric position immediately below  
 
the polar region of one hemisphere, owing to the increase of  
 
the formative cytoplasm and its segregation in the other  
 
hemisphere.  
 
  
  
The ripe ovum of Dasyurus thus possesses a polarity which  
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The ripe ovum of Dasyurus thus possesses a polarity which in its way is equally as striking as that of the Monotreme egg. Towards the one pole the main mass of the ovum is composed of dense, slightly vacuolated formative cytoplasm, in which the polar spindle is situated peripherally, but nearer the equator than the formative pole. Toward the opposite pole and practically reaching the surface is a rounded mass of greatly vacuolated deutoplasmic cytoplasm. Eoughly, the formative cytoplasm constitutes about two-thirds of the bulk of the ripe egg, the deutoplasmic the remaining third. Such being the structure of the ripe ovarian egg, if we classify it at all, we must place it, it seems to me, with eggs of the telolecithal type. My view of the significance of this marked polar differentiation of the constituent materials of the ripe ovum of Dasyurus I shall presently indicate. Meantime I would lay special emphasis on the fact that the eccentric mass of deutoplasmic cytoplasm represents matei'ial, surplus deutoplasmic material which has not been utilised in the upbuilding of the formative cytoplasm.
in its way is equally as striking as that of the Monotreme  
 
egg. Towards the one pole the main mass of the ovum is  
 
composed of dense, slightly vacuolated formative cytoplasm,  
 
in which the polar spindle is situated peripherally, but nearer  
 
the equator than the formative pole. Toward the opposite  
 
pole and practically reaching the surface is a rounded mass  
 
of greatly vacuolated deutoplasmic cytoplasm. Eoughly,  
 
the formative cytoplasm constitutes about two-thirds of the  
 
bulk of the ripe egg, the deutoplasmic the remaining third.  
 
Such being the structure of the ripe ovarian egg, if we  
 
classify it at all, we must place it, it seems to me, with eggs  
 
of the telolecithal type. My view of the significance of this  
 
marked polar differentiation of the constituent materials of  
 
the ripe ovum of Dasyurus I shall presently indicate. Meantime I would lay special emphasis on the fact that the  
 
eccentric mass of deutoplasmic cytoplasm represents matei'ial,  
 
surplus deutoplasmic material which has not been utilised in  
 
the upbuilding of the formative cytoplasm.  
 
  
  
The fact of the occurrence in the Eutherian ovum of a  
+
The fact of the occurrence in the Eutherian ovum of a polar differentiation of its constituent materials is now definitely established, thanks especially to the valuable researches of Prof. 0. Van der Stricht and his pupils - H. Lams and the late J. Doorme. In this connection I wish to refer here in some detail to the extremely interesting observations of Van der Stricht ['03, '05] on the structure and polarity of tlie ovum of the bat (Vesperugo noctula), since these observations are in essential agreement with my own on the ovum of Dasyurus, and enable me to affirm that the polar differentiation herein recorded for the first time for the Marsupial ovum is attained as the result of vitellogenetic processes, which essentially correspond with those of the ovum of the bat. Van der Stricht, as is well known, has made a special study of the process of vitellogenesis in the Eutherian ovum, and is, indeed, at the present time the foremost authority on this particular subject, so that his views are worthy of all respect.
polar differentiation of its constituent materials is now  
 
definitely established, thanks especially to the valuable  
 
researches of Prof. 0. Van der Stricht and his pupils - H.  
 
Lams and the late J. Doorme. In this connection I wish to  
 
refer here in some detail to the extremely interesting observations of Van der Stricht ['03, '05] on the structure and  
 
polarity of tlie ovum of the bat (Vesperugo noctula), since  
 
these observations are in essential agreement with my own  
 
on the ovum of Dasyurus, and enable me to affirm that the  
 
polar differentiation herein recorded for the first time for the  
 
Marsupial ovum is attained as the result of vitellogenetic  
 
processes, which essentially correspond with those of the ovum  
 
of the bat. Van der Stricht, as is well known, has made a  
 
special study of the process of vitellogenesis in the Eutherian  
 
ovum, and is, indeed, at the present time the foremost  
 
authority on this particular subject, so that his views are  
 
worthy of all respect.  
 
  
  
Study of the oocyte of Vesperugo during the period of  
+
Study of the oocyte of Vesperugo during the period of growth shows, according to Van der Stricht, that “ a un moment donne du developpement du jeune oeuf, les boyaux et amas vitellogenes [derived, according to him, from ‘ une couche vitellogene, mitochoudriale,' present in the young oocyte in the first stage of growth] disparaissent au profit du vitellus, dont la structure pseudo - alveolaire s'accentue graduellement.” The full-grown oocyte at the stage just prior to the appearance of the first polar spindle is characterised by the presence of this “ pseudo-alveolar structui'e ” throughout the extent of its cytoplasmic body. The alveoli or vacuoles are of variable size, are filled by a clear liquid, and “ correspondent incontestablement au deutoplasma de I'oeuf. A ce stade du developpement de I'oocyte, ce vitellus nutritif, auquel s'ajoutent bientot des granulations graisseuses, est repandu uniformement dans toutes les profondeurs du cytoplasme. Nulle part on ne constate une zone deutoplasmique distincte d'une zone de vitellus plastique.” In Dasyurus the stage in vitellogenesis which almost exactly coiTesponds with that of the full-grown oocyte of Vesperugo just described is seen in oocytes not quite full-gi*own. In fig. 4 is shown an oocyte of Dasyurus (•26 x ‘20 inm. in diameter), in which the same pseudo-alveolar sti'ucture as described by Van der Stricht for the Vesperugo oocyte is perfectly distinct. Here, however, fatty particles are not apparent, and the peripheral portion of the cytoplasm tends to be free from vacuoles. In Dasyurus the formation of these deutoplasmic vacuoles begins in oocytes about '2 mm. or less in diameter. This characteristic pseudo-alveolar ” stage is followed in both Vesperugo and Dasyurus by one in which there is recognisable in the cytoplasmic body of the ovum a differentiation into a dense peripheral zone and a central vacuolated area. In Vesperugo this stage is attained about the time of appearance of the first polar spindle, whilst in Dasyurus it is attained somewhat earlier, always prior to the formation of the latter. So close is the agreement between the two forms that Van der Stricht's desci'iption of the bat's egg at the time of appearance of the first polar spindle might equally well be applied to the full-grown ovum of Dasyurus. He writes ['03, p. 43] : “Vers I'epoque de I'apparition du premier fuseau de maturation, le vitellus prend un autre aspect. La partie centrale deutoplasmique conserve une structure pseudo-alveolaire, mais dans le voisinage immediat du premier fuseau et daus toute I'etendue de la couche peripherique du protoplasme, apparait une mince zone de vitellus compact et dense, plus ou moins homogene ou les vesicules claii'es font defaut. . . . A ce moment, on
growth shows, according to Van der Stricht, that “ a un  
 
moment donne du developpement du jeune oeuf, les boyaux  
 
et amas vitellogenes [derived, according to him, from ‘ une  
 
couche vitellogene, mitochoudriale,' present in the young  
 
oocyte in the first stage of growth] disparaissent au profit du  
 
vitellus, dont la structure pseudo - alveolaire s'accentue  
 
graduellement.” The full-grown oocyte at the stage just  
 
prior to the appearance of the first polar spindle is characterised by the presence of this “ pseudo-alveolar structui'e ”  
 
throughout the extent of its cytoplasmic body. The alveoli  
 
or vacuoles are of variable size, are filled by a clear liquid,  
 
and “ correspondent incontestablement au deutoplasma de  
 
I'oeuf. A ce stade du developpement de I'oocyte, ce vitellus  
 
nutritif, auquel s'ajoutent bientot des granulations graisseuses,  
 
est repandu uniformement dans toutes les profondeurs du  
 
cytoplasme. Nulle part on ne constate une zone deutoplasmique distincte d'une zone de vitellus plastique.” In  
 
Dasyurus the stage in vitellogenesis which almost exactly  
 
coiTesponds with that of the full-grown oocyte of Vesperugo  
 
just described is seen in oocytes not quite full-gi*own. In  
 
fig. 4 is shown an oocyte of Dasyurus (•26 x ‘20 inm. in  
 
diameter), in which the same pseudo-alveolar sti'ucture as  
 
described by Van der Stricht for the Vesperugo oocyte is  
 
perfectly distinct. Here, however, fatty particles are not apparent, and the peripheral portion of the cytoplasm tends  
 
to be free from vacuoles. In Dasyurus the formation of these  
 
deutoplasmic vacuoles begins in oocytes about '2 mm. or less  
 
in diameter. This characteristic pseudo-alveolar ” stage is  
 
followed in both Vesperugo and Dasyurus by one in which  
 
there is recognisable in the cytoplasmic body of the ovum a  
 
differentiation into a dense peripheral zone and a central  
 
vacuolated area. In Vesperugo this stage is attained about  
 
the time of appearance of the first polar spindle, whilst in  
 
Dasyurus it is attained somewhat earlier, always prior to the  
 
formation of the latter. So close is the agreement between  
 
the two forms that Van der Stricht's desci'iption of the bat's  
 
egg at the time of appearance of the first polar spindle might  
 
equally well be applied to the full-grown ovum of Dasyurus.  
 
He writes ['03, p. 43] : “Vers I'epoque de I'apparition du  
 
premier fuseau de maturation, le vitellus prend un autre  
 
aspect. La partie centrale deutoplasmique conserve une  
 
structure pseudo-alveolaire, mais dans le voisinage immediat  
 
du premier fuseau et daus toute I'etendue de la couche  
 
peripherique du protoplasme, apparait une mince zone de  
 
vitellus compact et dense, plus ou moins homogene ou les  
 
vesicules claii'es font defaut. . . . A ce moment, on  
 
  
  
distingue daus I'odcyte de V. noctula une zone centi'ale  
+
distingue daus I'odcyte de V. noctula une zone centi'ale tres etendue, riche en deutoplasme et une zone corticale tres mince, riche en vitellus plastique.” This centrolecithal phase, as we may term it, is followed in Vesperugo during fertilisation and the separation of the second polar body by a telolecithal phase characterised by a distinct polarity. “ La zone de vitellus plastique s'epaissit encore, mais surtout a un pole de I'oeuf, a celui oppose au pole ou se detachent les deux globules polaires. Ce pole, ou s'accumule graduellement le vitellus formateur, merite le nom de pole animal. II est oppose au pole d'expulsion des globules polaires, vers lequel est refoule le deutoplasme, et qui se comporte desormais comme le pole vegetatif. Pendant que les deux pronucleus male et femelle se ferment, le vitellus plastique augmente graduellement en abundance au pole animal, tandis qu'il diminue an p61e vegefcafcif, et le deutoplasme, parseme d'un plus grand nombre de boules graiss6uses, constitue une masse spberique excenfcriqne, voisine des deux globules polaires” (Van der Stricbfc, '03, pp. 44 - 45). It is evident, then, that the fertilised ovum of Yesperugo exhibits a polarity comparable with that of the ripe ovarian ovum of Dasyurus, and that the vitellogenetic processes in the ova of these two widely separated forms proceed along lines almost identical, at all events so far as their broad outlines are concerned. In both we find during growth a progressive vacuolisation of the egg-cytoplasm consequent on tlie elaboration of a deutoplasmic fiuid. In both, the '^pseudo-alveolar ” condition so engendei-ed is followed by one in which there is recognisable a differentiation into a peripheral "formative ” zone rich in deutoplasmic granules, and a central " deutoplasmic ” zone rich in fluid yolk, and finally in both there occurs a segregation of the granular "formative” and fiuid yolk-constituents to opposite I'egions of the egg, with resulting attainment of a definite polarity. In view of the close general agreement in the vitellogenetic processes, and in the constitution of the ova in Vesperugo and Dasyurus, it might be expected that the poles would accurately correspond, but such is not the case if Van der Stricht's determination of the poles in the ovum of Vesperugo is correct. In the latter, according to Van der Stricht, the deutoplasm is located at that pole from which the polar bodies are given off; at the opposite pole the "plastic” vitellus accumulates, and close to it the two pronuclei unite and the first cleavage spindle is formed. Accordingly Van der Stricht concludes that "le premier pole correspond au pole vegetatif, le second au p61e animal des oeufs a deutoplasme polaire (0. Hertwig).” In Dasyurus, on the other hand, I am perfectly convinced (and adequate reason for my conviction will be forthcoming in the course of my description of the processes of cleavage and germ-layer formation) that the pole of the ripe ovum in relation to the mass of deutoplasmic cytoplasm is not the vegetative pole, but represents morphologically the upper or animal pole of the egg, the opposite pole in relation to which the formative cytoplasm is situated being' the lower or vegetative. The deutoplasmic cytoplasm thus lies in the upper hemisphere, whilst the formative cytoplasm occupies the lower. If Van der Stricht's determination of the poles of the ovum of Vesperugo be accepted, then we must conclude that the poles of the Dasyurus ovum are exactly reversed as compared with those of the bat^s egg. In this connection it may be recalled that Lams and Doorme ['07] have demonstrated the occurrence in Cavia of an actual reversal of the original polarity of the ovum, pi'ior to the beginning of cleavage. These facts may well give us pause before we proceed to attach other than a purely secondary significance to the exact location of the formative and deutoplasmic constituents in the Metatherian and Eutherian ovum. But besides this apparent difference in the location of the deutoplasmic constituents of the ova of Dasyurus and Vesperugo, there exists yet another which concerns the fate of these constituents in the respective eggs. In Vesperugo, Van der Stricht shows that the “ deutoplasm ” remains an integral part of the egg, and I'etains its polar distribution in the blastomeres up to at least the 4-celled stage. ^ In Dasyurus, on the other hand, the fate of the deutoplasmic mass is a very different, and, indeed, a very remarkable one. It does not remain an integral part of the segmenting egg as in Vesperugo, but prior to the completion of the first cleavage furrow it becomes bodily separated off, apparently by a process of abstriction, from the formative cytoplasm as a clear rounded mass which takes no further direct part in the developmental processes. As soon as its elimination is effected, the remainder of the cytoplasmic body of the ovum, formed of the formative cytoplasm alone, divides into the first two equal-sized blastomeres, the first cleavage plaue being coincident with the polar diameter and at right angles to the plane of separation of the deutoplasmic mass, or “yolk-body ” as we may term it (PI. 2, figs. 14-16, 19, ij.b.), so that it is this formative zone of the ovum which is filone concerned in tlie production of the embryo and its foetal membranes.
tres etendue, riche en deutoplasme et une zone corticale  
 
tres mince, riche en vitellus plastique.” This centrolecithal phase, as we may term it, is followed in Vesperugo  
 
during fertilisation and the separation of the second polar  
 
body by a telolecithal phase characterised by a distinct  
 
polarity. “ La zone de vitellus plastique s'epaissit encore,  
 
mais surtout a un pole de I'oeuf, a celui oppose au pole ou se  
 
detachent les deux globules polaires. Ce pole, ou s'accumule  
 
graduellement le vitellus formateur, merite le nom de pole  
 
animal. II est oppose au pole d'expulsion des globules  
 
polaires, vers lequel est refoule le deutoplasme, et qui se  
 
comporte desormais comme le pole vegetatif. Pendant que  
 
les deux pronucleus male et femelle se ferment, le vitellus  
 
plastique augmente graduellement en abundance au pole animal, tandis qu'il diminue an p61e vegefcafcif, et le deutoplasme, parseme d'un plus grand nombre de boules graiss6uses,  
 
constitue une masse spberique excenfcriqne, voisine des deux  
 
globules polaires” (Van der Stricbfc, '03, pp. 44 - 45). It is  
 
evident, then, that the fertilised ovum of Yesperugo exhibits a  
 
polarity comparable with that of the ripe ovarian ovum of  
 
Dasyurus, and that the vitellogenetic processes in the ova of  
 
these two widely separated forms proceed along lines almost  
 
identical, at all events so far as their broad outlines are concerned. In both we find during growth a progressive  
 
vacuolisation of the egg-cytoplasm consequent on tlie elaboration of a deutoplasmic fiuid. In both, the '^pseudo-alveolar ”  
 
condition so engendei-ed is followed by one in which there  
 
is recognisable a differentiation into a peripheral "formative ” zone rich in deutoplasmic granules, and a central  
 
" deutoplasmic ” zone rich in fluid yolk, and finally in both  
 
there occurs a segregation of the granular "formative” and  
 
fiuid yolk-constituents to opposite I'egions of the egg, with  
 
resulting attainment of a definite polarity. In view of the  
 
close general agreement in the vitellogenetic processes, and in  
 
the constitution of the ova in Vesperugo and Dasyurus, it  
 
might be expected that the poles would accurately correspond,  
 
but such is not the case if Van der Stricht's determination of  
 
the poles in the ovum of Vesperugo is correct. In the latter,  
 
according to Van der Stricht, the deutoplasm is located at  
 
that pole from which the polar bodies are given off; at the  
 
opposite pole the "plastic” vitellus accumulates, and close to  
 
it the two pronuclei unite and the first cleavage spindle is  
 
formed. Accordingly Van der Stricht concludes that "le  
 
premier pole correspond au pole vegetatif, le second au p61e  
 
animal des oeufs a deutoplasme polaire (0. Hertwig).” In  
 
Dasyurus, on the other hand, I am perfectly convinced (and  
 
adequate reason for my conviction will be forthcoming in the  
 
course of my description of the processes of cleavage and  
 
germ-layer formation) that the pole of the ripe ovum in  
 
relation to the mass of deutoplasmic cytoplasm is not the  
 
vegetative pole, but represents morphologically the upper or animal pole of the egg, the opposite pole in relation to which  
 
the formative cytoplasm is situated being' the lower or  
 
vegetative. The deutoplasmic cytoplasm thus lies in the  
 
upper hemisphere, whilst the formative cytoplasm occupies  
 
the lower. If Van der Stricht's determination of the poles of  
 
the ovum of Vesperugo be accepted, then we must conclude  
 
that the poles of the Dasyurus ovum are exactly reversed as  
 
compared with those of the bat^s egg. In this connection it  
 
may be recalled that Lams and Doorme ['07] have demonstrated the occurrence in Cavia of an actual reversal of the  
 
original polarity of the ovum, pi'ior to the beginning of  
 
cleavage. These facts may well give us pause before we  
 
proceed to attach other than a purely secondary significance  
 
to the exact location of the formative and deutoplasmic constituents in the Metatherian and Eutherian ovum. But  
 
besides this apparent difference in the location of the deutoplasmic constituents of the ova of Dasyurus and Vesperugo,  
 
there exists yet another which concerns the fate of these constituents in the respective eggs. In Vesperugo, Van der  
 
Stricht shows that the “ deutoplasm ” remains an integral  
 
part of the egg, and I'etains its polar distribution in the  
 
blastomeres up to at least the 4-celled stage. ^ In Dasyurus,  
 
on the other hand, the fate of the deutoplasmic mass is a very  
 
different, and, indeed, a very remarkable one. It does not  
 
remain an integral part of the segmenting egg as in Vesperugo,  
 
but prior to the completion of the first cleavage furrow it  
 
becomes bodily separated off, apparently by a process of  
 
abstriction, from the formative cytoplasm as a clear rounded  
 
mass which takes no further direct part in the developmental  
 
processes. As soon as its elimination is effected, the remainder  
 
of the cytoplasmic body of the ovum, formed of the formative  
 
cytoplasm alone, divides into the first two equal-sized blastomeres, the first cleavage plaue being coincident with the polar  
 
diameter and at right angles to the plane of separation of the  
 
deutoplasmic mass, or “yolk-body ” as we may term it (PI. 2,  
 
figs. 14-16, 19, ij.b.), so that it is this formative zone of the ovum which is filone concerned in tlie production of the  
 
embryo and its foetal membranes.  
 
  
  
 +
Vide, however, “Addendum” (p. 121).
  
* Vide, however, “Addendum” (p. 121).  
+
We have but to recall the conclusion already reached that the clear vacuolated zone at the upper pole of the ripe ovum of Dasyurus consists of surplus material, mainly in the form of fluid of deutoplasmic nature which has not been utilised in the upbuilding of the formative cytoplasm, and the signiflcance of this remarkable and, so far as the Mammalian ovum is concerned, absolutely unique occurrence becomes at once manifest.'^ We have to do here with an actual elimination of surplus deutoplasmic material by the Marsupial ovum - a phenomenon only paralleled elsewhere, so far a,s I am awai'e, and even then but distantly, by the curious temporary separation of the so-called yolk-lobe which occurs during the cleavage of the yolk-laden eggs of certain Molluscs (Nassa, Ilyanassa, Modiolaria, Aplysia, Dentalium) and Annelids (Myzostoma, Chjetopterus) . In these forms cleavage of the ovum into the first two blastomeres is accompanied by the sepai'ation of a portion of the ovular substance in the form of a non-nucleated mass or so-called yolk-lobe. This latter, which has been shown to be connected Avith the formation of determined organanlagen, reunites Avith one of the tAvo blastomeres, and then the same process of abstriction and reunion recurs at the second cleavage.^ We have here evidently a purely adaptive phenomenon, the object of which no doubt is to permit of the total cleavage of the yolk-laden ovum on Avhat are presumably the old ancestral lines, and I believe a comparable explanation Avill be found applicable to the elimination of surplus yolkmaterial by the Marsupial ovum.
  
 +
As regards the significance of the occurrence of the deutoplasmic zone in the ovum of Dasyurus, holding the views that I do as to the phylogeny of the Marsupialia (viz. that the Metatheria and Eutheria are the divergent branches of a common stock, itself of Prototlierian derivation), and bearing in mind the occurrence of an undoubted repi'esentative of the shell round the Marsupial ovum, I venture to see in the fluidmaterial of the deutoplasmic zone the partial and vestigial equivalent of the yolk-mass of the monotreme egg. In other words, I would regard the deutoplasmic fluid as the product of an abortive attempt at the formation of such a solid yolkmass. The objection will no doubt be forthcoming that this interpretation cannot possibly be correct since the supposed equivalent of the yolk-mass in the Dasyure ovum is located, on my own showing, at the wrong pole - at the upper instead of at the lower. But its precise location does not seem to me to be a matter to which we need attach any great importance, since it has doubtless been adaptively determined in correlation with the special character of the cleavage process.
  
We have but to recall the conclusion already reached that
 
the clear vacuolated zone at the upper pole of the ripe ovum
 
of Dasyurus consists of surplus material, mainly in the form of
 
fluid of deutoplasmic nature which has not been utilised in
 
the upbuilding of the formative cytoplasm, and the signiflcance of this remarkable and, so far as the Mammalian ovum
 
is concerned, absolutely unique occurrence becomes at once
 
manifest.'^ We have to do here with an actual elimination of
 
surplus deutoplasmic material by the Marsupial ovum  -  a phenomenon only paralleled elsewhere, so far a,s I am awai'e, and
 
even then but distantly, by the curious temporary separation
 
of the so-called yolk-lobe which occurs during the cleavage
 
of the yolk-laden eggs of certain Molluscs (Nassa, Ilyanassa,
 
Modiolaria, Aplysia, Dentalium) and Annelids (Myzostoma,
 
Chjetopterus) . In these forms cleavage of the ovum into the
 
first two blastomeres is accompanied by the sepai'ation of a
 
portion of the ovular substance in the form of a non-nucleated
 
mass or so-called yolk-lobe. This latter, which has been shown
 
to be connected Avith the formation of determined organanlagen, reunites Avith one of the tAvo blastomeres, and then
 
the same process of abstriction and reunion recurs at the
 
second cleavage.^ We have here evidently a purely adaptive
 
phenomenon, the object of which no doubt is to permit of the
 
total cleavage of the yolk-laden ovum on Avhat are presumably
 
the old ancestral lines, and I believe a comparable explanation
 
Avill be found applicable to the elimination of surplus yolkmaterial by the Marsupial ovum.
 
  
As regards the significance of the occurrence of the deutoplasmic zone in the ovum of Dasyurus, holding the views that
+
Yide “Addendum” (p. 121), in Avliich reference is made to the discovery by Prof. Van der Stricht of the elimination of deutoplasm in the ovum of Yesperugo.
I do as to the phylogeny of the Marsupialia (viz. that the Metatheria and Eutheria are the divergent branches of a common stock, itself of Prototlierian derivation), and bearing
+
® Of. Korschelt u. Heider, ‘Lehrbuch d. vergl. Entwicklungsgeschichte,' Lief. 3, p. 107, 1909.
in mind the occurrence of an undoubted repi'esentative of the
 
shell round the Marsupial ovum, I venture to see in the fluidmaterial of the deutoplasmic zone the partial and vestigial
 
equivalent of the yolk-mass of the monotreme egg. In other
 
words, I would regard the deutoplasmic fluid as the product
 
of an abortive attempt at the formation of such a solid yolkmass. The objection will no doubt be forthcoming that this
 
interpretation cannot possibly be correct since the supposed
 
equivalent of the yolk-mass in the Dasyure ovum is located,  
 
on my own showing, at the wrong pole  -  at the upper instead
 
of at the lower. But its precise location does not seem to me
 
to be a matter to which we need attach any great importance,  
 
since it has doubtless been adaptively determined in correlation with the special character of the cleavage process.  
 
  
  
* Yide “Addendum” (p. 121), in Avliich reference is made to the discovery by Prof. Van der Stricht of the elimination of deutoplasm in the ovum of Yesperugo.  
+
The belief that the minute yolk-poor ovum of the Eutheria is no pure primarily holoblastic one, but that it has only secondarily arrived at the total type of cleavage as the result of the all but complete loss of the yolk ancestrally present in it, consequent on the substitution of the intra-uterine mode of development for the old oviparous habit, is now widely held amongst Mammalian embryologists. Hubi'echt, however, is an exception, wedded as he is to a belief in the direct derivation of the Eutheria from Protetrapodous ancestors with yolkpoor, holoblastic eggs. Whether the interpretation I have put forward, viz. that the non-formative or deutoplasmic zone of the Dasyure ovum is the reduced and partial equivalent of the yolk-mass of the Monotreme egg, be accepted or not, I venture to think that my discovery of an actual elimination of deutoplasmic material by the Marsupial ovum affords a striking confirmation of the truth of the prevailing conception as to the phylogeny of the Eutherian ovum, and I further venture to think that the facts I have brought forward in the preceding ])ages justify us in' regarding the ripe ovarian ovum of Dasyurus as being potentially of the yolk-laden, telolecithal type, and the uterine ovum, by bodily casting out the superfluous part of its deutoplasm, as becoming at the same time secondarily lioniolecithal and secondarily lioloblastic. Ihe Marsupial ovum presents itself to my mind as tlie victim of tendencies conditioned by its ancestry, and in particular it appears as if its inherited tendency to elaborate yolk had not yet been brought into accurate correlation with the other changes (reduction in size, intra-uterine development), which it has undergone in the course of phylogeny. As the consequence it manufactures more yolk than it can utilise, and so finds itself under the necessity of getting rid of the surplus. Whether or not a comparable elimination of deutoplasmic material occurs in the ova of other Marsupials, future investigation must decide. I should be quite prepared to find variation in this regard, correlated perhaps with the size of the egg. In the case of Phascolarctus, Caldwell gives the diameter of the ovum as '17 mm., and his figure of a (horizontal ?) section of the uterine ovum (here produced as text-fig. 1, p. 27) shows a differentiation of the cytoplasmic body of that into vacuolated and granular zones quite comparable with that of the Dasyure ovum. From the few measurements of ova of other marsupials that I have been able to make, it would appear that the ovum of Trichosurus approximates in size to that of Dasyurus, whilst that of Perameles and pi-obably also that of Macropus are smaller. From Selenka's figure I have calculated that the ovum of Didelphys measures about 'IS x •12 mm. in diameter. In the smaller ova it is quite likely that yolk-formation may not proceed so far as in the relatively large ovum of Dasyurus.
  
® Of. Korschelt u. Heider, ‘Lehrbuch d. vergl. Entwicklungsgeschichte,' Lief. 3, p. 107, 1909.  
+
===2. Maturation and Ovulation===
  
 +
The details of the maturation process have not been fully worked out, owing to lack of material. As in the Eutheria (Sobotta, Van der Stricht, Lams and Doorme, and others), the first polar body is separated off in the ovary, the second apparently in the upper part of the- Fallopian tube where entrance of the sperm takes place. The first polar figure (late anaphase observed, fig. 5) lies in the formative cytoplasm, close below and afc right angles to the zona. Its exact site is subject to some slight variation, and is best described as adjacent to the equatorial region of the egg, sometimes nearer the lower pole, more usually, perhaps, nearer the upper. Centrosomes and polar radiations were not observed. The heterotypical chromosomes (gemini) have the form of somewhat irregular, more or less angular granules. I have not been able to determine their number. The figure is barrel-shaped, and almost as broad as long, measuring
  
 +
015 X ‘OIS mm. The first polar body (fig. 6, is small
 +
relatively to the size of the egg, its diameters varying round •03 X '01 mm., and its shape is that of a flattened bi-convex disc. In uterine eggs thex'e is some evidence pointing to the probability of its having undergone division.
  
The belief that the minute yolk-poor ovum of the Eutheria
+
The second polar spindle (figs. 3 and 7) lies immediately subjacent to the first polar body in the fully ripe ovarian ovum. It is shorter than the first, measuring '013 mm., and xnuch narrower. The second polar body measures about '015 X '01 mm. in diameter, and is thus smaller than the first. I have only seen the second polar body in uterine ova, and therefore can only presume that it is sepai'ated off in the upper part of the Fallopian tube, subsequently to the penetration of the sperm, as in Eutheria.
is no pure primarily holoblastic one, but that it has only
 
secondarily arrived at the total type of cleavage as the result
 
of the all but complete loss of the yolk ancestrally present in
 
it, consequent on the substitution of the intra-uterine mode of
 
development for the old oviparous habit, is now widely held
 
amongst Mammalian embryologists. Hubi'echt, however, is
 
an exception, wedded as he is to a belief in the direct derivation of the Eutheria from Protetrapodous ancestors with yolkpoor, holoblastic eggs. Whether the interpretation I have put
 
forward, viz. that the non-formative or deutoplasmic zone of
 
the Dasyure ovum is the reduced and partial equivalent of the
 
yolk-mass of the Monotreme egg, be accepted or not, I venture
 
to think that my discovery of an actual elimination of deutoplasmic material by the Marsupial ovum affords a striking
 
confirmation of the truth of the prevailing conception as to
 
the phylogeny of the Eutherian ovum, and I further venture
 
to think that the facts I have brought forward in the preceding
 
])ages justify us in' regarding the ripe ovarian ovum of
 
Dasyurus as being potentially of the yolk-laden, telolecithal
 
type, and the uterine ovum, by bodily casting out the superfluous part of its deutoplasm, as becoming at the same time secondarily lioniolecithal and secondarily lioloblastic. Ihe
 
Marsupial ovum presents itself to my mind as tlie victim of
 
tendencies conditioned by its ancestry, and in particular it
 
appears as if its inherited tendency to elaborate yolk had not
 
yet been brought into accurate correlation with the other
 
changes (reduction in size, intra-uterine development), which
 
it has undergone in the course of phylogeny. As the consequence it manufactures more yolk than it can utilise, and so
 
finds itself under the necessity of getting rid of the surplus.  
 
Whether or not a comparable elimination of deutoplasmic
 
material occurs in the ova of other Marsupials, future investigation must decide. I should be quite prepared to find variation
 
in this regard, correlated perhaps with the size of the egg.
 
In the case of Phascolarctus, Caldwell gives the diameter of
 
the ovum as '17 mm., and his figure of a (horizontal ?) section
 
of the uterine ovum (here produced as text-fig. 1, p. 27) shows
 
a differentiation of the cytoplasmic body of that into vacuolated and granular zones quite comparable with that of the  
 
Dasyure ovum. From the few measurements of ova of other
 
marsupials that I have been able to make, it would appear
 
that the ovum of Trichosurus approximates in size to that of
 
Dasyurus, whilst that of Perameles and pi-obably also that
 
of Macropus are smaller. From Selenka's figure I have
 
calculated that the ovum of Didelphys measures about 'IS x
 
•12 mm. in diameter. In the smaller ova it is quite likely
 
that yolk-formation may not proceed so far as in the relatively
 
large ovum of Dasyurus.  
 
  
2. Maturation and Ovulation.  
+
Ovulation takes place irrespective of whether copulation has occurred or not, and it is a fact Avorbhy of record that, even if the ova be not fertilised, the pouch and mammary glands undergo the same series of growth changes as are characteristic of, at all events, the earlier stages of normal pregnancy.
  
The details of the maturation process have not been fully
+
The follicular cells of the discus proligerus investing the ovum are already in the ripe follicle in a state of disruption, and I believe they separate completely from the ovum at the moment of dehiscence, so that, except for the zona, the ova are quite naked Avhen they enter the tube. I have no evidence of the existence outside the zona of a layer of proalbumen such as Caldwell describes round the ovum of Phascolarctus. Apparently the ova are shed almost simultaneously, and they must pass with considerable rapidity down the tubes to the uteri wliere cleavage begins, for I have only once found a tubal ovum, and that one had evidently been retarded for some reason, and was polyspermic.
worked out, owing to lack of material. As in the Eutheria
 
(Sobotta, Van der Stricht, Lams and Doorme, and others),  
 
the first polar body is separated off in the ovary, the second
 
apparently in the upper part of the- Fallopian tube where
 
entrance of the sperm takes place. The first polar figure
 
(late anaphase observed, fig. 5) lies in the formative cytoplasm, close below and afc right angles to the zona. Its exact
 
site is subject to some slight variation, and is best described
 
as adjacent to the equatorial region of the egg, sometimes
 
nearer the lower pole, more usually, perhaps, nearer the
 
upper. Centrosomes and polar radiations were not observed.
 
The heterotypical chromosomes (gemini) have the form of
 
somewhat irregular, more or less angular granules. I have  
 
not been able to determine their number. The figure is
 
barrel-shaped, and almost as broad as long, measuring
 
*015 X ‘OIS mm. The first polar body (fig. 6, is small
 
  
relatively to the size of the egg, its diameters varying round
 
•03 X '01 mm., and its shape is that of a flattened bi-convex
 
disc. In uterine eggs thex'e is some evidence pointing to the
 
probability of its having undergone division.
 
  
The second polar spindle (figs. 3 and 7) lies immediately
+
===3. The Secondary Egg-membranes: Albumen and Shell membrane===
subjacent to the first polar body in the fully ripe ovarian
 
ovum. It is shorter than the first, measuring '013 mm., and
 
xnuch narrower. The second polar body measures about
 
'015 X '01 mm. in diameter, and is thus smaller than the first.
 
I have only seen the second polar body in uterine ova, and
 
therefore can only presume that it is sepai'ated off in the
 
upper part of the Fallopian tube, subsequently to the penetration of the sperm, as in Eutheria.
 
  
Ovulation takes place irrespective of whether copulation has
+
During the passage of the ovum down the tube it is fertilised, and becomes enclosed externally to the zona by two secondary layers formed as secretions by the cells of the oviducal lining. First of all, the ovum becomes surrounded by a transparent to semi-transparent laminated layer of albumen, •015 to ■02'2 mm. in thickness, composed of numerous very delicate concentric lamellae, and having, normally, numbers of sperms imbedded in it (figs. 8-11, alb., sp.). Then outside the albumen layer there is laid down a definite, but at first very thin, double-contoured membrane (figs. 8 and 10, s.m.), which, following Caldwell, I have no hesitation in homologising with the shell-membrane of the Monotreme egg. Caldwell in 1887 described and figured a definite membrane enclosing the uterine ovum of Phascolarctus, externally to, and quite distinct from the albumen, which he interpreted as the representative of the shell-membrane of the Monotremata, but owing apparently to the fact that Selenka altogether failed to recognise its true nature in Didelphys, since he regarded it as a derivative of the follicular epithelium, and termed it the “ granulosa-membran,” this highly significant discovery of Caldwell has been largely ignox'ed. Such a membrane is constantly present and easily recognisable in all the Marsupials (Dasyurus, Perameles, Trichosurus, Macropus, Petrogale, Phascologale, Acrobates, Phascolarctus, Bettongia), of which I have had the opportunity of studying early developmental stages. It is laid down in the Fallopian tube, is perfectly distinct from the albumen, and increases in thickness in the uterus, and if it has not the significance which Caldwell has suggested, then I must leave it to those who decline to accept Caldwell's interpretation to put forward an alternative one, since I am unable to do so.
occurred or not, and it is a fact Avorbhy of record that, even
 
if the ova be not fertilised, the pouch and mammary glands
 
undergo the same series of growth changes as are characteristic of, at all events, the earlier stages of normal
 
pregnancy.  
 
  
The follicular cells of the discus proligerus investing the
+
The shell-membrane of Dasyurus (PI. 1, figs. 8-11; PI. 2, figs. 17, 18, s.m.) is a ti*ansparent, perfectly homogeneous layer, highly refractive in character and of a faint yellowish tint. When fully formed it possesses firm, resistant properties, recalling those of chitin, and is doubtless composed of a keratin base. It is distinguishable at once from the albumen by its optical characters and staining reactions, so that there is not the slightest justification for the supposition that it may represent simply the specially differentiated outermost portion of that layer. In ova which have just passed into the uterus (fig. 10) the shell-membrane is extremely delicate, its thickness being only about '0016 mm., but even before cleavage begins it has increased to ”002 mm. (fig. 12) ; in the 2-celled stage (fig. 18) it has reached ‘005 mm., in the 4-celled stage (fig. 22) '0072 mm., whilst in the 16-celled stage (figs. 24-26) it has practically attained its maximum thickness, viz., ‘0075'008 mm. Caldwell's measurements in the case of Phascolarctus agree closely with the above (shell of unsegmented ovum from the uterus, '0015 mm. thick, that of the '3 mm. ‘‘ovum,” '01 mm.). Its presence renders the thorough penetration of ova and early blastocysts with pai'affin a capricious and frequently troublesome operation, and its resistant shell-like nature becomes only too obvious in the process of section-cutting, since it cracks with the utmost readiness (cf. PI. 3, figs. 32, 37).
ovum are already in the ripe follicle in a state of disruption,  
 
and I believe they separate completely from the ovum at the
 
moment of dehiscence, so that, except for the zona, the ova
 
are quite naked Avhen they enter the tube. I have no evidence
 
of the existence outside the zona of a layer of proalbumen
 
such as Caldwell describes round the ovum of Phascolarctus.  
 
Apparently the ova are shed almost simultaneously, and they must pass with considerable rapidity down the tubes to the
 
uteri wliere cleavage begins, for I have only once found a
 
tubal ovum, and that one had evidently been retarded for
 
some reason, and was polyspermic.  
 
  
  
3. The Secondary Egg-membranes: Albumen and Shell membrane
+
The occurrence of a shell-membrane round the Marsupial ovum is a feature of considerable phyletic significance, as I need hardly point out. It shows us that the ancestors of the Metatheria must have been oviparous, or must themselves have come from an oviparous stock, which there is no valid reason for supposing was other than Prototherian in its characters. It also renders untenable the views of Hubrecht to the effect that the Metatheria are the descendants of Eutheria, whilst the Eutheria themselves have been directly derived from some presumed viviparous group of hypothetical Protetrapods, unless we are to suppose that the Metatheria are even novv on the way to acquire secondarily the oviparous habit, much in the same way as the Mouotremes, according to Hubi-echt, have long since succeeded in doing.
  
During the passage of the ovum down the tube it is
 
fertilised, and becomes enclosed externally to the zona by
 
two secondary layers formed as secretions by the cells of the
 
oviducal lining. First of all, the ovum becomes surrounded by
 
a transparent to semi-transparent laminated layer of albumen,
 
•015 to ■02'2 mm. in thickness, composed of numerous very
 
delicate concentric lamellae, and having, normally, numbers
 
of sperms imbedded in it (figs. 8-11, alb., sp.). Then outside
 
the albumen layer there is laid down a definite, but at first
 
very thin, double-contoured membrane (figs. 8 and 10, s.m.),
 
which, following Caldwell, I have no hesitation in homologising with the shell-membrane of the Monotreme egg.
 
Caldwell in 1887 described and figured a definite membrane
 
enclosing the uterine ovum of Phascolarctus, externally to,
 
and quite distinct from the albumen, which he interpreted
 
as the representative of the shell-membrane of the Monotremata, but owing apparently to the fact that Selenka
 
altogether failed to recognise its true nature in Didelphys,
 
since he regarded it as a derivative of the follicular epithelium, and termed it the “ granulosa-membran,” this highly
 
significant discovery of Caldwell has been largely ignox'ed.
 
Such a membrane is constantly present and easily recognisable
 
in all the Marsupials (Dasyurus, Perameles, Trichosurus,
 
Macropus, Petrogale, Phascologale, Acrobates, Phascolarctus,
 
Bettongia), of which I have had the opportunity of studying
 
early developmental stages. It is laid down in the Fallopian
 
tube, is perfectly distinct from the albumen, and increases in
 
thickness in the uterus, and if it has not the significance
 
which Caldwell has suggested, then I must leave it to those who decline to accept Caldwell's interpretation to put forward
 
an alternative one, since I am unable to do so.
 
  
The shell-membrane of Dasyurus (PI. 1, figs. 8-11; PI. 2,
+
The occurrence of a shell-membrane round the Marsupial ovum has also an important ontogenetic significance in relation to the mode of formation of the blastocyst, as I shall endeavour presently to show.
figs. 17, 18, s.m.) is a ti*ansparent, perfectly homogeneous
 
layer, highly refractive in character and of a faint yellowish
 
tint. When fully formed it possesses firm, resistant properties,
 
recalling those of chitin, and is doubtless composed of a
 
keratin base. It is distinguishable at once from the albumen
 
by its optical characters and staining reactions, so that there
 
is not the slightest justification for the supposition that it
 
may represent simply the specially differentiated outermost
 
portion of that layer. In ova which have just passed into the
 
uterus (fig. 10) the shell-membrane is extremely delicate, its
 
thickness being only about '0016 mm., but even before cleavage
 
begins it has increased to ”002 mm. (fig. 12) ; in the 2-celled
 
stage (fig. 18) it has reached ‘005 mm., in the 4-celled stage
 
(fig. 22) '0072 mm., whilst in the 16-celled stage (figs. 24-26)
 
it has practically attained its maximum thickness, viz., ‘0075'008 mm. Caldwell's measurements in the case of Phascolarctus agree closely with the above (shell of unsegmented
 
ovum from the uterus, '0015 mm. thick, that of the '3 mm.
 
‘‘ovum,” '01 mm.). Its presence renders the thorough
 
penetration of ova and early blastocysts with pai'affin a
 
capricious and frequently troublesome operation, and its
 
resistant shell-like nature becomes only too obvious in the
 
process of section-cutting, since it cracks with the utmost
 
readiness (cf. PI. 3, figs. 32, 37).  
 
  
The occurrence of a shell-membrane round the Marsupial
 
ovum is a feature of considerable phyletic significance, as I
 
need hardly point out. It shows us that the ancestors of the
 
Metatheria must have been oviparous, or must themselves
 
have come from an oviparous stock, which there is no valid
 
reason for supposing was other than Prototherian in its
 
characters. It also renders untenable the views of Hubrecht to
 
the effect that the Metatheria are the descendants of Eutheria,
 
whilst the Eutheria themselves have been directly derived
 
from some presumed viviparous group of hypothetical Protetrapods, unless we are to suppose that the Metatheria are
 
even novv on the way to acquire secondarily the oviparous
 
habit, much in the same way as the Mouotremes, according
 
to Hubi-echt, have long since succeeded in doing.
 
  
q'be occurrence of a shell-membrane round the Marsupial
+
===4. The Uterine Ovum===
ovum has also aai important ontogenetic significance in relation to the mode of formation of the blastocyst, as I shall
 
endeavour presently to show.  
 
  
 +
The unsegmented ovum from the uterus (figs. 8-13) consists of the following parts :
  
4. The Uterine Ovum.  
+
# The shell-membrane externally, 'OOlfi - '002 mm. in thickness.
 +
# The laminated layer of albumen, '015 - "022 mm. or more in thickness.
 +
# The zona, about -0016 mm. in thickness.
 +
# The perivitelline space, between the zona and the ovum, occupied by a clear fluid which coagulates under the action of certain fixatives, e. g. Hermann's fluid (fig. ll,p.s.), and which has diffused in from the uterus. The minute polar bodies lie in this space, usually nearer the upper pole than the lower.
 +
# The ovum proper.
  
The unsegmented ovum from the uterus (figs. 8-13)
 
consists of the following parts :
 
  
(1) The shell-membrane externally, 'OOlfi -  '002 mm. in
+
The entire egg is spherical in form, and varies in diameter in the fresh state from about '3 mm. to '36 mm. (average about ‘32 mm.).
thickness.  
 
  
(2) The laminated layer of albumen, '015 -  "022 mm. or
 
more in thickness.
 
  
(3) The zona, about -0016 mm. in thickness.  
+
The ovum itself is ovoidal, its polar diameter always slightly exceeding the equatorial. Its average diametrical measurements in the fresh state run about '25 x '24 mm., though I have records of ova measuring as much as ‘3 x ‘29 mm., and I find that there is an undoubted slight variation iu the size of the ova of even one and the same batch, as well as iu those from different females.
  
(4) The perivitelline space, between the zona and the
 
ovum, occupied by a clear fluid which coagulates under the
 
action of certain fixatives, e. g. Hermann's fluid (fig. ll,p.s.),
 
and which has diffused in from the uterus. The minute
 
polar bodies lie in this space, usually nearer the upper pole
 
than the lower.
 
  
(5) The ovum proper.  
+
The uterine ovum exhibits the same marked polarity as cliaracterises the ripe ovarian ovum (the upper pole being marked by the vacuolated deutoplasmic zone (figs. 8-11, d.z.), and so far as its cytoplasmic body is concerned it shows no essential difference from that.
  
The entire egg is spherical in form, and varies in diameter
 
in the fresh state from about '3 mm. to '36 mm. (average
 
about ‘32 mm.).
 
  
The ovum itself is ovoidal, its polar diameter always slightly
+
Examined fresh in normal salt solution, the formative cytoplasm forming the bulk of the ovum appears dense, finely granular, and of a very faint lightish-brown tint, its opacity being such that the two pronuclei situated in its central region ' can just be made out. In section, this central region is distinguishable from the peripheral zone by its uniform, more finely granular character and by the absence of the fluid-filled vacuolar spaces which are generally present in the latter figs. 10 and 12). The deutoplasmic zone at the upper pole, which is only partially visible in the entire egg owing to the way in which it is enclosed by the formative cytoplasm (figs. 8, 9, d.z.), presents a characteristically clear or semi-transparent vacuolated appearance in the fresh state, but may have einbedded in it a small dense mass (fig. 8, cf. also figs. 11 and 14), evidently formed by the transformation of a portion of its fluid constitutent into the solid state, and so to be regai'ded as compai'able with a bit of formative cytoplasm.
exceeding the equatorial. Its average diametrical measurements in the fresh state run about '25 x '24 mm., though I
 
have records of ova measuring as much as ‘3 x ‘29 mm., and  
 
I find that there is an undoubted slight variation iu the size
 
of the ova of even one and the same batch, as well as iu those
 
from different females.  
 
  
The uterine ovum exhibits the same marked polarity as cliaracterises the ripe ovarian ovum (the upper pole being
 
marked by the vacuolated deutoplasmic zone (figs. 8-11, d.z.),
 
and so far as its cytoplasmic body is concerned it shows no
 
essential difference from that.
 
  
Examined fresh in normal salt solution, the formative cytoplasm forming the bulk of the ovum appears dense, finely
+
In most of the unsegmented uterine ova at my disposal the male and female pronuclei have attained approximately tlie same size and lie in proximity in the central more homogeneous region of the formative cytoplasm (figs. 10-12). The transformation of the sperm-head into the male pronucleus probably takes place during the passage of the ovum down the tube, and was not observed, and I am as yet uncertain whether the pronuclei unite to form a single cleavage nucleus or give origin directly to the chromosomes of the first cleavage figure.
granular, and of a very faint lightish-brown tint, its opacity
 
being such that the two pronuclei situated in its central region '
 
can just be made out. In section, this central region is distinguishable from the peripheral zone by its uniform, more
 
finely granular character and by the absence of the fluid-filled
 
vacuolar spaces which are generally present in the latter
 
figs. 10 and 12). The deutoplasmic zone at the upper pole,
 
which is only partially visible in the entire egg owing to the way
 
in which it is enclosed by the formative cytoplasm (figs. 8, 9,
 
d.z.), presents a characteristically clear or semi-transparent
 
vacuolated appearance in the fresh state, but may have einbedded in it a small dense mass (fig. 8, cf. also figs. 11 and 14),
 
evidently formed by the transformation of a portion of its fluid
 
constitutent into the solid state, and so to be regai'ded as compai'able with a bit of formative cytoplasm.  
 
  
In most of the unsegmented uterine ova at my disposal the
 
male and female pronuclei have attained approximately tlie
 
same size and lie in proximity in the central more homogeneous region of the formative cytoplasm (figs. 10-12). The
 
transformation of the sperm-head into the male pronucleus
 
probably takes place during the passage of the ovum down
 
the tube, and was not observed, and I am as yet uncertain
 
whether the pronuclei unite to form a single cleavage nucleus
 
or give origin directly to the chi'omosomes of the first cleavage
 
figure.
 
  
Caldwell figures ('87, PI. 30, fig. 5) a section through the  
+
Caldwell figures ('87, PI. 30, fig. 5) a section through the uterine ovum of Phascolarctus which I reproduce here as Text-fig. l,in order to facilitate comparison with my figs. 11 and 12, with which it shows an essential agreement, apart from the pi'esence of follicular cells in the albumen which I have never observed in Dasyurus, and making allowance for the difference in sectional plane. The figure is stated to represent “the seventeenth section of a vertical longitudinal seines of thirty-five sections through the segmenting ovum, containing two nuclei, taken from the uterus and measuring T7 mm. in diameter.” Caldwell has, I think, fallen into several errors in his interpretation of the structural features seen in this figure. In the first place, the sectional plane appears to me not to be vertical as in my own figs. 11 and 12, but horizontal, and to have passed through the lower portion of the deutoplasmic zone, shown in the figure as a central markedly vacuolated area. Then there is no evidence to be derived from the figure in support of the description of the ovum as segmenting. The part inside the zona {vm.) labelled and described as protoplasm with finest jolk-granules,” I would interpret simply as coagulum in the perivitelline space, whilst the so-called “ segmentation nuclei ” Ua) situated in it are probably the polar bodies or their derivatives. The part labelled y«, and designated “ white yolk,” I would regard as the ovum itself. It exhibits an obvious differentiation into a central vacuolated area and a peripheral, dense, granular zone with scattered vacuoles, and I thiuk there can be little doubt but that the former corresponds to the deutoplasmic zone of the Dasyure ovum, the latter to the formative zone. It is these errors of intei-pretatiou apparently which misled Caldwell into making the statement, now widely quoted in the text-books, that cleavage in Phascolarctus is of the meroblastic type.
uterine ovum of Phascolarctus which I reproduce here as  
 
Text-fig. l,in order to facilitate comparison with my figs. 11 and  
 
12, with which it shows an essential agreement, apart from  
 
the pi'esence of follicular cells in the albumen which I have  
 
never observed in Dasyurus, and making allowance for the difference in sectional plane. The figure is stated to represent  
 
“the seventeenth section of a vertical longitudinal seines of  
 
thirty-five sections through the segmenting ovum, containing  
 
two nuclei, taken from the uterus and measuring T7 mm. in  
 
diameter.” Caldwell has, I think, fallen into several errors  
 
in his interpretation of the structural features seen in this  
 
  
Text-fig. 1.
 
  
  
Section of uterine ovnni of Phascolarctus cinereus. (After Caldwell.)
 
  
figuie. In the first place, the sectional plane appears to me
+
Text-fig. 1. Section of uterine ovnni of Phascolarctus cinereus. (After Caldwell.)
not to be vertical as in my own figs. 11 and 12, but horizontal,
+
 
and to have passed through the lower portion of the deutoplasmic zone, shown in the figure as a central markedly
 
vacuolated area. Then there is no evidence to be derived
 
from the figure in support of the description of the ovum as
 
segmenting. The part inside the zona {vm.) labelled and
 
described as protoplasm with finest jolk-granules,” I would interpret simply as coagulum in the perivitelline space, whilst
 
the so-called “ segmentation nuclei ” Ua) situated in it are
 
probably the polar bodies or their derivatives. The part
 
labelled y«, and designated “ white yolk,” I would regard as
 
the ovum itself. It exhibits an obvious differentiation into a
 
central vacuolated area and a peripheral, dense, granular
 
zone with scattered vacuoles, and I thiuk there can be little
 
doubt but that the former corresponds to the deutoplasmic
 
zone of the Dasyure ovum, the latter to the formative zone.
 
It is these errors of intei-pretatiou apparently which misled
 
Caldwell into making the statement, now widely quoted in
 
the text-books, that cleavage in Phascolarctus is of the
 
meroblastic type.
 
  
  
 
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A personal message from Dr Mark Hill (May 2020)  
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I have decided to take early retirement in September 2020. During the many years online I have received wonderful feedback from many readers, researchers and students interested in human embryology. I especially thank my research collaborators and contributors to the site. The good news is Embryology will remain online and I will continue my association with UNSW Australia. I look forward to updating and including the many exciting new discoveries in Embryology!

Hill JP. The Early Development of the Marsupialia, with Special Reference to the Native Cat (Dasyurus Viverrinus). (1910) Quart. J. Micro. Sci. 56(1): 1-134.

  Contents: 1 Review of Previous Observations | 2 The Ovum of Dasyurus | 3 Cleavage and Blastocyst | 4 Blastocyst Growth Ectoderm Entoderm | 5 Early Stages of Perameles and Macropus | 6 Summary and Conclusions | 7 Early Mammalia Ontogeny | Explanation of Plates
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Eastern quoll
Eastern quoll
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This historic 1910 paper by James Peter Hill describes marsupial development in the native cat (Dasyurus Viverrinus)



Note that native cat, eastern native cat, are historic names for the eastern quoll Dasyurus Viverrinus (D. viverrinus). The eastern quoll is a medium-sized carnivorous marsupial native to Australia.

  • Dasyurus - "hairy tail"

dasyurid

Modern Notes:

Australian Animal: echidna | kangaroo | koala | platypus | possum | Category:Echidna | Category:Kangaroo | Category:Koala | Category:Platypus | Category:Possum | Category:Marsupial | Category:Monotreme | Development Timetable | K12
Historic Australian Animal  
Historic Embryology: 1834 Early Kangaroo | 1880 Platypus Cochlea | 1887 Monotremata and Marsupialia | 1910 Eastern Quoll | 1915 The Monotreme Skull | 1939 Early Echidna

The Hill Collection contains much histology of echidna and platypus embryonic development.

Embryology History | Historic Disclaimer

Other Marsupials  
Monito del Monte Development | Opossum Development
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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)

Chapter II. - The Ovum of Dasyueus

1. Structure of the Ovarian Ovum

The full-grown ovarian ovum of Dasyurus (PI. 1, fig. 1) appears as a rounded, or more usually, ovalish cell, the diameter of which varies in section in ten eggs measured from '28 X ‘126 mm. to '27 x '26 mm. (average, '24 mm.), and is therefore large relatively to the ova of Eutheria. It is enclosed by a tliin, but very definite I'efractive membrane or zona (vitelline membrane of Caldwell) of an approximate thickness of '002 mm. (fig. 1, z-p-), on which the cells of the discus proligerus (fig. I, d.p.) directly abut, a differentiated corona radiata and syncytial layer being absent. It appears to be identical in its relations and optical characters with the membrane investing the monotreme ovum, and never shows in section any trace of radial striations (though I believe I have detected an extremely faint appearance of such in the fresh zona), or of the extension into it of protoplasmic processes from the adjacent cells of the discus ‘proligerus, such as Caldwell figures in the case of the ovum of Phascolarctus (cf. his PI. 29, fig. 5). Within the zona the peripheral cytoplasm of the ovum is differentiated to form an exceedingly thin but distinct bounding layer or egg-membrane (vitelline membrane, sensu stricto).


The cytoplasmic body of the ovum exhibits a very obvious and striking differentiation into two regions in correspondence with the presence in it of two definitely localised varieties of deutoplasmic material, respectively granular and fluid. Peripherally it consists of a relatively narrow cytoplasmic zone of practically uniform width, dense and finely granular in appearance owing to the presence in it of numei'ous particles of deutoplasmic nature. This we may distinguish as the foi'mative zone (fig. 1, f.z.). In it lies embedded the large vesicular nucleus (about ‘06 x ‘03 mm. in diam.). Centrally and forming the main bulk of the ovum is a mass of greatly vacuolated cytoplasm presenting the appearance of a clear wide-meshed reticulum. Its framework is coarser peripherally where it passes over without definite limit into the formative zone, with which it is structurallv identical, but much finer and wider-meshed centrally, so fine, indeed, that it almost invariably breaks down under the action of fixatives, and appears in sections as an irregular space, perhaps crossed by a few fine interlacing strands (fig. 1, d.z.). The meshes of this reticulum are occupied by a clear fluid which must be held to constitute the central deutoplasm of the egg. We may accordingly designate this central reticular area as the deutoplasmic zone.


If we pass now from the full-grown to the ripe ovarian ovum (PI. 1, figs. 2 and 3), i. e. an ovum in which either thefirst polar spindle has appeared or the first polar body hasalready been separated off, it at once becomes evident that important changes have occurred in the disposition and relative proportions of the two constituent regions of the eggcytoplasm. The full-grown ovum is of the centrolecithal type, the central deutoplasmic zone forming its main bulk and being completely surrounded by the thin formative zone. The ripe ovum, on the other hand, exhibits an obvious and unmistakable polarity, and is of the telolecithal type, as the following facts show. The cytoplasmic body evidently consists of the same two regions as form that of the full-gi-own ovum, but here the dense formative region now forms its main bulk, and no longer surrounds the clear deutoplasmic region as a uniform periphei*al layer. It has not only increased considerably in amount as compared with that of the full-grown egg, and at the expense apparently of the moreperipheral coarser portion of the deutoplasmic zone, but it has undergone polar segregation, with the result that it now occupies rather more than one hemisphere of the egg as a dense finely granular mass, with vacuoles of varying sizesparsely scattered through it (figs. 2 and 3, /.z.). It accordingly defines one of the ovular poles. The opposite pole is just as markedly chai'acterised by the presence immediately below it of a more or less rounded clear mass, eccentrically situated, and composed of an extremely finecytoplasmic reticulum with wide fluid-filled meshes. It is completely surrounded by formative cytoplasm (though over the polar region the enclosing layer is so extremely thin that it here almost reaches the sui'face), and its cytoplasmic framework is perfectly continuous with the same, the line of junction of the two being abrupt and well defined. So delicate, however, is this framewoi'k that it breaks down more or less completely under the action of fixatives of such excellence even as the fluids of Flemming and Hermann, and thus in sections usually all that represent it ai'e a few irregular cytoplasmic strands crossing a large, sharply defined clear space (figs. 2 and 3, d.z.). The mass in question has thus all the characters of the deutoplasmic zone of the fullgrown ovum, and it must undoubtedly be held to represent the central portion of that which has not been utilised in the upbuilding of the formative cytoplasm, and which has been forced to take up an excentric position immediately below the polar region of one hemisphere, owing to the increase of the formative cytoplasm and its segregation in the other hemisphere.


The ripe ovum of Dasyurus thus possesses a polarity which in its way is equally as striking as that of the Monotreme egg. Towards the one pole the main mass of the ovum is composed of dense, slightly vacuolated formative cytoplasm, in which the polar spindle is situated peripherally, but nearer the equator than the formative pole. Toward the opposite pole and practically reaching the surface is a rounded mass of greatly vacuolated deutoplasmic cytoplasm. Eoughly, the formative cytoplasm constitutes about two-thirds of the bulk of the ripe egg, the deutoplasmic the remaining third. Such being the structure of the ripe ovarian egg, if we classify it at all, we must place it, it seems to me, with eggs of the telolecithal type. My view of the significance of this marked polar differentiation of the constituent materials of the ripe ovum of Dasyurus I shall presently indicate. Meantime I would lay special emphasis on the fact that the eccentric mass of deutoplasmic cytoplasm represents matei'ial, surplus deutoplasmic material which has not been utilised in the upbuilding of the formative cytoplasm.


The fact of the occurrence in the Eutherian ovum of a polar differentiation of its constituent materials is now definitely established, thanks especially to the valuable researches of Prof. 0. Van der Stricht and his pupils - H. Lams and the late J. Doorme. In this connection I wish to refer here in some detail to the extremely interesting observations of Van der Stricht ['03, '05] on the structure and polarity of tlie ovum of the bat (Vesperugo noctula), since these observations are in essential agreement with my own on the ovum of Dasyurus, and enable me to affirm that the polar differentiation herein recorded for the first time for the Marsupial ovum is attained as the result of vitellogenetic processes, which essentially correspond with those of the ovum of the bat. Van der Stricht, as is well known, has made a special study of the process of vitellogenesis in the Eutherian ovum, and is, indeed, at the present time the foremost authority on this particular subject, so that his views are worthy of all respect.


Study of the oocyte of Vesperugo during the period of growth shows, according to Van der Stricht, that “ a un moment donne du developpement du jeune oeuf, les boyaux et amas vitellogenes [derived, according to him, from ‘ une couche vitellogene, mitochoudriale,' present in the young oocyte in the first stage of growth] disparaissent au profit du vitellus, dont la structure pseudo - alveolaire s'accentue graduellement.” The full-grown oocyte at the stage just prior to the appearance of the first polar spindle is characterised by the presence of this “ pseudo-alveolar structui'e ” throughout the extent of its cytoplasmic body. The alveoli or vacuoles are of variable size, are filled by a clear liquid, and “ correspondent incontestablement au deutoplasma de I'oeuf. A ce stade du developpement de I'oocyte, ce vitellus nutritif, auquel s'ajoutent bientot des granulations graisseuses, est repandu uniformement dans toutes les profondeurs du cytoplasme. Nulle part on ne constate une zone deutoplasmique distincte d'une zone de vitellus plastique.” In Dasyurus the stage in vitellogenesis which almost exactly coiTesponds with that of the full-grown oocyte of Vesperugo just described is seen in oocytes not quite full-gi*own. In fig. 4 is shown an oocyte of Dasyurus (•26 x ‘20 inm. in diameter), in which the same pseudo-alveolar sti'ucture as described by Van der Stricht for the Vesperugo oocyte is perfectly distinct. Here, however, fatty particles are not apparent, and the peripheral portion of the cytoplasm tends to be free from vacuoles. In Dasyurus the formation of these deutoplasmic vacuoles begins in oocytes about '2 mm. or less in diameter. This characteristic pseudo-alveolar ” stage is followed in both Vesperugo and Dasyurus by one in which there is recognisable in the cytoplasmic body of the ovum a differentiation into a dense peripheral zone and a central vacuolated area. In Vesperugo this stage is attained about the time of appearance of the first polar spindle, whilst in Dasyurus it is attained somewhat earlier, always prior to the formation of the latter. So close is the agreement between the two forms that Van der Stricht's desci'iption of the bat's egg at the time of appearance of the first polar spindle might equally well be applied to the full-grown ovum of Dasyurus. He writes ['03, p. 43] : “Vers I'epoque de I'apparition du premier fuseau de maturation, le vitellus prend un autre aspect. La partie centrale deutoplasmique conserve une structure pseudo-alveolaire, mais dans le voisinage immediat du premier fuseau et daus toute I'etendue de la couche peripherique du protoplasme, apparait une mince zone de vitellus compact et dense, plus ou moins homogene ou les vesicules claii'es font defaut. . . . A ce moment, on


distingue daus I'odcyte de V. noctula une zone centi'ale tres etendue, riche en deutoplasme et une zone corticale tres mince, riche en vitellus plastique.” This centrolecithal phase, as we may term it, is followed in Vesperugo during fertilisation and the separation of the second polar body by a telolecithal phase characterised by a distinct polarity. “ La zone de vitellus plastique s'epaissit encore, mais surtout a un pole de I'oeuf, a celui oppose au pole ou se detachent les deux globules polaires. Ce pole, ou s'accumule graduellement le vitellus formateur, merite le nom de pole animal. II est oppose au pole d'expulsion des globules polaires, vers lequel est refoule le deutoplasme, et qui se comporte desormais comme le pole vegetatif. Pendant que les deux pronucleus male et femelle se ferment, le vitellus plastique augmente graduellement en abundance au pole animal, tandis qu'il diminue an p61e vegefcafcif, et le deutoplasme, parseme d'un plus grand nombre de boules graiss6uses, constitue une masse spberique excenfcriqne, voisine des deux globules polaires” (Van der Stricbfc, '03, pp. 44 - 45). It is evident, then, that the fertilised ovum of Yesperugo exhibits a polarity comparable with that of the ripe ovarian ovum of Dasyurus, and that the vitellogenetic processes in the ova of these two widely separated forms proceed along lines almost identical, at all events so far as their broad outlines are concerned. In both we find during growth a progressive vacuolisation of the egg-cytoplasm consequent on tlie elaboration of a deutoplasmic fiuid. In both, the '^pseudo-alveolar ” condition so engendei-ed is followed by one in which there is recognisable a differentiation into a peripheral "formative ” zone rich in deutoplasmic granules, and a central " deutoplasmic ” zone rich in fluid yolk, and finally in both there occurs a segregation of the granular "formative” and fiuid yolk-constituents to opposite I'egions of the egg, with resulting attainment of a definite polarity. In view of the close general agreement in the vitellogenetic processes, and in the constitution of the ova in Vesperugo and Dasyurus, it might be expected that the poles would accurately correspond, but such is not the case if Van der Stricht's determination of the poles in the ovum of Vesperugo is correct. In the latter, according to Van der Stricht, the deutoplasm is located at that pole from which the polar bodies are given off; at the opposite pole the "plastic” vitellus accumulates, and close to it the two pronuclei unite and the first cleavage spindle is formed. Accordingly Van der Stricht concludes that "le premier pole correspond au pole vegetatif, le second au p61e animal des oeufs a deutoplasme polaire (0. Hertwig).” In Dasyurus, on the other hand, I am perfectly convinced (and adequate reason for my conviction will be forthcoming in the course of my description of the processes of cleavage and germ-layer formation) that the pole of the ripe ovum in relation to the mass of deutoplasmic cytoplasm is not the vegetative pole, but represents morphologically the upper or animal pole of the egg, the opposite pole in relation to which the formative cytoplasm is situated being' the lower or vegetative. The deutoplasmic cytoplasm thus lies in the upper hemisphere, whilst the formative cytoplasm occupies the lower. If Van der Stricht's determination of the poles of the ovum of Vesperugo be accepted, then we must conclude that the poles of the Dasyurus ovum are exactly reversed as compared with those of the bat^s egg. In this connection it may be recalled that Lams and Doorme ['07] have demonstrated the occurrence in Cavia of an actual reversal of the original polarity of the ovum, pi'ior to the beginning of cleavage. These facts may well give us pause before we proceed to attach other than a purely secondary significance to the exact location of the formative and deutoplasmic constituents in the Metatherian and Eutherian ovum. But besides this apparent difference in the location of the deutoplasmic constituents of the ova of Dasyurus and Vesperugo, there exists yet another which concerns the fate of these constituents in the respective eggs. In Vesperugo, Van der Stricht shows that the “ deutoplasm ” remains an integral part of the egg, and I'etains its polar distribution in the blastomeres up to at least the 4-celled stage. ^ In Dasyurus, on the other hand, the fate of the deutoplasmic mass is a very different, and, indeed, a very remarkable one. It does not remain an integral part of the segmenting egg as in Vesperugo, but prior to the completion of the first cleavage furrow it becomes bodily separated off, apparently by a process of abstriction, from the formative cytoplasm as a clear rounded mass which takes no further direct part in the developmental processes. As soon as its elimination is effected, the remainder of the cytoplasmic body of the ovum, formed of the formative cytoplasm alone, divides into the first two equal-sized blastomeres, the first cleavage plaue being coincident with the polar diameter and at right angles to the plane of separation of the deutoplasmic mass, or “yolk-body ” as we may term it (PI. 2, figs. 14-16, 19, ij.b.), so that it is this formative zone of the ovum which is filone concerned in tlie production of the embryo and its foetal membranes.


Vide, however, “Addendum” (p. 121).

We have but to recall the conclusion already reached that the clear vacuolated zone at the upper pole of the ripe ovum of Dasyurus consists of surplus material, mainly in the form of fluid of deutoplasmic nature which has not been utilised in the upbuilding of the formative cytoplasm, and the signiflcance of this remarkable and, so far as the Mammalian ovum is concerned, absolutely unique occurrence becomes at once manifest.'^ We have to do here with an actual elimination of surplus deutoplasmic material by the Marsupial ovum - a phenomenon only paralleled elsewhere, so far a,s I am awai'e, and even then but distantly, by the curious temporary separation of the so-called yolk-lobe which occurs during the cleavage of the yolk-laden eggs of certain Molluscs (Nassa, Ilyanassa, Modiolaria, Aplysia, Dentalium) and Annelids (Myzostoma, Chjetopterus) . In these forms cleavage of the ovum into the first two blastomeres is accompanied by the sepai'ation of a portion of the ovular substance in the form of a non-nucleated mass or so-called yolk-lobe. This latter, which has been shown to be connected Avith the formation of determined organanlagen, reunites Avith one of the tAvo blastomeres, and then the same process of abstriction and reunion recurs at the second cleavage.^ We have here evidently a purely adaptive phenomenon, the object of which no doubt is to permit of the total cleavage of the yolk-laden ovum on Avhat are presumably the old ancestral lines, and I believe a comparable explanation Avill be found applicable to the elimination of surplus yolkmaterial by the Marsupial ovum.

As regards the significance of the occurrence of the deutoplasmic zone in the ovum of Dasyurus, holding the views that I do as to the phylogeny of the Marsupialia (viz. that the Metatheria and Eutheria are the divergent branches of a common stock, itself of Prototlierian derivation), and bearing in mind the occurrence of an undoubted repi'esentative of the shell round the Marsupial ovum, I venture to see in the fluidmaterial of the deutoplasmic zone the partial and vestigial equivalent of the yolk-mass of the monotreme egg. In other words, I would regard the deutoplasmic fluid as the product of an abortive attempt at the formation of such a solid yolkmass. The objection will no doubt be forthcoming that this interpretation cannot possibly be correct since the supposed equivalent of the yolk-mass in the Dasyure ovum is located, on my own showing, at the wrong pole - at the upper instead of at the lower. But its precise location does not seem to me to be a matter to which we need attach any great importance, since it has doubtless been adaptively determined in correlation with the special character of the cleavage process.


Yide “Addendum” (p. 121), in Avliich reference is made to the discovery by Prof. Van der Stricht of the elimination of deutoplasm in the ovum of Yesperugo. ® Of. Korschelt u. Heider, ‘Lehrbuch d. vergl. Entwicklungsgeschichte,' Lief. 3, p. 107, 1909.


The belief that the minute yolk-poor ovum of the Eutheria is no pure primarily holoblastic one, but that it has only secondarily arrived at the total type of cleavage as the result of the all but complete loss of the yolk ancestrally present in it, consequent on the substitution of the intra-uterine mode of development for the old oviparous habit, is now widely held amongst Mammalian embryologists. Hubi'echt, however, is an exception, wedded as he is to a belief in the direct derivation of the Eutheria from Protetrapodous ancestors with yolkpoor, holoblastic eggs. Whether the interpretation I have put forward, viz. that the non-formative or deutoplasmic zone of the Dasyure ovum is the reduced and partial equivalent of the yolk-mass of the Monotreme egg, be accepted or not, I venture to think that my discovery of an actual elimination of deutoplasmic material by the Marsupial ovum affords a striking confirmation of the truth of the prevailing conception as to the phylogeny of the Eutherian ovum, and I further venture to think that the facts I have brought forward in the preceding ])ages justify us in' regarding the ripe ovarian ovum of Dasyurus as being potentially of the yolk-laden, telolecithal type, and the uterine ovum, by bodily casting out the superfluous part of its deutoplasm, as becoming at the same time secondarily lioniolecithal and secondarily lioloblastic. Ihe Marsupial ovum presents itself to my mind as tlie victim of tendencies conditioned by its ancestry, and in particular it appears as if its inherited tendency to elaborate yolk had not yet been brought into accurate correlation with the other changes (reduction in size, intra-uterine development), which it has undergone in the course of phylogeny. As the consequence it manufactures more yolk than it can utilise, and so finds itself under the necessity of getting rid of the surplus. Whether or not a comparable elimination of deutoplasmic material occurs in the ova of other Marsupials, future investigation must decide. I should be quite prepared to find variation in this regard, correlated perhaps with the size of the egg. In the case of Phascolarctus, Caldwell gives the diameter of the ovum as '17 mm., and his figure of a (horizontal ?) section of the uterine ovum (here produced as text-fig. 1, p. 27) shows a differentiation of the cytoplasmic body of that into vacuolated and granular zones quite comparable with that of the Dasyure ovum. From the few measurements of ova of other marsupials that I have been able to make, it would appear that the ovum of Trichosurus approximates in size to that of Dasyurus, whilst that of Perameles and pi-obably also that of Macropus are smaller. From Selenka's figure I have calculated that the ovum of Didelphys measures about 'IS x •12 mm. in diameter. In the smaller ova it is quite likely that yolk-formation may not proceed so far as in the relatively large ovum of Dasyurus.

2. Maturation and Ovulation

The details of the maturation process have not been fully worked out, owing to lack of material. As in the Eutheria (Sobotta, Van der Stricht, Lams and Doorme, and others), the first polar body is separated off in the ovary, the second apparently in the upper part of the- Fallopian tube where entrance of the sperm takes place. The first polar figure (late anaphase observed, fig. 5) lies in the formative cytoplasm, close below and afc right angles to the zona. Its exact site is subject to some slight variation, and is best described as adjacent to the equatorial region of the egg, sometimes nearer the lower pole, more usually, perhaps, nearer the upper. Centrosomes and polar radiations were not observed. The heterotypical chromosomes (gemini) have the form of somewhat irregular, more or less angular granules. I have not been able to determine their number. The figure is barrel-shaped, and almost as broad as long, measuring

015 X ‘OIS mm. The first polar body (fig. 6, is small relatively to the size of the egg, its diameters varying round •03 X '01 mm., and its shape is that of a flattened bi-convex disc. In uterine eggs thex'e is some evidence pointing to the probability of its having undergone division.

The second polar spindle (figs. 3 and 7) lies immediately subjacent to the first polar body in the fully ripe ovarian ovum. It is shorter than the first, measuring '013 mm., and xnuch narrower. The second polar body measures about '015 X '01 mm. in diameter, and is thus smaller than the first. I have only seen the second polar body in uterine ova, and therefore can only presume that it is sepai'ated off in the upper part of the Fallopian tube, subsequently to the penetration of the sperm, as in Eutheria.

Ovulation takes place irrespective of whether copulation has occurred or not, and it is a fact Avorbhy of record that, even if the ova be not fertilised, the pouch and mammary glands undergo the same series of growth changes as are characteristic of, at all events, the earlier stages of normal pregnancy.

The follicular cells of the discus proligerus investing the ovum are already in the ripe follicle in a state of disruption, and I believe they separate completely from the ovum at the moment of dehiscence, so that, except for the zona, the ova are quite naked Avhen they enter the tube. I have no evidence of the existence outside the zona of a layer of proalbumen such as Caldwell describes round the ovum of Phascolarctus. Apparently the ova are shed almost simultaneously, and they must pass with considerable rapidity down the tubes to the uteri wliere cleavage begins, for I have only once found a tubal ovum, and that one had evidently been retarded for some reason, and was polyspermic.


3. The Secondary Egg-membranes: Albumen and Shell membrane

During the passage of the ovum down the tube it is fertilised, and becomes enclosed externally to the zona by two secondary layers formed as secretions by the cells of the oviducal lining. First of all, the ovum becomes surrounded by a transparent to semi-transparent laminated layer of albumen, •015 to ■02'2 mm. in thickness, composed of numerous very delicate concentric lamellae, and having, normally, numbers of sperms imbedded in it (figs. 8-11, alb., sp.). Then outside the albumen layer there is laid down a definite, but at first very thin, double-contoured membrane (figs. 8 and 10, s.m.), which, following Caldwell, I have no hesitation in homologising with the shell-membrane of the Monotreme egg. Caldwell in 1887 described and figured a definite membrane enclosing the uterine ovum of Phascolarctus, externally to, and quite distinct from the albumen, which he interpreted as the representative of the shell-membrane of the Monotremata, but owing apparently to the fact that Selenka altogether failed to recognise its true nature in Didelphys, since he regarded it as a derivative of the follicular epithelium, and termed it the “ granulosa-membran,” this highly significant discovery of Caldwell has been largely ignox'ed. Such a membrane is constantly present and easily recognisable in all the Marsupials (Dasyurus, Perameles, Trichosurus, Macropus, Petrogale, Phascologale, Acrobates, Phascolarctus, Bettongia), of which I have had the opportunity of studying early developmental stages. It is laid down in the Fallopian tube, is perfectly distinct from the albumen, and increases in thickness in the uterus, and if it has not the significance which Caldwell has suggested, then I must leave it to those who decline to accept Caldwell's interpretation to put forward an alternative one, since I am unable to do so.

The shell-membrane of Dasyurus (PI. 1, figs. 8-11; PI. 2, figs. 17, 18, s.m.) is a ti*ansparent, perfectly homogeneous layer, highly refractive in character and of a faint yellowish tint. When fully formed it possesses firm, resistant properties, recalling those of chitin, and is doubtless composed of a keratin base. It is distinguishable at once from the albumen by its optical characters and staining reactions, so that there is not the slightest justification for the supposition that it may represent simply the specially differentiated outermost portion of that layer. In ova which have just passed into the uterus (fig. 10) the shell-membrane is extremely delicate, its thickness being only about '0016 mm., but even before cleavage begins it has increased to ”002 mm. (fig. 12) ; in the 2-celled stage (fig. 18) it has reached ‘005 mm., in the 4-celled stage (fig. 22) '0072 mm., whilst in the 16-celled stage (figs. 24-26) it has practically attained its maximum thickness, viz., ‘0075'008 mm. Caldwell's measurements in the case of Phascolarctus agree closely with the above (shell of unsegmented ovum from the uterus, '0015 mm. thick, that of the '3 mm. ‘‘ovum,” '01 mm.). Its presence renders the thorough penetration of ova and early blastocysts with pai'affin a capricious and frequently troublesome operation, and its resistant shell-like nature becomes only too obvious in the process of section-cutting, since it cracks with the utmost readiness (cf. PI. 3, figs. 32, 37).


The occurrence of a shell-membrane round the Marsupial ovum is a feature of considerable phyletic significance, as I need hardly point out. It shows us that the ancestors of the Metatheria must have been oviparous, or must themselves have come from an oviparous stock, which there is no valid reason for supposing was other than Prototherian in its characters. It also renders untenable the views of Hubrecht to the effect that the Metatheria are the descendants of Eutheria, whilst the Eutheria themselves have been directly derived from some presumed viviparous group of hypothetical Protetrapods, unless we are to suppose that the Metatheria are even novv on the way to acquire secondarily the oviparous habit, much in the same way as the Mouotremes, according to Hubi-echt, have long since succeeded in doing.


The occurrence of a shell-membrane round the Marsupial ovum has also an important ontogenetic significance in relation to the mode of formation of the blastocyst, as I shall endeavour presently to show.


4. The Uterine Ovum

The unsegmented ovum from the uterus (figs. 8-13) consists of the following parts :

  1. The shell-membrane externally, 'OOlfi - '002 mm. in thickness.
  2. The laminated layer of albumen, '015 - "022 mm. or more in thickness.
  3. The zona, about -0016 mm. in thickness.
  4. The perivitelline space, between the zona and the ovum, occupied by a clear fluid which coagulates under the action of certain fixatives, e. g. Hermann's fluid (fig. ll,p.s.), and which has diffused in from the uterus. The minute polar bodies lie in this space, usually nearer the upper pole than the lower.
  5. The ovum proper.


The entire egg is spherical in form, and varies in diameter in the fresh state from about '3 mm. to '36 mm. (average about ‘32 mm.).


The ovum itself is ovoidal, its polar diameter always slightly exceeding the equatorial. Its average diametrical measurements in the fresh state run about '25 x '24 mm., though I have records of ova measuring as much as ‘3 x ‘29 mm., and I find that there is an undoubted slight variation iu the size of the ova of even one and the same batch, as well as iu those from different females.


The uterine ovum exhibits the same marked polarity as cliaracterises the ripe ovarian ovum (the upper pole being marked by the vacuolated deutoplasmic zone (figs. 8-11, d.z.), and so far as its cytoplasmic body is concerned it shows no essential difference from that.


Examined fresh in normal salt solution, the formative cytoplasm forming the bulk of the ovum appears dense, finely granular, and of a very faint lightish-brown tint, its opacity being such that the two pronuclei situated in its central region ' can just be made out. In section, this central region is distinguishable from the peripheral zone by its uniform, more finely granular character and by the absence of the fluid-filled vacuolar spaces which are generally present in the latter figs. 10 and 12). The deutoplasmic zone at the upper pole, which is only partially visible in the entire egg owing to the way in which it is enclosed by the formative cytoplasm (figs. 8, 9, d.z.), presents a characteristically clear or semi-transparent vacuolated appearance in the fresh state, but may have einbedded in it a small dense mass (fig. 8, cf. also figs. 11 and 14), evidently formed by the transformation of a portion of its fluid constitutent into the solid state, and so to be regai'ded as compai'able with a bit of formative cytoplasm.


In most of the unsegmented uterine ova at my disposal the male and female pronuclei have attained approximately tlie same size and lie in proximity in the central more homogeneous region of the formative cytoplasm (figs. 10-12). The transformation of the sperm-head into the male pronucleus probably takes place during the passage of the ovum down the tube, and was not observed, and I am as yet uncertain whether the pronuclei unite to form a single cleavage nucleus or give origin directly to the chromosomes of the first cleavage figure.


Caldwell figures ('87, PI. 30, fig. 5) a section through the uterine ovum of Phascolarctus which I reproduce here as Text-fig. l,in order to facilitate comparison with my figs. 11 and 12, with which it shows an essential agreement, apart from the pi'esence of follicular cells in the albumen which I have never observed in Dasyurus, and making allowance for the difference in sectional plane. The figure is stated to represent “the seventeenth section of a vertical longitudinal seines of thirty-five sections through the segmenting ovum, containing two nuclei, taken from the uterus and measuring T7 mm. in diameter.” Caldwell has, I think, fallen into several errors in his interpretation of the structural features seen in this figure. In the first place, the sectional plane appears to me not to be vertical as in my own figs. 11 and 12, but horizontal, and to have passed through the lower portion of the deutoplasmic zone, shown in the figure as a central markedly vacuolated area. Then there is no evidence to be derived from the figure in support of the description of the ovum as segmenting. The part inside the zona {vm.) labelled and described as protoplasm with finest jolk-granules,” I would interpret simply as coagulum in the perivitelline space, whilst the so-called “ segmentation nuclei ” Ua) situated in it are probably the polar bodies or their derivatives. The part labelled y«, and designated “ white yolk,” I would regard as the ovum itself. It exhibits an obvious differentiation into a central vacuolated area and a peripheral, dense, granular zone with scattered vacuoles, and I thiuk there can be little doubt but that the former corresponds to the deutoplasmic zone of the Dasyure ovum, the latter to the formative zone. It is these errors of intei-pretatiou apparently which misled Caldwell into making the statement, now widely quoted in the text-books, that cleavage in Phascolarctus is of the meroblastic type.



Text-fig. 1. Section of uterine ovnni of Phascolarctus cinereus. (After Caldwell.)



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Contents: 1 Review of Previous Observations | 2 The Ovum of Dasyurus | 3 Cleavage and Blastocyst | 4 Blastocyst Growth Ectoderm Entoderm | 5 Early Stages of Perameles and Macropus | 6 Summary and Conclusions | 7 Early Mammalia Ontogeny | Explanation of Plates


Cite this page: Hill, M.A. (2020, July 7) Embryology Paper - Contributions to the embryology of the marsupialia 4-2. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_Contributions_to_the_embryology_of_the_marsupialia_4-2

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