Proc, Indian Acad. ScL, Vol. 87 B (Animal Sciences-2), No.6, June 1978, pp. 91-104, © printed in India

Early development, implantation and amniogenesis in the Indian vampire bat, Megaderma lyra lyra (Geoffroy)

A GOPALAKRISHNA and M S KHAPARDE* Department of Zoology, Institute of Science, Nagpur 440 001 ·Present address: NCERT, New Delhi

MS received 22 December 1977

Abstract. The embryo of Megaderma lyra lyra enters the uterus as a morula. The blastocyst establishes contact with the uterine wall circumferentially at the level of implantation. The orientation of the embryonic mass at early stages is variable, but the flat embryonic disc at later stages is invariably mesometrially oriented. The pre­ implantation response of the uterus is markedly evident in the enormous expansion of the uterine glands and the hypertrophy of their cells. After the attachment of the blastocyst there is a rapid degeneration of the uterine glands and the cells of the endo­ metrial stroma resulting in the formation of a thick zone of symplasma into which roots of the trophoblast enter. The maternal blood capillaries increase in number and their endothelial cells undergo hypertrophy. The amniotic cavity is formed by cavitation, but with the expansion of the embryonic disc the original roof of the am­ nion undergoes rupture and loss. The definitive amnion is formed by the develop­ ment of folds from the margin of the embryonic disc.

Keywords. Early development; implantation and amniogenesis; Megaderma lyra lyra.

1. Introduction

Among the seventeen extant families of Chiroptera incorporating over 150 genera the details of early embryology are known with respect to a few species of Vesperti­ lionidae (Van Beneden 1880, 1888a, b; Van Beneden and Julin 1880a,b; Duval 1894, 1895, 1896; Ramaswarni 1933; Wimsatt 1944; Gopalakrishna 1949; Potts and Racey 1971; Phansalkar 1972; Sapkal 1973), and a few stages of early development have been described in one or two species among Pteropidae (Moghe 1951, 1956),Rhinopo­ matidae (Srivastava 1952; Gopalakrishna 1958), Emballonuridae (Gopalakrishna 1958), Rhinolophidae (Bhiwgade 1976), Hipposideridae (Gopalakrishna 1958), Phyllostomatidae (Rasweiler IV 1972; Bonilla and Rasweiler IV 1974), Desmodonti­ dae (Wimsatt 1954) and Molossidae (Sansom 1932; Hamlett 1934; Stephens 1962). Since there is no information on the early embryology ofany member of the family Megadermatidae it was felt that a detailed study of the embryology of Megaderma lyra lyra would be of considerable interest and value. The present report embodies observations on the early stages of development of this species. The development of the foetal membranes and placentation in this species will be reported later.

91 92 .A Gopa/akrishna and M S Khaparde

2. Material and methods

The specimens of Megaderma lyra lyra were collected at Aurangabad, Maharashtra State at frequent intervals during the months from November to April. All the adult females in the colony conceive in the latter half of November and deliver a young each during the second half of the following April. Although the uterus is bicornuate and morphologically symmetrical, only the left cornu carries the pregnancy during each cycle except in rare cases where the right cornu also carries an embryo (Ramaswami and Anand Kumar 1963; Gopalakrishna et al 1977).· The specimens were killed by chloroform and their genitalia were fixed in one of the following fixatives-neutral formalin, Bouin's, Carney's and Rossman's fixative. In cases of very advanced pregnancy the uteri were slit open for the proper penetration of the fixative. The tissues were dehydrated by passing through graded ethanol, cleared in xylol, embedded in paraffin and sectioned at 5 to 8iL thickness. For routine histologi­ cal examination the sections were stained with Ehrlich's haematoxylin and counter­ stained with eosin. A few selected sections from each series were stained by the PAS procedure (Pearse 1968) with or without prior salivary digestion, and some sections were also stained by Heidenhain's azan procedure.

3. Observations

3.1. The female genitalia

The ovary is ellipsoidal in shape and is completely enclosed by an ovarian capsule. The fallopian tube arises from the median side of the ovarian capsule, and, after taking a circuitous course round the cranial aspect of the ovarian capsule, bends caudally on the lateral side of the capsule and opens into the cranial end of the res­ pective uterine cornu. The two uterine cornua are symmetrically placed and form the lateral limbs of a 'Y '-shaped structure with the vagina forming the median limb of the' Y ', The following descriptions refer to the histological structure of the prospective functional uterine cornu (the left one) at estrus. In transverse sections (figure I) the uterus has a slightly oblong outline with the dorso-ventral axis being slightly longer than the lateral axis. The two layers of the myometrium imperceptibly merge into each other since numerous muscle fibres run criss-cross between the two layers. Numerous simple tubular uterine glands, some of which have coiled distal ends, are present uniformly on all the sides of the uterus and extend deep into the endometrium. The epithelium of the glands consists of cuboidal cells each with a centrally located spherical nucleus. The endometrial stroma consists of spindle­ shaped cells, connective tissue fibres and blood capillaries. The uterine epithelium is made up of tall columnar cells with basally situated darkly staining nuclei. The uterine lumen has an irregular shape due to the presence of a few endometrial ridges projecting into it.

3.2. Preimplantation stages ofdevelopment

An unfertilized ovum (figure 2) was noticed near the ovarian end of the fallopian tube of a specimen collected on 24 November 1964. The slight distortion of the Early stages ofdevelopment ofMegaderma lyra lyra 93

shape of the ovum is an artifact caused during the preparation of the sections. It measures 85 fL across its largest diameter and is surrounded by a distinct zona pellu­ cida. The cytoplasm of the ovum is granular and the nucleus is vesicular and eccen­ tric. The ovum is surrounded by numerous small cells of the cumulus layer of the Graafian follicle which had undergone ovulation. An embryo consisting of eight cells was found near about the middle of the length of the fallopian tube of a specimen collected on 24 November 1964. The eight cells are so arranged that four cells lie in one plane (figure 3) in the form of a cross, and two cells lie on either surface of the cross. Hence, all the cells are not visible in the same section. The zona pellucida is not seen. The nuclei of the blastomers are located in the centre of the cells and are darkly stained. The embryo enters the uterus in this species at the morula stage of development (figure 4) as in most bats studied so far. Altogether six morulae have been observed at nearly the same stage of development, and most of the embryos have become artifactually distorted in shape during the preparation of the stained sections. In none of the morulae was the zona pellucida seen. Some of the cells in each morula were in active division. Two free unilaminar blastocysts at slightly different stages of development were available for the present study. The younger of the two blastocysts (figure 5)measures 90fL in dia and its inner cell mass is composed of 26 irregularly shaped cells, some of which contain mitotic stages. The trophoblast layer is composed of nearly cubical cells each with a spherical centrally placed nucleus. A distinct zona pellucida surrounds the blastocyst. The second blastocyst (figure 6), which is slightly more advanced in development than the one described above, measures 96 fL in dia and the embryonic mass is composed of 38 cells. The cells of the trophoblast layer are dis­ tinctly cuboidal and each contains a large, darkly staining, centrally placed nucleus. The zona pellucida is not present in this blastocyst. The next stage of development of the embryo available for this report consists of seven free unilaminar blastocysts at approximately the same stage of development (figure 7), but more advanced than the two described above. In all the cases the blastocyst lies in about the middle of the length of the uterus. The trophoblast layer is made of cubical cells and the embryonic mass is spherical in shape and con­ sists of numerous polygonal cells each with a central nucleus. Whereas the number of cells has increased in the inner cell mass the individual cells are smaller than the cells of the embryonic mass of the earlier blastocysts. The wall of most of the blasto­ cysts appears to have partially collapsed artifactually.

3.3. Progestational changes in the uterus

Whereas the cranial half of the uterine cornu exhibits marked progestational res­ ponse, the caudal half does not show much change. Hence, the following descrip­ tions pertain to the changes occurring only in the cranial halfof the uterus. Further, in the cranial half of the uterus the progestational changes occur uniformly on all the sides, that is on the mesometrial, antimesometrial and the lateral sides of the uterus. Evidently, there does not seem to be any specific locus in the cranial half of the uterus which is preformed for the attachment of the blastocyst. During the preimplantation period of the development of the embryo there is a progressive increase in the thickness of the left uterine cornu, and in freshly dissected 94 .A Gopa/akrishna and M S Khaparde specimens the left cornu appears to be more richly vascularized than the right cornu. Histological examination reveals that the most obvious change seems to occur with respect to the uterine glands which become progressively wider and extend deeper into the endometrium (figures 8 and 9). The cells of the gland epithelium become progressively taller and their cytoplasm vacuolated. With the widening of the lumina ofthe glands the endometrial stromal tissue gets compressed into thin strands ofconnective tissue consisting of scattered fusiform cells, numerous fluid-filled spaces and blood capillaries. There is also a progressive increase in the thickness and vascularity of the myometrium. The uterine lumen gets slightly enlarged and the uterine epithelium increases in height.

3.4. Development ofthe bilaminar blastocyst and implantation

Several bilaminar blastocysts of Megaderma lyra lyra at closely graded stages of development have been studied for the present report. The blastocyst expands enor­ mously after hatching out of the zona pellucida and undergoes implantation at the bilaminar stage of development. In all the cases the blastocyst occupies the entire uterine lumen at the level of implantation and comes into contact with the uterine wall on all its sides (figures 10-12). Since this species exhibits certain peculiarities so far not noticed in any bat, and since this is the first report on the early embryology ofa member of the family Megadermatidae, these stages are described in some detail. The blastocysts have been classified into five stages and one specimen representing each stage has been described.

3.4.1. Stage I

Seventeen early implanting blastocysts at nearly the same stage of development have been examined, and it was noticed that the orientation of the embryonic mass varies with respect to the morphology ofthe uterus. The embryonic mass is oriented mesometrially in five, laterally in eight, between the mesometrial and lateral sides in three and antimesometrially in one. This interesting feature was reported earlier in a short note (Gopalakrishna and Khaparde 1973). In all the blastocysts the embryonic mass is spherical (figures 10, 16, 18) and consists of closely arranged cells, some of which contain mitotic stages. The trophoblast (figures 16, 17) is composed of a single layer of cubical cells containing spherical centrally located nuclei on all the sides except where the trophoblast sends out short conical projections which either come into contact with the subepithelial endometrium or enter the wide lumina ofthe glands. In these regions the trophoblast is two to three cell thick. The endoderm layer is made up ofa layer of squamous cells (which appear spindle­ shaped in sectional views) lining the inner surface of the wall of the blastocyst. In some places the endodermal layer seems to have shrunk from its original position. This is evidently an artifact caused during the preparation of the stained sections. The uterine epithelium has been lost from most places and its place is occupied by the trophoblast layer (figures 10, 16, 17). The most obvious histological change is noticed in the uterine glands, which have become very wide (figure 10), and the cells of the gland epithelium have lost their cell walls in most places and form. a symplasma, and their nuclei lie scattered near the distal border of the symplasma (figures 16, 17). The epithelial lining of the glands can be easily recognized since it occurs Early stages ofdevelopment ofMegaderma lyra lyra 95

6

Figures 1-7. I. Transverse section of the uterus of Mcgaderma lyra lyra at estrus. Note the numerous uterine glands which arc present uniformly on all the sides. ".: 48. 2. Section of an unfertil ized ovum. Note the distinct zona pcllucida surrounding the ovum and man y small cells of the cumulus layer lying ncar the ovum. The nucleus is not included in the figure. x 240. 3. Section passing through the center of an embryo at the 8-celled stage of development. Only four cells are seen in the sec­ tion. : 240. 4. Section of an uterine morula. The distorted shape is art ifactual. 240. S..,Free.... uterine unilam inar blastocyst with a. distinct... . zona pellucida . 6. Free 96 A Gopalakrishna and M S Khaparde

Figures 8-11. 8 and 9. Sections of the uterus containing a free morula and a free uni­ laminar blastocyst respectively. Note the enlargement of the uterine glands resulting in the reduction of the stromal tissue into thin strands . x 48. 10. Early implanted blastocyst described under stage 1. See text for description. Arrow points towards the mesometr ium. x 48. 11. Implanted bilaminar blastocyst described under stage II. See text for description. Arrow points towards the mesometrium x 48. Early stages ofdevelopment ofMegaderma lyra lyra 97 as an irregular, lightly staining zone in contrast with the more darkly stained endo­ metrial stromal tissue.

3.4.2. Stage II (figure 11)

The embryonic mass is nearly biconvex and lies oriented towards the mesometrial side of the uterus. On the side towards the blastocyst cavity the cellsof the embryonic mass are more or less compactly arranged whereas in the central part and towards the trophoblast layer the cesll are loosely arranged and there are many small inter­ cellular species. The cells of the trophoblast are cuboidal and each has a large spherical nucleus and granular cytoplasm or a mitotic nucleus. The trophoblast is multilayered in places where it has invaded the uterine wall. The endodermal cells underlying the embryonic disc appear spindle-shaped whereas the rest of the endodermallayer, which has shrunk in some places from its original position underlying the trophoblast, is composed of squamous cells with large nuclei. The uterine epithelium has been lost from all the sides and its position is occupied by the basal trophoblastic layer. The proximal segments of most of the uterine glands have degenerated (figure 19) and their cells have become masses of cytoplasm containing freely floating, irregularly scattered, darkly staining fragments of nuclear material. These cytoplasmic masses nearly fil1 up the original lumen of the glands. The distal regions of the glands near the myometrial border, however, retain the identity of their cells and have wide lumina (figures 11, 19).

3.4.3. Stage III

Examination of serial sections indicates that the embryonic mass has expanded laterally to form a biconvex disc and has several small intercellular spaces-the forerunners of the amniotic cavity-ofwhich the one in the centre isprominent (figure 12). The embryonic disc is oriented towards the mesometrial side of the uterus. The trophoblast has penetrated the endometrium deeper than in the previous stage and has established a firm attachment to the uterus on al1 sides. The cell boundaries are not seen in the invading trophoblast towards the deeper portions of the endo­ metrium whereas the basal trophoblast consisting of cuboidal cells has distinct cell boundaries. The uterine changes noticed in the previous stage are further augmented, and the proximal segments of most of the glands have broken down to form large masses of cytoplasm containing irregularly scattered fragments of nuclei. The endometrial connective tissue between the glands also show signs of degeneration and occur as patches of necrotic tissue between the gland syrnplasma in many places. There is, however, a considerable increase in the number of blood capillaries.

3.4.4. Stage IV

The blastocyst cavity increases in size, and the embryonic disc is oriented towards the mesometrial side of the uterus. The amniotic cavity has expanded considerably resulting in the stretching of the roof of the amniotic cavity into a thin unilaminar membrane underlying the trophoblast layer (figure 13). The floor of the amniotic 98 A Gopalakrishna and M S Khaparde cavity is formed by the embryonic disc, which is composed of three or four layers of small, compactly arranged cells with darkly staining nuclei. There is no change in the trophoblast except that the syncytial trophoblast has penetrated deeper into the endometrium than in the previous stage and the zone of symplasma has increased in thickness. The maternal blood capillaries have distinctly hypertrophied nearly cubical endothelial cells (figure 13). The endoderm layer composed of a layer ofsquamous cells lies artifactually shrunk from its original position underlying the trophoblastic layer. In a blastocyst, which is slightly more advanced than the one described above, the amniotic cavity has expanded further stretching the roof into a thin layer which has ruptured in several places indicating the early stages of the loss ofthis layer.

3.4.5. Stage V

The embryonic mass occurs as a flat disc forming the base of a wide amniotic cavity (figure 14) containing some cell debris, which is presumably the remnants of the cells of the embryonic mass and the cells of the primitive amnion in the process of degene­ ration. The embryonic disc is oriented towards the mesometrial side. The original roof ofthe amniotic cavity has been lost from most places and folds of the embryonic disc-the definitive amniotic folds-have formed and have grown over the sides of the amniotic cavity. Examination of serial sections reveals that, while in some places the folds have grown completely and have fused on the roof of the amniotic cavity to form the ectodermal component of the definitive amnion, in certain other places the folds have not yet grown completely thereby leaving the amniotic cavity roofed over by the basal trophoblastic layer itself. The trophoblastic layer has proliferated and has entered into the endometrium in the form of numerous solid root-like projections, some of which have branched in the deeper regions. The disintegration of most of the uterine glands and the major part of the endometrial stroma results in the formation ofa thick zone of sym­ plasma in which the roots of the trophoblast are embedded. Numerous maternal blood capillaries with hypertrophied endothelial cells are present in the symplasma, and the capillaries at the foetal border of the symplasma are surrounded by tropho­ blast. Whereas the basal layer of trophoblast is distinctly cellular, the penetrating roots of the trophoblast soon lose their cell boundaries and become syncytial. Numerous remnants of the fundic regions of the uterine glands with wide lumina and distinct epithelial lining occur near the myometrial border of the endometrium. The endodermal layer, which is artifactually peeled away from its original position, is composed of nearly cubical cells and lies freely in the blastocyst cavity except in a few places where it maintains its contact with the basal cytotrophoblastic layer. At a little later stage of development (figure 15) the definitive amnion has formed completely and has been reinforced by the mesodermal layer which has differentiated and extended between the amniotic ectodermal layer and the basal trophoblast.

4. Discussion

4.1. Implantation

One of the unique features of the early embryology of Megaderma lyra lyra is the Early stages ofdevelopntcnt of Megaderma lyra lyra 99

Figures 12-15. 12. Advanced implanted bilaminar blastocyst described under stage Ill. Note the presence of small intercellular clefts in the embryonic mass. See text for description. ;-.: 80. 13. Advanced implanted bilaminar blastocyst described under stage IV. Note the thin primitive amn ion form ing the roof of the amniotic cavity. Maternal blood capillaries have hypertrophied endothelial cells (arrow). See text for description. x 240. 14. Late implanted bilaminar blastocyst. Note the large amniotic cavity containing some cell debris. Definitive amniotic fold (arrow head) has formed at the margin of the disc on one side. The trophoblast has entered the endometrium as solid roots (arrow). x 96. IS. Part of a late im­ planted trilaminar blastocyst to show the definitive amnion (arrow head) supported 100 A Gopalakrishna and M S Khaparde

Figures 16-19. 16. Part of 10 enlarged to show the spherical embryonic mass and the darkly staining trophoblastic layer which has replaced the uterine epithelium. x 360. 17. Part of 10 enlarged to show the early stage of the degeneration of the uterine glands (arrow) and the endometrial stromal tissue (Darkly stained) between the glands. x 360. 18. Implanted bilaminar blastocyst with spherical embryonic mass oriented antimesometriaJly. The lumina of the glands persist only i'n the distal parts of the uterine glands in the deeper regions of the endometrium. Arrow points towards the mesometrium. x 48. 19. Late implanted bilaminar blastocyst described under stage II. Note the hypertrophy of the epithelium of the uterine glands and the beginnings of the formation of the symplasma, x 60. Early stages ofdevelopment ofMegaderma lyra lyra 101 variable orientation of the embryonic mass with reference to the morphology of the uterus during early stages of implantation when the embryonic mass is still spherical. Equally interesting and unique is the fact that in the more advanced stages, when the embryonic mass becomes expanded into a disc, the embryonic disc is invariably oriented mesometrially. In most mammals, whose embryology has been studied the orientation of the embryonic mass of the blastocyst has a specific relationship to the morphology of the uterus. Mossman (1937) recognized this specificity and mentioned that, 'the orientation of the disc with reference to the uterus is usually quite constant in all groups known to be closely related.' He utilized this feature as one of the criteria for determining the inter-relationships of the different orders of mammals. However, in Chiroptera the available information reveals that there are considerable differences in the orientation of the embryonic disc among the different families so far studied. Among the Megachiroptera the disc is at first lateral and later becomes mesometrial in Pteropus giganteus giganteus (Moghe 1951), mesometrial in Cynopterus sphinx gangeticus (Moghe 1956) and directed cranially in the uterus and lies adjacent to the opening of the fallopian tube in Rousettus leschenaulti (Karim 1971). Among the Microchiroptera the orientation of the disc varies not only in different families but in different species within the family, and, in some cases, in different specimens of the same species. The embryonic mass is lateral during early stages, but the disc becomes mesometrial during later stages in Rhinopoma kinneari (Srivastava 1952; Gopalakrishna 1958). In Taphozous longimanus (Gopala­ krishna 1958) the embryonic mass is oriented mesometrially during early stages, but during the neural groove stage the disc is lateral. In Noctillio labialis minor (Ander­ son and Wimsatt 1963) the embryonic disc has been shown to be oriented laterally. In Rhinolophus rouxi (Bhiwgade 1976; Ramakrishna 1977) the embryonic mass has a variable orientation. In Hipposideros bicolor pal/idus (Gopalakrishna 1958) the embryonic disc is mesometrial, but in Hipposideros fulvus fulvus (Gopalakrishna and Karim 1973) the early embryonic disc has a variable orientation. In Desmodontidae (Wimsatt 1954) and Vespertilionidae (Wimsatt 1944; Gopalakrishna 1949) the embryonic disc is invariably antimesometrial. In Phyllostomidae the uterus is simplex and the disc is oriented towards the fundic side (Hamlett 1935; Rasweiler IV 1972; Bonilla and Rasweiler IV 1974). Among the molossid bats the disc is lateral in Molossus rufus and Molossus obscurus (Sansom 1932)while it has been described as being mesometrial in Tadarida brasiliensis cynocephala (Stephens 1962). With regard to the depth of implantation, completely interstitial implantation occurs in Desmodontidae (Wimsatt 1954) and Phyllostomatidae (Wislocki and Fawcett 1941; Hamlett 1934, 1935). Superficial and circumferential implantation has been noticed in Cynopterus marginatus (Keibel 1922), Cynopterus sphinx gangeticus (Moghe 1956) among Megachiroptera, and in Rhinopoma kinneari (Srivastava 1952; Gopalakrishna 1958), Taphozous longimanus (Gopalakrishna 1958), Rhinolophus rouxi (Bhiwgade 1976; Ramakrishna 1977), Hipposideros bicolor pal/idus (Gopala­ krishna 1958; Gopalakrishna and Moghe 1960), Hipposideros fulvus fulvus (Gopala­ krishna and Karim 1976) and Tadarida brasiliensis cynocephala (Stephens 1962). Evidently, in all these cases the expanding blastocyst establishes contact with the uterine wall on all the sides thereby obliterating the uterine lumen at the level of nidation. In all the vespertilionids (Wimsatt 1944; Gopalakrishna 1949; Phansalkar 1972; Sapkal 1973) implantation is superficial and antimesometrial so that the blastocyst lies freely hanging in the persistent uterine lumen on the mesometrial 102 A Gopalakrishna and M S Khaparde

side. Partially interstitial implantation occurs in Pteropus giganteus giganteus (Moghe 1951) among Megachiroptera and possibly in Noctilio labialis minor (Anderson and Wimsatt 1963)among the Microchiroptera. In Megaderma lyra lyra the blastocyst comes into contact with the uterine wall on all its sidesand thus obliterates the uterine lumen at the level of implantation. Hence, implantation in this species is superficial and circumferential.

4.2. Uterine response

The uterine changes preceeding and during the implantation of the blastocyst in Megaderma lyra lyra are unlike any bat so far described. The uterine glands on all the sides of the uterus expand enormously and cause the relative reduction of the endometrial stroma even before the blastocyst comes into contact with the uterine wall. The process of implantation is accompanied by an hypertrophy of the epi­ thelium of the glands followed by the break down of the cell walls thereby forming a sheet of cytoplasm in which the nuclei lie scattered. This is followed by the necrosis of the endometrial stromal tissue, which forms a part of the syrnplasma into which the roots of the trophoblast of the implanting blastocyst penetrate. Among the other bats, whose, embryology has been studied in detail, the uterine changes during early development are known in members of'Vespertilionidae (Wimsatt 1944;Gopala­ krishna 1949) in which a small area on the antimesometrial side of the uterus exhibits a prominent edema resulting in the shifting of the glands from this side towards the lateral sides. Evidently in Megaderma lyra lyra, where implantation is circumferen­ tial and where the early orientation of the embryonic mass is variable, all the sides of the uterus exhibit equally pronounced changes, and hence the glands undergo hypertrophy on all the sides.

4.3. Amniogenesis

Among the Microchiroptera three different methods of amnion formation have been described-(I) The amniotic cavity arises as inter-cellular clefts in the embryonic mass, and these clefts coalesce to form a large cavity-the amniotic cavity. The roof of the amniotic cavity is formed by the cells of the embryonic mass itself, and conti­ nues as the ectodermal component of the definitive amnion after it is reinforced by extra-embryonic mesoderm. In such a mode of amnion formation there is no distinc­ tion between the primitive amnion and the definitive amnion. This method of amnion formation has been reported to occur in Glossophaga soricina (Hamlett 1934, 1935), Scotophilus wroughtoni (Gopalakrishna 1949), Desmodus rotundus murinus (Wimsatt, 1954) and possibly in Noctilio labialis minor (Anderson and Wimsatt 1963). (2) The early amniotic cavity arises as in (I) above, and is at first roofed over by the cells of the embryonic mass itself. However, as the amniotic cavity expands the roof of the amniotic cavity is lost so that the amniotic cavity is roofed over by the basal layer of trophoblast of the blastocyst for some time. The definitive amnion is later formed by the up growth from the margin of the embryonic disc of folds which arch over the amniotic cavity. Such a method of amniogenesis has been reported to occur in Vespertilio murinus (Van Beneden 1888a), Miniopterus schreibersii (Da Costa 1920), Molossus rufus and Molossus obscurus '(Sansom 1932), Vesperugo leisleri (Ramaswami 1933) and Myotis lucifugus lucifugus (Wimsatt 1944). Early stages ofdevelopment of Megaderma lyra lyra 103

(3) The amnion of Tadarida brasiliensis cynocephala has been described as developing exclusively by the formation of folds (Mossman 1937; Stephens 1962). The present study reveals that the mode of formation of the amnion in Megaderma lyra lyra is similar to that described under type (2) above.

References

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P.(B)-2 104 A Gopalakrishna and M S Khaparde

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