Observations on Some Early Stages of Development and Implantation of the Blastocyst of Rhinolophus Rouxi (Temminck)*

Proe. Indian Aead. Sci., Vol. 84 B, No. 6, 1976, pp. 201-209

Observations on some early stages of development and implantation of the blastocyst of Rhinolophus rouxi (Temminck)*

D. A. BHIWGADE Department of Zoology, Institute of Science, Nagpur 440001 MS received 9 June 1976 ; revised 13 September 1976

ABSTRACT The embryo enters the uterus at the morula stage of development. The l;rogestational response of the uterus is characterized by the enlarge- ment of the uterine lumen near the centre of the right uterine cornu into a large spherical chamber in which the blastocyst undergoes nidation, and by a pronounced hypertrophy of the utL rine glands on all the sides of the implantation chamber. The implantation of the blastocyst is circumferential, and the embryonic disc is directed towards the side of the uterus which is between the mesometrial and the lateral sides. The primitive amniotic cavity is formed by cavitation and the definitive amnion is later formed by the formation of folds from the margin of the embryonic disc.

1. INTRODUCTION

ALTHOUGH Chiroptera is one of the largest orders of mammals irtcluding over a hundred extant genera of bats incorporated in seventeen families, 1 some det~.ils of embryology are available with reference to a few species belonging to a few families only and even amongst these, the details regarding the early development and placentation are lacking in most cases. Among the Megachiroptera, which includes a single family, Pteropidae, details of the embryology of a few species are known through the work of Gohre, ~ Selenka, 3 Keibel, 4 Van der Sprenkel, 5 Kohlbrugge, e Moghe,V,g Wimsatt a and Karim. TM Among the Microchiroptera the embryology of members belonging to the family Vespertilioniclae has been studied exten- sively.a,n-17 The other families of Microchiroptera, about which some details of embryology are known, are Rhinopomatidae,15, TM Emballonuridae,15,1a Noctilionidae, 2° Megadermatidae, ~5,~1,22 H'ipposideridae, 15,23 Phyllosto- matidae, 24-27 Desmodontidae28, ~9 and Molossidae.9, 3°-37

* Part of the Ph.D. thesis submitted to Nagpur University. 201

BI-Dec. 76 202 D.A. BHIWGADE

There is very little information concerning the embryology of Rhinolo- phidae. Van der Sprenkel5 made a brief mention about the structure of the yolk sac of Rhinolophus hipposideros. H~.mlett 38 mentioned that the allantoic- placenta is discoidal and endotheliochorial in rhinolophids. Gopalakrishna and Bhiwgade39 gave a description of the arrangement of the foetal membranes in Rhinolophus rouxi at a late stage of gestation. Since there is practically no information concerning the early embryology and development of foetal membranes of any member of the family Rhinolophidae it was felt that a detailed study of the embryology of Rhinolophus rouxi would be of cansiderable interest and value. In the present paper, the early development and implantation of the blastocyst in this species are recorded. The development of the foetal membranes and placentation will be reported separately.

2. MATERIALSAND METHODS Specimens of Rhinolophus rottxi were collected at frequent intervals for two years (1971 and 1972) from a railway tunnel near Khandala in Maha- rashtra State. The specimens were killed either by decapitation or by chloro- form, and the genitalia were fixed in various fixatives such as alcoholic Bouin's fluid, neutral formalin, Carnoy's fixative and preserved in 70~o ethanol where necessary. After following the usual procedure of dehydration through graded series of ethanol and embedding in paraffin, the tissues were cut at a thickness of 6-8 tz. For routine histological examination the sections were stained with Ehrlich's haematoxylin and counterstained with eosin, Some slides in each series were also stained by the PAS procedure. 40 Altogether 95 pregnant females are examined. Table 1 includes the various stages studied with details of collection record.

3. OBSERVATIONS

THE FEMALE GENITALIA The breeding habits and the structure of the genit~.lia in the non-pregnant female of Rhinolophus rouxi have been described by Gopalakrishna and Rao. 41 Only the right ovary releases a single ovum during each cycle and pregnancy is invariably borne in the right uterine cornu. At oestrus the uterus (Plate I--figure 1) has an oblong outline in trans. verse sections, the mesometrial-antimesometrial axis being longer than the lateral axis. The muscle layers occapy nearly a quarter of the thickness of the uterine wall. The endometrium is nearly of uniform thickness cn all the sides of the uterus, and the uterine glands are long and extend deep into the endometrium and are evenly distributed on all the sides. The uterine epithelium is made up of columnar cells about 30 u in height. The uterine lumen is slit-like in consonance with the general shape of the uterus. DEVELOPMENT AND IMPLANTATION OF Rhinolophus rouxi 203. Table 1. Collection record and pertinent notes

Date No. of specimens Stage of pregnancy

7-1-1971 1 Morula 1-2-1971 9 Preimplantation stages 4-2-1971 12 Preimplantation stages" 12-2-1971 2 Preimplantation stages 23-2-1971 2 Implanted bilaminar blastccyst 26-2-1971 1 Implanted bila.minar blastccYst 16-3-1971 18 Neural groove Stages and early limb-buds, stages 23-3-1971 12 Late limb-buds stages and mid-pregr_~,r.cy stages 1--4-1971 9 Mid-pregnancy .stages 21-4-1971 1 Mid-pregnancy stage 26-4-1972 20 Advanced pregn~.ncy stz.ges 25-5-1971 8 Full term pregnancy ste.ge Total 95

PREIMPLANTATION STAGES OF DEVELOPMENT The earliest stage of development available for the present study is a free uterine morula of 48 cells and having a distinct envelope of zona pellucida (text-figure 1 ; Plate I--figure 2). The cells of the morula have fine granular cytoplasm, and vesicular nuclei each containing one or two nucleoli and darkly staining chromatin granules. A few small intercellular spaces, which are apparently the fore-runners of the blastocyst cavity, are present in the morula. Six free unilaminar blastocysts at progressively advanced stages of development, and which had hatched out of the zona pellucida, were noticed in different specimens collected on February 1, 1971. In each case the blastocyst was present in an enlarged pocket of the uterus--the prospective implantation chamber--at about the middle of the length of the uterine cornu. The youngest blastocyst (Plate I--figures 3 and 4) had a nearl~ spherical inner cell mass composed of 68 cells. Two blastocysts, which were slightly more adw, nced thart the one described above, had each a slightly flattened inner cell mass made up of 80 cells. The most advanced of these blasto=ysts (Plate II--figures 5 and 6) had a nearly biconvex inner cell mass composed of numerous cells. In all the blastocysts the trophoblast layer is composed of 204 D.A. BHIWGADE

~(a) (b). (e) (d}

I, O-05 mm I Text. Figure 1. Camera lucida drawings of all the sections of the embryo shown in Plate In figure 2. nearly cubicalcells e~ch witha vesicular nucleus, and the inner cell mass is composed of small polygonal or irregularly shaped compactly arranged cells with darkly staining nuclei. In the most advanced of these blastocysts a few flat endodernml cells underlie the embryonic disc. Six free bilaminar blastocysts arc available for the present study (Plate II-- figures 7 and 8; Plate III--figurcs 9 znd 10). In all the cases the bIastocyst wall is composed of an outer layer of trophobIast made up of nearly cubical cells and an inner layer of endoderm composed of squamous cells. The embryonic mass, which is composed of round or polygonal cells, becomes progressively flattened into a disc.

PREIMPLANTATION CHANGES IN THE UTERUS The progestational changes are more pronounced in the middle third of the,length of uterus than in the rest of regions. This segment of the uterus increases m its diameter across the dorso-ventral axis from 895/~ at the morula stage to 1580/z at the bilaminar blastoeyst stage. The uterine lumen in this region expands into a large spherical chamber at this level to accommodate the iapidly enlarging blastoeyst (Plate II--figures 7 and 8; Plate III--figures 9 and 10). The progressive expansion of the implantation chamber is accom- panied by a reduction in the height of the epithelium, especially on the lateral side of the uterus. Table 2 gives the measurements of the various parts of uterus during the growth of the embryo during the preimplarttation period. D. .4. Bhiwgade Proc. Indian Acad. Sci., Plate I 1Iol. 84 B, No. 6, 1976, pp. 201-209

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:i ~" f~" 2-~

Figures 1-4. (1) Transverse section of the uterus at cestrus. The uterir el urnen is slit- like, × 54. (2). Section of the uterus containing a morula, × 54. (3). Section of tie uterus conta.ining a. free unile.minar bl~.stocyst. The ler~g, cciled glards wilh wide lumira, × 54. (4). Magnified view of the blastocyst shown in figure 3, × 290. (facing page 204 ) D. A. Bhiwgade Proc. lndian Acad. Sci., Plate H Vol. 84 B, No. 6, 1976, pp. 201-209

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Figures 5-8. (5). Section of the uterus to show a free unilaminarblastocyst in a large implantation chamber of the uterine lumen, × 54. (6). Magnified picture of the blastocyst shown in figure5. Note the endodermalcells (arrow), × 300. (7). Section ofthe uterus containing a free bilaminar blastocyst in a large implantation chamber. A distinct, but collapsed endcder- real layer is seen in the blastocyst cavity(arrow), × 75. (8). Section of the utelus contain- irrg a free bilamin.ar blastocyst lying in the implantation chamber, × 75. ( facing page 205 ) DEVELOPMENT AND IMPLANTATION OF Rhinolophus rouxi 205

Table 2. Measurements of the uterus and its part during the preimplantation stages of the development of the embryo. i Height of the uterine epithelium in /~ Stage of deveiopment Diameter of Thickness of of the embryo the uterus the myome- Mesometrial Lateral Antimeso- in/~ trium in 5 side side metrial side

1, Morula 895 240 30 28--30 32 2. Unilaminar tz blastocyst (I) 900 230 30 25-30 30 (II) 975 230 30 25-30 30 (III) 1000 230 25 25-30 28 (IV) 1050 190 25 20--25 25 (V) 1050 180 25 20-25 25 (VI) 1050 160 25 20-25 25 3. Bilaminar blastocyst (I) 1100 110 25 20-25 25 (II) 1200 96- 20 20 25 (III) 1200 96 20 18-20 25 (IV) 1350 80 18 15-18 20 (V) 1350 75 18 15 20 (VI) 1380 60 15 10-15 20

During the morula stage the uterine epithelium is composed of roll columnar cells with nuclei situated in the middle of the cells. As the embryo reaches the unilaminar blastocyst stage the cells of the uterine epithelium becomes closely crowded, and their nuclei occur at different levels, and at the bilaminar blastocyst stage they are euboidal on the lateral sides and columnar on the rest of the sides of the uterus, and the nuclei oceur near the basal regions of the cells. A noteworthy feature of the stage is the accumulation of considerable quantity of fluid between the epithelial lining and the subjacent endometrial tissue making this region loose and edematous. The most prominent preimplantation change in the uterus, as seen i.n stained sections, seems to Occur with respect to the uterine glands. During the morula stage (Plate I--figure 2) the uterine glands occur evenly on all the sides of the uterus although their number appears to be more on the antimesometrial side than on the other sides. Most of the uterine glands are nearly straight, and extend to about three-fourths of the thickness of 206 D.A. BHIWGADE the endometrium. The epithelium of the glands consists mostly of cubical cells with homogeneous cytoplasm and vesicular nuclei situated rear the basal regions of the cells. Progressively the glands increase in length and number, become coiled and undergo progressive widening (Plate I--figure 3 ; Plate II--figure 5) and their lumina contain copious amount of eosinoohilie secretion. The cells of the gland epithelium become tall columnar and w, cuolated with basaUy situated vesicular nuclei except towards their fundie regions, where the cells remain cubical and contain darkly staining nuclei. Concomitant with the widening of the uterine glands the endometrizl stroma between the glands gets progressively compressed into thin strands composed of fusiform cells having darkly staining nuclei, fine fibres and blood capillaries. The myometrium is progressively reduced in its thickness from 230/~ in the m orula stage to 60/~ in the bilaminar blastocyst stage.

FURTHER DEVELOPMENT OF THE BLASTOCYST AND IMPLANTATION The following descriptions are based on the examination of the three bilaminar blastocysts at different stages of implantation. The bilaminar blastocyst establishes its contact with the uterine wall commencing from the e.mb~/onic regior~ and progressively extending tO the lateral side of the blastocyst. In the earliest stage (Plate III--figures ll'and 12)the embryonic mass faces the lateral side of the uterus, and processes emanating from the cells of the trophoblast overlying the embryonic mass extend towards the uterine epithelium (Plate III--figure 12). The uterine epithelium is intact on all the sides except or~ the side adjacent to the embryonic mass, where the cells of the uterine epithelium appear to be undergoing destruction as evidenced by the factthat the cell boundaries have disappeared, and the nuclei have become fragmented into small darkly staining bodies. The uterine glands become further coiled, and undergo further widening resulting in the further reduction of the endometrial connective tissue. At a slightly more advanced stage of development the embryonic disc is oriented between the lateral and the mesometrial sides of the uterus, and occurs as a multilayered nearly flat disc, which forms the floor of a spacious cavity--the primitive amniotic cavity (Plate IV--figures 13 and 14). The edges of the embryonic disc are curved dorsally in the form of folds underlying the trophoblast. These are apparently the folds of the definitive amnion. The endoderm consisting of squamous cells underlies the embryonic disc and the trophoblast. As the trophoblastie layer progressively establishes eont2.ct with the uterine stroma on all the sides, the uterine epithelium disappears from all the Sides of the implantation chamber, and the trophoblastic layer undergoes proliferation and enters the endometrium in the form of conical irrpushing D. A. Bhiwgade Proc. Indian Acad. Sci., Plate Ili VoL 84 B, No. 6, 1976, pp. 20i-209

M I 12

Figures 9-12. (9). Section of the uterus containing a late free bilaminal blastocyst, × 54. (10). Part of the blastocyst shown in figure 9, × 380. (11). Section of the uterus with an ~atly implanting bilaminar blastocyst (arrow points mesometrically), × 80. (12). Enlarged part of the blastocyst shown in figure 11. The uterine epithelium which shows signs ot destrttc- tion (alrow), × 380. (facing page 206 ) D. A. Bhiwgade Prec. Indian Acad. Sci., Plate IV VoL 84 B, No. 6, 1976, pp. 201-209

Figures 13-17. (13). Section of the uterus containing an implanted bilaminar blaslccyst (arrow points mesometrically). The endodermal layer (arrow head) has been artifaclually peekd away from its original position, x 54. (14). Magnified view of llae embrycnic regicn cf the blastocyst shown in figure 13. Note the early amniotic folds (arrows), X 350. (15). Part of the uterine wall at implanted bilaminar blastccyst stage shown in figure 13. Solid cords of trophoblast (arrow) push into the endometrium, × 180. (16). Section of the uterus containing an implanted bilaminar blastocyst (arrow points mesometrically), × 54. (17). Enlarged part of the uterine wall to show the finger-shaped intrusions (arrow) of the trophoblast into the endometrium~ × 360, (facing page 207) DEVELOPMENT AND IMPLANTATION OF Rhinolophus rouxi 207

(Plate IV--figure 15). Thus, the blastocyst gets firmly anchored to the uterine wall on its entire surface. The uterine glands seem to undergo rapid disintegration in the proximal region (that is near the uterine lumen). Only the fundic parts of the glands persist near the myometrial border of the endometrium. A late implanted bilaminar blastocyst is shown in Plate IV-- figure 16. The embryonic disc lies facing between the lateral and the mesometrial sides of the uterus. The endodermal lining, both in the region of the embryonic disc and the extraembryonic region, is a distinct layer of lightly staining cells, which are nearly cubical in the region of the embryonic disc, but squamous in the extra embryonic regions. The primitive trophobl,~st on the mesometrial and the lateral sides of the uterus has entered the endometrium in the for.m of finger-shaped cords (Plate IV-- figure 17) which, in many places, are branched z.nd the branches are inter- connected. The endonletrial tissue is loose and spongy near the myometrial border, and contains numerous patches of necrotic tissue. A few remnants of the uterine glands are present in the deeper regions of the endometrium.

4. DISCUSSION

An unusual feature of the early development of Rhinolophus rouxi is that the embryonic m~_ss, which is oriented towards the lateral side of the uterus during early stages, appears to change towards the mesometrial side as the embryo advances in development. The position of the embryonic disc in rela- tion to the uterus is constant for a species, and in many mammalian orders, it is constant for members of the family. While evaluating the phylogenetic importance of the foetal membranes, Mossman 31 stated: " the orientation of the embryonic disc with reference to the uterus is usually constant in all groups known to be closely related ". In a later review Mossman4z reite- rated the same hypothesis and mentioned, "In most major taxonomic groups of eutherian mammals, available data show that at the time of first attach- ment of the blastocyst to the endometrium the inner cell mass or the embryonic disc has an almost constantly specific directional orientation to the uterus." Regarding the bats he mentioned, "The literature on the Micro- chiroptera (Insectivorous bats) indicated wide variation similar to those in Insectivores. It is particularly confusing since it often indicates lack of consistency even within smaller groups, that is superfamilies and familie~ ." Rhinolophus rouxi, therefore, appears to differ from all the other wild mammalian species so far studied except Megaderma lyra lyra 22 where the embryonic mass has a variable orientation. The most prominent progestational reaction of the uterus in all the bats so far studied concerns the formation of an edema in a specific region 208 D.A. BHIWGADE

of the uterus, 11, 13 or circumferential on all the sides of the uterus. 1° In Rhinolophus rouxi, on the other hand, the most prominent progestationaL uterine reaction appears to concern the glands which undergo enormous hypertrophy on all the sides of the uterus around the implantation chamber. By the time the blastocyst begins to implant the endometrial stromal tissue is reduced to thin strands pressed between the large uterine glands. Per- haps, the circumferential implantation of the blastocyst is in some way related to the diffuse progestational changes.

ACKNOWLEDGEMENT I am deeply indebted to Dr. A. Gopalakrishna, Institute of Science, Nagpur, for his valuable guidance throughout this work.

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