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.