Ultrastructural study of the keratinization of the dorsal epithelium of the tongue of Middendorff's bean goose Anser fabalis middendorffii (Anseres Antidae)код для вставкиСкачать
THE ANATOMICAL RECORD 247:85–101 (1997) Formation of Reticulated Endoderm, Reichert’s Membrane, and Amniogenesis in Blastocysts of Captive-Bred, Short-Tailed Fruit Bats, Carollia perspicillata NILIMA K. BADWAIK,1 JOHN J. RASWEILER IV,1* AND SERGIO F. OLIVEIRA2 1Department of Obstetrics and Gynecology, Cornell University Medical College, New York, New York 2Department of Histology and Embryology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil ABSTRACT Background and Methods: As part of an effort to develop the short-tailed fruit bat (Carollia perspicillata) as a new animal model for the study of interstitial implantation and trophoblast–uterine interactions, early embryogenesis was examined histologically and ultrastructurally in captive-bred females at different intervals after the first appearance of spermatozoa in daily vaginal smears (day 1 postcoitum [p.c.]). Results: In most of the early uterine embryos examined on days 16–18 p.c., much of the endoderm appeared as a reticulated meshwork; however, a unilocular yolk sac was formed prior to the development of any mesoderm. Early blastocysts of Carollia were also unusual in that endoderm surrounded much of the inner cell mass (ICM), Reichert’s membrane continued over the dorsal side of the ICM, and basal laminalike material was observed around many of the endoderm and epiblast cells. A primordial amniotic cavity was formed between days 19 and 26 p.c. by cavitation. The first mesoderm appeared between days 23 and 26 p.c., concommitant with the development of an embryonic shield. Conclusion: The unusual reticulated appearance of early endoderm in Carollia, which is reminiscent of that seen in early human blastocysts, may be attributable to constraints imposed on growth of the blastocyst by the site and mode of implantation, temporary retardation of trophoblastic invasion by the basal laminae of endometrial epithelial elements, and endodermal proliferation in anticipation of rapid yolk sac expansion. Reichert’s membrane appears to play an important role in this species in tethering the ICM and embryonic shield to the developing placenta prior to the formation of significant amounts of mesoderm. Anat. Rec. 247:85–101 r 1997 Wiley-Liss, Inc. Key words: reticulated endoderm, reichert’s membrane, amniogenesis, embryogenesis, fruit bat Early blastocysts of the human and chimpanzee are unusual in possessing a reticulated meshwork that was initially identified as precociously formed extraembryonic mesoderm (Heuser, 1940; Hertig and Rock, 1941; Hertig, 1945). Luckett (1974, 1978) subsequently suggested that the reticulated meshwork of cells that fills much of the blastocyst cavity in the 11-day-old human and the 10.5-day-old chimpanzee is actually extraembryonic endoderm. Such a meshwork is absent in rhesus monkey and baboon blastocysts; however, cells appear between the primitive endoderm and trophoblast of rhesus monkey blastocysts and progressively develop mesenchymal characteristics. This finding led Enders and King (1988) to conclude that endoderm gives rise to the first extraembryonic mesoderm in the rhesus monkey. They also suggested that the ‘‘endodermal reticur 1997 WILEY-LISS, INC. lum’’ in the human may actually constitute forming mesenchymal cells and hence the first extraembryonic mesoderm. In the course of studies intended primarily to examine implantation, placental development and trophoblast-uterine interactions in captive-bred, short-tailed fruit bats (Carollia perspicillata), early blastocysts were seen to develop a reticulated meshwork much like that observed at comparable stages in the human. Although the cells making up this meshwork initially Received November 6, 1995; accepted August 19, 1996 *Correspondence to: Dr. John J. Rasweiler IV, Department of Obstetrics and Gynecology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021. Contract grant sponsor NIH; Contract grant number HD28592. 86 N.K. BADWAIK ET AL. have some mesenchymal characteristics, they soon give rise to a unilocular yolk sac lined by an endodermal monolayer. This transformation occurs prior to amniogenesis and is sharply separated in a temporal sense from the formation of any mesoderm. Several other aspects of early blastocysts in this bat are also unusual: endoderm is not limited to the lining of the yolk sac but rather surrounds much of the inner cell mass; Reichert’s membrane is not associated just with the parietal endoderm (as in rodents) but continues over the dorsal side of the inner cell mass; and basal laminalike material is commonly observed on other surfaces of the endoderm cells and between some of the epiblast cells. Early embryonic development in Carollia is also of interest because the morphological organization of the blastocyst, mode of initial formation of the yolk sac, and differentiation of cytotrophoblast within the preplacenta differ significantly from that observed in another bat (Noctilio albiventris; Rasweiler and Badwaik, 1996) belonging to the same superfamily. This difference indicates that, even in relatively closely related mammals, early cell lines can exhibit considerable interspecies diversity in their phenotypic and functional differentiation. The timing of the developmental events reported here has assumed considerable importance because pregnancy in Carollia can be substantially prolonged under some circumstances both in captivity and the wild. In captivity, this prolongation is due primarily to the retardation of conceptus development after implantation is completed (Rasweiler and Badwaik, in press). MATERIAL AND METHODS Source of the Animals All of the C. perspicillata used in this study were collected on the island of Trinidad in the West Indies. Animal Maintenance The animals were kept in rooms with a controlled light cycle (12 hr light and 12 hr dark), with the dark phase set to commence at 1600 hr to facilitate feeding just before its onset. The temperature was maintained between 24 and 27°C. The bats were housed in bipartite cages, measuring approximately 170 cm wide 3 81 cm high 3 77 cm deep, which permitted the animals to fly. The cages were divided into an open feeding area (92 cm wide) and a darkened roosting box connected by an access hole (15 cm high 3 30 cm wide). The bats were maintained in sexually segregated groups composed of 8–12 males or 10–20 females until the time of breeding. They were fed a fruit-based liquid diet prepared from readily available canned and powdered components (peach nectar, canned peaches, ground monkey chow, dibasic calcium phosphate, corn oil, an emulsifier, and a multivitamin preparation). This diet was occasionally supplemented with small amounts of apple, banana, or melon as treats. The animals were fed every night without exception, and the diet was served cold, usually no more than 60–90 min before the room lights went off, to minimize microbial growth. Water was provided ad libitum in chick waterers. Further details of the animal husbandry procedures may be found elsewhere (Rasweiler and Badwaik, 1996). During the course of this work, it was discovered that lab-bred C. perspicillata exhibited substantial variation in gestation length (.2 months). This variation was more pronounced in wild-caught animals bred during their first year in captivity than during their second. Furthermore, most of the developmental delays occurred after the initiation of implantation (Rasweiler and Badwaik, in press, unpublished observations). The extent to which the rate of development may differ during the preimplantation period and implantation has not yet been determined. For this reason, it should be pointed out that the material preserved for light microscopic examination in this study was obtained from animals killed at the following intervals after capture in the wild: 4–9 months, 17 bats; 12–18 months, 22 bats; and 24–27 months, 36 bats. All of the specimens examined with the electron microscope were obtained from females that had been maintained in captivity for more than 12 months. Timing of Reproductive Stages For breeding purposes, a single male with prominent testes was added to groups of 10–18 females. On subsequent mornings, between 0530 and 0900 hr, a small quantity of distilled water was aspirated in the vagina of each female with a microeyedropper. The aspirate was then dried and examined for spermatozoa. Most females exhibited a single period of spermpositive vaginal aspirates that usually varied from 1 to 5 days in duration (Rasweiler and Badwaik, 1996). In a few cases, females exhibited two separate periods of sperm- positive smears. For these females, the probable time of conception could be identified from an examination of their breeding records and the stage of development of their embryos. Mated females were killed between 0630 and 1030 hr at different intervals after the first morning on which spermatozoa were detected in their vaginal aspirates (day 1 postcoitum [p.c.]). The animals were killed by administering an intraperitoneal injection of sodium pentobarbital at a dosage of approximately 90 mg/kg body weight. Histological Procedures The reproductive tracts were fixed in Zenker’s fluid for 8–10.5 hr, immersed in 2.5% aqueous potassium dichromate for an additional 2 hr, washed overnight in running tap water, dehydrated through graded ethyl alcohols, cleared overnight in warm cedar wood oil (37°C) followed by Histosol or Histoclear II (National Diagnostics, Atlanta, GA), and embedded in paraffin wax. The tracts were then serially sectioned (in a frontal plane) at 6 µm. The histological sections were stained with hematoxylin and eosin, Weigert’s resorcinfuchsin (Clifford and Taylor, 1973) followed by Masson’s trichrome procedure (modified from Humason, 1972), or by the periodic acid-Schiff (PAS) technique and counterstained with hematoxylin. In the modified Masson’s trichrome procedure, mordanting in iron alum was omitted, and the nuclei were stained with stabilized iron chloride hematoxylin (Lillie, 1965). Also, when the sections were washed in running water after 87 BLASTOCYSTS OF THE SHORT-TAILED FRUIT BAT TABLE 1. Location of the embryo during early pregnancy in the short-tailed fruit bat, Carollia perspicillata Day postcoitum Developmental stage and location 12 13 14 15 16 Oviductal blastocyst (1ZP) Oviductal blastocyst (2ZP) Uterine blastocyst close to uterotubal junction Uterine blastocyst within usual implantation zone Implanting uterine blastocyst 1 1 1 3 — — — 1 5 1 — 1 — 1 3 — — — 1 7 — 1 — — 6 1ZP, zona pellucida present; 2ZP, zona pellucida not present. being stained with acid fuchsin and ponceau de xylidine, the wash time and water temperature (23°C) were precisely controlled. Some sections of each tract were incubated for 1 hr at 37°C in 0.1% a-amylase (1,4a-D-glucan-glucanohydrolase; Sigma Chemical Company, St. Louis, MO) dissolved in a 0.02 M phosphate buffer (pH 6) to remove any glycogen before being processed according to the PAS procedure. Parallel sections were incubated under similar conditions in the buffer alone and then stained. OBSERVATIONS Location of Embryos The location of the embryos examined histologically on days 12–16 p.c. is shown in Table 1. From day 17 p.c. on, all of the blastocysts were found in the uterus. The first clear evidence of implantation (trophoblastic penetration of the uterine epithelium) was observed in some animals on day 14 p.c. However, earlier phases of the process may have been disrupted by shrinkage of the specimens during histological processing. Specimens Examined Histologically Uterine Anatomy and Site of Implantation The reproductive tracts from 5–8 mated females per day were examined for embryos on days 12–25. The tracts from 2–3 mated females per day were examined on days 26, 28, and 30. Although Carollia has a simplex uterus, relatively narrow tubular segments are interposed between the distal end of each oviduct and the main uterine cavity. These segments are clearly uterine because they are lined by endometrium, and some uterine glands open into their lumina. They appear to be homologous to the cranial ends of the uterine horns in related species with bicornuate uteri and have therefore been referred to as ‘‘intramural uterine cornua’’ (Rasweiler, 1979, 1993). A detailed description of implantation is beyond the scope of this paper, but a brief summary of the process is presented because of its possible relevance in understanding some unusual aspects of the early embryogenesis of Carollia. Although implantation usually occurs within one of the intramural uterine cornua, it sometimes takes place from the main uterine cavity in immediately adjacent areas of endometrium. Initial trophoblastic attachment within the intramural uterine cornu is circumferential, and trophoblastic penetration of the endometrium is temporarily retarded when the basal laminae of the uterine luminal and glandular epithelia are reached. Much of the trophoblast then grows along these basal laminae for a while before passing into the endometrial stroma. Even when the blastocyst first enters the main uterine cavity, there appear to be significant spatial constraints during the implantation process because of closure of the uterine lumen and rapid engulfment of the embryo by the decidual reaction. Regardless of the site of initial attachment, the blastocyst becomes interstitially implanted and covered by a decidua capsularis (Rasweiler, 1979; unpublished observations). Electron Microscopy To confirm some of the light microscopic observations, five blastocysts obtained on days 16 and 17 p.c. were examined ultrastructurally. To obtain these specimens, pregnant females were anesthetized between 800 and 1000 hr with an intraperitoneal injection of sodium pentobarbital at a dosage of approximately 90 mg/kg body weight. The animals were then perfused via the left ventricle with approximately 15 ml of lactated Ringer’s solution followed immediately by cold fixative consisting of 2.5% glutaraldehyde and 2% formaldehyde (methanol-free) in 0.1M cacodylate buffer (pH 7.4) containing 2.5 mM CaCl2. The uterus was then removed, immersed in fixative, its maximum diameter measured, and cut in half in a frontal plane. Implantation sites could be readily identified as rounded hemorrhagic sites. The uterine pieces were immersion fixed for an additional 24 hr at 4°C, rinsed for 2 hr in three changes of the same buffer, and then postfixed for 2 hr at room temperature in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) containing 2.5 mM CaCl2. The specimens were dehydrated through a graded series of acetones and embedded in PolyBed 812 (Polysciences, Warrington, PA). Semithin sections were cut and stained with 1% toluidine blue in 1% aqueous sodium borate for light microscopic examination. Thin sections were picked up on copper grids and stained for 30 min with methanolic uranyl acetate followed by 3 min with lead citrate. Sections were then examined with a JEOL-JEM 100CXII electron microscope at an accelerating voltage of 80 kV. Endoderm The first clear examples of endoderm cells were found histologically in a blastocyst examined on day 14 p.c. 88 N.K. BADWAIK ET AL. Figs. 1–4. BLASTOCYSTS OF THE SHORT-TAILED FRUIT BAT 89 These were located on the ventral surface of the inner cell mass and also partially lined the interior of the mural trophoblast. The endoderm cells had smaller nuclei and darker cytoplasm than did the epiblast cells. On day 15 p.c., endoderm could be positively identified in three-quarters of the blastocysts (Fig. 1). In one of these, a small unilocular yolk sac had developed, and this sac contained an acidophilic, moderately PAS1, granular coagulum. All six of the uterine blastocysts examined on day 16 p.c. contained endoderm, which in most had a mesenchymelike appearance (Fig. 2). The endoderm was most abundant on the ventral side of the inner cell mass, but it also extended up and around much of the mass. Apparent gaps were sometimes noted in the supraembryonic endodermal layer at this stage; however, these were often quite small (i.e., the approximate size of a few endoderm cells). Ultrastructural studies would be required to confirm that endoderm was not still present but was highly attenuated and/or less differentiated in such regions. The interstitial spaces within the endodermal meshwork ventral to the inner cell mass contained a granular coagulum, and in three of six blastocysts these spaces were dilated to a considerable extent with fluid. On day 17 p.c. reticulated endoderm was present but not abundant in one small implanting blastocyst. The interstitial spaces in the endoderm of this specimen also lacked any granular coagulum. In three other blastocysts obtained on this day, the endodermal meshwork was more extensive and contained dilated pockets of fluid (Fig. 3). Another day 17 p.c. specimen had a unilocular yolk sac cavity lined by an endodermal monolayer, but some of the endoderm surrounding the epiblast was still multilayered and mesenchymal in appearance (Fig. 4). Several lines of evidence clearly indicated that the cells on the dorsal and lateral sides of the inner cell mass were endoderm. In histological sections, they appeared similar in size and staining characteristics to the visceral and parietal endoderm cells (Figs. 2–4). At the ultrastructural level, both the parietal and supraembryonic endoderm cells typically contained many elon- Fig. 1. Early implanting blastocyst, day 15 p.c. The arrowheads indicated darker-staining endoderm cells. Reichert’s membrane had developed between the parietal endoderm and the trophoblast. Masson’s trichrome. 3403. Fig. 2. Early implanting blastocyst, day 16 p.c. By this stage, endoderm (E) surrounded much of the epiblast (EP) and had formed a meshwork on the ventral side of the inner cell mass. Reichert’s membrane was present between the endoderm and the cytotrophoblast (e.g., at arrowheads). Deposits of PAS1, basal laminalike material, were also frequently evident between many of the endoderm cells and epiblast, around some of the endoderm cells, and to a lesser extent between some epiblast cells. The granular coagulum present in pockets within the endodermal meshwork was less intensely stained by the PAS reaction. Amylase-PAS-hematoxylin. 3287. Figs. 3, 4. Implanting blastocysts, day 17 p.c. The endoderm on the ventral side of the inner cell mass of one embryo (Fig. 3) contained several fluid-filled pockets, and that of the other (Fig. 4) had formed a unilocular yolk sac. These pockets again held flocculent material that was moderatedly PAS1. In both embryos, endoderm (E) dorsal and lateral to the epiblast (EP) was often multilayered and mesenchymal in appearance. Portions of Reichert’s membrane are indicated by arrowheads. Deposits of basal laminalike material were distributed as in the blastocyst shown in Figure 2. Fig. 3: Amylase-PAS- hematoxylin, Fig. 4: PAS-hematoxylin. 3287. Fig. 5. Portion of the parietal wall of the yolk sac in an implanting blastocyst, day 17 p.c. Reichert’s membrane (R) is evident between the endoderm (E) and the mural cytotrophoblast (C). In this region, Reichert’s membrane consists of two layers (inset). The outer layer appears to be the basal lamina of the cytotrophoblast. The inner layer may be of endodermal origin because it is present on all surfaces of the endoderm cells (e.g., at arrowheads) and in the yolk sac cavity (YSC). Very similar material is present in the cisternae of the rER (arrows) in the endoderm cells. 39,579. Inset, 322,320. Fig. 6. Embryonic pole of an implanting blastocyst, day 16 p.c. Reichert’s membrane (R) passes between the polar cytotrophoblast (C) at the top of the figure and a layer of supraembryonic endoderm cells (E). The endoderm cells contain a moderate abundance of rER with anastomosing cisternae, as is the case with the parietal endoderm (Fig. 5). Rough ER is much less abundant in the epiblast cells (EP). Basal laminalike material is also present as a coating on exposed surfaces of the endoderm and epiblast cells (arrowheads), and larger masses (arrows) frequently occur between these cells. Apoptotic bodies (A) are evident, and two of these have been phagocytosed by epiblast cells. 35,035. Fig. 7. Embryonic pole of an implanting blastocyst, day 16 p.c., showing three examples of well-differentiated supraembryonic endoderm cells (E) with their characteristic content of rER. In general, much less rER is present in the epiblast cells (EP; cf. Fig. 9). Material similar to Reichert’s membrane (R) is also present on other surfaces of the endoderm and epiblast cells (e.g., at arrowheads). C, polar cytotrophoblast; R, Reichert’s membrane. 36,270. Fig. 8. Higher power view of the portion of Reichert’s membrane (R) shown in Figure 7. In this region, Reichert’s membrane does not appear to consist of two layers, in contrast to what has been observed between the parietal endoderm and mural trophoblast (cf. Fig. 5). C, polar cytrotrophoblast; E, endoderm. 319,143. 92 N.K. BADWAIK ET AL. Fig. 9. Mass of basal laminalike material (arrow) between epiblast cells in an implanting blastocyst, day 16 p.c. This mass is composed of at least two components of different electron densities. A thinner coat of extracellular material is also present on other portions of the epiblast cells (e.g., at arrowheads). 311,970. gated, branching cisternae of rough endoplasmic reticulum (rER), whereas epiblast cells contained much less rER (Figs. 5–9). The endoderm cells in both locations were closely associated with Reichert’s membrane and an abundance of extracellular material with similar histological and ultrastructural characteristics. Two of the implanting blastocysts examined on day 18 p.c. were quite small. In one, much of the epiblast was surrounded by a single layer of endodermal cells with a mesenchymal appearance. In the other, the endoderm was multilayered on the ventral side of the epiblast. No dilated pockets of fluid were seen between the endoderm cells of either embryo. Three other day18-p.c. blastocysts were much larger and contained unilocular yolk sacs. In one case, however, the yolk sac still had several outpocketings (Fig. 10). In the other two embryos, some of the endoderm surrounding the epiblast still appeared mesenchymal. A multilocular yolk sac was seen only in two blastocysts examined on days 19–21 p.c. (Table 2). All of the remaining blastocysts examined on those days and on days 22–25 p.c. had unilocular yolk sacs lined by an endodermal monolayer (Fig. 11). In two of the specimens examined on days 20 and 21 p.c., however, some of the endoderm dorsolateral to the epiblast still appeared mesenchymal and contained small pockets of extracellular fluid (Fig. 12). Reichert’s Membrane and Extracellular Matrix Material In three of the blastocysts examined histologically on day 15 p.c. and in all six uterine blastocysts examined on day 16 p.c., a prominent acidophilic and PAS1 basal lamina (Reichert’s membrane) had developed between the parietal endoderm and the cytotrophoblast (Figs. 1 & 2). This basal lamina continued up between the endoderm surrounding much of the epiblast and the cytotrophoblast. At the ultrastructural level, Reichert’s membrane between the parietal endoderm and mural trophoblast could be seen to consist of two components (Fig. 5). One appeared to be the basal lamina of the cytotrophoblast, and the other was identical to extracellular material coating free surfaces of the endoderm cells. Clumps of similar material were present in the yolk sac cavity along with many small, dense granules. Dorsal to the inner cell mass (Figs. 6–8), Reichert’s membrane appeared to consist of just one broad, homogeneous layer composed of material similar to that found on the surface of the endoderm cells. A transition in Reichert’s membrane to a bipartite structure occurred at about the level of the junction of the parietal and visceral endoderm cells. In paraffin sections, extracellular masses of glycoprotein-rich material (i.e., strongly PAS1 after amylase digestion) were commonly seen along the interface of the endoderm (visceral and supraembryonic) and the epiblast (see Fig. 12). In tangential sections of the endoderm–epiblast interface, much of this basal laminalike material was found to be actually part of a meshwork rather than a continuous layer. This meshwork extended inward between some of the epiblast cells (particularly those on the periphery) and outward between some of the endoderm cells to Reichert’s membrane. Additional clumps or cords of strongly PAS1 material were observed between some of the BLASTOCYSTS OF THE SHORT-TAILED FRUIT BAT 93 Fig. 10. Blastocyst, day 18 p.c. This specimen has a unilocular yolk sac with outpocketings (*) that, because of the plane of section, appear to be separate sacs. Some portions of the endoderm (E) dorsal and lateral to the epiblast (EP) are multilayered and organized into small sacs. Reichert’s membrane is present between the endoderm and cytotrophoblast (e.g., at arrowheads). Amylase-PAS-hematoxylin. 3200. Fig. 11. Implanted blastocyst, day 19 p.c. This embryo possesses a unilocular yolk sac lined by an endodermal monolayer. Endoderm (E) also surrounds much, if not all, of the epiblast (EP). Masson’s trichrome. 3200. Fig. 12. Implanted blastocyst, day 20 p.c. Prominent, extracellular accumulations of glycoproteins strongly stained by Schiff’s reagent are present in many places (e.g., at the arrowheads) between the endoderm and epiblast or around endoderm cells. Reichert’s membrane (below R) passes dorsal to the inner cell mass. Many of the epiblast cells seem to have assumed a similar concentric orientation (e.g., around asterisk). Amylase-PAS- hematoxylin. 3450. epiblast cells, even in the center of the inner cell mass. Masses and cords of basal laminalike material were found in comparable locations at the ultrastructural level (Figs. 6, 7, 9). These masses and cords contained of at least two components differing in electron density (Fig. 9). In many areas, the epiblast cells also had a thin coat of extracellular material of variable thickness. As development progressed, trophoblast-lined clefts developed on the interior of the preplacenta dorsal and lateral to the inner cell mass or embryonic shield, and 94 N.K. BADWAIK ET AL. TABLE 2. Summary of major developmental events during early embryogenesis in the short-tailed fruit bat, Carollia perspicillata Number of embryos exhibiting developmental characteristic Day postcoitum n Endodermal meshwork Unilocular yolk sac Extensive apoptosis and multiple cavities within epiblast 16 17 18 19 20 21 22 23 24 25 26 28 30 6 5 5 4 6 5 3 4 5 5 3 2 2 4 4 2 1 — 1 — — — — — — — 2 1 3 3 6 4 3 4 5 5 3 2 2 — — — — 1 — 1 — 3 1 — — — Large primordial amniotic cavity containing cellular debris Embryonic shield present; formation of mesoderm — — — — — — 2 1 1 3 1 — — — — — — — — — 1 — 1 2 21 22 1In both embryos, mesoderm had begun to grow along the amniotic ectoderm, and part of the yolk sac wall had been converted into a trilaminar omphalopleure. 2In one embryo, the amnion had been formed, and part of the yolk sac wall had been converted into a trilaminar omphalopleure. Reichert’s membrane inserted into these clefts (Figs. 13, 17, 19, 20). Cytotrophoblastic Villi Cytotrophoblast extended into the preplacenta from the base of these clefts and elsewhere in the form of villi (Fig. 20). The cells constituting these villi appeared relatively undifferentiated, were not disposed in a highly ordered fashion, and lacked obvious basal laminae. Ultrastructural studies in progress have confirmed that when cytotrophoblast cells initially penetrate portions of the developing placenta of Carollia in the form of primary villi (i.e., lacking mesoderm) they lack basal laminae (Badwaik and Rasweiler, unpublished observations). Amniogenesis and the First Appearance of Mesoderm Commencing on day 19 p.c., it was common to see groups of epiblast cells with a similar orientation pattern, indicative of changing cell shape and possibly increased motility, that appears to portend the initiation of amniogenesis (Fig. 12). Amniogenesis in Carollia occurs by cavitation. At first this involved the apoptosis of scattered cells within the epiblast, the rearrrangement and polarization of epiblast cells in the forming embryonic shield, the formation of bulges and indentations on some portions of the surface of the epiblast (apparently associated with morphogenetic cell movements), and the development of small cavities within the epiblast (Figs. 13–16). The apoptotic epiblast cells generally exhibited cytoplasmic shrinkage, nuclear condensation, and fragmentation, and there was no associated inflammatory reaction (Fig. 16). Many of the epiblast cells bordering on the small cavities that developed in the embryonic mass had a radial orientation and had clearly become polarized (Figs. 14–15). These spaces appear very similar to the primordial amniotic cavities observed in another bat (Molossus ater; Sansom, 1932; Rasweiler, 1990), the rhesus monkey, and human (Luckett, 1975; Enders et al., 1986). Although some apoptosis was observed within the epiblast during implantation (Fig. 6), this became much more extensive when sizeable cavities appeared within the embryonic mass (Figs. 17–19). These coalesced and enlarged to form the primordial amniotic cavity. The timing of amniogenesis exhibited some variation between embryos (Table 2), but for the most part it commenced after the formation of a unilocular yolk sac. Amniogenesis was also associated with extensive disruption of the supraembryonic endoderm (Figs. 13– 15, 19). Mesoderm formation was first noted when the epiblast of the inner cell mass had been converted into a curved embryonic shield and amniotic ectoderm (Fig. 19). Mesoderm was present in some specimens examined on days 23–25 p.c. (Fig. 21) and in all examined on days 26–30 p.c. DISCUSSION In most mammals, the primitive endoderm (hypoblast) develops initially on the basal (inner) surface of the inner cell mass. It then spreads peripherally to line the interior of the blastocyst cavity, thereby creating the yolk sac. In Carollia, the primitive endoderm also differentiates on the surface of the inner cell mass, but it proliferates to create a meshwork of mesenchymelike cells in the blastocystic cavity and initially forms a covering for much, if not all, of the inner cell mass. The endoderm dorsal and lateral to the inner cell mass also often has a reticulated appearance. Concomitant with formation of the endodermal meshwork, a granular coagulum begins to accumulate in its extracellular spaces, and a prominent PAS1 basal lamina (Reichert’s membrane) develops between the peripheral cells of the meshwork and the cytotrophoblast. Reichert’s membrane is also produced dorsal and lateral to the inner cell mass. BLASTOCYSTS OF THE SHORT-TAILED FRUIT BAT 95 Figs. 13, 14. Two sections through the inner cell mass of an implanted blastocyst, day 22 p.c. Some of the epiblast cells have become organized as a columnar epithelium (between the arrowheads in Fig. 13), in what presumably would have become part of the embryonic shield. Others have become polarized around small cavities that had formed between other epiblast cells (e.g., at tip of arrow in Fig. 14). There are also some scattered apoptotic cells within the inner cell mass. Part of the mass at the top of the figures lacks a covering of darker-staining endoderm cells (E), apparently due to partial disruption of the supraembryonic endoderm at this stage. Examination of all sections of this embryo suggests that some of the epiblast has grown outward (e.g., in the direction of the arrowheads in Fig. 14) to partially engulf endoderm cells and basal laminalike material originally at the endoderm–epiblast interface. Reichert’s membrane (R) extends into a cleft in the preplacenta (P) in Figure 13. Amylase-PAS-hematoxylin. 3274. Although much of the endoderm in early implanting Carollia blastocysts has a mesenchymal phenotype, there is no question about the identity of these cells. The multilocular yolk sac composed of such cells is soon converted into a unilocular one lined by an endodermal monolayer. Furthermore, the first mesoderm does not appear until several days later, when a large primordial amniotic cavity has formed and conversion of the epiblast into an embryonic shield and amniotic ectoderm is nearly complete. Carollia resembles another bat (Glossophaga) belonging to the same family (the Phyllostomidae) in exhibiting the formation of the yolk sac from a reticulated endodermal meshwork, but this is not seen in the bat Noctilio, a member of the closely allied family Noctilionidae. Our comparative studies suggest that there may Fig. 15. Higher power view of part of the section shown in Figure 14. The radial arrangement of epiblast cells around small intercellular spaces (e.g., at arrow) portends imminent development of the primordial amniotic cavity. Only one cluster of endoderm (E) cells is evident at the top of the figure, on the dorsal surface of the inner cell mass. Prominent PAS1 cords of basal laminalike material (e.g., at the arrowheads) can be seen to extend into the epiblast, and these were found in serial sections to be linked to Reichert’s membrane. AmylasePAS-hematoxylin. 3996. 96 N.K. BADWAIK ET AL. Fig. 16. Inner cell mass in an implanted blastocyst, day 24 p.c. The onset of amniogenesis is indicated by the reorganization of a small group of epiblast cells (next to asterisk) into a columnar epithelium (presumptive embryonic shield) and the relative abundance of apoptotic cells and bodies (e.g., at arrowheads). The apoptotic cells are characterized by cytoplasmic shrinkage and nuclear condensation or fragmentation. Many other epiblast cells are undergoing mitosis (e.g., at arrows). E, endoderm; YSC, yolk sac cavity. Amylase-PAShematoxylin. 3566. be several reasons for this difference. Implantation in Carollia and Glossophaga usually takes place in a relatively constricted site (i.e., in one of the intramural uterine cornua) that is interposed between the end of each oviduct and the main cavity of a simplex uterus. Furthermore, during the early phases of implantation in Carollia, blastocyst expansion appears to be constrained by (1) closure of the uterine lumen, (2) growth of the decidua over the abembryonic pole of the embryo, and (3) retardation of trophoblastic invasion of the endometrial stroma by the basal laminae of the uterine luminal and glandular epithelial cells (unpublished observations). These three conditions exist even when implantation occurs from the main uterine cavity rather than within an intramural uterine cornu. Although proliferation of the endoderm continues at this time in both Carollia and Glossophaga, such spatial constraints at the implantation site may serve to prevent rapid early expansion of the yolk sac. In contrast, in Noctilio, implantation occurs at the cranial end of a uterine horn, and the yolk sac can (and does) expand into a more spacious uterine lumen (Rasweiler, 1979, 1993; also see Figs. 5 and 7 in Anderson and Wimsatt, 1963). The depth of implantation in Carollia and Glossophaga is ultimately interstitial, whereas in Noctilio it is only partially interstitial, in part because of early expansion of the yolk sac. Early proliferation of the endoderm may be important in Carollia and Glossophaga because it creates a cellular reservoir that can be used for subsequent rapid growth of the yolk sac. The large yolk sac that soon develops in all three species presumably plays an important role in early physiological exchange between the embryo and mother, and it provides space for further growth of the conceptus. The development of an endodermal meshwork is quite rare among mammals. In addition to Carollia and Glossophaga, this has been reported to occur only in the human, chimpanzee, and a prosimian primate, Demidoff’s bushbaby (Galago demidovii). Small patches of reticulated endoderm also are present in implanted blastocysts of three other prosimian primates: the Senegal bushbaby (Galago senegalensis), the slender loris (Loris tardigradus), and the slow loris (Nycticebus cougang; Luckett, 1974). The present study provides additional comparative support for Luckett’s suggestion that an endodermal meshwork forms in early human and chimpanzee blastocysts because of spatial constraints imposed by an interstitial mode of implantation. Such a meshwork is not seen in blastocysts of the rhesus monkey or baboon, which exhibit central implantation. However, Enders and King (1988) obtained strong evidence that extraembryonic mesoderm can differentiate from endoderm in the rhesus monkey, and they go on to suggest that the endodermal reticulum in the human may actually constitute forming mesenchymal cells and hence the first extraembryonic mesoderm. Envelopment of much of the inner cell mass by endoderm is unusual but not unique to Carollia. This BLASTOCYSTS OF THE SHORT-TAILED FRUIT BAT Fig. 17. Implanted blastocyst, day 20 p.c. Amniogenesis has progressed further in this specimen by apoptosis and the enlargement of cavities within the epiblast (EP). The arrowheads indicate points at which Reichert’s membrane is tethered to clefts in the preplacenta (P). The supraembryonic endoderm (E) appears less organized and not in the form of an epithelial monolayer like the visceral and parietal endoderm. YSC, yolk sac cavity. PAS-hematoxylin. 3166. 97 Fig. 18. Implanted blastocyst, day 25 p.c. The inner cell mass has differentiated into an epiblastic shield (EP) and amniotic ectoderm (AE). The primordial amniotic cavity (AC) contains a large mass of epiblast cells, most of which are undergoing apoptosis. In other sections, this mass was still connected to the shield and developing amnion; however, in view of the location of the mass and its degenerate condition, it seems unlikely that most of its cells would have survived amniogenesis. VE, visceral endoderm; YSC, yolk sac cavity. Masson’s trichrome. 3487. 98 N.K. BADWAIK ET AL. Figs. 19–20 BLASTOCYSTS OF THE SHORT-TAILED FRUIT BAT 99 Fig. 21. Section through the caudal end of the embryonic shield of a blastocyst, day 25 p.c. Abundant mesoderm (M) is present between the shield epiblast (EP) and the visceral endoderm (VE). AC, primordial amniotic cavity; AE, amniotic ectoderm; P, preplacenta; YSC, yolk sac cavity. Masson’s trichrome. 3242. envelopment had been reported for another microchiropteran bat (Glossophaga soricina; Rasweiler, 1974) belonging to the same family and for a megachiropteran bat (Haplonycteris fischeri; Heideman, 1989). It also may occur in the vampire bat (Desmodus rotundus), where it was identified as precociously formed extraembryonic mesoderm (Wimsatt, 1954), the disc-winged bat (Thyroptera tricolor; Wimsatt and Enders, 1980), and several other megachiropterans (Selenka, 1892; Kohlbrugge, 1913; Keibel, 1922; Moghe, 1956). In rats and mice, Reichert’s membrane develops between the parietal endoderm and mural trophoblast and is generally considered to be secreted by the endoderm (Hogan et al., 1980, 1982, 1984; Mazariegos et al., 1987). Recent evidence indicates, however, that the mural trophoblast may also be involved in its initial formation (Salamat et al., 1995). Both the endoderm and cytotrophoblast probably contribute to the formation of Reichert’s membrane in Carollia. At the ultra- Fig. 19. Implanted blastocyst, day 23 p.c. Cavitation has progressed still further in this specimen, and the inner cell mass has differentiated into a curved epiblastic shield (EP) and amniotic ectoderm (AE). Apoptotic epiblast cells containing condensed and fragmented masses of chromatin are still evident in the primordial amniotic cavity. Some disorganized supraembryonic endoderm (E) persists, and Reichert’s membrane extends into two deep clefts (arrowheads) in the preplacenta (P). Some mesoderm (e.g., at arrows) is present between the epiblast (EP) and visceral endoderm (VE). YSC, yolk sac cavity. Amylase-PAS-hematoxylin. 3242. Fig. 20. Higher power view of Reichert’s membrane (R) inserting into the two clefts in the preplacenta shown in Figure 19. These clefts are lined by cytotrophoblast (C). The deepest points of penetration by Reichert’s membrane are indicated by the arrows. Although two villous projections of the cytotrophoblast (CV) extended much deeper into the preplacenta, these lacked obvious, central basal laminae. Syncytiotrophoblast (S) on the periphery of each villus has been labeled. E, endoderm; MBS, maternal blood space. Amylase-PAShematoxylin. 3521. structural level, Reichert’s membrane is most intimately associated with the cytotrophoblast of early blastocysts and, in the parietal wall of the yolk sac, consists of two layers. One layer appears to be the basal lamina of the trophoblast. The other layer may be of endodermal origin because similar material is commonly observed all around the endoderm cells and in cisternae of their rER. For reasons that are still unclear, the outer layer of Reichert’s membrane (corresponding to what is apparently the basal lamina of the cytotrophoblast) is not evident where this structure passes between the inner cell mass and polar trophoblast. Reichert’s membrane has also been observed dorsal and lateral to the inner cell mass in other phyllostomid bats (Glossophaga soricina and Macrotus californicus), a thyropterid bat (Thyroptera tricolor), and a noctilionid bat (Noctilio albiventris; Rasweiler, 1974; Bleier, 1975; Wimsatt and Enders, 1980; Rasweiler and Badwaik, 1996). The situation in Noctilio is of additional interest because, during initial development of Reichert’s membrane dorsal to the inner cell mass, no endoderm is evident in that region. Therefore, the supraembryonic portion of Reichert’s membrane in Noctilio must be a product of the epiblast and/or cytotrophoblast (Rasweiler and Badwaik, 1996). Masses and cords of basal laminalike material were also observed deep within the inner cell mass of Carollia and Noctilio blastocysts (Rasweiler and Badwaik, 1996). This finding raises the possibility that the material is secreted by the epiblast cells. Basal lamina material has been seen to a more limited extent between epiblast cells in baboon blastocysts (Enders et al., 1990). In view of the disposition of Reichert’s membrane in Carollia and Noctilio and its linkage to a meshwork of basal laminalike material at the endoderm–epiblast 100 N.K. BADWAIK ET AL. interface and extending into both layers, this structure probably plays an important mechanical role in tethering the inner cell mass and the embryonic shield to the preplacenta. This tethering may be important because, at least in comparison with primates like the human and rhesus monkey (Hamilton and Mossman, 1972; Luckett, 1978; Enders et al., 1986; Enders and King, 1988), these components of the embryo become unusually large in Carollia and Noctilio before mesoderm appears and contributes to the early development of a connecting stalk. In both bats, cytotrophoblast proliferates into the syncytiotrophoblast of the preplacenta in the form of villous projections. There are, however, substantial species differences in the structure of these cytotrophoblastic villi until at least the early primitive streak stage. In Noctilio, all of the villi consist of a bilayer of clearly polarized cytotrophoblast cells, with central basal laminae that stain prominently with the PAS reaction. These basal laminae appear to be continuous, or fused, with Reichert’s membrane in the clefts that develop on the interior of the preplacenta. In Carollia, Reichert’s membrane appears to end abruptly within the clefts. Furthermore, the cytotrophoblast cells of villi extending deep into the preplacenta are much less regularly arranged and, at least initially, lack basal laminae. The significance of these differences in cytotrophoblastic development in the preplacentae of these bats remains to be elucidated. Comparative studies of Carollia and Noctilio (Rasweiler and Badwaik, 1996) have now established that their early embryos differ in a number of very significant ways. In Noctilio, a typical inner cell mass only forms and becomes properly oriented after the initiation of implantation. Their embryos also exhibit differences in the distribution of endoderm, mode of formation of the yolk sac, and the differentiation of cytotrophoblast in the preplacenta. These differences are particularly noteworthy because Carollia and Noctilio are generally considered to be closely related, being assigned to the same superfamily (the Noctilionoidea; Koopman, 1994), and this classification is strongly supported by reproductive similarities observed between these species (Rasweiler, 1979, 1993). This study has established that amniogenesis in Carollia occurs by cavitation. This process involves both the apoptosis of some cells within the inner cell mass and the coalescence of spaces that develop between the epiblast cells. This process is very similar to what has been observed in other phyllostomid bats (Glossophaga: Hamlett, 1935; Rasweiler, 1974; and probably Desmodus: Wimsatt, 1954), Noctilio (Rasweiler and Badwaik, 1996), Thyroptera (Wimsatt and Enders, 1980), and several megachiropteran bats (Selenka, 1892; Kohlbrugge, 1913; Keibel, 1922; Moghe 1951, 1956; Heideman, 1989). In most other microchiropteran bats, a primordial amniotic cavity is formed within the inner cell mass. The epiblastic roof of this space is then lost, creating a trophoepiblastic cavity, and the definitive amnion is subsequently formed by folding (Gopalakrishna and Karim, 1979, 1980; Luckett, 1980). Although there are substantial differences between these two modes of amniogenesis, there are also some similarities. The black mastiff bat, Molossus ater, exhibits amniogenesis by the second method; however, formation of the primordial amniotic cavities in Molossus involves the radial arrangement and polarization of epiblast cells in the inner cell mass, as has been observed in Carollia. Furthermore, apoptosis appears to occur in the epiblast at this time in both species (Sansom, 1932; Rasweiler, 1990). ACKNOWLEDGMENTS The technical assistance of Yarka Chvojka, Anita Piccolie, Andrea O’Neill, and Simeon Williams in different aspects of this work is gratefully acknowledged. Thanks also go to the staff of the Department of Zoology, University of the West Indies, St. Augustine, Trinidad for their generous assistance and the use of departmental facilities during field work required for this study. This research was supported by departmental funds and National Institutes of Health grant HD-28592. Thanks are due to the United States Educational Foundation in India and the Council for International Exchange of Scholars for their support and assistance to N.K. Badwaik as a Visiting Scholar under the Fulbright Scholar Program and to the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo for a fellowship (grant 94/2348-1) provided for S.F. Oliveira. LITERATURE CITED Anderson, J.W., and W.A. Wimsatt 1963 Placentation and fetal membranes of the Central American noctilionid bat, Noctilio labialis minor. Am. J. Anat., 112:181-202. Bleier, W.J. 1975 Early embryology and implantation in the California leaf-nosed bat, Macrotus californicus. Anat. Rec., 182:237-254. 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