Embryonic staging of bats with special reference to Vespertilio sinensis and its cochlear development.

BACKGROUND: How bats deviate heterochronically from other mammals remains largely unresolved, reflecting the lack of a quantitative staging framework allowing comparison among species. The standard event system (SES) is an embryonic staging system allowing quantitative detection of interspecific developmental variations. Here, the first SES-based staging system for bats, using Asian parti-colored bat (Vespertilio sinensis) is introduced. General aspects of normal embryonic development and the three-dimensional development of the bat cochlea were described for the first time. Recoding the embryonic staging tables of 18 previously reported bat species and Mus musculus into the SES system, quantitative developmental comparisons were performed. RESULTS: It was found that limb bud development of V. sinensis is relatively late among 19 bat species and late limb development is a shared trait of vespertilionid bats. The inner ear cochlear canal forms before the semicircular canal in V. sinensis while the cochlear canal forms after the semicircular canal in non-volant mammals. CONCLUSIONS: The present approach using the SES system provides a powerful framework to detect the peculiarities of bat development. Incorporating the timing of gene expression patterns into the SES framework will further contribute to the understanding of the evolution of specialized features in bats.

Embryonic evidence uncovers convergent origins of laryngeal echolocation in bats.

Bats are the second-most speciose group of mammals, comprising 20% of species diversity today. Their global explosion, representing one of the greatest adaptive radiations in mammalian history, is largely attributed to their ability of laryngeal echolocation and powered flight, which enabled them to conquer the night sky, a vast and hitherto unoccupied ecological niche. While there is consensus that powered flight evolved only once in the lineage, whether laryngeal echolocation has a single origin in bats or evolved multiple times independently remains disputed. Here, we present developmental evidence in support of laryngeal echolocation having multiple origins in bats. This is consistent with a non-echolocating bat ancestor and independent gain of echolocation in Yinpterochiroptera and Yangochiroptera, as well as the gain of primitive echolocation in the bat ancestor, followed by convergent evolution of laryngeal echolocation in Yinpterochiroptera and Yangochiroptera, with loss of primitive echolocation in pteropodids. Our comparative embryological investigations found that there is no developmental difference in the hearing apparatus between non-laryngeal echolocating bats (pteropodids) and terrestrial non-bat mammals. In contrast, the echolocation system is developed heterotopically and heterochronically in the two phylogenetically distant laryngeal echolocating bats (rhinolophoids and yangochiropterans), providing the first embryological evidence that the echolocation system evolved independently in these bats.

Role of putrescine in ovary and embryo development in fruit bat Cynopterus sphinx during embryonic diapause.

The aim of this study was to evaluate the effect of putrescine on ovarian activity and the rate of embryonic development in Cynopterus sphinx during delayed development. The result showed the presence of a rate-limiting enzyme, ornithine decarboxylase-1, in both ovary and utero-embryonic unit of C. sphinx suggests a synthesis of putrescine in these sites. The corpus luteum showed increased, whereas utero-embryonic unit showed decreased production of putrescine during delayed development as compared with the normal development. The bat treated in vivo with putrescine during delayed development showed increase in progesterone and estradiol synthesis, correlated with increased expression of luteinizing hormone receptor, steroidogenic acute receptor protein, and 3ß-hydroxysteroid dehydrogenase through extracellular signal-regulated kinase (ERK1/2)-mediated pathway in the ovary; but showed increase in the weight and expression of progesterone receptor (PR), B-cell lymphoma 2, proliferating cell nucleus antigen, and vascular endothelial growth factor proteins in utero-embryonic unit. The in vitro treatment of putrescine showed stimulatory whereas treatment with an inhibitor of putrescine, 2-difluoromethylornithine caused an inhibitory effect on ovarian progesterone synthesis and cell proliferation, and cell survival in the utero-embryonic unit. In conclusion, the putrescine showed two separate roles during embryonic diapause, high concentration of putrescine in the ovary may support corpus luteum and basal synthesis of progesterone, whereas a low level of putrescine causes retarded embryonic development by inhibiting cell proliferation in the utero-embryonic unit. The bat treated with putrescine either directly promotes cell proliferation, cell survival, and angiogenic activities or acts indirectly increasing PR on utero-embryonic unit thereby activating development in delayed embryo in C. sphinx.

Normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum, with special reference to nose leaf formation.

The order Chiroptera (bats) is the second largest group of mammals, composed of more than 1,300 species. Although powered flight and echolocation in bats have attracted many biologists, diversity in bat facial morphology has been almost neglected. Some bat species have a "nose leaf," a leaf-like epithelial appendage around their nostrils. The nose leaf appears to have been acquired at least three times independently in bat evolution, and its morphology is highly diverse among bats species. Internal tissue morphology of nose-leaves has been investigated through histological analyses of late-stage fetuses of some bat species possessing the nose leaf. However, the proximate factors that bring about chiropteran nose-leaves have not been identified. As an initial step to address the question above, we describe the normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum, and examine development of the tissues associated with their nose leaf during embryogenesis through histological analyses. We found that the nose leaf of R. ferrumequinum is formed through two phases. First, the primordium of the nose leaf appears as two tissue bulges aligned top and bottom on the face at embryonic stages 15-16. Second, the sub-regions of the nose leaf are differentiated through ingrowth as well as outgrowth of the epithelium at stage 17. In embryogenesis of Carollia perspicillata, a phyllostomid species with a nose leaf, the nose leaf primordium is formed as a small tissue bulge on the nostril at stage 17. This tissue bulge grows into a dorsally projected thin epithelial structure. Such differences in the nose leaf developmental process between chiropteran lineages may suggest that distinct developmental mechanisms have been employed in each lineage's nose leaf evolution.

Postcranial heterochrony, modularity, integration and disparity in the prenatal ossification in bats (Chiroptera).

BACKGROUND: Self-powered flight is one of the most energy-intensive types of locomotion found in vertebrates. It is also associated with a range of extreme morpho-physiological adaptations that evolved independently in three different vertebrate groups. Considering that development acts as a bridge between the genotype and phenotype on which selection acts, studying the ossification of the postcranium can potentially illuminate our understanding of bat flight evolution. However, the ontogenetic basis of vertebrate flight remains largely understudied. Advances in quantitative analysis of sequence heterochrony and morphogenetic growth have created novel approaches to study the developmental basis of diversification and the evolvability of skeletal morphogenesis. Assessing the presence of ontogenetic disparity, integration and modularity from an evolutionary approach allows assessing whether flight may have resulted in evolutionary differences in the magnitude and mode of development in bats. RESULTS: We quantitatively compared the prenatal ossification of the postcranium (24 bones) between bats (14 species), non-volant mammals (11 species) and birds (14 species), combining for the first time prenatal sequence heterochrony and developmental growth data. Sequence heterochrony was found across groups, showing that bat postcranial development shares patterns found in other flying vertebrates but also those in non-volant mammals. In bats, modularity was found as an axial-appendicular partition, resembling a mammalian pattern of developmental modularity and suggesting flight did not repattern prenatal postcranial covariance in bats. CONCLUSIONS: Combining prenatal data from 14 bat species, this study represents the most comprehensive quantitative analysis of chiropteran ossification to date. Heterochrony between the wing and leg in bats could reflect functional needs of the newborn, rather than ecological aspects of the adult. Bats share similarities with birds in the development of structures involved in flight (i.e. handwing and sternum), suggesting that flight altriciality and early ossification of pedal phalanges and sternum are common across flying vertebrates. These results indicate that the developmental modularity found in bats facilitates intramodular phenotypic diversification of the skeleton. Integration and disparity increased across developmental time in bats. We also found a delay in the ossification of highly adaptable and evolvable regions (e.g. handwing and sternum) that are directly associated with flight performance.

Comparative Study of Prenatal Development Between Myotis albescens (Chiroptera: Vespertilionidae) and Eumops patagonicus (Chiroptera: Molossidae): The Chorionic Vesicle and Extraembryonic Membranes Considerations.

We presented a comparative study of two species of South American bats, Myotis albescens and Eumops patagonicus, about prenatal development. This study was carried out using 60 specimens, which were measured and photographed, and the embryonic stage was assigned by the staging system for Carollia perspicillata. We observed that the chorionic vesicle showed similarities in the disposition of the extraembryonic membranes, but they differed in characteristics of their yolk sac; in E. patagonicus, it was more glandular than M. albescens. M. albescens presented a well-developed discoid placenta with a caudal antimesometrial position, but E. patagonicus presented a diffuse placenta, which persists until the end of gestation and a discoid placenta in the uterus-tubal junction. In the embryogenesis, early stages, middle stages, and late stages were defined. In the early stage, the embryonic morphology is similar in the two species. The middle stage is characterized by the muzzle and pinna formation, fore and hind limb regionalization, and the formation of the patagium primordium. In the late stage, the overall growth of the embryo occurs. Its fore and hind limbs, patagium, and the typical craniofacial features are configured. We conclude that in early stages of development, the embryonic morphology of M. albescens and E. patagonicus is similar, while in late stages differences are evident; mainly the craniofacial structures and uropatagium configuration characteristics that allow their classification at the family level. Moreover, differences in time of fusion of maxillary and mandibular process were registered. This could be related to the morphology of the muzzle of each species. Anat Rec, 301:1527-1543, 2018. © 2018 Wiley Periodicals, Inc.

Prenatal cranial bone development of Thomas's horseshoe bat (Rhinolophus thomasi): with special reference to petrosal morphology.

Cochlear morphology has been regarded as one of the key traits to understand the origin and evolution of echolocation in bats, given its functionality and performance for receiving echolocation sonar. While numerous researchers have compared adult-stage morphology, few have studied the prenatal development of the cochlea. Here, we provide the first detailed three-dimensional description of the prenatal cranial development in bats, using Rhinolophus thomasi as a model, with particular interest to the petrosal which houses the cochlea. Results revealed that among all cranial bones the onset of the ossification of the petrosal is earlier in R. thomasi when compared to other reported mammals. Generally, the cochlea reaches adult size and shape before or around birth in placental mammals including bats, but we found that its shape and size growths continue until maturity in Rhinolophus species. The relationship of cochlear size and skull size is maintained constant throughout the postnatal ontogeny to adulthood in Rhinolophus, a pattern previously reported neither in any other bats nor other mammals. The peculiar developmental pattern in Rhinolophus possibly allows them to form their characteristically large cochlea and facilitate their distinctive echolocation behavior. A recent study reported that non-echolocating Pteropodidae shares a similar prenatal cochlear size to laryngeal echolocating bats. The apparent resemblance of fetal cochlear size was proposed to be a vestigial signal of large cochlear size in the last common ancestor of bats and thus as supporting evidence for the single origin of laryngeal echolocation. However, results from the present observations suggest that limited aspects of the cochlear development were captured in this previous investigation and that the resulting interpretations may be questionable. We point out that diversity and patterns of cochlear development among bats are still not resolved, and the controversy on the origins of laryngeal echolocation is still open to discussion.

Adiposity associated changes in serum glucose and adiponectin levels modulate ovarian steroidogenesis during delayed embryonic development in the fruit bat, Cynopterus sphinx.

The aim of the present study was to evaluate the mechanism by which embryonic development in Cynopterus sphinx is impaired during the period of increased accumulation of white adipose tissue during winter scarcity of food. The change in the mass of white adipose tissue during adipogenesis showed significant positive correlation with the circulating glucose level. But increase in circulating glucose level during the adipogenesis showed negative correlation with circulating progesterone and adiponectin levels. The in vivo study showed increased glucose uptake by the adipose tissue during adipogenesis due to increased expression of insulin receptor (IR) and glucose transporter (GLUT) 4 proteins. This study showed decline in the adiponectin level during fat accumulation. In the in vitro study, ovary treated with high doses of glucose showed impaired progesterone synthesis. This is due to decreased glucose uptake mediated decrease in the expression of luteinizing hormone-receptor, steroidogenic acute regulatory protein, IR, GLUT4 and AdipoR1 proteins. But the ovary treated with adiponectin either alone or with higher concentration of glucose showed improvement in progesterone synthesis due to increased expression of IR, GLUT4 and AdipoR1 mediated increased glucose uptake. In conclusion, increased circulating glucose level prior to winter dormancy preferably transported to white adipose tissue for fat accumulation diverting glucose away from the ovary. Consequently the decreased availability of adiponectin and glucose to the ovary and utero-embryonic unit may be responsible for impaired progesterone synthesis and delayed embryonic development. The delayed embryonic development in Cynopterus sphinx may have evolved, in part, as a mechanism to prevent pregnancy loss during the period of decreased energy availability.

Prenatal development in greater mouse-eared bat, Myotis myotis (Borkhausen, 1797) (Chiroptera, Vespertilionidae).

Order Chiroptera is the second largest mammal group after rodents. An understanding of the development of the bats, which is a very special mammal group in terms of their lifestyles, morphology and their ability to fly, is very important because most of the adult anatomical differences characterizing species occur during organogenesis. In this study, developmental stages were determined for Myotis myotis species based on external morphological characteristics from embryos obtained from wild-caught pregnant females. The developmental stages of M. myotis were comparable with those of other bat species.

Gonadal morphogenesis and establishment of the germline in the phyllostomid bat Sturnira lilium.

In vertebrates such as the mouse and the human, primordial germ cells (PGCs) arise at the base of the allantois and are carried to the epithelium of the posterior intestine, to later migrate to the primordial gonads. In the case of bats, almost nothing is known about this process. To clarify the dynamics of PGCs during gonadal morphogenesis in the phyllostomid bat Sturnira lilium, the proteins for the Ddx4, Sox9 and Mis genes were detected in the gonads of embryos at different stages of development. We identified 15 stages (St) of embryonic development in Sturnira lilium. We found that the formation of the genital ridge and the establishment of the undifferentiated gonad take place between stages 11 and 14. The onset of morphological differentiation in the gonad is first detected in the male gonads at St17. The first PGCs in meiosis are detected in the ovary at St19, whereas in the testicles, the PGCs were in mitotic arrest. Structural changes leading to testicular and ovarian development in Sturnira lilium are observed to be similar to those described for the mouse; however, differences will be established concerning the time taken for these processes to occur.

Prolactin modulates luteal activity in the short-nosed fruit bat, Cynopterus sphinx during delayed embryonic development.

The aim of this study was to evaluate the role of prolactin as a modulator of luteal steroidogenesis during the period of delayed embryonic development in Cynopterus sphinx. A marked decline in circulating prolactin levels was noted during the months of November through December coinciding with the period of decreased serum progesterone and delayed embryonic development. The seasonal changes in serum prolactin levels correlated positively with circulating progesterone (P) level, but inversely with circulating melatonin level during first pregnancy showing delayed development in Cynopterus sphinx. The results also showed decreased expression of prolactin receptor-short form (PRL-RS) both in the corpus luteum and in the utero-embryonic unit during the period of delayed embryonic development. Bats treated in vivo with prolactin during the period of delayed development showed significant increase in serum progesterone and estradiol levels together with significant increase in the expression of PRL-RS, luteinizing hormone receptor (LH-R), steroidogenic acute receptor protein (STAR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in the ovary. Prolactin stimulated ovarian angiogenesis (vascular endothelial growth factor) and cell survival (B-cell lymphoma 2) in vivo. Significant increases in ovarian progesterone production and the expression of prolactin-receptor, LH-R, STAR and 3ß-HSD proteins were noted following the exposure of LH or prolactin in vitro during the delayed period. In conclusion, short-day associated increased melatonin level may be responsible for decreased prolactin release during November-December. The decline in prolactin level might play a role in suppressing P and estradiol-17ß (E2) estradiol levels thereby causing delayed embryonic development in C. sphinx.

Transcriptomic and epigenomic characterization of the developing bat wing.

Bats are the only mammals capable of powered flight, but little is known about the genetic determinants that shape their wings. Here we generated a genome for Miniopterus natalensis and performed RNA-seq and ChIP-seq (H3K27ac and H3K27me3) analyses on its developing forelimb and hindlimb autopods at sequential embryonic stages to decipher the molecular events that underlie bat wing development. Over 7,000 genes and several long noncoding RNAs, including Tbx5-as1 and Hottip, were differentially expressed between forelimb and hindlimb, and across different stages. ChIP-seq analysis identified thousands of regions that are differentially modified in forelimb and hindlimb. Comparative genomics found 2,796 bat-accelerated regions within H3K27ac peaks, several of which cluster near limb-associated genes. Pathway analyses highlighted multiple ribosomal proteins and known limb patterning signaling pathways as differentially regulated and implicated increased forelimb mesenchymal condensation in differential growth. In combination, our work outlines multiple genetic components that likely contribute to bat wing formation, providing insights into this morphological innovation.

Role of adiponectin in delayed embryonic development of the short-nosed fruit bat, Cynopterus sphinx.

The aim of this study was to evaluate the role of adiponectin in the delayed embryonic development of Cynopterus sphinx. Adiponectin receptor (ADIPOR1) abundance was first observed to be lower during the delayed versus non-delayed periods of utero-embryonic unit development. The effects of adiponectin treatment on embryonic development were then evaluated during the period of delayed development. Exogenous treatment increased the in vivo rate of embryonic development, as indicated by an increase in weight, ADIPOR1 levels in the utero-embryonic unit, and histological changes in embryonic development. Treatment with adiponectin during embryonic diapause showed a significant increase in circulating progesterone and estradiol concentrations, and in production of their receptors in the utero-embryonic unit. The adiponectin-induced increase in estradiol synthesis was correlated with increased cell survival (BCL2 protein levels) and cell proliferation (PCNA protein levels) in the utero-embryonic unit, suggesting an indirect effect of adiponectin via estradiol synthesis by the ovary. An in vitro study further confirmed the in vivo findings that adiponectin treatment increases PCNA levels together with increased uptake of glucose by increasing the abundance of glucose transporter 8 (GLUT8) in the utero-embryonic unit. The in vitro study also revealed that adiponectin, together with estradiol but not alone, significantly increased ADIPOR1 protein levels. Thus, adiponectin works in concert with estradiol to increase glucose transport to the utero-embryonic unit and promote cell proliferation, which together accelerate embryonic development.

Differential expression of Meis2, Mab21l2 and Tbx3 during limb development associated with diversification of limb morphology in mammals.

Bats are the only mammals capable of self-powered flight using wings. Differing from mouse or human limbs, four elongated digits within a broad wing membrane support the bat wing, and the foot of the bat has evolved a long calcar that spread the interfemoral membrane. Our recent mRNA sequencing (mRNA-Seq) study found unique expression patterns for genes at the 5' end of the Hoxd gene cluster and for Tbx3 that are associated with digit elongation and wing membrane growth in bats. In this study, we focused on two additional genes, Meis2 and Mab21l2, identified from the mRNA-Seq data. Using whole-mount in situ hybridization (WISH) we validated the mRNA-Seq results for differences in the expression patterns of Meis2 and Mab21l2 between bat and mouse limbs, and further characterize the timing and location of the expression of these two genes. These analyses suggest that Meis2 may function in wing membrane growth and Mab21l2 may have a role in AP and DV axial patterning. In addition, we found that Tbx3 is uniquely expressed in the unique calcar structure found in the bat hindlimb, suggesting a role for this gene in calcar growth and elongation. Moreover, analysis of the coding sequences for Meis2, Mab21l2 and Tbx3 showed that Meis2 and Mab21l2 have high sequence identity, consistent with the functions of genes being conserved, but that Tbx3 showed accelerated evolution in bats. However, evidence for positive selection in Tbx3 was not found, which would suggest that the function of this gene has not been changed. Together, our findings support the hypothesis that the modulation of the spatiotemporal expression patterns of multiple functional conserved genes control limb morphology and drive morphological change in the diversification of mammalian limbs.

Patterns of postcranial ossification and sequence heterochrony in bats: life histories and developmental trade-offs.

The recently increased interest in studies on sequence heterochrony has uncovered developmental variation between species. However, how changes in developmental program are related to shifts in life-history parameters remains largely unsolved. Here we provide the most comprehensive data to date on postcranial ossification sequence of bats and compare them to various boreoeutherian mammals with different locomotive modes. Given that bats are equipped with an elongated manus, we expected to detect characteristic heterochronies particularly related to wing development. Although heterochronies related to wing development were confirmed as predicted, unexpected heterochronies regarding the pedal digits were also found. The timing of ossification onset of pedal phalanges is earlier than other mammals. Particularly, bats deviate from others in that pedal phalanges initiate ossification earlier than manual phalanges. It is known that the foot size of new born bats is close to that of adults, and that it takes several weeks to month until the wing is developed for flight. Given that the foot is required to be firm and stable enough at the time of birth to allow continued attachment to the mother and/or cave walls, we suggest that the accelerated development of the hind foot is linked to their unique life history. Since the forelimb is not mature enough for flight at birth and requires extended postnatal time to be large enough to be fully functional, we postulate that bats invest in earlier development of the hindlimb. We conclud that energy allocation trade-offs can play a significant role in shaping the evolution of development.

[Key morphofunctional transformations in the evolution of chiropterans (Bats, Chiroptera)].

Study on the morphology and morphogenesis of wing membranes in Bats has revealed some peculiarities in their structure and development. Understanding the embryogenesis of these animals, as well as attraction of data obtained on their molecular genetics and paleontology, allows one to single out some factors that could have initiated evolutionary modifications in development programs. A scenario of the key morphofunctional transformations in the forelimbs during the evolution of chiropterans is given.

Establishment of cell line from embryonic tissue of Pipistrellus ceylonicus bat species from India & its susceptibility to different viruses.

BACKGROUND & OBJECTIVES: Pipistrellus ceylonicus bat species is widely distributed in South Asia, with additional populations recorded in China and Southeast Asia. Bats are the natural reservoir hosts for a number of emerging zoonotic diseases. Attempts to isolate bat-borne viruses in various terrestrial mammalian cell lines have sometimes been unsuccessful. The bat cell lines are useful in isolation and propagation of many of the viruses harboured by bats. New stable bat cell lines are needed to help such investigations and to assist in the study of bat immunology and virus-host interactions. In this study we made an attempt to develop a cell line from P. ceylonicus bats. METHODS: An effort was made to establish cell line from embryo of P. ceylonicus species of bat after seeding to Dulbecco's modified eagle medium (DMEM) supplemented with 10 per cent foetal bovine serum; a primary cell line was established and designated as NIV-BtEPC. Mitochondrial DNA profile analysis was done using cyt-b and ND-1 gene sequences from the cell line. Phylogenetic tree was constructed using neighbour-joining algorithm for cyt-b and ND-1 genes with 1000-bootstrap replicates. RESULTS: NIV-BtEPC cell line was susceptible to Chandipura (CHPV) and novel adenovirus (BtAdv-RLM) isolated from Rousettus leschenaulti from India but did not support multiplication of a number of Bunyaviruses, Alphaviruses and Flavivirus. This might be useful for isolation of a range of viruses and investigation of unknown aetiological agents. INTERPRETATION & CONCLUSIONS: In this study, a new bat cell line was developed from P. ceylonicus. This cell line was successfully tested for the susceptibility to Chandipura and BtAdv-RLM virus isolated from bats. The approach developed and optimised in this study may be applicable to the other species of bats and this established cell line can be used to facilitate virus isolation and basic research into virus-host interaction.

The developmental basis of bat wing muscle.

By acquiring wings, bats are the only mammalian lineage to have achieved flight. To be capable of powered flight, they have unique muscles associated with their wing. However, the developmental origins of bat wing muscles, and the underlying molecular and cellular mechanisms are unknown. Here we report, first, that the wing muscles are derived from multiple myogenic sources with different embryonic origins, and second, that there is a spatiotemporal correlation between the outgrowth of wing membranes and the expansion of wing muscles into them. Together, these findings imply that the wing membrane itself may regulate the patterning of wing muscles. Last, through comparative gene expression analysis, we show Fgf10 signalling is uniquely activated in the primordia of wing membranes. Our results demonstrate how components of Fgf signalling are likely to be involved in the development and evolution of novel complex adaptive traits.

Early oogenesis in the short-tailed fruit bat Carollia perspicillata: transient germ cell cysts and noncanonical intercellular bridges.

The ovaries of early embryos (40 days post coitum/p.c.) of the bat Carollia perspicillata contain numerous germ-line cysts, which are composed of 10 to 12 sister germ cells (cystocytes). Variability in the number of cystocytes within the cyst and between the cysts (defying the Giardina rule) indicates that the mitotic divisions of the cystoblast are asynchronous in this bat species. Serial section analysis showed that the cystocytes are interconnected via intercellular bridges that are atypical, strongly elongated, short-lived, and rich in microtubule bundles and microfilaments. During slightly later stages of embryonic development (44-46 days p.c.), somatic cells penetrate the cyst, and their cytoplasmic projections separate individual oocytes. Separated oocytes surrounded by a single layer of somatic cells constitute the primordial ovarian follicles. The oocytes of C. perspicillata are similar to mouse oocytes and are asymmetric. In both species, this asymmetry is clearly recognizable in the localization of the Golgi complexes. The presence of germ-line cysts and intercellular bridges (although noncanonical) in the fetal ovaries of C. perspicillata suggest that the formation of germ-line cysts is an evolutionarily conserved phase in the development of the female gametes in a substantial part of the animal kingdom.

Ovulation, fertilization, and early embryonic development in the menstruating fruit bat, Carollia perspicillata.

To characterize periovulatory events, reproductive tracts were collected at 12 hr intervals from captive-bred, short-tailed fruit bats, Carollia perspicillata, on days 1-3 post coitum and examined histologically. Most bats bred readily. Graafian follicles developed large antra and exhibited preovulatory expansion of the cumulus oophorus. Ovulation had occurred in some on the morning, and in most by the evening, of day 1. The single ovum was released as a secondary oocyte and fertilized in the oviductal ampulla. Ovulated secondary oocytes were loosely associated with their cumulus cells, which were lost around the initiation of fertilization. Supernumerary spermatozoa were occasionally noted attached to the zonae pellucidae of oviductal ova, but never within the perivitelline space. By day 2, most ova had reached the pronuclear stage and by day 3, early cleavage stages. Several lines of evidence indicate that C. perspicillata is a spontaneous ovulator with a functional luteal phase. Most newly mated females had recently formed, but regressing corpora lutea, and thickened (albeit menstrual) uteri despite having been housed with males only for brief periods (<23 days). Menstruation is usually periovulatory in this species. Furthermore, the interval between successive estrus periods in most mated females that failed to establish ongoing pregnancies at the first was 21-27 days. Menstruation involved substantial endometrial desquamation, plus associated bleeding, and generally extended to the evening of day 3, the last time point studied. In nearly all females with a recent corpus luteum (n = 24 of 25; 96%), the preovulatory or newly ruptured follicle was in the opposite ovary.