Ovulation Fertilization and Early Embryonic Development in the Menstruating Fruit Bat Carollia perspicillata.код для вставкиСкачать
THE ANATOMICAL RECORD 294:506–519 (2011) Ovulation, Fertilization, and Early Embryonic Development in the Menstruating Fruit Bat, Carollia perspicillata JOHN J. RASWEILER IV,1* NILIMA K. BADWAIK,2 1 AND KIRANMAYI V. MECHINENI 1 Department of Obstetrics and Gynecology, State University of New York Downstate Medical Center, Brooklyn, New York 2 Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York, New York ABSTRACT 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 ¼ 24 of 25; 96%), the preovulatory or newly ruptured follicle was in the opposite ovary. Anat Rec, C 2010 Wiley-Liss, Inc. 294:506–519, 2011. V Key words: ovulation; ovum maturation; fertilization; early embryology; menstruation; uterus Grant sponsor: The National Institutes of Health; Grant number: HD28592. *Correspondence to: John J. Rasweiler IV, Department of Obstetrics and Gynecology, State University of New York Downstate Medical Center, 450 Clarkson Avenue, Box #24, Brooklyn, NY 11203-2098. Tel.: 1-718-270-8262. Fax: 1-718-270-2067. E-mail: email@example.com C 2010 WILEY-LISS, INC. V Received 14 September 2010; Accepted 30 September 2010 DOI 10.1002/ar.21304 Published online 2 December 2010 in Wiley Online Library (wileyonlinelibrary.com). PERIOVULATORY EVENTS IN THE FRUIT BAT INTRODUCTION The short-tailed fruit bat, Carollia perspicillata, is a robust, moderate-sized bat (approximate adult weight: 14–24 g) found widely and often in abundance in forested areas of the lowland tropics of the New World. C. perspicillata also adapts readily to captivity, where it can be maintained and bred using simple and inexpensive husbandry procedures. Thus, C. perspicillata now provides a convenient laboratory representative for the bats, one of the largest (>1,116 species), but most inadequately studied, mammalian groups (Rasweiler et al. 2009). Many aspects of C. perspicillata’s reproductive and developmental biology are of interest. Like humans (in most cases), this bat is monovular, has a simplex uterus, exhibits true menstruation, displays interstitial implantation of the blastocyst within a preferred region of the uterus, has highly invasive trophoblast, and forms a discoidal, hemochorial placenta (Badwaik and Rasweiler, 2000; Rasweiler and Badwaik, 2000). By the primitive streak stage, C. perspicillata’s embryo has assumed a planar form, which is much more primatelike than the cup-shaped embryos of rodents (Rasweiler and Badwaik, 1997; Eakin and Behringer, 2004). C. perspicillata is also unusual among mammals, in that it sometimes takes embryos into prolonged periods of delay (diapause) after implantation, at the primitive streak stage. The delays can be either facultative or obligate, as they occur in response to stress in captivity and seasonally in the wild. These can last from weeks to months (Rasweiler and Badwaik, 1997; Badwaik and Rasweiler, 2001). The studies presented here focus on periovulatory events in captive-bred C. perspicillata. These include ovulation, fertilization, early embryonic development, and associated uterine changes. They expand on some of the earliest studies of Carollia sp. (de Bonilla and Rasweiler, 1974; Rasweiler and de Bonilla, 1992) and were pursued to provide a foundation for work on the reproductive endocrinology of female C. perspicillata. In the previous work, many of the animals were slow to breed and frequently the mated females failed to conceive. Far better breeding results were obtained in these studies. Finally, they provide additional, relevant observations on the same females in which sperm transport, temporary storage, and disposition have been examined (Rasweiler et al. 2010). This work is of broader signiﬁcance, because C. perspicillata is one of the few nonprimates conclusively shown to menstruate, and the adaptive value of this process has been subject to recent debate (Rasweiler and Badwaik, 2000). It is therefore of importance to establish the physiological circumstances under which menstruation occurs in C. perspicillata and its contribution to the reproductive ﬁtness of the species. MATERIALS AND METHODS Source of animals. The animals used in these studies were born and raised in a laboratory colony, to females of Trinidadian (West Indian) origin or stock. As far as is known, C. perspicillata is the only member of the genus Carollia occurring on that island. All bats uti- 507 lized for the studies of periovulatory events had been captive-reared and -bred. Animal maintenance. The captive colony was maintained in centralized animal facilities at the Weill Medical College of Cornell University and subsequently the State University of New York Downstate Medical Center in accordance with National Institute of Health Guidelines for the Care and Use of Laboratory Animals. Protocols for the studies were approved by Institutional Animal Care and Use Committees at these universities. The bats were kept in rooms with a controlled light cycle (12 hrs light: 12 hrs dark), and the dark phase was set to commence at 16:00 or 15:00 hrs at the respective institutions. The dark phase was set 1 hr earlier at the Downstate Medical Center in an effort to further minimize microbial growth in the bats’ diet after placement in their cages on weekends and holidays. The temperature was maintained between 24 C and 27 C. The bats were housed in bipartite cages, each having an open feeding area and a darkened roosting compartment. Both parts were large enough to permit the animals to ﬂy. Before being bred, the females were maintained in groups of 10–15 animals. The males were housed in groups of 8–12 animals or, more recently, singly (because some post-pubertal males will harass and injure other males in a caged environment). The bats were routinely fed a fruit-based diet prepared from readily available canned and powdered components (peach or apricot nectar, pureed 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 sliced apple, banana, or melon as treats. The animals were fed every night, and the diet was served cold, not more than 60–90 mins before the room lights went off, to minimize microbial growth. Water was provided ad libitum in chick waterers (Rasweiler and Badwaik, 1996; Rasweiler et al., 2009). Animal breeding and timing of reproductive stages. For breeding purposes, a single male with prominent testes was housed with each group of 10–15 females. This is in accord with the known propensity of C. perspicillata to organize into harems consisting of one male and an excess of females (Fleming, 1988). On subsequent mornings, between 5:30 and 8:30 AM, a small quantity of distilled water was aspirated in the vagina of each female with a microeyedropper (Rasweiler et al., 2009). The aspirate was then spread on a slide, dried, and examined for spermatozoa. The ﬁrst day on which spermatozoa were observed in an aspirate was considered to be day 1 post coitum (p.c.). Nonpregnant cycle length. As part of this study, observations were made on old corpora lutea (CLs), presumably from previous cycles, and the uterine condition of the bats. To further understand these, data are presented on nonpregnant cycle lengths exhibited by captive animals. These represent the intervals observed between the onset of successive periods of sperm-positive smears for animals that bred, failed to establish ongoing pregnancies, and then bred again. These data were collected from the records of all animals bred in the colony 508 RASWEILER ET AL. since its inception in 1991. Such data are relatively limited in number because, in recent years, mated animals have generally been transferred to new, female-only cages on day 5 p.c. Thus, many animals that failed to establish ongoing pregnancies would not have had the opportunity to soon breed again. Animal and tissue processing. To more precisely time ovulation, fertilization and the early stages of embryonic development, mated females were euthanized at 12-hr intervals (between 09:00 and 10:00 hrs or 21:00 and 22:00 hrs) on days 1–3 p.c. by administering an intraperitoneal injection of sodium pentobarbital at a dosage of about 90 mg/kg body weight. During dissection, the reproductive tract was removed as one unit above approximately the level of the vaginal-cervical junction. Manipulation of the tract during severance of its mesenteric attachments and transfer to ﬁxative was done primarily by holding the urinary bladder with a ﬁne forceps. There was no squeezing or manipulation of the uterus or oviducts. The tracts were ﬁxed in Zenker’s ﬂuid for 8–10.5 hrs, immersed in 2.5% aqueous potassium dichromate for an additional 2 hrs, washed overnight in running tap water, dehydrated through graded ethyl alcohols, cleared overnight in warm cedar wood oil (37 C) followed by Histosol (National Diagnostics, Atlanta, GA), and embedded in parafﬁn wax. The tracts were then serially sectioned in a frontal plane at 6 lm. The histological sections were stained with hematoxylin and eosin, or Weigert’s resorcin-fuchsin for elastic ﬁbers (Clifford and Taylor, 1973) followed by Masson’s trichrome procedure (modiﬁed from Humason, 1972). Powdered Weigert’s resorcin-fuchsin stain (#1A 294) was obtained from Chroma Gesellschaft Schmid Gmbh & Co (48161 Münster, Germany). In the version of Masson’s procedure used, 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 being stained with acid fuchsin and ponceau de xylidine, the wash time and water temperature (23 C) were precisely controlled in order to regulate cytoplasmic staining. Uterine measurements. The thickness of the endometrium (EN) was measured on the fundic and lateral walls of the uterine corpus. RESULTS Breeding success. Of the 33 females processed on days 1–3 p.c., six carried large preovulatory follicles (Table 1). Five of the follicles, found on day 1 p.c., appeared normal. The sixth animal (CW 26, day 2 p.c.) exhibited greater luteinization of the granulosa cells in its largest follicle than was the case with any of the normal preovulatory or postovulatory day 1 and 2 animals. This was sufﬁciently atypical to suggest that the follicle might not have been destined to rupture normally. Twenty-ﬁve of the 33 females processed on days 1–3 p.c. had recently ruptured follicles (RFs). Most of the reproductive tracts (23 of 25) from these animals appeared normal in all respects and carried a secondary oocyte, an ovum in the process of being fertilized, or an early cleavage stage embryo, all in the oviductal ampullae. Two of the 25 newly ovulated females were unusual. In one (CW 27), processed on the morning of day 2 p.c., the egg had already been transported into the uterus. Normally, embryos of C. perspicillata are transported into the uterus between days 11 and 13 p.c. (Oliveira et al., 2000). The other newly ovulated female (CW 21) had exhibited two periods of estrus. She carried an implanted blastocyst on the right side of her uterus and a pronuclear stage ovum in the contralateral oviduct (Rasweiler et al., 2010). Two of the 33 mated females were nonpregnant and possessed reproductive tracts inappropriate for their postcoital timings. In both cases, their oviducts exhibited no signs of normal periovulatory stimulation (i.e., no epithelial cell hypertrophy), and their endometria were compact with small epithelial cells and a very dense stroma. This indicates that females can sometimes be inseminated when not in a periovulatory condition. No further consideration will be given to these two specimens, and they have been omitted from Table 1. Final maturation, ovulation, and fertilization of the ovum. Although females with preovulatory follicles were collected on days 1–3 p.c., most (4 of 5) examined on the evening of day 1 p.c. had ovulated. One examined on day 2 (bat CW 26), that had not yet ovulated, possessed the luteinizing follicle. Another examined on day 3 p.c. (CL 99) had a normal preovulatory follicle, but probably would have ovulated late. A third bat (CW 32) examined on day 3 p.c. carried an oviductal secondary oocyte and had apparently become sexually receptive early or ovulated late. Major stages of ovum maturation and fertilization were obtained. One female (CP 40) processed on the morning of day 1 p.c. carried a preovulatory follicle with a primary oocyte at the germinal vesicle stage (Fig. 1A). This exhibited no expansion of the cumulus oophorus, little amorphous extracellular material therein, and only modest elongation of the cells of the corona radiata. Another female (CW 31) processed on the morning of day 1 p.c. carried a follicle with an oocyte in metaphase of the ﬁrst meiotic division (Fig. 2A,B). This and a similar follicle in another bat possessed more expanded cumuli, signiﬁcant extracellular material between the cumulus cells (CC), and elongated coronal cells. In follicles with ova in anaphase or telophase of the ﬁrst meiotic division or at the secondary oocyte stage, the cumulus oophorus was greatly expanded, exhibited pronounced muciﬁcation of its matrix, and had elongated coronal cells (Fig. 3A,B). All the preovulatory Graaﬁan follicles examined possessed large antra. This is not typical of all bat species (Rasweiler and Badwaik, 2000). Several females carried recently ovulated secondary oocytes or ova in the process of being fertilized. With the exception of one unusual oocyte (fertilization status uncertain) found in the uterine lumen on day 2, all of these were located in the ampulla of the oviduct and were loosely associated with their CC. Figure 4A depicts a secondary oocyte (found in female CP 18, day 1 p.c.), with the closest association of CC. When consideration is given to the probable shrinkage caused by histological processing, the cumulus–oocyte complex may TABLE 1. Ovulation, fertilization, embryonic development, and uterine condition in captive-bred fruit bats, Carollia perspicillata Postcoital time and animal # Ovarian status Total days housed with male Preovulatory Postovulatory Day 1 AM CW 1 10 CW 17 12 CW 25 CW 31 12 9 þ CP 40 8 þ þ Old CL present þ þ þ þ þ None þ Day 1 PM CR 16 9 þ None CP 17 CP 18 CW 24 10 17 15 þ þ þ þ þ þ CW 34 22 þ Day 2 AM CP 13 CW 26 6 6 þ CW 27 þ þ þ þ 12 þ CA only CW 28 CB 32 11 17 þ þ þ þa Day 2 PM CW 12 CW 18 7 9 þ þ 7 18b þ þ þ þ Condition of oocyte or embryo Secondary oocyte separating from follicle wall Telophase of second meiotic division Secondary oocyte Metaphase of ﬁrst meiotic division Primary oocyte, germinal vesicle stage Pronuclear stage Pronuclear stage Secondary oocyte Telophase of second meiotic division Anaphase of ﬁrst meiotic division Pronuclear stage Metaphase of ﬁrst meiotic division Dead ovum in uterine lumen Pronuclear stage Telophase of second meiotic division Uterine status Endometrial condition (thickness) Menstrual (370–640 lm) þ Menstrual (490–540 lm) þ Menstrual (510–830 lm) Shallow and proliferative (270–420 lm) Menstrual (420–490 lm) þ þ Shallow and proliferative (200–390 lm) Menstrual (270–640 lm) Menstrual (370–660 lm) Menstrual (270–610 lm) þ Menstrual (200–220 lm) Menstrual (490–510 lm) þ þ Shallow and proliferative (100–250 lm) Menstrual (510–640 lm) Shallow, late menstrual – early proliferative (320–420 lm) þ 15 18 þ þ þ þ 15 15 22 13 þ þ þ þ þ þ þ þa Two cell Eight cell Pronuclear stage Secondary oocyte CL 99 5 þ Telophase of ﬁrst meiotic division þ þ þ þ þ Eight cell Four cell Two cell Eight cell Eight cell Day 3 PM CR 2 CW 2 CW 6 CW 7 CW 9 14 23 5 14 21 þ þ þ þ þ þ þ þ CW 23 CY 63 Day 3 AM CW 8 CW 16 CR 21 CW 32 þ þ Shallow, late menstrual – early proliferative (170–220 lm) Menstrual (510–590 lm) Shallow, late menstrual – early proliferative (120–290 lm) Pronuclear stage Menstrual (510–540 lm) Pronuclear stage þ Most of endometrium early implanted shallow and proliferative blastocyst conceived (270 lm), but with during prior estrus small, superﬁcial areas of breakdown; endometrium at implantation site thick and healthy (660 lm) Pronuclear stage Menstrual (390–490 lm) Pronuclear stage Menstrual (510–760 lm) CP 19 CW 21 (also day 18 p.c.) Luminal dilatation þ 3 cell Probable Pronuclear stage CA þ þ þ Menstrual (440–490 lm) Menstrual (340–510 lm) Menstrual (540–740 lm) Shallow and proliferative (170–220 lm) Menstrual (510–710 lm) þ Menstrual (370–510 lm) Menstrual (440–760 lm) Menstrual (590–710 lm) Menstrual (370–490 lm) Proliferative with minor, residual epithelial sloughing (440–510 lm) þ Abbreviations: CA, corpus albicans; CL, corpus luteum. a Regression of the old CL was advanced. Many of its lutein cells had been lost or were severely shrunken, and few intact ones persisted. b Female CW 21 exhibited sperm-positive vaginal aspirates on days 1–4 and 17–18 after the introduction of a stud male (day 0 ¼ the day on which the male was added). Both estrus periods were associated with the ovulation and fertilization of single oocytes. 510 RASWEILER ET AL. Fig. 1. (A) Section of a preovulatory follicle, with its ovum at the germinal vesicle stage, found on the morning of day 1 p.c. in bat CP 40. (B) Section of menstrual uterus found in the same bat. This portion of the endometrium (EN) was devoid of a luminal epithelium, and sub- stantial tissue had been sloughed into the uterine lumen. Note the partially dissolved remnants of endometrial glands (around *). Hematoxylin and eosin. Scale bars ¼ 100 lm (A) and 50 lm (B). signiﬁcantly block the oviductal lumen at this stage, thereby facilitating successful fertilization. An ovum in telophase of the second meiotic division, found in female CW 17 (day 1 p.c.) was almost completely devoid of attached coronal cells (although many free CC were nearby). The ﬁrst pronuclear stage ova were observed on the evening of day 1 p.c. (Fig. 5A). The recently RF (Fig. 5B) that had released this ovum was evident in one of the ovaries. Pronuclear stage ova were found more frequently on day 2 p.c., although in one female embryonic development had progressed to the 3-cell stage. In each of these animals, CC (usually in small clumps) were scattered in the oviductal ampulla and isthmus. By the evening of day 2 p.c., 3 of 5 RFs had become redistended with ﬂuid and a variable amount of blood (Fig. 6). By day 3 p.c. most females (n ¼ 7 of 10) carried cleaving embryos in their oviductal ampullae (Fig. 7), with their CC clumped separately. On day 3 p.c., the RFs in 4 of 9 females were also redistended with ﬂuid or ﬂuid and blood. All of the RFs associated with cleaving embryos exhibited early luteinization of their granulosa cells. in the perivitelline space of ova or within the zonae pellucidae of early embryos. Supernumerary spermatozoa. A few supernumerary spermatozoa were observed near to, or probably bound to, the exterior surfaces of the zonae pellucidae surrounding three oviductal ova or embryos. One additional ovum, in the process of being fertilized, had a supernumerary spermatozoon partially embedded in its zona pellucida. Many spermatozoa were adherent to the zona pellucida of the ovum found in the uterine lumen on day 2 p.c. No supernumerary sperm were ever seen Evidence of female reproductive cycling and menstruation. Most periovulatory females (n ¼ 23 of 31) had large CLs (Table 1), that had presumably been formed in the previous, nonpregnant cycle (when breeding males were not present) and menstrual endometria (Figs. 1, 3–5, 7, 8). These CLs had lutein cells that were generally much smaller than those found in CLs around the time of implantation (Rasweiler and de Bonilla, 1992; Rasweiler and Badwaik, unpublished observations). On day 1 p.c., most of the lutein cells still appeared viable. By day 3, however, some cells (probably lutein) with pyknotic nuclei were also generally present in the CLs. In many of these animals, the EN was still quite thick (presumably reﬂecting growth that had occurred during the previous, nonpregnant cycle), but was being sloughed with associated bleeding (Table 1). In others, the EN had been substantially reduced in thickness, but abundant debris and blood were still evident in the uterine lumen (Figs. 1B, 3C, 4B). This menstrual debris normally undergoes extensive dissolution within the lumen. As a result, external or vaginal evidence of menstruation was usually absent or minimal. Occasionally, periovulatory vaginal aspirates were red-tinted with blood, and vaginal plugs (which were found and dislodged only rarely) exhibited some red surface coloration. Of the 26 females conﬁrmed to be menstrual in this study (Table 1), only ﬁve exhibited blood-tinged vaginal aspirates and then not on every PERIOVULATORY EVENTS IN THE FRUIT BAT 511 Fig. 2. (A) Section of a preovulatory follicle, with its ovum in metaphase of the ﬁrst meiotic division, found on the morning of day 1 p.c. in bat CW 31. (B) Higher power view of the ovum. The vitelline chromosomes are indicated by the arrowhead. (C) Section of the uterus from the same bat depicting its shallow, proliferative endometrium (EN), with an intact epithelial covering, and many spermatozoa in the uterine lumen. (D) Higher power view of spermatozoa (e.g., at arrowhead) and some intermingled leukocytes in the uterine lumen. Hematoxylin and eosin. M, myometrium. Scale bars ¼ 100 lm (A), 25 lm (B), 50 lm (C), and 20 lm. periovulatory day. Two females (CW 1 – day 1 p.c. and CW 2–day 3 p.c.) exhibited blood-tinged vaginal aspirates on the day before their ﬁrst sperm-positive aspirate. Endometrial thickness often varied considerably within individual uteri (Table 1) because of regional differences in the extent of endometrial sloughing. The overall thickness of the EN must have also been reduced 512 RASWEILER ET AL. Fig. 3. (A) Section of a preovulatory follicle, with its ovum in anaphase of the ﬁrst meiotic division and the cumulus-oocyte complex separating from the follicle wall. This was found on the evening of day 1 p.c. in bat CW 34. (B) Higher power view of the ovum. The vitelline chromosomes are indicated by the arrowhead. (C) Section of the uterus from the same bat depicting its late menstrual—early prolifera- tive endometrium (EN). This was generally shallow and only partially covered by a luminal epithelium (LE) that varied from being continuous to disorganized to completely absent (e.g., above *) from sizeable areas. Some residual sloughing of epithelial elements is evident here (e.g., at arrowheads). Hematoxylin and eosin. M, myometrium. Scale bars ¼ 100 lm (A), 25 lm (B), and 50 lm (C). Fig. 4. (A) Section of oviductal ampulla containing a secondary oocyte still loosely associated with its cumulus cells. This was found on the evening of day 1 p.c. in bat CP 18. The vitelline chromosomes (arrowhead) are in metaphase of the second meiotic division. (B) Sec- tion of the menstrual uterus found in the same bat. Much of the luminal epithelium and sizeable areas of the underlying endometrium (EN) had been desquamated. Hematoxylin and eosin. Scale bars ¼ 25 lm (A) and 50 lm (B). PERIOVULATORY EVENTS IN THE FRUIT BAT Fig. 5. (A) Section of a pronuclear stage ovum found on the evening of day 1 p.c. in the oviductal ampulla of bat CP 17. Some cumulus cells (CC) are nearby. (B) Section of ovary from the same bat showing the recently ruptured follicle (RF). (C) Section of the menstrual 513 uterus found in the same bat. The endometrium (EN) here is still relatively thick, but loss of the luminal epithelium and partial dissolution of the endometrial stroma are evident. Hematoxylin and eosin. UC, uterine cavity. Scale bars ¼ 25 lm (A), 100 lm (B) and 50 lm (C). somewhat in many menstrual uteri (n ¼ 15 of 21) on days 1 and 2 p.c. because of dilatation of their lumina with ﬂuid. Menstruation generally extended to day 3 p.c. (the latest stage examined), although in one animal (CW 9) this had been reduced to minor epithelial sloughing from an otherwise proliferative EN. Most animals (7 of 10) on day 3 p.c. carried cleaving embryos (Fig. 7). In many menstrual uteri, spermatozoa had penetrated the endometrial stroma, sometimes to a signiﬁcant depth (Fig. 8). Two females with relatively intact, old CLs were exceptional. In one (CW 34), the CL was more regressed than most observed during the periovulatory period. This bat had a shallow EN that was late menstrual to early proliferative (Fig. 3C). The other (CW 21) carried an implanted blastocyst, as well as an oviductal pronuclear stage ovum. The lutein cells in the corpus luteum associated with the blastocyst still appeared healthy and potentially secretory. The EN of this bat was intact and thick immediately around the implanted blastocyst, as would be typical of that stage of pregnancy. The remaining EN was shallow and proliferative, but included small, superﬁcial areas of endometrial breakdown. Some sperm had penetrated into the stroma in these areas. Females lacking intact, old CL. The remaining females (n ¼ 6 of 31) fell into three groups: (1) Two had old CLs with signiﬁcantly fewer (CB 32), or very few (CW 32), intact lutein cells. The ﬁrst animal had a shallow EN that was late menstrual to early proliferative, whereas the second had a shallow proliferative EN. (2) Two (CW 18 and CW 27) had corpora albicantia containing no intact lutein cells and shallow, proliferative endometria. (3) Two (CR16 and CW 31) had no regressing CLs and were presumably pubertal animals or coming out of an anestrous period. These females also had Fig. 6. Section of a redistended, ruptured follicle (RF) found on the evening of day 2 p.c. in bat CW 21. This was the origin of a pronuclear stage ovum in the ipsilateral oviductal ampulla. Hematoxylin and eosin. Scale bar ¼ 100 lm. 514 RASWEILER ET AL. Fig. 7. (A) Section of an eight-cell embryo and a mass of its cumulus cells (CC) found in the oviductal ampulla on the evening of day 3 p.c. in bat CR 2. Masson’s trichrome and Weigert’s resorcin fuchsin. (B) Section of the menstrual uterus found in the same bat. Its endometrium (EN) was still relatively thick, with a very dense stroma, and exhibited only small areas of stromal breakdown. Much of the luminal epithelium had been sloughed (e.g., above *), and abundant blood was present in the uterine lumen. Hematoxylin and eosin. Scale bars ¼ 25 lm (A) and 50 lm (B). Fig. 8. (A) Section of the menstrual uterus found on the morning of day 1 p.c. in bat CW 17. A major gap is evident in the luminal epithelium (LE) and some dissolution of the underlying endometrial stroma has occurred. Note that many spermatozoa are present in the uterine cavity (e.g., around *), and some had penetrated deeply into the semi- intact endometrial stroma to the level of the white arrowheads. (B) Higher power view of spermatozoa (e.g., at arrowhead) in the endometrial stroma to the left of the endometrial gland (G) also evident in A. This female carried an ovum in the process of being fertilized. Hematoxylin and eosin. Scale bars ¼ 25 lm (A) and 10 lm (B). 515 PERIOVULATORY EVENTS IN THE FRUIT BAT shallow, proliferative endometria (Fig. 2). In all six of these females, the uterine glandular epithelial cells generally appeared stimulated (i.e., more hypertrophied than during menstruation and with frequent mitotic ﬁgures), and their uterine lumina were dilated with ﬂuid. Evidence for spontaneous ovulation. All of the females with old CLs or corpora albicantia had been housed with males only for relatively short periods (5–23 days) (Table 1). It is apparent from studies of many early pregnant Carollia sp. that it takes about 13–14 days for CLs to develop to their full secretory appearance typical of the peri-implantation period (Rasweiler and de Bonilla, 1992; Badwaik et al., 1997, unpublished observations). It therefore seems likely that all of the regressing CLs observed in this study had been formed by spontaneous ovulation before the introduction of the males. Nonpregnant cycle length. Two lines of evidence obtained from other animals in the colony indicate that C. perspicillata has a moderately long reproductive cycle—in most cases probably in excess of 20 days. Some of these animals had bred, but failed to establish ongoing pregnancies. They then soon bred again and successfully became pregnant. These animals are best considered further in two groups: 1) Females in one group exhibited a period of spermpositive vaginal aspirates that commenced on the ﬁrst morning after they were housed with males. Such females frequently did not establish ongoing pregnancies in the ﬁrst estrus, possibly in some cases because breeding had been initiated too late for normal conceptions to occur (Rasweiler and Badwaik, 1996). Four females in this group exhibited intervals between the onset of successive periods of sperm-positive aspirates of 22, 22, 22, or 25 days. Because these females may have begun to breed late in their ﬁrst estrus, the interval between the onset of successive periods of estrus (and thus their nonpregnant cycle length) could have actually been slightly longer. 2) Females in the second group commenced breeding more than 24 hrs after being placed with males. Such females had a better chance of establishing ongoing pregnancies than those in the ﬁrst group (Rasweiler and Badwaik, 1996). Females in this second group exhibited intervals between the onset of successive periods of sperm-positive vaginal aspirates of 13, 19, 21, 22, 22, 24, 26, and 27 days. The normal duration of estrus in C. perspicillata is not known; however, spermatozoa have usually been seen in daily aspirates for 2–4 days. Rarely this has extended to a ﬁfth day or, in a very few cases, longer. Mated females are only infrequently found with vaginal plugs. The occasional discovery of new vaginal plugs on day 2 p.c., when they were not present on day 1 p.c., indicates that estrus can extend for more than 24 hours (Rasweiler et al., 2009). The breeding pattern observed after placing nonpregnant, adult females with stud males also suggests that C. perspicillata’s cycle is long and within the range indicated above. Most females breed within 1 month, although the onset of mating activity diminishes after about the 25th day with males (Table 1; also see Rasweiler and Badwaik, 1996). Alternation of successive ovulations. In most periovulatory females (n ¼ 24 of 31), the recent, regressing CL was in the opposite ovary from the preovulatory or newly RF. This was signiﬁcantly different (P < 0.001) from a random distribution of such CLs and follicles in the ovaries in successive cycles. In one exceptional case (CW 26), the CL was in the same ovary with a large preovulatory follicle that exhibited premature luteinization. Four other bats lacked a recent regressing CL and instead carried a much older CL (two bats) or a corpus albicans (two bats). Two bats carried no CLs. DISCUSSION In the initial efforts to breed Carollia in a research setting, many of the females were slow to mate, and the conception rate (even assuming that all of the preovulatory and newly ovulated females examined had become pregnant) would have been a rather poor 64% (de Bonilla and Rasweiler, 1974; Rasweiler and de Bonilla, 1992). New efforts were therefore initiated to breed C. perspicillata using modiﬁed husbandry procedures (Rasweiler and Badwaik, 1996). Females can now be bred much more readily—generally, within one month of placing nonpregnant adults with stud males—and the longterm conception rate for females bred in the colony is slightly more than 94%. Because several husbandry procedures were changed, it is impossible to identify with certainty why the breeding results are now better. Possible factors of importance include the following: (1) The early work was conducted with a colony that may have contained two different species, C. perspicillata and Carollia brevicauda (de Bonilla and Rasweiler, 1974). This raises the possibility that reproductive isolating mechanisms may have contributed to the suboptimal breeding results. This study involved only C. perspicillata. (2) In the early studies, efforts were made to breed wild-caught animals relatively soon (3–8 months) after capture. Some of these may not have been mature or adequately adapted to captivity when initially placed together to breed. In this study, all of the animals had been born and raised in captivity, although much-improved captive breeding results have also been achieved with wild-caught animals. (3) In the early studies, two or three males were placed with each group of females for breeding purposes. Discord between these males, housed together in a relatively small cage environment, may have interfered with breeding performance. It is now known that C. perspicillata organizes into harem groups that usually contain one mature male and from 2–10 females (Fleming, 1988). Since the establishment of the present colony, only a single mature male has been housed with each group of females for breeding purposes. Cycling females in this study also exhibited a higher incidence (96%) of alternation (or likely alternation) of successive ovulations between their two ovaries than the 78% observed previously (de Bonilla and Rasweiler 1974; Rasweiler, 1979). This may have been due to females in the recent work running more regular nonpregnant cycles and, as a result, to a stronger inhibitory inﬂuence of CLs upon folliculogenesis in the same ovary. 516 RASWEILER ET AL. Interestingly, one large preovulatory follicle found in this study exhibited abnormal early luteinization and may not have been destined to ovulate. This was located in the same ovary with a regressing CL. This study provides further evidence that C. perspicillata is a spontaneous ovulator with a functional luteal phase. Although all females had been killed within only 1–3 days of having commenced breeding activity, most carried regressing CLs and thickened (albeit menstrual) endometria. These must have been formed after spontaneous ovulations in the bats’ previous cycle. Most of these females had also been housed with breeding males for less than C. perspicillata’s probable nonpregnant cycle length, which appears in most cases to be about 21–27 days. Indeed, a number of females with old CLs and menstrual uteri had been housed with males for as little as 5–7 days. This and previous studies of captivemaintained Carollia sp., sexually isolated Glossophaga soricina, and unmated Molossus rufus (Rasweiler, 1979, 1988, 1991; Rasweiler and de Bonilla, 1992; Rasweiler and Badwaik, 2000) do not support the recent assertion that menstruation in microchiropteran bats is ‘‘coitus-dependent’’ (Zhang et al., 2007). In this study, in fact, four newly mated females had proliferative (rather than menstrual) uteri. In three other females, the uteri were in very late stages of menstruation, but this may have been attributable to more advanced regression of the CLs from prior cycles than to recent coital activity. Although endometrial sloughing and associated bleeding are extensive in C. perspicillata, most menstrual debris appears to undergo dissolution (observed histologically) within the uterine cavity. As a result, external signs of the menstruation (e.g., in the form of a vaginal discharge) have been observed only on very rare occasions and never during handling of the present series of animals. Sometimes vaginal aspirates were red-tinted with blood or vaginal plugs had some adherent blood; however, both were encountered too infrequently to provide a reliable indication that females were menstruating or of the duration of menstruation. Histological studies of the uteri collected in this study indicate that menstruation in C. perspicillata probably lasts for at least 3 days. Nearly all animals with old CLs examined on days 1 and 3 p.c. had menstrual uteri. Furthermore, in most of those examined on the morning of day 1 p.c., the process was already well under way. Two females had blood-tinged vaginal aspirates 24 hrs before their ﬁrst sperm-positive vaginal aspirates. As in many other mammals, ova are released from the ovaries of C. perspicillata as secondary oocytes, and fertilization occurs in the ampulla of the oviduct. In most other bats, for which data are available, fertilization also occurs in the ampulla. There are, however, several bat species in which it takes place sooner—in the ovarian bursa or possibly on initial passage into the oviduct (Uchida, 1953; Karim, 1975; Gopalakrishna et al., 1988; Krutzsch and Crichton, 1991). Adherence of the cumulus oophorus to the ovum in C. perspicillata appears to be quite tenuous, and by the initiation of fertilization this has largely been lost. This is a characteristic that varies considerably between mammals (Boyd and Hamilton, 1952; Austin, 1961; Bedford, 2004; Bedford et al., 2004) including bats. In some bat species, the cumulus oophorus surrounding the ovum becomes much reduced before ovulation, to a relatively thin corona radiata. In at least some of these, the corona appears to be lost soon after ovulation, although good temporal data are not always available (Gopalakrishna et al., 1974; Quintero and Rasweiler, 1974; Rasweiler, 1977; Mori and Uchida, 1981b; Ramakrishna et al., 1981; Gopalakrishna and Ramakrishna, 1983; Pendharkar and Gopalakrishna, 1983; Oh et al., 1985). In other bats, the CC are modestly more adherent after ovulation (Gopalakrishna and Khaparde, 1978; Rasweiler, 1982; Gopalakrishna et al., 1991). Finally, in some species of vespertilionid bats, preovulatory, and newly ovulated ova are surrounded by very prominent cumulus masses. In the case of the hibernating vespertilionids, glycogenrich CC are thought to play an important role in the prolonged maintenance of oocytes in Graaﬁan follicles (Pearson et al., 1952; Uchida, 1953; Wimsatt and Kallen, 1957; Rasweiler and Badwaik, 2000). A variety of functions have been proposed for the cumulus oophorus during and immediately after ovulation (Bedford, 2004). In light of its appearance in C. perspicillata, at a minimum, these might include facilitating extrusion of the small egg from the follicle, as well as its pick-up by the ﬁmbriae and transport into the oviduct. It may also function as a target or trap for the few spermatozoa passing into the ampulla, the site of fertilization. The diameter of the cumulus-oocyte complex soon after ovulation in C. perspicillata approaches that of the ampullary lumen. The block to polyspermic fertilization in C. perspicillata appears to lie at the zona pellucida, as supernumerary sperm were occasionally observed attached to the zona, but never within the perivitelline space. It must be cautioned, however, that only a few spermatozoa were ever seen in the oviductal ampullae of these animals (Rasweiler et al., 2010). Thus, the efﬁciency of establishing the zona block may not have been frequently tested in the specimens examined. The location of this block varies in bats. In Rhinolophus ferrumequinum, it seems to reside in the zona pellucida (Oh et al., 1985), whereas in Miniopterus schreibersii and Peropteryx kappleri it appears to develop at the level of the vitelline membrane. In the latter species, supernumerary spermatozoa were sometimes observed in the perivitelline space around fertilized ova (Mori and Uchida, 1981a; Rasweiler, 1982). Although there is no question that C. perspicillata exhibits true menstruation at the end of nonpregnant cycles, its periovulatory timing is unusual. This works successfully for C. perspicillata and some related bat species (e.g., Glossophaga soricina and Desmodus rotundus), presumably because they share several other reproductive adaptations. These include (1) a temporal overlapping of folliculogenesis, as well as preparation of the oviduct for fertilization and maintenance of the early embryo, with uterine regression, (2) a cycle in which most endometrial regrowth is postovulatory, and (3) holding the embryo in the oviduct for an unusually long time (until at least 11–12 days p.c. in the case of C. perspicillata) and to an advanced state of development (the expanded, zona-free blastocyst stage) during endometrial regeneration (Badwaik et al., 1997; Badwaik and Rasweiler, 2000; Oliveira et al. 2000; Rasweiler and Badwaik, 2000). Another bat, P. kappleri (family Emballonuridae), exhibits reproductive similarities and differences to PERIOVULATORY EVENTS IN THE FRUIT BAT C. perspicillata which are helpful in elucidating why some of these adaptations may have evolved (Rasweiler and Badwaik, 2000; Rasweiler, 1982). Although C. perspicillata has a simplex uterus, P. kappleri possesses a bicornuate uterus with long cornua. Both species are monovular and exhibit strong tendencies to alternate successive ovulations between their ovaries. C. perspicillata also usually shows preferential stimulation of the oviduct on the side of ovulation during the periovulatory period (de Bonilla and Rasweiler, 1974; Rasweiler et al., 2010), but no suggestion of a unilateral effect has ever been noted at the level of its simplex uterus. In contrast, P. kappleri exhibits both unilateral oviductal and endometrial reactions. Particularly during early pregnancy, preferential stimulation is evident at the prospective implantation site at the cranial end of the uterine cornu ipsilateral to the CL. It has been suggested that following ovulation and fertilization failures, or early embryonic losses, P. kappleri may then ovulate from the opposite ovary and locally stimulate the adjacent uterine cornu. This prepares the uterus for another attempt at establishing a pregnancy, but circumvents the problem of having an EN in the ﬁrst cornu that may be too highly differentiated for normal sperm transport, or the maintenance and implantation an early embryo. As C. perspicillata has a simplex uterus, this option is not available. Instead, following similar reproductive failures, C. perspicillata utilizes what appears to be the alternative strategy. It quickly eliminates the highly differentiated EN at the prospective implantation site by menstruating and develops a new one that would be properly synchronized with a new embryo. These differing patterns of renewing or recycling the uterus after reproductive failures would presumably be of considerable adaptive signiﬁcance to C. perspicillata and P. kappleri. As both species have low reproductive potentials (producing only single young after long gestation periods) and restricted breeding seasons (Rasweiler, 1982, 1991; Rasweiler and Badwaik, 2000; Badwaik and Rasweiler, 2001), the ability to quickly establish new pregnancies should enhance reproductive ﬁtness. In contrast to higher primates, C. perspicillata and P. kappleri exhibit endometrial cycles in which most growth is post ovulatory, and embryonic development within the oviducts is advanced (usually to the blastocyst stage in both species). These may be advantageous in at least two respects. C. perspicillata exhibits a postpartum estrus (Rasweiler and Badwaik, 1996). In the event of conception at such an estrus, prolonged retention of the embryo in the oviduct may provide the necessary time for the mother’s simplex uterus to involute and regenerate a new lining that is suitable for blastocyst implantation. Furthermore, both bats exhibit implantation within relatively small, predetermined endometrial zones. Development to the blastocyst stage in the oviduct may facilitate proper attachment on ﬁrst passage of the embryo into one of these areas (Rasweiler, 1982; Badwaik and Rasweiler, 2000; Oliveira et al., 2000; Rasweiler and Badwaik, 2000). In neither species have unattached embryos or implanting embryos ever been observed distal/caudal to the normal implantation zones (Rasweiler, unpublished observations). This may serve to ensure that placental development occurs at optimally vascularized sites (Badwaik and Rasweiler, 2000). In humans improper positioning of the implanta- 517 tion site too far caudally in the uterus is relatively common and can give rise to serious complications of pregnancy (e.g., partial or complete placenta previa). Menstruation has also been reported to occur in bats belonging to three other families—Molossus rufus (family Molossidae) (Rasweiler, 1991, 1992), Rousettus leschenaulti (family Pteropodidae) (Zhang et al., 2007), and Myotis ricketti (family Vespertilionidae) (Wang et al., 2008). Although M. rufus is usually monovular and possesses a bicornuate uterus, alternate preparation and usage of the two uterine cornua is not an option for this species in the event of reproductive failures. That is because ovulation only occurs from the right ovary in M. rufus, and its single conceptus is normally carried only in the right uterine horn. In the event of the types of reproductive failures discussed above, M. rufus deals with the problem of having a very highly differentiated, luteal phase EN at the prospective implantation site by menstruating like C. perspicillata. This may provide female M. rufus with another chance at establishing pregnancies at a still opportune time within the same breeding season. This would presumably contribute signiﬁcantly to the ﬁtness of females that may have a reproductive potential of only two young per year (Rasweiler, 1988, 1991, 1992). Reports of menstruation in R. leschenaulti and M. ricketti (Zhang et al., 2007; Wang et al., 2008) require conﬁrmation, as convincing histological evidence of extensive endometrial sloughing with associated bleeding has not been provided for either species. Vaginal bleeding was observed in all female R. leschenaulti collected in the wild on two dates separated by 33 days, but what this signiﬁed is unclear. If true menstruation, it would indicate a remarkable degree of reproductive synchronization for that species. Furthermore, it raises the question of why so many females in the wild colony were apparently running successive, nonpregnant cycles, particularly when both sexes usually live together (Bates and Harrison, 1997). Zhang et al. (2007) also illustrated the endometrial cycle of R. leschenaulti with transverse histological sections taken at undeﬁned levels from the uteri in captive-maintained animals. Although they asserted that the uteri cornua were symmetrical, R. leschenaulti is known to exhibit preferential stimulation of the cranial end of the cornu ipsilateral to the CL (Gopalakrishna and Karim, 1971; Gopalakrishna and Choudhari, 1977), as is the case in some other pteropodid bats (Marshall, 1953; Badwaik and Gopalakrishna, 1990). The section of a ‘‘menstrual’’ uterus provided by Zhang et al. (2007) appears to have been taken from the distal end of one of the uterine cornu and exhibits no sloughing or bleeding. Finally, they failed to address the hypothesis that R. leschenaulti and related species may use the alternation of successive ovulations between the ovaries and unilateral stimulation of the uterus as an alternative strategy to menstruation in coping with certain types of reproductive failures (Rasweiler, 1982; Rasweiler and Badwaik, 2000). The report of menstruation by M. ricketti (Wang et al., 2008) is equally problematic. The sections of a supposed menstrual uterus depict no clear evidence of endometrial sloughing and bleeding. Furthermore, apparently continous, but detached, epithelial layers running along the luminal side of the EN are not typical of menstrual 518 RASWEILER ET AL. uteri. These might actually be fetal membranes retained from an aborted pregnancy, which would be consistent with the date of specimen collection. It is important to clarify whether menstruation is widespread in the Chiroptera or not. If, as suggested, some bats menstruate and others do not (Rasweiler, 1982, 1991, 1992; Rasweiler and Badwaik, 2000), further comparative studies on members of this diverse order could ﬁrmly establish why menstruation evolved. In particular, more information is required on which bat families exhibit menstruation or unilateral stimulation of their uteri, how the latter are recycled in the event of reproductive failures (e.g., when precluded from breeding in captivity), endocrine controls, and how these adaptations serve to increase reproductive ﬁtness in the wild. Because some species are abundant in the wild and can also be maintained at reasonable cost in a research setting, bats are the one closely related group of mammals in which such studies are actually feasible. ACKNOWLEDGMENTS Thanks are due to the United States Educational Foundation in India and the Council for International Exchange of Scholars for their support of Dr. Nilima K. 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