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Ovulation Fertilization and Early Embryonic Development in the Menstruating Fruit Bat Carollia perspicillata.

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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: john.rasweiler@downstate.edu
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.
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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.
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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.
Badwaik as a Visiting Scholar under the Fulbright
Scholar Program. The assistance of Yarka Chvojka in
caring for the animals is also gratefully acknowledged.
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