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Polarized Ovaries of the Long-tongued Bat Glossophaga soricinaA Novel Model for Studying Ovarian Development Folliculogenesis and Ovulation.

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THE ANATOMICAL RECORD 290:1439–1448 (2007)
Polarized Ovaries of the Long-Tongued
Bat, Glossophaga soricina: A Novel
Model for Studying Ovarian
Development, Folliculogenesis,
and Ovulation
CAROLYN M. KOMAR,1* FRANCES ZACHARACHIS-JUTZ,1
CHRIS J. CRETEKOS,2 RICHARD R. BEHRINGER,2 AND JOHN J. RASWEILER IV3
1
Department of Animal Science, Iowa State University, Ames, Iowa
2
Department of Molecular Genetics, University of Texas, M.D. Anderson Cancer Center,
Houston, Texas
3
Department of Obstetrics and Gynecology, State University of New York Downstate
Medical Center, Brooklyn, New York
ABSTRACT
Glossophaga soricina is a spontaneously ovulating, monovular, polyestrous bat with a simplex uterus, exhibiting true menstruation. Studies conducted on reproductively active, captive-maintained animals established
that G. soricina also has polarized ovaries, with the ovarian surface epithelium (OSE) restricted to the medial side of the ovary, and primordial follicles
limited to an immediately adjacent zone. Follicles selected for further development are recruited from the medullary side of this zone, and ovulation is
restricted to the portion of the ovary covered by the OSE. To further develop
G. soricina as a model for studying ovarian development and physiology,
ovaries were collected from fetal, neonatal, and adult females and processed
for morphological and immunohistochemical analyses. The latter included
staining for factor VIII-related antigen (von Willebrand factor) to assess regional differences in ovarian vascularity. The ovarian structure in fetal and
neonatal animals was very similar to that in other species that do not have
polarized ovaries at comparable stages of development. This indicated that
polarization of the ovary does not occur during fetal development, but rather
sometime between the neonatal period and adulthood. Vascular elements
were abundant in areas of the ovary surrounding early growing follicles, but
sparse in the zone of the ovary containing primordial follicles. The polarized
nature of the ovaries in G. soricina suggests that this species might be used
as a model to investigate the formation, long-term maintenance, and activation of primordial follicles, and the role of the OSE in ovulation and folliculogenesis. Anat Rec, 290:1439–1448, 2007. Ó 2007 Wiley-Liss, Inc.
Key words: ovarian development; ovary; ovarian surface epithelium; ovulation; folliculogenesis
Grant sponsor: NSF foundation; Grant number: IBN
0220458; Grant number: GB-6435; Grant sponsor: NIH; Grant
number: HD00171; Grant number: TW00143; Grant sponsor:
the Population Council.
*Correspondence to: Carolyn M. Komar, Division of Functional Biology, West Virginia School of Osteopathic Medicine,
Ó 2007 WILEY-LISS, INC.
400 N. Lee Street, Lewisburg, WV 24901. Fax: 304-645-4859.
E-mail: ckomar@wvsom.edu
Received 17 April 2007; Accepted 22 August 2007
DOI 10.1002/ar.20602
Published online in Wiley InterScience (www.interscience.
wiley.com).
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KOMAR ET AL.
Glossophaga soricina is a microchiropteran bat and a
member of the family Phyllostomidae (American leafnosed bats). This bat is a common inhabitant of the lowland tropics of the New World from southern Sonora in
western Mexico, south to northeastern Argentina and
southeastern Brazil. They eat nectar, fruit, flower parts,
pollen, and insects (Rasweiler, 1975, 1977; Alveraz et al.,
1991). G. soricina is important ecologically; several
night-blooming plants rely on them for pollination (Ramirez et al., 1984; Silva and Peracchi, 1999; Zortea, 2003).
The physiology of reproduction in G. soricina is similar in many respects to that of anthropoid primates
(Hamlett, 1934, 1935; Rasweiler, 1974, 1979a; Rasweiler
and Badwaik, 2000). G. soricina is a polyestrous, monovular species with a simplex uterus. Ovulation occurs
spontaneously and can take place from either ovary. The
ovarian cycle is 22–26 days with a functional luteal
phase (Rasweiler, 1972). Nonpregnant cycles are terminated by true menstruation, characterized by extensive
breakdown and shedding of the superficial lamina functionalis of the endometrium with associated bleeding.
Menstruation is correlated with, and presumably caused
by, luteal regression. Implantation of the embryo is interstitial, and ultimately a discoidal, hemochorial placenta is formed. These animals can be easily maintained
in captivity, and captive females cycle throughout the
year (Rasweiler and de Bonilla, 1972; Rasweiler, 1973).
Observations presented herein indicate that the ovaries of G. soricina are also of interest because, in adult
animals, they exhibit an unusual polarity of structure
and function not seen in the more commonly studied
mammals. The ovarian surface epithelium (OSE) covers
only one part of the ovary, and the primordial follicles
are restricted to a zone underneath this epithelial layer.
As the follicles grow and mature, they occupy more of
the ovarian medulla. Interestingly, ovulation always
occurs in the area where the OSE covers the ovary. The
polarity of the ovaries in G. soricina suggests that this
animal may be a revealing model to study local factors
supporting maintenance of primordial follicles in a quiescent state, selection of follicles for further growth and
maturation, and development of the eventual site of
follicular rupture. The following study was initiated to
better define when the ovaries polarize and to further
develop G. soricina as a model for studies of ovarian
biology.
MATERIALS AND METHODS
Source and Maintenance of Captive Bats
In part to study ovulation and luteal development, tissues were collected from captive-maintained colonies of
G. soricina. Bats worked with in a colony established at
Cornell University (Ithaca, New York) had been captured on the West Indian island of Trinidad. Bats
worked with in a colony established at the Universidad
del Valle (Cali, Colombia) had been captured in rural
areas of the Departamento del Valle, Colombia. The animals were housed in bipartite cages of modest dimensions (with enclosed roosting and open feeding areas)
and fed fortified diets using canned peach nectar (Cornell University) or canned guava pulp (Universidad del
Valle) as palatable bases (Rasweiler and de Bonilla,
1972: Rasweiler, 1973, 1975). All of the work with captive-maintained animals was conducted before any
national laws in the United States or Colombia governing the care and use of laboratory animals. The work
was conducted humanely, however, using methods that
would now easily meet legislation of this type.
Tissue Collection
Reproductive tracts were collected from 36 sexually
segregated adult females killed at various times during
their 22–26 day cycle and 98 mated females killed
between days 1 and 32 post coitum (p.c.; Rasweiler,
1972, 1974). Animals were killed for these studies with
diethyl ether. Nonpregnant cycles had been timed by
using changes in vaginal cytology to identify the approximate time of ovulation. The distribution of cycling
specimens examined was as follows, with day 1 being
the first day on which a newly ovulated oocyte was present in one of the oviducts: day 1 (n 5 2 bats), 2 (1), 3
(1), 4 (1), 5 (1), 6 (1), 7 (2), 11 (2), 12 (2), 13 (3), 14 (2),
16 (1), 17 (2), 18 (1), 20 (3), 21 (2), 22 (2), 23 (4), and 24
(3). Mating was determined by the daily examination of
vaginal aspirates for spermatozoa (Rasweiler, 1972). The
first day of a sperm-positive aspirate (day 1 p.c.) correlated with the presence of a newly fertilized oocyte in
one of the oviducts. The distribution of pregnant specimens examined was as follows: day 1 p.c. (n 5 3 bats), 2
(3), 3 (2), 4 (3), 5 (2), 6 (2), 7 (2), 8 (2), 9 (2), 10 (2), 11
(2), 12 (3), 13 (3), 14 (8), 15 (7), 16 (5), 17 (2), 18 (5), 19
(2), 20 (5), 21 (2), 22 (3), 23 (2), 24 (6), 25 (2), 26 (3), 28
(5), 30 (4), and 32 (5). Some mated specimens were
found to be nonpregnant upon examination. These were
obtained on the following days: day 11 p.c. (n 5 1), 14
(1), 16 (1), 19 (1), 20 (1) 21 (1), 22 (2), and 24 (1). For
histological examination, each reproductive tract was
removed as a unit, with or without the vagina attached.
The tracts were manipulated during dissection and processing primarily by means of the urinary bladder and/
or the cervix and vagina, although a slit was also made
in the uterine isthmus to facilitate penetration of the fixative. There was no handling of the ovaries or oviducts.
Tissues were fixed in Helly’s, Carnoy’s, or Zenker’s fluid,
or acetic acid–mercuric chloride–formalin, and subsequently embedded in paraffin.
This work was undertaken primarily to confirm
reports, based upon field-collected specimens, that G.
soricina menstruated and exhibited interstitial implantation of a single blastocyst in a simplex uterus (Hamlett, 1934, 1935). This necessitated the development of
methods for introducing G. soricina into captivity, maintaining them in a reproductively active state, and timing
nonpregnant reproductive cycles and pregnancies. Histological studies were then pursued to (1) characterize
major ovarian, oviductal, and endometrial changes during the cycle and early pregnancy; (2) determine the distribution of Graafian follicles and corpora lutea between
the two ovaries (because bats frequently exhibit functional asymmetries of the ovaries; Rasweiler and Badwaik, 2000); and (3) characterize embryonic development
from fertilization through formation of the early embryonic shield in carefully timed pregnancies. Similar
studies had not previously been conducted with any
other bat in captivity. This strategy required a large
number of G. soricina for several reasons: (1) this species’ nonpregnant cycle proved to be relatively long (22–
26 days); (2) early embryonic development was unusu-
1441
POLARIZED OVARIES OF G. SORICINA
ally slow (implantation commencing between days 12
and 14 p.c.), and in some respects unusual, when compared with more commonly studied mammals; and (3)
there was some variability between animals in the timing of major reproductive events (Rasweiler, 1972, 1974,
1979a,b, 1993; Badwaik and Rasweiler, 2000). The polarized nature of G. soricina’s ovaries was discovered in the
course of this work. This played no role in determining
the number of animals used.
Female G. soricina were also collected from the field
on the island of Trinidad using techniques described previously for the closely related Carollia perspicillata
(with which G. soricina frequently roosts; Evarts et al.,
2004). Briefly, females were collected with hand nets in
their diurnal roosts during the months of January and
May 2004–2005. All procedures for collecting and handling animals in the field were approved by the Iowa
State University’s Institutional Animal Care and Use
Committee. The relative ages and sexual maturity of the
animals were determined by observations of pelage
(darker for juveniles than adults), area around nipples
(if bare, indicating current or recent lactation), and
gross examination of the reproductive tract. Neonatal
animals were identified as those still nursing and unable
to fly on their own. Animals with large, antral follicles
and/or luteal tissue in the ovaries were considered
adults. Females carrying young, or with bare nipples
from which milk could readily be expressed, were classified as lactating. These animals were euthanized by cervical dislocation.
Intact reproductive tracts were collected from neonatal
(n 5 3) and adult (n 5 12) animals, including 2 pregnant bats and their female fetuses. Tissues were fixed in
Bouin’s solution or 4% methanol-free formaldehyde buffered with 0.1 M Dulbecco’s phosphate-buffered saline at
48C overnight and then transferred into 70% ethanol.
After fixation, tissues were embedded in paraffin for
morphological or immunohistological analyses.
Morphological Analysis
Reproductive tracts from field-collected G. soricina
and fixed in Bouin’s solution were processed to paraffin
and sectioned at 5 mm. These sections were stained with
Gill’s hematoxylin (Fisher Scientific), or the periodic acid
Schiff procedure and counterstained with fast green. A
minimum of 4 tissue sections were examined from fetal,
neonatal, and adult field-caught animals. Tissue sections
chosen for analysis were collected from the central portion of the ovary, which represent more of the organ
than peripheral sections.
Tracts collected from the captive-maintained animals
were fixed as noted above, processed to paraffin, and
serially sectioned at 5–7 mm. The sections were stained
with a variety of routine histological and histochemical
techniques (Rasweiler, 1972, 1974). All slides were
mounted with Permount.
Serial sectioning was necessary to (1) adequately characterize advanced follicular development, ovulation, and
fertilization; (2) examine the distribution of normally
developing follicles, new and regressing corpora lutea,
within and between the two ovaries; (3) locate and
describe the single embryo at progressive stages of early
pregnancy; (4) determine the fate of the zona pellucida,
which is usually shed by the blastocyst and then
retained within one of the oviducts; (5) attempt to
account for all new eggs ovulated by mated females, as
well as the remnants of degenerating, ovulated ova typically retained in the oviducts from prior nonfertile
cycles; (6) compare corresponding segments of the two
oviducts, because G. soricina is unusual in exhibiting
their differential stimulation during the periovulatory
period; and (7) look for regional differences in endometrial development, because G. soricina apparently exhibits implantation only in very limited and predictable
uterine sites (Rasweiler, 1972, 1974, 1979a,b, 1993).
Immunohistological Analysis
Immunolocalization of factor VIII-related antigen (von
Willebrand factor) was used to enhance the visibility of
vascular elements and assess the relative vascularity of
various zones within the ovaries of G. soricina (n 5 4
adult animals). Paraffin-embedded tissues (5 mm) were
deparaffinized, rehydrated, and endogenous peroxidase
activity was quenched with 1.5% hydrogen peroxide in
methanol. Sections were rinsed in phosphate buffered
saline (PBS; pH 7.2) and subsequently blocked with 10%
normal goat serum (Zymed Laboratory), followed by
overnight incubation at 48C with antiserum directed
against factor VIII-related antigen (1:250; rabbit antihuman factor VIII-related antigen; Sigma). The primary
antiserum was omitted as a control. After rinsing
with PBS, sections were incubated with a biotinylated
secondary antibody (1:100; goat anti-rabbit), and subsequently incubated with a peroxidase/streptavidin complex (Vector). To visualize the immunocomplex, sections
were treated with 3,3-diaminobenzidine tetrahydrochloride (Zymed Laboratory). Sections were counterstained
with Gill’s hematoxylin, dehydrated, cleared, and mounted
with Permount.
Female reproductive tissues from fetal (n 5 1) and
adult (n 5 4) G. soricina were also processed for the
immunodetection of cytokeratins. Paraffin-embedded tissues were processed as described above with the following modifications. The primary antiserum used was a
mouse monoclonal antibody directed against Type II cytokeratins 1, 5, 6, and 8 from human epidermis (Sigma
catalog no. C1801). This antiserum has been shown to
cross-react with cytokeratins in tissues from a variety of
vertebrates (mammals, chicken, lizard, snake, and carp).
Tissues were incubated with the primary antiserum
diluted at 1:400 with diluent supplied in the Mouse on
Mouse Immunodetection Kit (Vector). For negative controls, the primary antiserum was omitted and tissues
were incubated in diluent alone.
RESULTS
The subject of this study, G. soricina, is a small microchiropteran bat (Fig. 1) that weighs approximately 9–12
grams when adult females are nonpregnant or in early
pregnancy (i.e., up to day 32 p.c., the latest stage of
pregnancy examined in captive-maintained specimens).
G. soricina’s small size is significant because it facilitates very thorough morphological studies of the reproductive tract and early embryos in situ that would be
impractical with many larger species. These bats are
specialized, in part, for hovering over night-blooming
flowers and taking nectar with an elongated tongue.
1442
KOMAR ET AL.
Fig. 1. An adult, female Glossophaga soricina carrying a neonate,
which appears underneath and is gray in color.
Figures 2–5 illustrate some major histological features
of G. soricina’s ovaries at fetal, neonatal, and adult
stages of development. The fetal ovary shown was obtained from a specimen corresponding to stage 22 of embryonic development for the closely-related bat, Carollia
perspicillata (approximately 80 days p.c. with a gestation length of 113–120 days; Cretekos et al., 2005). A
comparable, carefully-timed staging sequence has not
yet been generated for G. soricina beyond the early embryonic disc stage (Rasweiler, 1974). At this stage of development, the OSE (Fig. 2a), luminal epithelia of the
oviducts and uterus, and ordinary mesothelia of ovaries,
oviducts, and uterus were reactive with the pan-cytokeratin antiserum. The ovary contained an abundance of
primordial follicles, located principally in the cortex, and
a substantial epithelial component located in the medulla (Fig. 2a,b). The latter included anastomotic elements of the rete ovarii, as well as possibly some medullary tubules and early developing (primary) follicles,
containing oocytes. These epithelial cells generally
exhibited a high nuclear to cytoplasmic ratio and did not
contain much cytoplasm. In some cases, epithelial cells
surrounding oocytes had become cuboidal, but the
oocytes did not appear to have enlarged or exhibit other
signs of activation. The mesenchymal component of the
fetal ovary at this stage was relatively inconspicuous.
In neonatal females, primordial follicles still predominated in the cortex, and the ovaries were not yet polar-
ized (Fig. 3a). In contrast to the fetal ovary, the medulla
contained several activated oocytes (denoted by their
larger size; Fig. 3a,b), frequently clustered together
(Fig. 4). Most of these appeared to be within the rete
ovarii, as well as possibly some medullary tubules or primary follicles. Activation of oocytes appeared to occur
preferentially in or near elements of the rete ovarii. Particularly in sections stained with the periodic acid–Schiff
procedure, zonae pellucidae could also be seen around
many of these activated oocytes. During neonatal life,
many of the epithelial cells in the rete ovarii and adjacent medullary structures appeared more active than in
the fetal ovary (cf., Figs. 2b, 3b). These cells appeared
larger, with more cytoplasm and bigger, more euchromatic nuclei than in fetal ovaries. Also, the nuclear to
cytoplasmic ratio of these cells generally appeared to be
lower than in fetal life.
By the time females had reached adulthood, a striking
regionalization of the ovaries was apparent (Fig. 5).
Only a cap-shaped region on the medial surface of each
ovary was covered by the OSE (Fig. 6). This was equivalent to the mesothelial-derived, surface epithelium as
described by Mossman and Duke (1973). The remainder
of the ovary was covered by a less specialized, squamous
mesothelium (Fig. 6). It was highly unlikely that any of
the OSE might have been artifactually removed during
dissection and processing, because the ovaries were
never mechanically manipulated and were also well-protected within bursal sacs (albeit, each with an opening;
Rasweiler, 1972). The ovarian surface epithelial cap was
opposite the ovarian hilus. Primordial follicles were limited to a broad zone immediately adjacent to the OSE
(Figs. 5, 6). In all histological specimens examined, follicles that had been selected for further growth and development were observed only on the medullary side of
the primordial follicle zone. Follicles containing multiple
oocytes were also occasionally observed in ovaries from
adult G. soricina.
Despite that preovulatory follicles had large antra and
developed well within the ovaries of G. soricina (Rasweiler, 1972), ovulation ultimately took place only in the
region of the ovary covered by the OSE. This finding
was determined by observing follicle rupture points in
serial sections of the ovaries from a large series of nonpregnant, cycling, or early pregnant bats (Figs. 5, 7). In
females examined on days 1 or 2 of their cycle or pregnancy that had recently ovulated, rupture points (stigmata) could be readily identified on the ovarian surface.
Immediately after ovulation, the still partially swollen
cortex around the ruptured follicle was responsible for
some attenuation of the overlying surface epithelium
(Fig. 7); however, the presence of primordial follicles in
these swollen areas, to either side of the stigmata, indicated that this epithelium was most likely OSE (rather
than ordinary mesothelium). In sections containing
areas where the OSE had healed, the shape and proximity of the corpus luteum to the OSE indicated the probable rupture point (Figs. 5, 7). Often the corpus luteum
had a tapering neck, or was funnel-shaped, on this side.
In no case was there any suggestion that ovulation had
occurred through the extensive portion of the ovaries
covered by an ordinary, squamous mesothelium or their
broad mesenteric attachments. It is interesting to note
that this situation prevailed, even though preovulatory
Graafian follicles in G. soricina became very large and
1443
POLARIZED OVARIES OF G. SORICINA
Fig. 2. Sections of an ovary from a fetal Glossophaga soricina. a:
A fetal ovary and adjacent tissues processed for immunolocalization
of cytokeratins 1, 5, 6, and 8, counterstained with hematoxylin. As
depicted by the brown reaction product, the pan anti-cytokeratin antiserum labeled the ovarian surface epithelium (OSE) and ordinary mesothelium. Much of the ovarian cortex is occupied by primordial fol-
licles. Arrowheads denote epithelial components in the medulla. b:
The medulla and adjacent cortex depicting the anastomotic, epithelium-lined channels of the rete ovarii (arrow heads). Although some
oocytes (arrows) in this region are surrounded by a cuboidal epithelium, none exhibit clear signs of having been activated. Section was
stained with hematoxylin. Scale bars 5 250 mm in a, 50 mm in b.
sometimes closely approached this ordinary mesothelium
(see Fig. 1 in Rasweiler, 1972).
All of the ovaries from the adult, captive-maintained
G. soricina contained an abundance of atretic follicles,
many of which were large and antral. Three large follicles in relatively early stages of atresia are evident
along the bottom of the ovary shown in Figure 7a. Each
contained some granulosa cells with pyknotic nuclei, or
which were necrotic and had been sloughed off into the
antrum. In the case of the middle follicle, the cumulus
had also been completely lost from around the oocyte.
These are some of the generally accepted signs of atresia
in other mammals (Ingram, 1962; Peters and McNatty,
1980). The cumulus was still present in large, normal
Graafian follicles approaching ovulation (see Fig. 1 in
Rasweiler, 1972). It also deserves to be noted that the
ovary shown in Figure 7a had been obtained on day 1
p.c., soon after ovulation. G. soricina normally ovulates
only one follicle per cycle. All antral follicles persisting
in the ovaries on that day, or during the first couple of
weeks of the nonpregnant cycle and early pregnancy,
were invariably atretic (Rasweiler, unpublished observations).
Immunolocalization of factor VIII-related antigen was
used to visualize regional differences in the density of vascular elements within ovarian tissue from adult G. soricina.
Vascular elements were quite abundant in the stroma surrounding developing follicles (Fig. 8a,c). In contrast, such
elements tended to be sparse in the primordial follicle
zone (Fig. 8a,b) and were most frequently localized there
to the major stromal partitions between clusters of follicles.
DISCUSSION
The histological structure of the ovary in G. soricina
during fetal and neonatal life did not differ dramatically
from that found during comparable stages of development in mammals with more ‘‘typical’’ ovaries, such as
those in primates, ruminants, rats, and mice, with most
of the surface covered by OSE (Mossman and Duke,
1973; Konishi et al., 1986; Hirshfield and DeSanti,
1995). By the time the female bats reached sexual maturity, the ovary had been transformed into a polarized
structure with well-defined regions of follicles at progressive stages of development. The recruitment of follicles for development was consistently observed to occur
1444
KOMAR ET AL.
Fig. 3. Ovary from a neonatal Glossophaga soricina stained with
hematoxylin. a: Much of the ovary is still occupied by primordial follicles (pf). Some oocytes (arrows) in the rete ovarii, as well as possibly
medullary cords or primary follicles, had been activated and have
begun to grow. b: Higher power view of part of the rete ovarii (arrowheads). In contrast to the fetal condition (see Fig. 2a,b), many of the
rete epithelial cells appeared larger and more active. Scale bars 5
100 mm in a, 50 mm in b.
only in a narrow zone along the medullary side of the
area occupied by primordial follicles. Similarly, clear evidence of oocyte activation (as indicated by an increase in
their size) was observed only in the central part of the
ovary in neonatal females. There was no evidence of follicular activation and development within most of the
primordial follicle zone closer to the OSE.
The current study indicated that polarization of the
ovaries in G. soricina occurred between birth and adulthood. The mechanisms whereby this was accomplished
remain unclear, but two possibilities seem deserving of
consideration. As each ovary grew, its broad mesenteric
attachment did as well. This finding may have displaced
the OSE and primordial follicles from the mesenteric
side. Alternatively, or in addition, regional differences in
follicular atresia may have been involved. In some neonatal animals, the activation (early growth) of follicles
on the medullary side of the primordial follicle zone was
indeed seen. This pattern of follicular activation has
been reported to occur in other species, with these activated follicles subsequently undergoing atresia (Hirshfield and DeSanti, 1995).
Fig. 4. Ovarian medulla and adjacent cortex from a neonatal Glossophaga soricina stained with hematoxylin. Multiple oocytes are evident within epithelial-lined channels of the rete ovarii (arrows). Scale
bar 5 100 mm.
POLARIZED OVARIES OF G. SORICINA
1445
Fig. 6. Portion of ovarian cortex from an adult Glossophaga soricina stained with hematoxylin. An arrowhead shows where the ovarian
surface epithelium (OSE) covering the region containing primordial follicles changes to a more ordinary mesothelium (mt). Arrows indicate
growing follicles on the medullary side of the primordial follicle zone
(pf). Scale bar 5 100 mm.
Fig. 5. Ovary obtained from an adult Glossophaga soricina on day
22 of her nonpregnant cycle and stained with hematoxylin and eosin.
By this age, the ovary had become polarized, and primordial follicles
were restricted to a zone (pf) on its medial side. cl, corpus luteum.
The asterisk denotes a probable area of follicular rupture at ovulation.
Scale bar 5 100 mm.
There is evidence that the rete ovarii might be responsible for at least some of the follicular activation that is
observed during ovarian development. Hence, the rete
ovarii may contribute to ovarian polarization in G. soricina. In neonatal bats, epithelial cells of the rete ovarii
appeared more active histologically than in the fetal
specimens. This was suggested by the generally greater
size of these cells and their nuclei in the neonatal specimens. Furthermore, activated oocytes—characterized by
their increased size compared with oocytes in primordial
follicles—were noted in and near elements of the rete
ovarii. Previous studies have shown that the rete ovarii
and medullary cords may be involved in early follicular
development and oocyte activation in other mammals
(Byskov, 1975; Byskov et al., 1977; Rajah et al., 1992;
Hirshfield and DeSanti, 1995). Presumably in all of
these cases, where oocyte activation occurs early in life,
it is followed by atresia.
Ovulation can take place from anywhere on the ovarian surface in most mammals, but occurred only in the
region of the ovary covered by the OSE in G. soricina.
This phenomenon is not limited to G. soricina, but has
also been noted in equids (mares, donkeys, zebras), armadillos (pp. 25, 362, and 381 in Mossman and Duke,
1973), and the cuis (Galea musteloides)—a cavid rodent
(Weir and Rowlands, 1974). In these animals, only limited portions of the ovaries are covered by OSE, and
these are the usual sites of ovulation. There are other
species of micro- and megachiropteran bats that have a
restricted distribution of OSE and primordial follicles
(Rasweiler and Badwaik, 2000), raising the possibility
that ovulation may be limited to a specific region of the
ovary in these species as well. Interestingly, however,
studies of two species of bats belonging to the same family (Phyllostomidae) as G. soricina—Desmodus rotundus
(common vampire bat) and Carollia perspicillata (shorttailed fruit bat)—have established that their ovaries exhibit the more generalized pattern of ovarian organization common to most mammals (de Bonilla and Rasweiler, 1974; Quintero and Rasweiler, 1974). In these
two species of bats, the OSE is much more extensive,
and ovulation can apparently take place from most of
the ovarian surface (Rasweiler, unpublished data).
It has been unclear why the extent of the ovary covered by the OSE varies so markedly between mammalian species. Upon reflection, however, it seems reasonable that follicular rupture should occur at such a surface, because ovulation and internal fertilization are the
culmination of a highly coordinated and carefully timed
sequence of events. Therefore, any attempt by follicles to
rupture at or near the hilus might delay the process,
and/or decrease the likelihood of successful conceptions.
Mechanisms to direct ovulation away from the hilus
would seem to be particularly important when attachment to the broad ligament or mesovarium is broad, the
female normally releases only a single oocyte during
each cycle, and the species has limited breeding seasons.
1446
KOMAR ET AL.
Fig. 7. Parallel sections of an ovary from a captive-bred adult
Glossophaga soricina collected on day 1 post coitum (p.c.), stained
with Masson’s trichrome procedure. a: The black arrow illustrates the
point of ovulation through the primordial follicle zone (pf). The white
arrow denotes a tangential section through the neck portion of an
older, regressing CL (cl) from the previous cycle, also depicted in b.
This image does not show the full width of the neck (which was narrower than the body of the CL), but indicates that follicle rupture presumably had occurred through the overlying ovarian surface epithelium (OSE). b: The box denotes early growing follicles on the medullary side of the primordial follicle zone. b, bursa; rf, newly ruptured
follicle; mt, mesothelium. Scale bar 5 150 mm.
G. soricina clearly exhibits the first two characteristics
and at least some populations breed seasonally, with a
maximum female reproductive potential of 2 young/year
(Fleming et al., 1972; Willig, 1985; Zortea, 2003). That
these characteristics are shared to a large extent by a
variety of unrelated species with polarized ovaries raises
the possibility that the direction of ovulation toward the
OSE may be a primitive mammalian reproductive characteristic that has been retained to promote successful
oocyte escape from the ovary. These considerations may
explain why there is evidence that the OSE also influences ovulation in some of the more commonly studied
mammals, such as rabbits, sheep, and pigs.
In several species, the OSE has been identified as a
source of proteolytic enzymes involved in stigma formation (Cajander and Bjersing, 1975; Colgin and Murdoch,
1997; reviewed by Murdoch and McDonnel, 2002). Raw-
Fig. 8. Relative vascularity of ovarian zones in adult Glossophaga
soricina as revealed by the immunolocalization of factor VIII-related
antigen in vascular endothelial cells. Endothelial cells containing factor
VIII-related antigen have been labeled by the brown reaction product.
Tissues were counterstained with hematoxylin. a,c: Blood vessels
lined by reactive endothelial cells (examples identified by arrowheads)
were more common around growing follicles and in major stromal partitions between clusters of primordial follicles. b: Relatively few such
vessels were seen in among the primordial follicles. pf, primordial follicle zone. Scale bars 5 100 mm in a, 50 mm in b, 200 mm in c.
son and Espey (1977) investigated the role of the OSE in
ovulation by removing this epithelial layer from one
ovary in rabbits after mating, but before ovulation, leaving the second ovary as an intact control. In that study,
ovulation rates were reduced 47% and 45%, if the ovaries were denuded of the OSE 2 or 5 hr p.c., respectively
(Rawson and Espey, 1977). Removal of the OSE in other
species such as frogs, sheep, and pigs, also disrupted
ovulation (reviewed by Murdoch and McDonnel, 2002).
These findings illustrated that the OSE was involved in
the ovulatory process and that compromising its integrity reduced ovulation efficiency. Because of the polarization of its ovaries, G. soricina should provide a unique
model to further define the possible role(s) of the OSE in
controlling follicular development and rupture.
It is noteworthy that over 90% of ovarian cancers are
believed to be derived from the OSE (reviewed by Auersperg et al., 2001). Therefore, more thorough elucidation
of the function(s) of this epithelial layer may enhance
POLARIZED OVARIES OF G. SORICINA
our understanding of the causes of ovarian cancer. The
identification of additional, function-related markers for
the OSE might also ultimately lead to better detection
and treatment methods.
The ovaries of G. soricina are of further interest
because initial follicular activation and growth were limited to a very narrow area along the medullary side of
the primordial follicle zone and away from the OSE. The
basis for this finding remains speculative, but several
reasonable possibilities come to mind. As the primordial
follicle zone was found to be relatively undervascularized (present study; ovarian cortex; Herrmann and Spanel-Borowski, 1998), follicular activation along its
medial edge could be stimulated by a higher oxygen tension and/or exposure to blood-borne factors due to the
presence of more vascular elements in the medullary
region of the ovary. Some factor(s) required for follicular
activation might also be produced in the latter region by
the vascular endothelial cells. It is now accepted that endothelial cells in different regions can vary substantially
in their functional and synthetic capabilities. Finally, we
must consider the possibility that the OSE may play a
critical role in maintaining primordial follicles in a quiescent state. During ovarian development, the OSE may
induce the formation of a distinct zone or microenvironment that promotes maintenance of primordial follicles
and inhibits follicular activation. Alternatively, the OSE
may continue to produce a factor that inhibits follicular
activation.
In summary, the current study better defined several
unique characteristics of the ovaries in G. soricina.
Their polarized structure may be exploited to answer
important questions regarding ovarian development and
function. These would include the investigation of local
factors involved in the long-term maintenance of primordial follicles in a quiescent state, recruitment of select
follicles for growth and development, and how the OSE
influences the process of ovulation. Finally, this animal
model has the advantage of being readily maintained,
bred, and worked with at low cost in modest facilities.
ACKNOWLEDGMENTS
The authors thank the personnel in the Department
of Life Sciences, University of the West Indies, St.
Augustine, Trinidad (particularly Dr. Indira OmahMaharaj) for generously providing assistance and the
use of Departmental facilities during the course of our
fieldwork. We also thank Simeon Williams, Dr. Karen
Sears, Dr. Scott Weatherbee, and Dr. Lee Niswander for
field assistance. We thank the Wildlife Section-Forestry
Division, Ministry of Agriculture, Land and Marine
Resources of the Republic of Trinidad and Tobago for
providing required collecting and export permits. R.R.B.
was funded by the NSF foundation, and J.J.R. was
funded by a grant from the Population Council.
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