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Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Jonathan L. Tilly, Ph.D., Massachusetts General Hospital, VBK137E-GYN, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: jtilly{at}partners.org
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-amanitin, 0.1 µg/ml) or protein synthesis
(cycloheximide, 1.0 µg/ml), whereas RA-mediated suppression of germ
cell apoptosis was not affected by cotreatment with either
macromolecular synthesis inhibitor (P > 0.05).
Moreover, cotreatment of fetal ovaries with 5 µM
LY294002, an inhibitor of phosphatidylinositol 3'-kinase, had no effect
on RA-promoted germ cell maintenance (P > 0.05).
By comparison, the antiapoptotic effects of insulin-like growth factor
I on germ cells in cultured fetal ovaries were significantly attenuated
by cotreating ovaries with LY294002 (P < 0.05) but
not with -amanitin or cycloheximide (P > 0.05).
Importantly, the effect of RA on the female germ line was also observed
in vivo because a single oral administration of 100
mg/kg RA to timed-pregnant female mice resulted in a significantly
(P < 0.05) larger endowment of primordial oocytes
in female offspring. That these actions were mediated, at least in
part, by specific retinoid receptors was demonstrated by the finding of
retinoic acid receptor protein in fetal female gonocytes, as assessed
by immunohistochemical localization experiments. Collectively, these
data indicate that RA can function, in vitro and
in vivo, as a potent germ cell survival factor and
mitogen during fetal oogenesis in the mouse. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the context of reproduction, vitamin A deficiency in male rats has
been known for many years to cause germ cell degeneration, whereas
female rats deprived of vitamin A are unable to reproduce, with
pregnancies typically ending in fetal death and resorption (11). More
recent work has shown that defective spermatogenesis in vitamin
A-starved male rats can be rescued by RA (12), suggesting that RA, and
not vitamin A per se, is the critical factor needed for germ
cell development. This proposal is supported by the findings of
extensive postnatal testicular degeneration in mutant mice lacking
expression of functional RA receptor (RAR)- (13) or retinoid X
receptor (RXR)-ß (14). Unfortunately, neither of these two studies
assessed the impact of RAR or RXRß mutation on development of the
female gonad, and very little else is currently known of RA actions in
female germ cells. It has been demonstrated in rats that RA inhibits
germinal vesicle breakdown in both denuded and cumulus-enclosed oocytes
(15). Moreover, RA has been reported to function as a potent growth
activator for murine primordial germ cells (PGC) maintained in
vitro without or with feeder cells (16).
To characterize factors involved in regulating female germ cell dynamics during fetal oogenesis, we recently developed an organ culture system for monitoring oogonium and oocyte proliferation and/or apoptosis in mouse fetal ovaries collected on embryonic day 13.5 (17). Using this system, we observed that apoptosis in germ cells caused by trophic hormone deprivation could be effectively suppressed by a combination of stem cell factor and leukemia inhibitory factor or by insulin-like growth factor I (IGF-I) alone (17). Furthermore, activation of the phosphatidylinositol 3'-kinase (PI3K) pathway, independent of p70 S6 kinase, is an essential component of female germ cell survival promoted by either stem cell factor and leukemia inhibitory factor or by IGF-I (17). In the present study, we employed this organ culture system to evaluate the possible role, and mechanisms of action, of RA in modulating fetal ovarian gametogenesis. Additionally, the ability of RA to alter primordial oocyte endowment in neonatal female mice following in utero exposure at embryonic day 13.5 was examined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Microsurgical isolation of genital ridges and organ cultures
Dissection of genital ridges was performed under sterile
conditions on e13.5, as originally described for studies of rodent
Müllerian duct regression (18) and as more recently detailed by
our laboratory (17). Once all cultures were prepared, two to three
genital ridges were immediately fixed as "Time 0" data points (see
below), and the remaining genital ridges were cultured for in a
humidified chamber gassed with 5% CO2–95% air in the
absence or presence of experimental treatments. In the first set of
experiments, cultures were carried out by treating ovaries for 72
h without or with 0.01–1.0 µM all-trans-RA
(Sigma Chemical Co., St. Louis, MO). To investigate the
mechanisms of action of RA in this model system, a second set of
experiments was performed by treating ovaries for 72 h without or
with 0.1 µM all-trans-RA in the absence or
presence of 0.1 µg/ml of the RNA polymerase II inhibitor,
-amanitin (Sigma Chemical Co.), 1.0 µg/ml of the
protein synthesis inhibitor, cycloheximide (Sigma Chemical Co.), or 5 µM of the PI3K inhibitor, LY294002
(Sigma Chemical Co.). The latter set of experiments with
LY294402 were included to determine if the seemingly broad requirement
for PI3K in female gonocyte survival mediated by other external stimuli
(i.e.. cytokines and growth factors; 17) is applicable to RA
as well. Selection of the final doses of inhibitors used was based on
previous studies (17, 19, 20), as well as on empirical dose-response
studies in our laboratory with fetal mouse ovaries in organ culture
(data not shown). As a control for potential macromolecular synthesis
inhibitor toxicity, fetal ovaries were also cultured without and with
50 ng/ml of human recombinant IGF-I (Promega Corp.,
Madison, WI) in the absence or presence of 0.1 µg/ml -amanitin or
1.0 µg/ml cycloheximide. This growth factor was selected based on our
recent observations that IGF-I, acting via PI3K, is a potent survival
factor for female gonocytes in cultured fetal ovaries (17).
Histology
Freshly isolated (Time 0, no culture) genital ridges or cultured
genital ridges at the conclusion of the experimental manipulation were
covered with 2% low-melting temperature agarose maintained at 44 C,
and the agarose was allowed to harden. The agarose-coated tissue was
then fixed in neutral-buffered 4% formaldehyde with 5% Bouin’s
fluid, dehydrated in ethanol, cleared in xylenes, embedded in paraffin,
and serially sectioned at 6 µm thickness. In some cases, every serial
section of the ovary was aligned in order on glass microscope slides
for hematoxylin and eosin (H/E) staining. These sections were used for
general histologic analysis of cellular morphology, as well as for
determination of germ cell counts (see below). In H/E-stained sections,
cells possessing lightly stained round nuclei along with a maintenance
of cytoplasmic volume and easily discernible spherical plasma membranes
were considered nonapoptotic, whereas cells showing nuclear
condensation (basophilia), cytoplasmic shrinkage, and convoluted plasma
membranes were considered apoptotic (21). As previously established for
studies of the fetal ovary (22, 23, 24), germ cells were distinguished from
somatic cells based on differences in cellular size (germ cells being
much larger than somatic cells) and morphology (germ cells and their
nuclei being spherical as opposed to somatic cells being squamous with
flattened nuclei). Furthermore, morphological identification of germ
cells was confirmed by alkaline phosphatase staining in preliminary
unpublished studies conducted during the validation of this culture
model (17).
Germ cell counts
Following histologic preparation and H/E-staining (see
Histology section), the total number of nonapoptotic germ
cells in sections taken at sites approximately one-third, one-half, and
two-thirds through the fetal ovary, along the long axis, were counted.
Each ovary was given a numerical code so that all germ cell counts were
conducted without knowledge of treatment group. After all counts were
completed, the mean number of germ cells per section was determined for
each ovary by taking the mean of the values from the three sections,
each ovary was decoded, and the values were then assigned to the
corresponding treatment group for analysis (17).
In situ DNA 3'-end labeling (ISEL)
The occurrence of apoptosis in germ cells was also assessed by
monitoring the presence of DNA fragmentation in situ, as
described previously (17, 25). Slides were analyzed by conventional
light microscopy after light counterstaining with hematoxylin, and
those cells exhibiting brown staining from the colorimetric reaction
were considered positive for DNA fragmentation (17, 25). Negative
controls, conducted by omitting the terminal deoxynucleotidyl
transferase enzyme, yielded no reaction product (data not shown).
Analysis of germ cell proliferation
To investigate further the effects of RA on mitosis, fetal
ovaries were cultured in the absence or presence of 0.1
µM RA, without or with 0.1 µg/ml -amanitin or 1.0
µg/ml cycloheximide, for 24 h, after which
5-bromo-2'-deoxyuridine (BrdU; Sigma Chemical Co.) was
added to each well at a final concentration of 30 µM. All
cultures were continued for an additional 2 h at 37 C
(pulse-labeling), after which tissues were fixed, embedded in paraffin,
section and analyzed by immunohistochemistry for sites of BrdU
incorporation as a marker of new DNA synthesis (26) under conditions
detailed previously (17, 27, 28, 29). Negative controls, conducted by
omitting the primary antibody, yielded no reaction product (data not
shown). Slides were analyzed by conventional light microscopy after
light counterstaining with hematoxylin, and those cells exhibiting
brown staining were considered positive for BrdU incorporation.
In vivo effects of RA on germ cell endowment
Single doses of vehicle (dimethylsulfoxide:corn oil, 1:4,
vol:vol) or 100 mg/kg RA were administrated to timed-pregnant female
mice on e13.5 by oral gavage (total volume of 0.4 ml/mouse), and the
mice were allowed to complete gestation and parturition. The dose of RA
used was based on previous studies of RA treatment in vivo
and was selected as one that does not cause caudal regression syndrome
(30). On the day of birth, neonatal ovaries were collected from female
offspring and fixed (0.34 N glacial acetic acid, 10%
formalin, 28% ethanol). Ovaries were then dehydrated, cleared in
xylenes, embedded in paraffin, and serially sectioned at 8 µm
thickness. Every serial section of the ovary was aligned in order on
glass microscope slides, stained and analyzed for the number of
primordial oocytes per section in every fifth section through the
entire ovary. Each ovary was given a numerical code so that all
primordial oocyte counts were conducted without knowledge of treatment
group. After all counts were completed, the total number of primordial
oocytes per ovary was calculated and used as a measure of germ cell
endowment (31, 32, 33).
Analysis of RAR and RXR expression
Fetal ovaries (or adult mouse uterus as a positive control; 34)
were processed for immunohistochemical localization of RAR and RXR
proteins using high-temperature antigen unmasking procedures detailed
previously (17, 27, 28, 29). For these experiments, a 1:50 dilution of a
rabbit polyclonal antiserum against the full-length human RAR
isoform, which detects all three isoforms of RAR (, ß, and ) of
human, mouse and rat origin (clone M-454; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a rabbit polyclonal
antiserum raised against the ligand binding domain of human RXR,
which detects all three isoforms of RXR (, ß and ) of human,
mouse and rat origin (clone 
N 197; Santa Cruz Biotechnology, Inc.) were used. Chromogenic detection of the sites of
antigen-primary antibody complexes was performed by incubating sections
for 1 h with a 1:200 dilution of a biotinylated goat antirabbit
IgG antibody (Vector Laboratories, Inc.), followed by
addition of avidin-biotin horseradish peroxidase complex components
(ABC kit; Vector Laboratories, Inc.) at 20 C for 45 min.
Sections were then washed and incubated with 0.5 mg/ml
3,3'-diaminobenzidine and 0.03% hydrogen peroxide for 1 min at 20 C,
and colorimetric reactions (generation of a brown reaction product)
were terminated by placing the slides in a buffer consisting of 10
mM Tris-HCl and 1 mM EDTA (pH 8.0). Negative
controls, conducted by omitting the primary antibody, yielded no
reaction product (data not shown). Slides were analyzed by conventional
light microscopy after light counterstaining with hematoxylin.
Data presentation and statistical analysis
In each experiment for the in vitro studies, two to
three genital ridges were used for each treatment group, and all
experiments were independently replicated at least three times. To
assess the in vivo effects of RA on primordial oocyte
endowment, four timed-pregnant female mice were used for each treatment
group, and one randomly selected ovary of the pair from one female
neonate in each litter was used to assess oocyte number. Therefore, the
quantitative data of germ cell numbers represent the mean ±
SEM of combined results obtained from analysis of a minimum
of six fetal ovaries or four neonatal ovaries in each treatment group.
One-way ANOVA was used to compare mean values of the various treatment
groups, followed by Scheffé’s F test to determine
significant differences at P < 0.05. Photomicrographs
of tissue histology (H/E-staining) or histochemistry (ISEL of DNA
integrity, immunohistochemical analysis of BrdU incorporation,
immunohistochemical analysis of retinoic acid receptors),
representative of results obtained in the replicate experiments, are
presented for qualitative analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and D). In vitro culture
of ovaries without trophic hormone support resulted in a time-dependent
increase in the number of germ cells displaying chromatin condensation,
cytoplasmic shrinkage, and DNA fragmentation such that most germ cells
remaining at 72 h were apoptotic (Fig. 1, B and E). Morphometric
analysis (germ cell counts) revealed that the number of healthy oogonia
and oocytes present in fetal ovaries after 72 h of culture was
significantly decreased to approximately 10% of the preculture (Time
0) mean number (Fig. 2).
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, C and F).
Furthermore, morphometric analysis indicated that the number of healthy
oogonia and oocytes remaining in ovaries after 72 h of culture
with 0.1 µM RA was 12.2 ± 1.0-fold greater than
that in untreated control ovaries cultured in parallel (Fig. 2).
Furthermore, the number of oogonia and oocytes in ovaries cultured for
72 h in the presence of higher doses of RA (0.1 and 1.0
µM) was significantly greater than preculture mean number
(P < 0.05) (Fig. 2), suggesting that RA not only
prevents apoptosis but also promotes mitosis in developing female germ
cells.
Stimulation of germ cell proliferation by RA
In fetal ovaries cultured for 24 h without RA, we could not
detect evidence of BrdU incorporation in cells of any section analyzed
(Fig. 1G). However, ovaries cultured in the presence of 0.1
µM RA possessed many BrdU-positive germ cells (Fig. 1H),
confirming that RA is a potent stimulator of germ cell proliferation in
cultured fetal ovaries.
Mechanisms of action RA in germ cells: mitosis vs.
apoptosis
The ability of RA to induce germ cell mitosis in cultured fetal
ovaries (Fig. 1H and Table 1) was
completely suppressed by cotreatment with 0.1 µg/ml -amanitin or
1.0 µg/ml cycloheximide (Table 1). However, the ability of RA to
prevent germ cell apoptosis was not affected by cotreatment with either
of these two macromolecular synthesis inhibitors (Table 1).
Morphometric analysis revealed that germ cell counts in fetal ovaries
cocultured for 72 h with RA and -amanitin or cycloheximide were
reduced to approximately 30% of those in fetal ovaries cultured for
72 h with RA alone (Fig. 3). In
contrast, LY294002 cotreatment did not alter the germ cell response to
RA following a 72-h culture (Fig. 3). As a control to test for
nonspecific toxicity associated with the macromolecular synthesis
inhibitors, we observed that IGF-I-promoted germ cell survival was not
altered by -amanitin or cycloheximide cotreatment (Fig. 3).
Furthermore, germ cell counts in ovaries treated with -amanitin or
cycloheximide were comparable to those obtained in control cultures
without inhibitor (P > 0.05) (Fig. 3).
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). Furthermore, gross histologic
analysis revealed no abnormalities in ovaries of neonates exposed to RA
during fetal gestation (Fig. 4C) as compared with vehicle controls
(Fig. 4B).
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, A and B), but not RXR (Fig. 5C), was
expressed in gonocytes at this stage of fetal development. In freshly
isolated ovaries, the majority of RAR was found in the cytoplasm of
germ cells (Fig. 5A), whereas a 24-h culture with 0.1 µM
RA caused a redistribution of RAR to the nucleus (Fig. 5B). In all
cases, low or no expression of RAR or RXR was observed in the
surrounding somatic cells (Fig. 5, A–C). To confirm that the RXR
immunostaining conditions were appropriate for detection of the
protein, RXR-positive stromal cells were easily detected in adult mouse
uterine tissue (Fig. 5D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-amanitin, and the protein synthesis inhibitor, cycloheximide,
abolish the effects on RA on oogonium proliferation but are without
effect on RA-mediated germ cell survival. Consequently, we can deduce
that out of the total number of germ cells present in fetal ovaries
cultured for 72 h with 0.1 µM RA, approximately
two-thirds of these are derived from mitogenesis (this is the
proportion of germ cells lost by cotreatment with macromolecular
synthesis inhibitors), whereas the remaining one-third are preserved as
a result of apoptosis inhibition.
The classic intracellular pathway of RA action involves receptors for
RA that mediate nuclear genomic responses. These receptors can be
classified into two major groups: RAR , ß, and ; and RXR ,
ß, and (35, 36, 37). Both RAR and RXR are ligand-dependent
transcriptional factors that homo- and heterodimerize to modulate
expression of target genes by binding with specific retinoic acid
response elements contained in the regulatory regions of these genes.
In addition to the receptors, however, there are two low molecular mass
cytoplasmic proteins involved in RA signal transduction that are
referred to as cellular RA-binding protein (CRABP) type I and CRABP
type II (38). The currently proposed functions of CRABPs include
"solubilization" of their hydrophobic ligand and, in some cases,
regulation of RA metabolism by restricting access of the vitamin A
metabolite to certain catabolic enzymes. One as yet unexplored possible
function for CRABPs may be comparable to that described for other
cytoplasmic steroid hormone receptors that transduce signals via a
variety of nongenomic actions including alterations in calcium flux and
cGMP accumulation (39, 40, 41, 42).
Many of the RAR and RXR isoforms, as well as CRABPs, are known to be expressed in the postnatal gonads and, in particular germ cells, of diverse species (for examples, see Refs. 43, 44, 45, 46). However, it remains to be determined if the reproductive defects observed in RAR and RXR mutant mice (13, 14) arise from a germ cell autonomous nature or indirectly via defective function the surrounding somatic cell lineages. Because germ cells express RARs, RXRs, and CRABPs, it may be that the genomic actions of RA observed herein (i.e. oogonium mitosis) are mediated via the classic nuclear RA receptors, whereas the antiapoptotic effects of RA are transduced via the cytosolic binding proteins that work independently of new gene expression. In support of the first stipulation of this hypothesis, we observed by immunohistochemical analyses that RAR, but not RXR, proteins were expressed in germ cells of fetal ovaries at e13.5. Interestingly, in freshly isolated gonads RAR immunoreactivity was essentially restricted to the cytosolic compartment of the gonocytes. Based on previous observations that unbound or free RAR exists in the cytoplasm (35, 47), these findings suggest that endogenous RA is low or absent at this time in fetal ovarian development. However, in vitro culture of fetal ovaries with RA resulted in nuclear translocation of RAR in gonocytes, consistent with the notion of receptor activation leading to new gene expression (35). Unfortunately, we were unable to obtain antibodies for CRABPI or -II to perform parallel investigations of fetal ovarian germ cell expression of these proteins. Moreover, because there are no reported CRABP-specific inhibitors or activators, generation of CRABP mutant mouse lines will be needed to provide the necessary tools to directly test the requirement for CRABPs as mediators of RA effects on female germ cell survival.
Of further interest, the antiapoptotic actions of IGF-I in female germ cells were not abrogated by inhibitors of new messenger RNA and protein synthesis, suggesting that the acute regulation of apoptosis in this model by diverse extracellular factors is executed via proteins and other factors preexistent in the developing oogonia and oocytes. Moreover, inhibition of PI3K with LY294002 (or wortmannin; data not shown) did not alter the mitogenic or survival response of germ cells to RA in cultured fetal ovaries, despite the fact that these inhibitors are known to be potent antagonists to fetal ovarian germ cell survival promoted by cytokines and IGF-I (17). Consequently, there must exist multiple signaling pathways, some requiring PI3K and others not, in developing female germ cells that are initially used as a response to external survival factors. However, based on our recent observations of a greater primordial follicle endowment in ovaries of neonatal female mice harboring a targeted disruption of a downstream death effector gene (i.e. Casp2; 29), it is likely that these early signaling pathways converge upon a final common pathway of apoptosis regulators in germ cells that either carry out or repress cell death execution (48, 49).
Lastly, it is important to note that the ability of RA to increase germ cell numbers in cultured fetal ovaries was replicated using an in vivo approach of RA exposure during fetal gestation. Aside from our recent observations with caspase-2 deficient mice (32), to our knowledge this is the only other report of an experimental manipulation that produces a surfeit of primordial oocytes in ovaries of neonatal mice. Furthermore, we observed no gross histopathologic alterations in ovaries of mice exposed to RA in utero, although it remains to be established if this increased primordial oocyte reserve will alter normal reproductive development and function, such as time to puberty and time to reproductive senescence. Based on our recent observations that the surplus of primordial follicles in young adult female mice produced as a result of Bax-deficiency does in fact lead to a dramatic extension of ovarian lifespan (33), these experiments will be important follow-up studies to support and extend the observations reported herein.
From these data, we conclude that RA can function as a potent survival factor and activator of germ cell proliferation in the developing fetal mouse ovary. Furthermore, the ability of in utero RA exposure to produce an overendowment of primordial oocytes in neonatal female mice supports the usefulness of the in vitro culture system and, more importantly, provides a unique model to explore the impact of excess follicle numbers at birth on functional lifespan of the female gonad. Specific modulation of new gene expression and protein synthesis by RA appears to play a crucial role in its actions as a mitogen for oogonia. However, the identity of the nongenomic intracellular pathways and mechanisms used by RA to prevent germ cell death during female gametogenesis remain to be elucidated.
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Acknowledgments |
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Footnotes |
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2 On leave from the Department of Obstetrics and Gynecology, Faculty
of Medicine, University of Tokyo, Tokyo 113, Japan, and supported by
the Japanese Society for the Promotion of Science.
Received September 29, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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