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REPRODUCTION-DEVELOPMENT |
Department of Anatomy (Y.I.), Institute of Basic Medical Sciences, University of Tsukuba, Ibaraki 305-8575, Japan; Comprehensive Reproductive Medicine (A.N.) and Section of Molecular Embryology (A.N., M.-a.I.), Graduate School, Tokyo Medical and Dental University, Tokyo 113-8549, Japan; and Graduate School of Integrated Science (S.H.), Yokohama City University, Yokohama 236-0027, Japan
Address all correspondence and requests for reprints to: Yayoi Ikeda, D.D.S., Ph.D., Department of Anatomy, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennohdai, Tsukuba, Ibaraki 305-8575, Japan. E-mail: yayoi-ik{at}md.tsukuba.ac.jp
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Introduction |
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Müllerian-inhibiting substance (MIS), also known as anti-Müllerian hormone, is expressed by Sertoli cells of the fetal testis and is an essential factor for male sexual differentiation, based on its ability to cause regression of the Müllerian duct, the anlagen of the uterus, oviducts, and the upper portion of the vagina, in the male (2). In the female, MIS is produced by granulosa cells in postnatal ovaries (3, 4, 5, 6). It is well known that MIS induces degeneration of granulosa cells, loss of oocytes, and appearance of testicular seminiferous tubule-like structures in the ovaries of freemartin, female fetuses that are exposed to excess MIS of a male twin by chorioallantoic anastomosis (7, 8). Similar ovarian degeneration was observed soon after birth in metallothionein-human MIS (MT-hMIS) transgenic mice that chronically overexpress human MIS (9). These reports suggest that an abnormally high MIS level inhibits ovarian differentiation. Recently, a role for MIS in the regulation of primordial follicle recruitment has been suggested by studies on the developing ovary of MIS-deficient mice (10). Thus, it is probable that MIS plays an important role in controlling follicular development during postnatal ovarian development (11, 12).
MIS gene expression appears to be regulated by several transcription
factors. Steroidogenic factor 1 (SF-1, officially designated NR5A1),
was originally found to be a transcriptional regulator of genes
required for steroid production (13, 14), but it also
activates MIS gene transcription in the fetal testis (15, 16). In recent studies, it has been further proposed that
transcriptional activation of the MIS gene by SF-1 is mediated through
synergistic interactions with other transcription factors such as WT-1
(Wilms’ tumor 1) (17), GATA-4 (a member of the GATA
family of transcriptional regulators) (18, 19), and
Sox9 (SRY HMG box related gene 9) (20, 21), in fetal
Sertoli cells. Because SF-1, WT-1, and GATA-4 are also expressed in
granulosa cells of postnatal ovaries (22, 23, 24, 25), these
transcription factors may play equally important roles in regulating
MIS gene expression in the ovary. Sox9 is normally not expressed in
postnatal ovaries. However, it has recently been reported that female
ß estrogen receptor knockout (ßERKO) mice, in which
both the ER- and ER-ß genes are disrupted, exhibited up-regulated
expression of MIS and Sox9 in the sex-reversed adult ovary
(26). This suggests that Sox9 expression in the ovary can
be induced under certain abnormal circumstances.
Neonatal female rats or mice exposed to a synthetic estrogen such as E2 benzoate (EB) or diethylstilbestrol, exhibit morphological and functional abnormalities in the adult ovary (27, 28). We previously reported that the expression levels of genes required for steroidogenesis were greatly reduced in the developing ovary of neonatally EB-exposed rats, suggesting that EB exposure inhibits theca/interstitial cell differentiation (29). The present study focuses on the molecular events accompanying the inhibition of follicular development; in this study, the expression of MIS and several of its related transcription factors in granulosa cells of the ovary after neonatal exposure to EB is examined. Our results suggested that the inhibitory effect of EB on early follicular growth is due to altered expression of MIS and SF-1, providing insights into a possible mechanism by which EB causes a delay in ovarian development and also a molecular mechanism that regulates postnatal ovarian development.
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Materials and Methods |
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Antibodies
For immunohistochemical analyses, the following antibodies were
used. The goat polyclonal MIS antibody (catalog no. sc-6886;
Santa Cruz Biotechnology, Inc., Santa Cruz, CA) is raised
against a carboxy terminal peptide of the precursor form of human MIS.
The rabbit polyclonal WT-1 antibody (catalog no. sc-192; Santa Cruz Biotechnology, Inc.) is raised against a carboxy terminal
peptide of human WT-1. The goat GATA-4 antibody (catalog no. sc-1237;
Santa Cruz Biotechnology, Inc.) is raised against a
carboxy terminal peptide of mouse GATA-4. The two rabbit polyclonal
antibodies to ER-ß, antibody no. PC168 (Oncogene Research Products, Boston, MA) and antibody no.
PAI-310 (Affinity BioReagents, Inc., Golden, CO), are both
raised against a synthetic peptide corresponding to the carboxy
terminal amino acid residues 467–485 of the rat ER-ß protein. For
detection of SF-1, the same antibody generated in a previous study
(29) was used.
In situ hybridization
Sense and antisense riboprobes for MIS (15), SF-1
(13), Sox9 (30), and ER-ß (31)
were generated with T7, T3, and SP6 RNA polymerases and a SureSite T7
RNA Probe Kit (Novagen Inc., Madison, WI). In situ
hybridization was performed using a SureSite Hybridization Reagents Kit
(Novagen). In brief, sections were dewaxed in xylene, rehydrated in
graded ethanols, treated with 0.2 N HCl, digested
with proteinase K (1 µg/ml) for 5 min, and acetylated with 0.25%
acetic anhydride. After prehybridization, the sections were hybridized
with the respective probe at 50 C overnight. Then, the sections were
rinsed in 2x SSC (standard saline citrate), treated with RNase
A (20 µg/ml) for 30 min, washed once in 2x SSC and twice in 1x SSC,
and air-dried. Sections were dipped in Kodak NTB-2 nuclear
tracking emulsion (Eastman Kodak Co., Rochester, NY) and
exposed at 4 C for 1 or 2 wk. The sections were developed in
Kodak D19, fixed in Kodak fixer, and
counterstained with methyl green (Sigma). To ensure that
in situ hybridization procedures were carried out under the
same conditions, sections from the control and treated animals were
mounted on the same slides. In situ hybridization analysis
was performed at least twice per tissue sample using each probe, and at
least two different samples were used per data point. Control studies
were performed using the sense probe and no signals higher than the
background level were detected.
Quantification of MIS mRNA levels
To quantify the MIS mRNA level, sections were observed
under bright-field illumination using an Olympus Corp.
AX80T microscope and an objective with a x60 magnification. The silver
grains in 10 random fields in each follicle were counted. Background
counts were determined by counting silver grains in 10 random fields in
regions of the same sections that were outside the ovary, and the
average of the grain counts in these regions was deducted from the
grain count in the follicle. The silver grains were counted at least
three times from three different litters for each treatment group, and
the group mean ± SEM was calculated.
Histology and immunohistochemistry
Sections were dewaxed in xylene and rehydrated in graded
ethanols. For histological examination, sections were stained with
hematoxylin and eosin (HE). For immunohistochemical analyses, sections
that were to be incubated with the antibodies against WT-1, GATA-4,
ER-ß, or SF-1 were subjected to antigen retrieval by microwaving in
10 mM citrate buffer (pH 6.0) on full power for 5 min, and
then cooled to room temperature. After washing in PBS, endogenous
peroxidase activity was blocked by immersing the sections in 3%
(vol/vol) hydrogen peroxidase in PBS for 5 min. The sections were
washed again in PBS and incubated in a blocking solution containing 5%
BSA in PBS at room temperature for 30 min. The sections were
subsequently incubated with the primary antibody diluted appropriately
with the blocking solution overnight at 4 C. The primary antibodies
included rabbit anti-SF-1 (1:10000 dilution), goat anti-MIS (1:5000
dilution), rabbit anti-WT-1 (1:50000 dilution), goat anti-GATA-4
(1:5000 dilution), and rabbit anti-ER-ß (1:200 or 1:500 dilution).
Biotinylated secondary antibodies (Vector Laboratories, Inc., Burlingame, CA) were detected using a
Vectastain ABC Elite kit (Vector Laboratories, Inc.) and a VectaDAB or NovaRED substrate kit (Vector Laboratories, Inc.). Negative controls were prepared by
substitution of the primary antibody with normal rabbit or goat IgG and
by omission of the primary and/or the secondary antibody. No staining
above the background was detected. The specificity of the MIS antibody
was further ascertained by incubating adjacent sections with the
antibody preabsorbed with the peptide against which the antibody had
been raised. Preabsorption of the MIS antibody with the immunizing
peptide abolished immunostaining for MIS in both the control and
EB-treated ovaries. To minimize the variation in staining, sections
from the control and treated animals of the same age were mounted on
the same slide, and immunohistochemical procedures for sections from
groups of different ages were carried out in parallel.
Quantitative evaluation of follicular development
Quantitative evaluation of follicular development in the
ovaries of the control and EB-treated P6 rats was performed. The entire
ovarian area in 6-µm-thick paraffin sections stained with HE was
observed under an Olympus Corp. (Hamburg, Germany) AX80T
microscope with an objective with x40 magnification. Secondary
follicles were counted in every 10th section of serial sections of an
ovary and are indicated as the sum. Each column in Fig. 1
represents the mean ±
SEM for five (Oil) or six (EB) ovaries per
group.
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Statistical analyses
Data on the number of multiple-layer follicles, the MIS mRNA
level, and the expression level of SF-1 protein were analyzed using
ANOVA (Single Factor; Microsoft Excel 97 SR-2,
Microsoft Corp., Redmond, WA), to evaluate the
significance of differences between the control and EB-treated rats.
P < 0.05 was considered to be significantly
different.
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Results |
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).
These results indicate that EB inhibits the formation of layered
follicles.
Expression of MIS mRNA and protein in EB-treated ovaries
The results shown in Fig. 1 indicate that EB may affect the
expression of factors required for follicular development. Therefore,
we examined the expression of MIS and several of its transcription
factors in EB-treated ovaries using in situ hybridization
and/or immunohistochemistry.
At P6, in situ hybridization signals for MIS mRNA were not
detected in primordial follicles, but intense signals were detected in
the primary and secondary follicles in both the control and EB-treated
ovaries (Fig. 2, A and B). The signal
intensity was apparently higher in the EB-treated ovaries than in the
control ovaries when secondary follicles of similar sizes were compared
(Fig. 2, A–D). At P14 and thereafter, the signals for MIS mRNA were
intense in smaller follicles at the stages ranging from primary to
small antral follicles but were less intense or nearly negative in
larger follicles such as the medium and large antral follicles in both
the EB-treated and control ovaries (Fig. 2, E and F, and data not
shown). The signal intensity was still higher in the EB-treated ovaries
than in the control ovaries at P14 (Fig. 2, E and F), but the signal
intensity of MIS mRNA in the two treatment groups were similar at P21
(data not shown). Quantitative analysis revealed that the number of
silver grains of MIS mRNA per secondary follicle in the
EB-treated ovaries was approximately 2 times higher than that in
the control ovaries (Fig. 3).
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, A–D). At later stages of development,
the level of MIS protein decreased in larger follicles such as medium
and large antral follicles (Fig. 4, E–H). The staining intensity was
apparently higher in the EB-treated ovaries than in the control ovaries
at P6 (Fig. 4, A–D) and P14 (Fig. 4, E and F), but became similar at
P21 (Fig. 4, G and H).
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Expression of SF-1 in granulosa cells of EB-treated ovaries
SF-1 is expressed in both theca/interstitial cells and granulosa
cells in postnatal ovaries. To study whether neonatal EB treatment
induces any change in the expression level of SF-1 in granulosa cells,
the cellular level of SF-1 was examined by immunohistochemistry and the
levels were quantitatively analyzed. The immunohistochemical results
were consistent with those of our previous study, demonstrating that
nuclear immunoreactivity for SF-1 was observed in some granulosa cells
with variable intensity in both the control and EB-treated ovaries
throughout the postnatal development (Ref. 29 and data not
shown). The percentage of intensely SF-1-immunoreactive granulosa
cells in the primary and secondary follicles was significantly higher
in the EB-treated ovaries than in the control ovaries at P6 (Fig. 5). At P14 and thereafter, there were no
significant differences in either the localization or the staining
intensity of SF-1 in granulosa cells between the two treatment groups
(Ref. 29 and data not shown).
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, A–D), and GATA-4 was detected in
granulosa cells, theca/interstitial cells and surface epithelial cells
(Fig. 6, E–H) of both the control and EB-treated ovaries. There was no
apparent difference in the expression profile of either of these two
factors between the control and EB-treated ovaries at all time points
examined (Fig. 6, and data not shown). Sox9 expression was examined by
in situ hybridization, but no signals for Sox9 mRNA were
detected in any of the control and EB-treated ovaries at any postnatal
day examined (data not shown).
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expression
is limited to theca/interstitial cells during early postnatal
development (29, 32, 33), ER-ß seems to mediate the
action of estrogen in granulosa cells. To determine whether estrogens
affect ER-ß expression, we examined the expression of ER-ß mRNA and
protein in EB-treated ovaries by in situ hybridization and
immunohistochemistry, respectively. Faint signals of ER-ß mRNA were
distributed diffusely over the inner ovarian part, where secondary
follicles are located, in both the control and EB-treated ovaries at P6
(Fig. 7, A and B). The signals became
intense and were localized in growing follicles at P14 and P21 (Fig. 7, C and D, and data not shown). There was no apparent difference in the
signal intensity of ER-ß mRNA between the control and EB-treated
ovaries at P6 (Fig. 7, A and B); however, at P14, the signals in small
secondary follicles of similar size were slightly more intense in the
EB-treated ovaries than in the control ovaries (Fig. 7, C and D). The
immunohistochemical results shown in Fig. 7 are based on studies using
antibody no. PC168, but comparable results were obtained using antibody
no. PAI-310. ER-ß protein was not found in any of the EB-treated and
control ovaries at P6 (data not shown). In both the control and
EB-treated ovaries at P14 and P21, immunoreactivity for ER-ß was
observed in some, but not all, granulosa cells of the growing follicles
and was localized in the cellular nucleus (Fig. 7, E–H). Some
cytoplasmic staining for ER-ß was detected in interstitial cells of
the control ovaries but not in those of the EB-treated ovaries
(Fig. 7, E and F).
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Discussion |
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The temporal and spatial expression patterns of MIS mRNA and protein during postnatal ovarian development are consistent with earlier findings (3, 4, 5, 6). The detection of very strong MIS expression in the primary and secondary follicles, where granulosa cells are actively proliferating, supports the idea that MIS plays a role in controlling early follicular growth (10).
The present results showed that the levels of both MIS mRNA and protein in granulosa cells were elevated in small growing follicles in the EB-treated ovaries for approximately 2 wk after birth. These results seem to be inconsistent with our previous results from RT-PCR in which there was no apparent difference in the total amount of MIS mRNA between the control and EB-treated ovaries at any developmental stage examined (29). This discrepancy must be due to the reduced number of follicles strongly expressing MIS in the EB-treated ovaries. The present results also seem to contradict the results reported by Baarends et al. (6) in which MIS expression was reduced in preantral and small antral follicles in the adult rat ovary after EB treatment. In adult animals, the effect of estrogens is thought to be secondary to the pituitary gonadotropins, with MIS expression hormonally regulated by a feedback system on the hypothalamic-pituitary-gonadal axis. However, ovaries are thought to be unresponsive to pituitary gonadotropins before 1 wk of age (34, 35), and therefore the effect of EB on the regulation of MIS expression in the early developing ovary is probably a direct action.
An abnormally high MIS level causes ovarian degeneration in freemartin twin females (7, 8), and in MT-hMIS transgenic female mice overexpressing human MIS during fetal and early postnatal development (9, 36). Furthermore, MT-hMIS transgenic female mice that have a targeted mutation in the MIS type II receptor (MISRII) gene, develop normal ovaries, suggesting that the ovarian degeneration caused by excess MIS in MT-hMIS transgenic mice is specifically mediated by the MISRII normally expressed in granulosa cells (37). Therefore, the inhibition of follicular development observed in the EB-treated ovaries may be due to the effect of abnormally high MIS levels mediated via MISRII in granulosa cells.
In males, the differentiation of Leydig cell precursors and the expression of steroidogenic enzyme mRNA were blocked in MIS transgenic male mice and in MIS-treated purified Leydig cells, suggesting a paracrine effect of MIS, which is expressed by Sertoli cells and binds to Leydig cells expressing MISRII (38, 39, 40, 41). In a previous in situ hybridization study (6), signals for MISRII mRNA were distributed evenly over the entire ovary during the early stages of postnatal development, indicating the possible expression of this gene in cells in the theca/interstitial region. Therefore, the reduction of the extent of steroidogenesis and the inhibition of theca/interstitial cell differentiation in EB-treated ovaries observed in our previous study (29) may be caused by excess MIS produced by granulosa cells, in a paracrine-mediated manner, acting via MISRII on theca/interstitial cells.
In contrast to the marked reduction in the number of SF-1-expressing cells among theca/interstitial cells in the EB-treated ovary, which was demonstrated in our previous study (29), our present results showed a significant increase in SF-1 expression in granulosa cells of EB-treated ovaries. These results also seem to be inconsistent with the reduction of the total expression level of SF-1 shown by RT-PCR analysis in the study of Morais da Silva et al. (30). Because the number of SF-1-expressing cells is larger and the expression level is higher in theca/interstitial cells than in granulosa cells (42, 43), the total expression level of SF-1 mainly reflects the expression level in theca/interstitial cells. Thus, it was revealed that estrogen has a different effect on SF-1 expression in granulosa cells and in theca/interstitial cells within an ovary. Although the regulation of MIS expression by SF-1 has not been proven in ovarian granulosa cells, the concomitant increase in the levels of these two factors indicates a functional correlation of these two factors in the developing ovary.
The colocalization of MIS and ER-ß in granulosa cells of growing follicles in the developing ovary and the presence of an estrogen-responsive halfsite element in the promoter region of the MIS gene (44) suggest that MIS acts on granulosa cells via ER-ß. In the present study, increased levels of both ER-ß mRNA and protein in secondary follicles of EB-treated ovaries were observed at P14 but not at P6. Because the level of ER-ß expression is very low in neonatal rats (45), it might be difficult to detect slight changes by in situ hybridization or immunohistochemistry. Therefore, the possibility that ER-ß directly or indirectly mediates the inhibitory effect of EB on follicular formation cannot be ruled out.
At present, the expression site of ER-ß in the ovary is controversial (46). Although ER-ß mRNA is strongly expressed in granulosa cells, several groups have reported that it is also expressed in theca cells by in situ hybridization (47, 48, 49). In our study, in situ hybridization signals for ER-ß were localized in granulosa cells and no signals were detected in the theca/interstitial region. We observed some cytoplasmic staining for ER-ß in interstitial cells of the control but not in those of the EB-treated ovaries by immunohistochemistry. Similar cytoplasmic staining of ER-ß was also reported by Sar and Welsch (32) using antibody no. PAI-310. In that report, the cytoplasmic staining was not completely blocked in sections that were incubated with preabsorbed ER-ß. In contrast, Fitzpatrick et al. did not detect cytoplasmic staining in interstitial cells using either antibody no. PC168 or antibody no. PAI-310 (33). These different results may be related to the technical differences such as tissue fixation, tissue preparation (frozen or paraffin sections), and permeabilization treatment (proteases, detergent, and microwaving). In the present study, no cytoplasmic staining was observed in the negative control sections in which the primary antibody was omitted. Because absorption is a more stringent indicator of specificity, we cannot yet evaluate the specificity quantitatively.
It has been suggested that ER- and ER-ß are functionally redundant
in ovarian folliculogenesis and that ER-ß plays an important role in
mediating the action of estrogen in stimulating granulosa cell
proliferation during follicular growth (46). Recently, the
importance of both ERs has been suggested in ßERKO female mice
(26, 50). The ovaries of adult ßERKO females showed
several features of sex-reversal, such as degeneration of granulosa
cells, loss of germ cells, appearance of Sertoli-like cells, and
elevated mRNA levels of marker genes for Sertoli cells (MIS, sulfated
glycoprotein-2, and Sox9). These results suggest that the sex-reversal
is due to aberrant expression of factors for both oocyte survival (such
as GATA-4) and Sertoli cell differentiation (such as MIS and Sox9)
caused by the lack of ERs (26). However, immunoreactivity
for MIS was not increased in the Sertoli-like cells of the ßERKO
ovaries, indicating that the appearance of Sertoli-like cells is
independent of increased MIS expression but is correlated with oocyte
loss (50). In the EB-treated ovaries, elevated MIS mRNA
levels were detected only temporarily during the early neonatal period,
and we did not observe any sign of sex-reversal such as appearance of
Sertoli-like cells, loss of oocytes or Sox9 expression. In spite of
these differences, altered expression of both ER- and ER-ß was
observed in the EB-treated ovaries, which was shown in the previous
(29) and present studies, respectively. These findings
indicate that both ERs are important for postnatal ovarian
differentiation, and the presence of a functional correlation between
MIS and ERs.
There were no differences in the expression profiles of WT-1 or GATA-4 between the control and EB-treated ovaries by immunohistochemistry. Because these factors were suggested to be involved in follicular development (22, 23), it is possible that subtle changes in the expression levels of these factors that were not detected by this method might have occurred in the EB-treated ovaries.
To our knowledge, the present study is the first to show that neonatal estrogen treatment increases the expression of MIS and inhibits follicular growth during early postnatal development of the rat ovary. These results indicate that exogenous estrogen inhibits ovarian follicular growth by altering the expression of MIS and that SF-1 and/or ER-ß may be involved in the estrogen-induced up-regulation of MIS expression in the developing ovary.
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Acknowledgments |
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Footnotes |
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Abbreviations: EB, E2 benzoate; ßERKO, ß estrogen
receptor knockout; GATA-4, a member of the GATA family of
transcriptional regulators; HE, hematoxylin and eosin; MIS,
Müllerian-inhibiting substance; MT-hMIS, metallothionein-human
MIS; P, postnatal day 1; SF-1, steroidogenic factor 1; SSC, standard
saline citrate; Sox9, SRY HMG box related gene 9; WT-1, Wilms’ tumor
1.
Received June 5, 2001.
Accepted for publication September 25, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene promoter. Mol Endocrinol 9:1356–1366 and ß. Science 286:2328–2331 in the rat ovary.
Endocrinology 140:963–971 (ER-) and ß
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Steroids 63:498–504[CrossRef][Medline]
and -ß mRNAs in the female reproductive
organ of the rat—an analysis by in situ hybridization. J
Endocrinol 167:363–369[Abstract]
(ER) and ß (ERß) on mouse reproductive phenotypes.
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