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Endocrinology Vol. 145, No. 12 5757-5766
Copyright © 2004 by The Endocrine Society

Developmental Expression of Estrogen Receptor (ER) {alpha} and ERß in the Hamster Ovary: Regulation by Follicle-Stimulating Hormone

Peixin Yang, Jinrong Wang, Yulei Shen and Shyamal K. Roy

Departments of Obstetrics and Gynecology (P.Y., J.W., S.K.R.), Pathology and Microbiology (Y.S.), and Cellular and Integrative Physiology (S.K.R.), University of Nebraska Medical Center, Omaha, Nebraska 68198-4515

Address all correspondence and requests for reprints to: Shyamal K. Roy, Ph.D., Durham Research Center 5013, Departments of Obstetrics and Gynecology and Physiology and Biophysics, University of Nebraska Medical Center, 984515 Nebraska Medical Center, Omaha, Nebraska 68198-4515. E-mail: skroy{at}unmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Perinatal expression of estrogen receptor (ER) protein and mRNA and the influence of FSH on this process were examined by immunofluorescence and RT-PCR using ovaries from fetal (d 13–15 of gestation) and postnatal [postnatal d 1–15 (P1–P15)] hamsters and from 8-d-old hamsters exposed in utero to an anti-FSH serum on d 12 of gestation and saline or equine chorionic gonadotropin (eCG) on P1. A few somatic cells expressing ER{alpha} immunoreactivity appeared first on d 14 of gestation and increased markedly by P8–P15 in the interstitial cells and granulosa cells of primordial follicles. In contrast, appreciable ERß immunoreactivity was localized on d 13 of gestation, and more cells expressed ERß immunoreactivity by P1–P8. By P7, ERß immunoreactivity was present in cells adjacent to the oocytes, and by P8, ERß was preferentially localized in the granulosa cells. Receptor immunoreactivities decreased markedly in P8 ovaries exposed in utero to the FSH antiserum but were reversed with postnatal eCG replacement. Oocytes and somatic cells expressed ER{alpha} and ERß mRNA, and levels of ER mRNA in the ovary increased by P7–P8, corresponding to the appearance of primordial follicles. Thereafter, only ERß mRNA levels increased progressively with postnatal ovary development. Similar to ER protein, mRNA levels decreased significantly in FSH antiserum-treated ovaries but were restored by eCG. These results indicate that both ER subtypes are expressed in undifferentiated somatic cells and the oocytes during perinatal ovary development in the hamster; however, ERß expression segregates with the differentiation of granulosa cells. Furthermore, ER expression and differentiation of somatic cells to granulosa cells depend on perinatal FSH action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE RAT OVARY, estrogens stimulate granulosa cell proliferation (1) and enhance the actions of FSH. Two types of estrogen receptor (ER), ER{alpha} and ERß, have been identified in estrogen target tissues (2, 3). Nuclear ERs are transcription factors, and their expression patterns in the adult ovary have been extensively investigated in a variety of species including the hamster (4, 5, 6). Impairment of follicular development has been reported for immature {alpha}ßER knockout mice (7), suggesting that both ERs are required for early ovarian folliculogenesis. Moreover, the postnatal sex reversal of the ovaries in {alpha}ßERKO mice indicates that both receptors are required for the maintenance of germ and somatic cells in the postnatal ovary (8). Both subtypes of ER are expressed differentially in the adult hamster ovary and regulated by steroid hormones and gonadotropins (6). Similarly, the up-regulation of ER by FSH was also found in cultured rat granulosa cells (9), suggesting that part of the FSH effect on ovarian cells may involve ER. Developmental expression of ER{alpha} and ERß in the ovary has been reported in the rat (5, 10, 11) and mouse (12). Furthermore, RT-PCR evaluation of human fetal ovaries has also revealed the presence of ER{alpha} and ERß mRNA (13). Immunohistochemical studies of mouse ovaries have revealed that ERß is exclusively present in the granulosa cells, whereas ER{alpha} is exclusively localized in the interstitial and theca cells (12). In contrast, both immunohistochemical and in situ hybridization studies of rat ovaries have revealed low levels of ER{alpha} and high levels of ERß mRNA in the granulosa cells of growing follicles (5, 10, 14). Data published so far seem to indicate that, whereas ER{alpha} levels remain relatively constant during the postnatal period, ERß expression increases with age and the number of granulosa cells (4, 15). Despite this knowledge, developmental expression of ER mRNA and protein in the hamster ovary during the formation of the first cohort of follicles is not known.

Hamster ovary during perinatal development represents a unique model because the formation of the first cohort of primordial follicles occurs after birth. In contrast to rats and mice, ovaries of newborn hamsters contain oocytes and scattered undifferentiated mesenchymal-epithelial (somatic) cells. Morphologically distinct primordial follicles are identifiable by postnatal d 8 (P8) (16). Whereas FSH action has been shown in vivo (16) and in vitro (17, 18) to be necessary for the formation of primordial follicles during perinatal ovarian development in the hamster, whether FSH influences ER expression during the differentiation of somatic cells to granulosa cells is not known. However, because FSH stimulates ER expression in the granulosa cells (6, 9) and serum FSH levels in perinatal hamsters show a unique profile with marked rises between P4 and P7 and a peak at P14 (19, 20, 21), it is logical to postulate that FSH may influence the expression of ER in the perinatal hamster ovary. Therefore, the objectives of the present study were to delineate the expression patterns of ER{alpha} and ERß in hamster ovaries during perinatal development and to examine whether FSH influenced ovarian ER expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
The monoclonal antibody to recombinant human ER{alpha} was from Novacastra Laboratories Ltd. (Newcastle, UK), and the specificity of the antibody for hamster ER{alpha} was previously validated using Western blotting and immunofluorescence localization (6). The antibody to ERß was generated in the rabbit by injecting a keyhole limpet hemocyanin-conjugated synthetic ERß peptide corresponding to amino acid residues 54–71 of the hamster ERß amino acid sequence, which was derived from the hamster ERß nucleic acid sequence (6), and was tested thoroughly for its specificity in parallel with the ERß antibody from Upstate Cell Signaling (Lake Placid, NY) by immunoblotting and immunofluorescence using ovaries from adult hamsters. Peptide with identical amino acid sequence was used to raise ERß antibody commercially (Upstate Cell Signaling), and we had successfully used such an antibody previously to detect ERß in the hamster ovary (6). Alexa-conjugated second antibodies were from Molecular Probes, Inc. (Eugene, OR). Optitran transfer membrane was from Schleicher & Schuell, Inc. (Dassel, Germany). Primers for real-time PCR and FAM- and Blackhole-modified oligonucleotide probes for hamster ER{alpha}, ERß, and ß-actin were designed using Primer Express software (PerkinElmer, Boston, MA) and synthesized in the Eppley Molecular Biology Core Facility at the University of Nebraska Medical Center. AmpliScribe T7-Flash transcription kit was from Epicenter (Madison, WI), and PCR chemicals were from Roche Molecular Biochemicals (Indianapolis, IN), Pharmacia Biotech Boehringer (Piscataway, NJ), and Promega (Madison, WI). [35S]-{alpha}-CTP (specific activity, 800 Ci/mmol) was from ICN Radiochemicals (Costa Mesa, CA), and the riboprobe synthesis kit was from Promega. Equine chorionic gonadotropin (eCG) was purchased from Sigma Chemical Company (St. Louis, MO). All other molecular-grade chemicals were purchased from Sigma Chemical Company, Fisher Scientific Company (Pittsburgh, PA), or United States Biochemical (Cleveland, OH). Rabbit polyclonal anti-FSH was generated in our laboratory, and the ability of this antibody to specifically neutralize endogenous FSH action in the hamster was verified (16).

Animals
Adult female and male golden hamsters (90–100 g body weight) were obtained from SASCO (Madison, WI) and kept under 14-h light, 10-h dark cycle in a climate controlled quarter with free access to food and water. Animals were maintained and used according to the United States Department of Agriculture and Institutional Animal Care and Use Committee guidelines, and the use of hamsters for the study was approved by the Institutional Animal Care and Use Committee. Females with at least three consecutive estrous cycles were used for breeding. On the evening of proestrous, one female was paired with two males, and the presence of sperm in the vaginal smear the next morning was considered as d 1 of pregnancy. Pregnant females were kept individually in separate cages. The gestation period of hamsters is 16 d, and delivery of pups occurred on d 16, which was considered as P1.

Determination of ER expression patterns in perinatal hamster ovaries
Ovaries were removed from 13- to 15-d-old fetal and 1- to 20-d-old postnatal hamsters, placed in the optimal cutting temperature (OCT) compound (Fisher Scientific), snap frozen in liquid nitrogen-cooled methylisopentene, and sectioned at 6 µm in a Leica cryostat microtome (Leica Instruments, Nussloch, Germany) for immunofluorescence localization and in situ hybridization detection of ER{alpha} and ERß protein and mRNA, respectively.

In a parallel experiment, three pairs of ovaries for each day of development were collected for RNA preparation, which was used for real-time quantification of the levels of ER{alpha} and ERß mRNA. No attempt was made to examine ER mRNA expression in antibody-treated ovaries by in situ hybridization.

Detection of ER mRNA in the primordial oocytes
Ovaries were collected from two 1-d-old (P1) hamsters, embedded in the OCT compound, frozen in liquid nitrogen-cooled methylisopentene, and sectioned at 6 µm in a Leica automatic cryostat microtome. Sections from each ovary were mounted on clean, uncoated SuperFrost+ glass slides (Fisher Scientific) and stored at –80 C until use. Oocytes from all sections of two ovaries from each hamster were collected and handled separately to obtain two samples. Before use, sections were fixed in 70% ethanol for 30 sec, stained lightly with hematoxylin, and dehydrated in 70, 95, and 100% ethanol. Slides were dipped twice in xylene for 2 min each and air-dried completely. Laser capture microscopy (LCM) was performed immediately using a PixCell II LCM system (Arcturus Engineering, Mountain View, CA). Microdissection was done under x400 magnification, with a spot size (size of the laser beam) of 7.5 µm, pulse duration of 600 µsec, and power of 65 mW, and the oocyte sections were collected in the caps of a microfuge tube and snapped onto a 1.5-ml microcentrifuge tube containing 500 µl Tri-reagent (Molecular Research Center, Inc., Cincinnati, OH) for RNA extraction. Only clearly identifiable oocytes were dissected out, and the laser beam vaporized any cellular material outside the cutting circle; hence, contamination from nonoocyte cells was completely eliminated.

Effect of anti-FSH serum on ovarian ER expression
Pregnant hamsters were injected sc with 200 µl of an anti-FSH serum on d 12 of gestation because previous studies indicated this time to be ideal for blocking the formation of primordial follicles (16). After the pups were born, one set of pups received 0.5% BSA in saline (vehicle for eCG), whereas the others were injected sc with 20 IU eCG on P1. Ovaries were collected on P8 for immunofluorescence and RT-PCR detection of ER{alpha} and ERß protein and mRNA expression, respectively. eCG was used because of its longer half-life in vivo, thus avoiding repeated injections to the small pups, and its ability to stimulate follicular development in the hamster (22, 23, 24). Moreover, eCG did not cross-react with the FSH-antiserum, and therefore, it could bypass the antibody neutralization; and LH receptors did not appear in postnatal hamster ovarian cells until P13 (Roy, S. K., unpublished observation), so contribution of LH receptors was not a concern.

Immunofluorescence detection of ER{alpha} and ERß
Immunofluorescence was done according to the method described by Yang et al. (6) with slight modifications. Briefly, for ER{alpha}, sections were fixed for 10 min in Zamboni’s fixative (6) at room temperature, whereas for ERß, sections were fixed sequentially in ice-cold methanol for 3 min, ice-cold ethanol for 3 min, and 4% freshly made paraformaldehyde in PBS (pH 7.4) for 10 min at room temperature and rinsed three times for 5 min each in PBS at room temperature (6). Nonspecific protein-binding sites were blocked with 10% donkey serum, 0.1% Triton X-100, and 0.1% sodium azide in PBS (pH 7.4) for 30 min at room temperature (ER{alpha}) or 1 h at 4 C (ERß) in a humidified chamber. Sections were briefly rinsed with ice-cold PBS and post-fixed in 1% ice-cold paraformaldehyde for 2 min. Sections were exposed overnight to optimal dilution of the receptor-specific antibody at 4 C in a humidified chamber. Sections were then rinsed twice for 5 min each in PBS and exposed to appropriate second antibody conjugated with Alexa-488 (green fluorescence) in a humidified chamber at room temperature. Nuclei were stained simultaneously with 4',6-diamidino-2-phenylindole (DAPI). After thorough rinsing, sections were mounted with Fluoromount G (Southern Biotechnology Associates, Inc., Birmingham, AL) and examined under epifluorescence in a Leica DMR research microscope equipped with a Retiga Fast1394 digital camera (Q-Imaging, Burnaby, British Columbia, Canada), and the images were captured using Openlab (Improvision, Lexington, MA) image analysis software.

The specificity of the ER{alpha} and ERß antibodies was verified by incubating sections of the ovaries from 15- and 8-d-old pups, respectively, with antibodies preabsorbed to 100-fold excess of recombinant human ER{alpha} (Panvera, Madison, WI) or a control ERß peptide (used to develop the antibody in the laboratory) and processed identically as mentioned earlier.

For digital image capturing, the exposure time was adjusted using sections incubated without the primary antibody to subtract any auto- or nonspecific fluorescence recording. Signal obtained after such background correction was considered as antigen-specific signal. All sections for a specific receptor antigen were evaluated under identical camera settings so that comparisons could be made between groups. Reproducibility of immunofluorescence localization was verified using ovarian sections from two different animals. Digital images of representative sections were captured, and images with ER signal were merged with those with nuclear signal using the Openlab software to detect the cellular site of the immunosignal. For contrast, blue fluorescence of DAPI was converted to red color. Photomicrographs were arranged using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA) without any further adjustment to maintain the true nature of the findings. To avoid redundancy, photomicrographs of ovaries reflecting distinct changes in the ER expression during perinatal development were presented.

In situ hybridization localization of ER{alpha} and ERß mRNA in developing hamster ovaries
In situ hybridization was done essentially as described previously in detail (6). Briefly, frozen sections of ovaries were fixed in 4% fresh paraformaldehyde in PBS (pH 7.4), acetylated, and prehybridized, followed by 4-h hybridization with [35S]-antisense or sense cRNA at 45 C. After rinsing and RNaseA digestion, sections were dehydrated with ascending grades of ethanol, coated with NTB2 nuclear track emulsion, exposed in the dark for optimum time, developed in Dektol (Kodak, Rochester, NY), and stained with hematoxylin and eosin. All sections were examined in a Leica DMR research microscope under bright- and dark-field illumination for morphology and silver grain distribution as an index of mRNA expression, and images were captured using a Leica digital camera and Openlab image analysis software (Improvision). In situ hybridization was repeated twice using sections from two different animals, and results were reproducible. However, for brevity, bright- and dark-field pictures of sections corresponding to critical immunofluorescence findings were presented.

Real-time RT-PCR quantification of the levels of ER{alpha} and ERß mRNA in developing hamster ovaries
Ovaries were collected in RNAlater solution, and RNA was isolated using the RNeasy mini kit (Qiagen Inc., Valencia, CA). The amount of RNA per microliter was quantified using the Ribogreen kit (Molecular Probes) according to the manufacturer’s protocol. For real-time quantification, mRNA standards for each ER subtype and for ß-actin were used in parallel with the sample RNA to obtain true quantitative values. PCR primers and probes were designed from hamster ER and ß-actin cDNA sequences, which were partially cloned by us (6). Sense strand of mRNA for each ER subtype and ß-actin was synthesized using linearized, hamster-specific cDNA (6) and the AmpliScribe T7-Flash transcription kit and quantified using the Ribogreen kit; its authenticity was verified by routine RT-PCR (6) using the forward and reverse primers designed specifically for real-time RT-PCR. A series of eight standards ranging from 0.1 fg to 1 ng (in 10-fold increments) of specific mRNA per 1.25 µl water was prepared. Duplicates of each standard were reverse-transcribed along with ovarian samples. Aliquots of reverse transcribed product of each ovarian sample were used for real-time quantification of ER{alpha}, ERß, and ß-actin using the Opticon thermocycler (MJ Research, Reno, NV). PCR reaction was continued for 35 cycles after an initial denaturation at 94 C for 15 min to activate the HotStatrt Taq polymerase (Qiagen). Each cycle of PCR consisted of 30 sec of denaturation at 94 C and 15 sec of annealing at 55 C, followed by a plate reading with a final extension at the end of 35 cycles for 10 min at 72 C. Because PCR product size for ER{alpha}, ERß, and ß-actin was kept at 96 bp, shorter annealing and extension time was adequate. Therefore, extension occurred during the time when temperature started to increase from the annealing temperature at the end of each cycle. This strategy helped us eliminating the extension step in each cycle, thus significantly reducing the total time of PCR. During optimization of the real-time RT-PCR, dedicated extension time of 1 min was included in each cycle; however, no difference in the output signal was noted when the step was omitted. Amount of specific mRNA was calculated from the linear portion of the amplification signal. The authenticity of the PCR signal was verified using tubes containing no RNA or RNA without reverse transcriptase. In addition, in preliminary experiments, the authenticity of the cDNA generated by real-time PCR was also verified by sequencing. The values were presented as femtograms of mRNA per microgram of total RNA. Because RNAs from developing ovaries were used, the amounts of ß-actin mRNA for each day of development were also presented to prove the specificity of ER gene expression during development. The sequences for forward and reverse primers and the probes were as follows: ER{alpha}: forward, 5'-CCAGAGTGGCCGAGAGAGACT-3'; reverse, 5'- CATAATGGTAGCCAGAGGCGTAGT-3'; probe, 6-FAM-5'-CCATGCTTCCTTTCTCGCTGCTGCT-3'-BLACKHOLE; ERß: forward, 5'-CTGTGCCAGCCCTGTCACTA-3'; reverse, 5'-CAGATGCATAATCGCTGCAAAC-3'; probe, 6-FAM-5'-CGCAGAAGTGAGCATCCCTCTTTGAACT-3'-BLACKHOLE; and ß-actin: forward, 5'-TGACCGAGCGTGGCTACAG-3'; reverse, 5'-CTTCTCTTTGATGTCACGCACAAT-3'; probe, 6-FAM-5'-TCACCACCACAGCCGAGAGGGA-3'-BLACKHOLE.

Statistical analysis
Immunofluorescence and in situ hybridization localization were done on sections from two ovaries from two hamsters for each day of perinatal development, and representative sections were presented. For fetal ovarian RNA samples, there were three pregnant hamsters for three samples for each indicated day of gestation. For each sample, ovaries from all fetuses from each pregnant female were pooled. There were three RNA samples for each postnatal day. Each sample was prepared from ovaries pooled from three postnatal hamsters. For LCM, oocytes were collected from ovarian sections from two different 1-d-old hamsters and analyzed separately. All quantitative data were analyzed by one-way ANOVA with Scheffé’s post hoc test using StatView software (SAS Institute, Inc., Cary, NC). The level of significance was set at 5%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ER{alpha} and ERß protein expression in developing ovary
The objectives of this study were to determine whether ER subtype expression in undifferentiated somatic cells of fetal ovaries would reflect any cell-type selectivity and would segregate with the differentiation of granulosa and interstitial cells and whether FSH plays any role in ER subtype expression during perinatal ovary development in the hamster. No ER{alpha}-specific immunoreactivity could be detected in sections incubated with the antibody, which was preneutralized with the receptor protein (Fig. 1AGo), thus verifying the specificity of the immunosignal. No ER{alpha} expression was apparent in 13-d-old fetal ovaries (Fig. 1BGo). Receptor protein was first visible in a few somatic cells in the ovaries of a 14-d-old fetus (Fig. 1CGo). The number of cells expressing ER{alpha} increased by P1 but remained steady up to P3 (Fig. 1Go, D and E). Noticeable increases in fluorescence intensity and cells expressing ER{alpha} were evident by P4 and P5, and ER{alpha}-positive cells were located between oocyte clusters (Fig. 1Go, F and G). The number of cells expressing ER{alpha} declined sharply by P6 (Fig. 1HGo), but marked increases in the intensity and number of positive cells occurred by P8 (Fig. 1IGo), when morphologically distinct primordial follicles could be identified (16). However, cells with stronger ER{alpha} immunoreactivity were located primarily in the interstitial cell compartment, whereas newly formed granulosa cells had low expression (Fig. 1IGo). Furthermore, P8 was the first time when an intense ER{alpha} immunosignal was present in the oocytes, regardless of the location (Fig. 1IGo). The number of interstitial cells showing strong ER{alpha} immunostaining increased markedly by P10, and granulosa cells of primordial follicles had increased expression (Fig. 1JGo). With further development of the follicles by P15, ER{alpha} expression remained restricted to interstitial cells and oocytes and granulosa cells of primordial follicles (Fig. 1Go, K and L). Neutralization of endogenous FSH with anti-FSH serum treatment on d 12 of gestation resulted in a dramatic reduction in ER{alpha}-positive interstitial cells, complete lack of ER{alpha}-positive oocytes, and significant absence of follicle-like structures on P8 relative to the untreated group (Fig. 1MGo). Treatment with eCG on P1 resulted in a resumption of the immunoreactivity in the interstitial cells and oocytes (Fig. 1LGo), almost to the level observed for untreated 8-d-old hamsters (compare Fig. 1IGo with Fig. 1NGo), and folliculogenesis. However, in contrast to granulosa cells in normal 8-d-old ovaries, eCG treatment on P1 also resulted in intense ER{alpha} expression in the granulosa cells of primordial and primary follicles (Fig. 1NGo).



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  		FIG. 1. Photomicrographs showing ER{alpha} immunofluorescence (yellow to green) in hamster ovarian cells during fetal through postnatal development under normal conditions (A–L) and after gestational exposure to an FSH antiserum with or without postnatal eCG treatment (M and N). Section of a 15-d-old hamster ovary exposed to the primary antibody that was preneutralized with ER{alpha} peptide (A). Sections of ovaries collected from 13-d-old (B) and 14-d-old (C) fetal hamsters; from 1-d-old (D), 3- to 6-d-old (E–H), 8-d-old (I), 10-d-old (J), and 15-d-old (K and L) postnatal hamsters; and from 8-d-old hamsters exposed in utero to an FSH antiserum on d 12 of fetal life that received vehicle (M) or eCG (N) on P1. Red color denotes nuclear fluorescence, whereas yellow to green color reflects ER fluorescence. Low-intensity ER{alpha} immunosignal appeared yellow upon image merger. GC, Granulosa cells; IC, interstitial cells; O, oocytes; OC, oocyte cluster (encircled area); S, somatic cells; S0, primordial follicle; S1, primary follicle; S3, secondary follicles. Bar, 10 µm. Immunofluorescence localization was repeated on sections of three ovaries (n = 3), and representative photographs were presented.

 
No ERß-positive immunostaining was present in any ovarian cells of 8-d-old hamsters when the sections were exposed to ERß antibody, which was preneutralized with ERß peptide (Fig. 2AGo). Distinct ERß immunoreactivity was evident in somatic cells as well as the oocytes of 13-d-old fetal hamsters, and some somatic cells had a stronger immunosignal (Fig. 2BGo). Signal intensity declined noticeably in all cell types in the ovaries of 14-d-old fetal hamsters (Fig. 2CGo), but a marked increase was evident on P1 (Fig. 2DGo). Interestingly, oocytes in some clusters showed intense immunosignal compared with other clusters (Fig. 2D). By P4, somatic cells adjacent to the oocytes showed strong immunosignal, but most of the somatic cells away from the oocyte clusters were negative (Fig. 2EGo). A similar trend was observed on P7 (Fig. 2FGo). By P8, intense ERß immunostaining was primarily associated with the granulosa cells of primordial and primary follicles and the oocytes, whereas interstitial cells showed low immunostaining (Fig. 2GGo). The patterns of ERß expression remained unchanged in subsequent days of development, although intensity of staining increased further (data not shown). Whereas gestational exposure to anti-FSH serum resulted in a virtual elimination of ERß immunostaining in the somatic cells on P8 and only a few scattered cells showed low immunostaining (Fig. 2HGo), marked ERß expression with associated appearance of primordial and primary follicles was evident in the granulosa cells and oocytes of animals that received eCG on P1 (Fig. 2IGo). However, similar to the expression observed for ER{alpha}, eCG treatment also caused appreciable ERß expression in the interstitial cells (Fig. 2IGo).



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  		FIG. 2. Photomicrographs showing ERß immunofluorescence (yellow to green) in hamster ovarian cells during fetal through postnatal development under normal conditions (A–G) and after gestational exposure to an FSH antiserum with or without postnatal eCG treatment (H and I). Section of an 8-d-old hamster ovary exposed to the primary antibody that was preneutralized with ERß peptide (A). Sections of ovaries collected from 13-d-old (B) and 14-d-old (C) fetal hamsters; from 1-d-old (D), 4-d-old (E), 7-d-old (F), and 8-d-old (G) postnatal hamsters; and from 8-d-old hamsters exposed in utero to an FSH antiserum on d 12 of fetal life that received vehicle (H) or eCG (I) on P1. Red color denotes nuclear fluorescence, whereas yellow to green color reflects ER fluorescence. Low-intensity ER{alpha} immunosignal appeared yellow upon image merger. GC, Granulosa cells; IC, interstitial cells; O, oocytes; OC, oocyte cluster (encircled areas); S, somatic cells; S0, primordial follicle; S1, primary follicle. Bar, 10 µm. Immunofluorescence localization was repeated on sections of three ovaries (n = 3), and representative photographs were presented.

 
Developmental changes of ER{alpha} and ERß mRNA levels
To determine whether developmental expression of ER protein in ovarian cells would be reflective of changes in the levels of corresponding ER mRNA, distribution and levels of ER{alpha} and ERß mRNA in developing ovaries were examined by in situ hybridization and real-time RT-PCR quantification, respectively. In general, levels of ER{alpha} mRNA were at least 2-fold higher than those of ERß (Fig. 3Go, A and B). ER{alpha} mRNA levels were relatively low in 13-d-old fetal ovaries, which corresponded to almost no expression of ER protein in any ovarian cell type (Figs. 1BGo and 3AGo). Marked increase in ER{alpha} mRNA levels, corresponding to the onset and increasing expression of receptor protein in the somatic cells, occurred on d 14 of gestation through P5 (Fig. 3AGo). ER{alpha} mRNA levels decreased markedly by P6, which also corresponded to very few ER{alpha}-positive cells in the ovary (Figs. 1HGo and 3AGo). ER{alpha} mRNA levels increased significantly by P7, showed a decreasing trend up to P9, then declined by more than 2-fold by P10, and remained steady afterwards (Fig. 3AGo). Decreases in the mRNA levels by P10 were in contrast with the remarkable increase in ER{alpha}-positive cells in the ovary (compare Fig. 1Go, J–L, with Fig. 3AGo).



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  		FIG. 3. Levels of (A) ER{alpha}, (B) ERß, and (C) ß-actin mRNA in hamster ovaries during perinatal development. ER mRNA levels were quantified by real-time RT-PCR using total ovarian RNA, and the results were presented in terms of total ovarian RNA. Bars with same letter in each panel were not significantly (P < 0.05) different from each other. Each bar in each panel represents three separate observations (n = 3).

 
ERß mRNA levels increased steadily to reach a peak on P10 (Fig. 3BGo), which more or less corresponded to increases in the number of cells expressing ERß protein. However, in contrast to ER{alpha}, low levels of mRNA expression on d 13 of gestation correlated well with ERß protein expression. ERß mRNA levels declined significantly by P11 and remained more or less steady, except for a marked increase on P14 (Fig. 3BGo); however, changes in mRNA levels from P10 and onward did not cause any noticeable difference in the overall expression of the receptor protein (data not shown).

Despite perinatal development of the ovary, overall levels of ß-actin mRNA remained steady throughout the period of the study (Fig. 3CGo), except for a small but significant increase on P7 (Fig. 3CGo).

Because the objective of in situ detection of ER mRNA was to examine cell type- and developmental-specific expression, ovaries from FSH antibody-treated hamsters were not included in the group. ER{alpha} mRNA expression remained scattered throughout the ovary (Fig. 4Go). ER{alpha} mRNA was present in 14-d-old fetal ovaries (Fig. 4Go, A and B), despite low protein expression, thus corroborating the RT-PCR data. Appreciable expression of ER{alpha} mRNA (Fig. 4IGo) was noted in all somatic cells and the oocytes before morphologically distinct primordial follicles appeared on P8 (Fig. 4JGo). After the formation of primary and secondary follicles by P9 (Fig. 4LGo) through P13 (Fig. 4NGo), ER{alpha} mRNA expression appeared to be somewhat concentrated in the interstitial cells, but ER mRNA expression was still evident in the granulosa cells (Fig. 4Go, K and M). Overall, ER{alpha} distribution correlated well with the quantitative data. Hybridization with sense cRNA did not yield any specific signal in sections of 9-d-old hamster ovary (Fig. 4Go, O and P), thus validating the specificity of the localization.



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  		FIG. 4. In situ hybridization detection of ER{alpha} mRNA expression in perinatal hamster ovaries. Dark-field (A, C, E, G, I, K, M, and O) photomicrographs with corresponding bright-field (B, D, F, H, J, L, N, and P) pictures were presented for morphological identification. Sections of ovaries collected from 14-d-old fetal hamster (A and B) and from 4-d-old (C and D), 6-d-old (E and F), 7-d-old (G and H), 8-d-old (I and J), 9-d-old (K and L), and 13-d-old (M and N) postnatal hamsters and hybridized with antisense ER{alpha} [35S]-cRNA. Sections of an ovary from a 13-d-old hamster hybridized with sense ER{alpha} [35S]-cRNA (O and P). Photographs are representatives of two separate hybridization experiments. GC, Granulosa cells; IC, interstitial cells; O, oocytes; OC, oocyte cluster; S, somatic cells; S0, primordial follicle; S1, primary follicle.

 
Similar to ER{alpha} mRNA, ERß mRNA was present in all cell types during early ovarian development (Fig. 5Go). Although granulosa cell-specific expression increased with the appearance of primordial follicles on P8 (Fig. 5Go, G and H) and primary follicles on P10 (Fig. 5Go, I and J), ERß expression in the interstitial cells remained prominent. ERß mRNA expression became more concentrated in the granulosa cells by 15 d of age (Fig. 5KGo), when large secondary follicles developed (Fig. 5LGo), and by that age, expression in the interstitial cells became relatively low. No ERß-specific signal was observed when sections of 15-d-old ovaries were hybridized with sense ERß cRNA (Fig. 5Go, M and N), indicating the specificity of the hybridization signal.



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  		FIG. 5. In situ hybridization detection of ERß mRNA expression in perinatal hamster ovaries. Dark-field (A, C, E, G, I, K, and M) photomicrographs with corresponding bright-field (B, D, F, H, J, L, and N) pictures were presented for morphological identification. Sections of ovaries collected from 14-d-old fetal hamster (A and B) and from 4-d-old (C and D), 7-d-old (E and F), 8-d-old (G and H), 10-d-old (I and J), and 15-d-old (K and L) postnatal hamsters and hybridized with antisense ERß [35S]-cRNA. Sections of an ovary from a 15-d-old hamster hybridized with sense ERß [35S]-cRNA (M and N). Photographs were representatives of two separate hybridization experiments. GC, Granulosa cells; IC, interstitial cells; O, oocytes; OC, oocyte cluster; S, somatic cells; S0, primordial follicle; S1, primary follicle; S2–S4, secondary follicles with two, three, or four layers of granulosa cells.

 
LCM combined with RT-PCR revealed that both ER{alpha} and ERß mRNA were expressed in the oocytes of postnatal hamsters (Fig. 6DGo), thus complementing ER subtype immunostaining in the oocytes.



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  		FIG. 6. RT-PCR detection of ER{alpha} and ERß mRNA in the oocytes of 1-d-old hamsters. Ovaries were collected from two hamsters, and oocytes from two ovaries of each animal were dissected by LCM and considered as one sample. A, Section of a 1-d-old hamster ovary showing oocytes (arrowheads). B, Section remaining after laser dissection and oocyte removal (laser-burned boundaries are clearly visible). C, Sections of some oocytes isolated by laser dissection. D, Southern hybridization signal (phosphor image) of ER{alpha} and ERß cDNA from two separate oocyte samples (n = 2). Bar, 10 µm.

 
FSH modulates ER{alpha} and ERß mRNA expression during postnatal ovary development
The objective was to reveal whether FSH action was necessary to maintain ovarian ER mRNA levels during perinatal development. Administration of FSH antiserum to pregnant females on d 12 of gestation resulted in a marked decrease in the levels of both ER{alpha} and ERß mRNA in the ovaries of the offspring by P8 (Fig. 7Go, A and B), which corresponded to negligible ER protein expression. However, eCG treatment of pups, which were gestationally exposed to the FSH antiserum, on P1 resulted in an almost complete reversal of the receptor mRNA levels (Fig. 7Go, A and B) and ER protein expression (Figs. 1NGo and 2IGo). In contrast to ER mRNA, levels of ovarian ß-actin were not altered appreciably either by the antiserum or eCG treatment (Fig. 7CGo), indicating the specificity of ER mRNA expression.



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  		FIG. 7. Levels of (A) ER{alpha}, (B) ERß, and (C) ß-actin mRNA in the ovaries of 8-d-old hamsters exposed in utero to FSH antiserum (FSH-AS) or nonimmune rabbit serum on d 12 of gestation and treated with eCG or saline on P1. Bars with same letter in each panel were not significantly (P < 0.05) different from each other. Each bar in each panel represents three separate observations (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study indicate that mRNA and protein of both ER{alpha} and ERß are expressed in hamster ovarian somatic cells and the oocytes long before any morphologically identifiable primordial follicles appear, which occurs by P8 (16). Steady levels of ß-actin mRNA suggest that changes in ER mRNA levels are specifically related to ovarian morphogenesis rather than due to a generalized growth effect. The differential expression pattern of ERß mRNA and protein compared with ER{alpha} mRNA and protein suggests that translation of ER{alpha} mRNA may be associated with the differentiation of somatic cells into interstitial cells, whereas cells expressing ERß primarily differentiate into the granulosa cells. This notion is further supported by in situ hybridization data showing that expression of receptor mRNA indeed occurs in ovarian cells when receptor protein expression cannot be detected. Whether the initiation of ER translation in ovarian cells during perinatal development requires specific levels of mRNA or whether specific translational regulations are imposed when cells differentiate into a particular lineage is not known, but a comparison of the patterns of protein and mRNA expression seem to suggest such possibilities. The increase in ER{alpha} mRNA levels and the number of interstitial cells expressing ER{alpha} by P15 coincide with the appearance of LHR, CYP11A1, and CYP17 in the hamster ovary (25) (Roy, S. K., unpublished observation). Although relatively more ERß mRNA is present in developing rat ovaries (5), marked increases in ERß mRNA levels in the rat ovary by P8 and steady high levels of ER{alpha} during the early part of the postnatal development (5) correlate well with the levels in the hamster. Similar increases in ERß mRNA levels have also been observed in postnatal mouse (12) ovaries. In contrast to neonatal hamsters, ovarian expression of ERß mRNA is higher than that of ER{alpha} in adult hamsters (6). In fact, such a tendency is noticeable with the development of larger secondary follicles by P15 (present study) when serum levels of FSH and LH start to increase (20, 26). The results provide evidence, for the first time, that FSH action during perinatal ovary development is needed for ER expression in undifferentiated somatic cells during their differentiation into respective cell types and that ER action may be necessary for the formation and functions of the granulosa cells during early ovarian morphogenesis. Absence of estrogen-mediated ER action in mice null with aromatase gene (ARKO) correlates with the lower number of primordial and primary follicles in the adult (27). Previously, we have demonstrated that the formation of primordial follicles is severely compromised when perinatal FSH is neutralized with an FSH antiserum (16). Furthermore, FSH induction of ER in the granulosa cells is well documented (28). Distinct expression of ER{alpha} and ERß protein, despite the decline in the levels of receptor mRNA, prompts us to speculate that once differentiation of ovarian somatic cells into specific lineages is complete, regulation of transcription and/or mRNA turnover become more precise. It is possible that the initiation of ER{alpha} translation requires a high rate of transcription, but once the expression of the receptor protein becomes adequate, a steady-state mechanism maintains the mRNA at moderate levels. It is noteworthy that the levels of ER{alpha} mRNA remain above the levels at d 13 of gestation when no ER{alpha} protein can be localized.

Whether estrogen action is needed for perinatal folliculogenesis in the hamster is not fully clear; however, the restoration of folliculogenesis with associated induction of ER mRNA and protein by eCG suggests the possibility. In support of this contention, recent findings have demonstrated that ARKO mice have a significantly low number of primordial and primary follicles (27). Moreover, replacement of estrogen in juvenile ARKO mice fails to correct the defect, indicating that estrogen-mediated ER activation may play a critical role in primordial and primary follicle formation. Combined deletion of ER{alpha} and ERß genes in female mice results in postnatal sex reversal with associated infertility (8). Likewise, deletion of the CYP19 gene in mice results in compromised antral follicle development with absence of ovulation (29). The localization of both ER{alpha} and ERß in hamster primordial granulosa cells corroborates earlier findings, which have demonstrated the presence of both ER subtypes in the somatic cells of fetal baboon ovaries as early as d 100 of gestation (30) and mRNA in neonatal rat (5, 10) and fetal human ovaries (13). Zachos et al. (30) have demonstrated that reduction of maternal and fetal serum estradiol levels in baboons by an aromatase inhibitor significantly reduces the number of primordial and primary-like follicles in fetal ovaries, and the loss can be prevented with estradiol replacement, thus providing the evidence to support the hypothesis that ER plays an important role during early folliculogenesis. Similar findings have been reported in mice by Britt et al. (27), with the exception that estradiol treatment cannot restore early folliculogenesis. Although evidence has accumulated to indicate that ER can be phosphorylated and activated by growth factors, such as epidermal growth factor (EGF) (31, 32), studies done on ARKO mice indicate that severe defects in folliculogenesis occur despite the presence of both ER{alpha} and ERß (27, 29, 33), thus implying that ligand-independent actions of ER may not be involved in ovarian follicular development. However, any supporting role of growth factors in estrogen- or ER-mediated folliculogenesis cannot be completely ruled out at present. Both EGF and EGF receptor are expressed in hamster granulosa cells (34, 35), and EGF receptor is expressed in the somatic cells of neonatal hamster ovaries (Wang, J., and S. K. Roy, unpublished observation).

Whereas the biological significance of ER expression in the hamster oocytes is less clear at present, our results provide strong evidence that ER genes are transcribed and translated in the oocytes. The early appearance of ERß and late appearance of ER{alpha} protein in the oocytes during perinatal development also indicates the specificity of ER expression. The presence of immunoreactive ERß in the oocytes of baboon fetal ovary (36) and the presence of ER mRNA in the mature oocytes of mouse (37) and human (38) ovaries have been documented. Recent studies on baboon fetal ovaries have demonstrated that estrogen maintains oocyte health by maintaining the number of microvilli (39), which are essential for oocyte-granulosa cell communication (40). Reduced formation of the primordial follicles (16) corresponding to attenuated expression of ER in FSH antiserum-treated ovaries and its reversal by eCG (present study) suggest that ER action may also be important for the development of the oocytes in the hamster.

Low ER{alpha} immunostaining in few granulosa cells of the secondary follicles confirms that granulosa cells are the primary site of ERß expression (4). Both subtypes of ER are present in fetal baboon ovarian cells (36), and low levels of ER{alpha} and high levels of ERß are present in the granulosa cells of adult hamster (6), rat (4, 41), monkey (42), and human (43, 44). Although both ER subtypes evoke estrogen-specific effect upon ligand binding (45), significant difference between ER{alpha} and ERß effects (46) and modulation of the effect of one subtype by the other have been documented (47, 48). Furthermore, ligand binding to one ER subtype may lead to either homo- or heterodimerization, and the latter possibility has been demonstrated in cells that possess both receptor subtypes (3, 49). These lines of evidence tempt one to speculate that a delicate balance of ER subtype interactions may ultimately determine the nature of estrogen signaling, which is critical for the differentiation of ovarian somatic cells into definitive granulosa and interstitial cells. The balance may shift toward ERß when follicles develop into large preantral stages and follicular steroidogenesis is established.

In summary, undifferentiated mesenchymal-epithelial cells and the oocytes in hamster ovaries during perinatal development express ER{alpha} and ERß immunoreactivities; however, ERß immunoreactivity segregates to the granulosa cells with the formation of primordial follicles. Furthermore, mRNA for both ER subtypes is expressed in ovarian cells as well as in the oocytes, and the onset of primordial follicle formation corresponds to increases in ER mRNA levels; however, mRNA expression declines to more stable levels when preantral folliculogenesis is established. Finally, perinatal FSH action is essential for ER expression in undifferentiated somatic cells and their subsequent differentiation into distinct ovarian cell lineage.


   Footnotes
 
This work was supported by Grant HD38468 (to S.K.R.) from the National Institute of Child Health and Human Development.

Abbreviations: ARKO, Null with aromatase gene; eCG, equine chorionic gonadotropin; EGF, epidermal growth factor; ER, estrogen receptor; LCM, laser capture microscopy; P1, postnatal d 1.

Received June 22, 2004.

Accepted for publication August 24, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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