Regulation by Gonadotropins and Estrogen in the ... - Oxford Academic

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Endocrinology 145(5):2433–2444 Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2003-1328

Small Nuclear RING Finger Protein Expression during Gonad Development: Regulation by Gonadotropins and Estrogen in the Postnatal Ovary SIRPA J. HIRVONEN-SANTTI, VENKATARAMAN SRIRAMAN, MIKKO ANTTONEN, SAIJA SAVOLAINEN, JORMA J. PALVIMO, MARKKU HEIKINHEIMO, JOANNE S. RICHARDS, ¨ NNE OLLI A. JA

AND

Biomedicum Helsinki, Institute of Biomedicine (Physiology) (S.J.H.-S., J.J.P., O.A.J.), Department of Clinical Chemistry (O.A.J.), Program for Developmental and Reproductive Biology (M.A., M.H.), University of Helsinki, FIN-00014 Helsinki, Finland; Hospital for Children and Adolescents (M.A., M.H.), Helsinki University Central Hospital, FIN-00290 Helsinki, Finland; Institute of Biomedicine, Department of Anatomy (S.S.), University of Turku, FIN-20520 Turku, Finland; and Department of Molecular and Cellular Biology (V.S., J.S.R.), Baylor College of Medicine, Houston, Texas 77030 Small nuclear RING finger protein (SNURF/RNF4) is a steroid receptor coregulator that is down-regulated in testicular germ cell cancer. In this work, we examined SNURF expression during murine fetal gonad development and postnatal ovarian folliculogenesis by in situ hybridization and immunohistochemical staining. SNURF mRNA was detectable in gonads of both sexes from embryonic 10.5 days post conception onward. SNURF protein localized to gonocytes and somatic Leydig and Sertoli cells of fetal testis and in oogonia and supporting cells of fetal ovary. In murine postnatal ovary, SNURF mRNA and protein were expressed throughout folliculogenesis, peaking in the oocytes of preantral follicles. Lower amounts of SNURF mRNA and protein were also present in granulosa cells of secondary, antral, and preovulatory follicles and in luteal glands. Exposure of immature female mice and rats to gonadotropin from pregnant mare

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HE INITIAL EVENT in murine gonad development is formation of a bipotential urogenital ridge from the thickening of ventrolateral surface of each mesonephros at embryonic (E) 10.5 d post conception (dpc) in both female and male mice (1, 2). At E10.5–E11.0 dpc, proliferating primordial germ cells migrate from the base of the allantois along the hindgut to the genital ridges, and the female oogonia become associated with supporting pregranulosa cells. Perinatally granulosa cells encircle primary oocytes as a single layer of squamous cells to form primordial follicles. During postnatal folliculogenesis, the primordial follicles develop into large preovulatory follicles that contain several layers of columnar granulosa cells surrounding the oocyte (3, 4). Granulosa cells provide nutrients and chemical messengers critical for the maturation of the egg, and the oocyte itself Abbreviations: AMH/MIS, Anti-Mu¨llerian hormone; AR, androgen receptor; DES, diethylstilbestrol; dpc, days post coitum; E, embryonic; ER, estrogen receptor; H, hypophysectomized rat; hCG, human chorionic gonadotropin; PMA, phorbol myristate; PMSG, gonadotropin from pregnant mare serum; PR, progesterone receptor; SNURF/RNF4, small nuclear RING finger protein; Sp1, specificity protein 1; WT1, Wilms’ tumor 1 gene product. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

serum and human chorionic gonadotropin did not change dramatically SNURF mRNA levels in ovary. SNURF mRNA expression was increased in ovaries of immature mice treated with diethylstilbestrol, an effect that was blocked by the pure antiestrogen ICI 182,780. SNURF protein was constitutively expressed in oocytes of hypophysectomized rats, and its content was augmented by estradiol in granulosa cells. In granulosa cell culture, SNURF mRNA accumulation was transiently increased by treatment with the LH agonists phorbol myristate and forskolin at 4 h after treatment and at 48 h in differentiated cells expressing markers of the preovulatory phenotype. These results suggest a role for SNURF in fetal germ cell development as well as in oocyte and granulosa cell maturation in an estrogen- and gonadotropin-regulated fashion. (Endocrinology 145: 2433–2444, 2004)

promotes granulosa cell proliferation and differentiation (4, 5). In addition, granulosa cells are the site of action of FSH and estradiol that are critical for the follicle maturation beyond the preantral stage (4, 5). Small nuclear RING finger protein (SNURF/RNF4) was originally identified as a nuclear receptor coregulator through its interaction with the androgen receptor (AR) in a yeast two-hybrid screen (6). In addition to steroid receptors, SNURF has subsequently been shown to interact with several transcription factors, including promoter specificity protein 1 (Sp1), POZ AT-hook zinc finger protein, Goosecoid-like, TATA-binding protein, and activator of stromelysin gene expression (6 –11). SNURF possesses a C-terminal RING finger, a motif typical for many tumor suppressor proteins and E3 ubiquitin ligases (12). In the postnatal rat testis, SNURF mRNA and protein accumulate in postmeiotic round and elongating spermatids, implicating a role for this protein in the last steps of spermatid maturation, during which vast amount of protein degradation and chromatin compaction take place (13). Moreover, it was recently reported that accumulation of SNURF mRNA and protein is attenuated in testicular germ cell cancer, a disease with probable origin in the fetal period (14). To date, the pattern and development of SNURF expression in the ovary are largely unknown.

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Some coregulator proteins have already been implicated in the maintenance of female fertility, as exemplified by nuclear receptor interacting protein 1 (Nrip1), the null mutants of which are infertile due to a complete ovulatory failure (15). Likewise, female mice lacking TAFII105, a TATA-binding protein-associated factor, are infertile because of a defect in folliculogenesis and a disrupted inhibin-activin signaling pathway (16). To gain insight into the potential role of SNURF coregulator in the murine gonad development, we examined the spatial and temporal expression pattern of SNURF mRNA and protein in murine fetal gonads and during postnatal ovarian folliculogenesis. Furthermore, we present evidence for potential regulation of SNURF transcripts by gonadotropins and estrogen in the rodent ovaries in vivo. Materials and Methods Reagents Cell culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY), Sigma-Aldrich Co. (St. Louis, MO), Research Organics (Cleveland, OH), Fisher Scientific (Fairlawn, NJ), Corning, Inc. (Corning, NY), and Hyclone Laboratories, Inc. (Logan, UT). Trypsin, soybean trypsin inhibitor, DNAase, phorbol myristate (PMA), ATP, dithiothreitol, 17␤-estradiol, and propylene glycol were purchased from Sigma-Aldrich. Electrophoresis and molecular biology grade reagents were procured from Sigma-Aldrich, Bio-Rad Laboratories, Inc. (Richmond, CA), and Roche Molecular Biochemicals (Indianapolis, IN). Oligonucleotides were purchased from Genosys (The Woodlands, TX).

Tissue samples Mice were mated, and gonads dissected at E10.5, 12.5, 13.5, 15.5, and 17.5, and at postnatal d 1, 7, 14, 25, and 59 as well as at 11 wk, as described previously (17). The sex of d 10 and 12 embryos was determined by PCR using Sry-specific primers and embryonic genomic DNA as a template under the conditions reported previously (18). Immature FVB female mice, aged 25 d (n ⫽ 2–3 in each treatment group), were primed with a single ip injection of 2.5 IU gonadotropin from pregnant mare serum (PMSG; Sigma-Aldrich) as described earlier (19). Some of the animals were also given 2.5 IU human CG (hCG; Sigma-Aldrich) 48 h later. Mice were killed 48 h after PMSG exposure or 5 or 20 h after hCG injection to obtain ovaries containing preovulatory and postovulatory follicles, respectively (20). In the postovulatory group, ovulation was confirmed by microscopic demonstration of oocytes in the oviduct. In additional experiments, immature FVB female mice (25 d old) were treated daily with sc injections of 125 ␮g diethylstilbestrol (DES; a synthetic estrogen; Sigma-Aldrich), 50 ␮g of the pure antiestrogen ICI 182,780 (Tocris, Bristol, UK), 125 ␮g DES, and 50 ␮g ICI 182,780 in combination or vehicle in 95% mineral oil and 5% ethanol for 3 d (19, 21). All the animal studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of the University of Helsinki. Immature intact (aged 23 d) and hypophysectomized (H) female rats were obtained from Harlan Sprague Dawley, Inc. (Chicago, IL). H rats were injected with 1.5 mg 17␤-estradiol in 0.2 ml propylene glycol (Sigma) sc once daily for 3 d to stimulate the growth of large preantral follicles (HE model). During the following 2 d, the rats were injected with 1.0 ␮g ovine FSH (oFSH-16, NIH, National Hormone and Pituitary Agency, Rockville, MD) in 0.1 ml PBS ip twice daily to further stimulate the growth of follicles (HEF model). On the following morning, the rats were treated with a single ovulatory dose (10 IU) of hCG (Organon Special Chemicals, West Orange, NJ) ip to stimulate ovulation and luteinization (HEF/hCG). For granulosa cell cultures, intact rats were primed with estradiol to increase the yield of cells. Animals were housed under a 16-h light, 8-h dark schedule in the Center for Comparative Medicine at Baylor College of Medicine (Houston, TX) and provided food and water ad libitum. Rats were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals, as approved by the Animal Care and Use Committee at Baylor College of Medicine.

Hirvonen-Santti et al. • SNURF/RNF4 in Developing Gonads

In situ hybridization The sections of murine fetal gonads and postnatal ovaries were deparaffinized and hybridized to antisense and sense murine SNURF RNA probes, which were prepared by linearizing pGEMT-Easy plasmid (Promega, Madison, WI) containing a SNURF cDNA insert (nt 156 –536, GenBank accession no. AF169300) with SpeI and NcoI restriction enzymes (Amersham Pharmacia Biotech, Aylesbury, UK), respectively, and synthesizing labeled RNA probes in the presence of [␣-35S]uridine 5-triphosphate (Amersham Pharmacia Biotech) using T7 and Sp6 polymerase (Promega), respectively. In situ hybridization and emulsion autoradiography were performed as described previously (22). In situ hybridization on rat ovarian samples was performed as reported (23, 24) using 35S-labeled antisense and sense rat SNURF RNA probes generated using linearized plasmids containing SNURF RT-PCR product inserts (see below) as templates. The 7-␮m paraffin-embedded rat ovarian sections were rehydrated, treated with 20 ␮g/ml proteinase K and 0.1 m triethanolamine/acetic anhydride, dehydrated, and hybridized with radiolabeled SNURF RNA probe overnight at 55 C. The slides were washed at high stringency, dried, and exposed to X-OMAT film (Kodak, Rochester, NY) overnight to assess hybridization signal intensity. Slides were dipped in photographic NTB-2 emulsion (Kodak) exposed at 4 C for 14 d, developed with D-19 developer and fixer (Kodak) and stained with hematoxylin.

Immunohistochemistry Paraffin-embedded sections of murine fetal gonads were used for immunohistochemical staining with polyclonal anti-SNURF antibody that was raised against SNURF in rabbits (6) (1:2000 dilution in Trisbuffered saline containing 1% BSA), and the positive cells were visualized using Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. As a control, 500 ng GST-SNURF were first incubated with the antibody (1:2000 dilution) for 16 h at 4 C, and the resulting preadsorbed anti-SNURF antibody was used to monitor the specificity of staining (25). Estrogen receptor (ER)␤ antigen was detected using anti-ER␤ 503 antibody that was raised against human ER␤ in chicken (a gift from Dr. Jan-Åke Gustafsson, Novum, Huddinge, Sweden) (diluted to 1:1000 in PBS with 3% BSA and 0.05% Tween 20) and peroxidase-conjugated rabbit antichicken IgG (1: 1000 dilution in PBS and 2% rat normal serum; Sigma-Aldrich). Peroxidase activity was visualized with diaminobenzidine as the substrate (Vector Laboratories), and the sections were stained with Mayer’s hematoxylin, dehydrated, and mounted. The rat ovarian sections were rehydrated, exposed to 0.1% hydrogen peroxide, and subjected to 10% nonimmune goat serum to block nonspecific sites. The sections were incubated with SNURF antiserum (1: 500) at 4 C overnight followed by incubation with biotinylated antirabbit antiserum (1:450, Vector Laboratories) for 1 h. Slides were washed, and streptavidin-conjugated horseradish peroxidase (Vector Laboratories) was applied for 30 min. Peroxidase activity was detected using diaminobenzidine (Vector Laboratories) for 2 min, and sections were dehydrated without counterstaining, and mounted.

Granulosa cell cultures Granulosa cells were harvested by needle puncture from immature rats (aged 26 d) treated with 1.5 mg estradiol in 0.2 ml propylene glycol on d 23–25 of age as described previously (25). The cells were cultured on serum-coated 12-well plates (0.5 ⫻ 106 cells per 1.5 ml) in DMEM/F12 containing 100 IU/ml penicillin and streptomycin. On the following day, the cells were supplemented with serum-free medium containing FSH (50 ng/ml; NIH oFSH-16), forskolin (10 ␮m), testosterone (10 ng/ml), and/or PMA (20 nm) and harvested after selected time intervals.

Semiquantitative RT-PCR Total RNA were extracted from whole ovaries of PMSG/hCG-primed intact immature rats and H rats primed with estradiol, FSH, and/or hCG as well as from granulosa cells and residual tissue using Trizol reagent (Life Technologies) according to the manufacturer’s instructions. Semiquantitative RT-PCR was performed as described previously (26), using primers specific for SNURF mRNA (forward: 5⬘-AACCGTTGGAGAT-


GAAATCG-3⬘ and reverse: 5⬘-GGAGGCACTGGCTACAGAAG-3⬘) and ribosomal protein L19 mRNA. Total RNA (500 ng) was reverse transcribed by using oligo-d(T) primer and AMV reverse transcriptase at 42 C for 75 min and 95 C for 5 min. DNA products were amplified in the presence of [␣-32P]dCTP and Taq polymerase for 22 cycles at 94 C for 1 min, 60 C for 2 min, and 72 C for 2 min. The cycle number was chosen by determining the linear range of amplification for the SNURF gene transcript. The amplified cDNA was resolved on a 5% polyacrylamide gel that was dried and exposed to x-ray film. The radioactive PCR products were quantified by using a Storm 860 PhosphorImager software (Molecular Dynamics, Sunnyvale, CA).

Results

To examine the pattern of SNURF mRNA accumulation during fetal gonad development and postnatal ovarian folliculogenesis, we prepared RNA probes corresponding to nt 156 –536 of murine SNURF cDNA (10). Using the antisense RNA probe, we detected SNURF mRNA at moderate levels in the fetal gonads of both sexes from E10.5 dpc onward (Fig.

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1). SNURF mRNA accumulated to high levels in the fetal testis by E15.5-E17.5 dpc, with the most intense signal being localized to the testicular cords (Fig. 1). Control in situ hybridization experiments with SNURF sense RNA probe revealed only background signal (Fig. 1). In addition, immunohistochemical staining using anti-SNURF antibody (6) detected SNURF protein in Leydig cells, some Sertoli cells, and male gonocytes in testis and in oogonia and somatic cells in the ovary at E17.5 dpc (Fig. 2). ER␤ protein colocalized with SNURF in gonocytes from E15.5 dpc onward in fetal testis and oogonia at E15.5 dpc in fetal ovary, as detected by immunohistochemical staining using anti-ER␤ antibody (Fig. 2). ER␤ protein was also detectable in some peritubular myoid cells. ER␤ immunoreactivity was not observed during E10.5-E13.5 dpc, and it could not be detected in the oogonia of fetal ovary after E15.5 dpc (data not shown). To examine SNURF mRNA expression pattern in murine

FIG. 1. Localization of SNURF mRNA during murine fetal gonad development at E10.5, E12.5, E13.5, E15.5, and E17.5 dpc by in situ hybridization using murine SNURF antisense RNA probe. Left, Bright-field and dark-field exposures of fetal testis. Right, Bright-field and dark-field exposures of fetal ovary at indicated time intervals. Control in situ hybridization experiments with SNURF sense RNA probe showed only background signal (indicated as sense).

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FIG. 2. Immunohistochemical staining of murine fetal testis (A) and fetal ovary (B) at E17.5 dpc using anti-SNURF antibody (1:2000 dilution). Positive immunoreactivity appears as brown color, and cell nuclei that are negative to SNURF protein are depicted by blue counterstain. C, Control staining of the fetal ovary using preadsorbed anti-SNURF antibody (1:2000 dilution). D and E, Immunohistochemical staining of murine fetal testis at E17.5 dpc and fetal ovary at E15.5 dpc, respectively, using anti-ER␤ 503 antibody (1:1000 dilution). Positive immunoreactivity is indicated by a brown color, and cell nuclei that are negative to ER␤ protein are visualized by blue counterstain. G, Male gonocyte; L, Leydig cell; O, oogonium; S, Sertoli cell.

postnatal ovary, ovarian sections from newborn, 7-d-old (7d, presenting primary follicles), 14-d-old (14d, secondary follicles), 25-d-old (25d, preantral follicles), 59-d-old (59d, antral follicles), and adult 11-wk-old mice were hybridized to SNURF antisense RNA probe. SNURF mRNA was expressed throughout the folliculogenesis: it peaked in oocytes of preantral follicles at 14 d (Fig. 3) and subsequently declined in oocytes of preovulatory follicles at 25 and 59 d (Figs. 3 and 4, A–D). In addition, SNURF mRNA was clearly detectable in granulosa cells of secondary, antral, and mature follicles and in luteal glands (Figs. 3 and 4). Immunohistochemical staining revealed the presence of SNURF protein in the oocytes and granulosa cells of primary, secondary, and preovulatory follicles and in luteal cells


(Fig. 4G). SNURF protein was also present at high levels in theca cells and at lower levels in interstitial cells and cells of atretic follicles, respectively (Fig. 4G). To determine whether the pituitary gonadotropins FSH and LH regulate SNURF mRNA levels in maturing ovarian follicles, 3-wk-old immature FVB female mice were treated with 2.5 IU PMSG followed by 2.5 IU hCG 48 h later (20). PMSG treatment (at 48 h) attenuated SNURF mRNA accumulation in oocytes and granulosa cells, whereas a subsequent injection of hCG (up to 20 h) increased SNURF mRNA accumulation to some extent (Fig. 5). To investigate whether SNURF mRNA levels were regulated by estrogen, immature female mice were treated with daily injections of 125 ␮g DES, 50 ␮g of the pure antiestrogen ICI 182,780, 125 ␮g DES and 50 ␮g of ICI 182,780 in combination, or vehicle for 3 d (19, 21). SNURF mRNA accumulation was increased to high levels in the oocytes of DES-treated ovaries; this effect was blocked by the antiestrogen ICI 182,780, suggesting that SNURF gene expression is controlled by ER-dependent signaling in the murine oocytes (Fig. 6). SNURF mRNA levels remained relatively constant in ovaries of female mice treated with ICI 182,780 alone, compared with the control mice (Fig. 6). To compare SNURF expression in the murine ovary with that in the rat and confirm hormone-regulated expression of this gene in detail, in situ hybridization, immunohistochemistry, and semiquantitative RT-PCR analyses were performed on ovarian samples derived from two in vivo rat models and granulosa cell cultures. As shown in Fig. 7A, SNURF mRNA is expressed in the oocytes of small and growing follicles of immature and PMSG-treated rats. In this intact rat model, SNURF mRNA accumulation was not dramatically regulated by PMSG or hCG treatments during preovulatory follicular growth and ovulation. However, SNURF mRNA content was reduced in the luteinized ovaries 24 – 48 h after hCG, indicating that SNURF is more prevalent in granulosa cells. In the H rat model, more dramatic changes in SNURF expression were observed, especially in granulosa cells. As shown by immunohistochemistry (Fig. 8A), SNURF protein was localized to granulosa cells and oocytes of small and growing follicles of the intact immature rat. In the H rat ovary, SNURF expression remained in oocytes, whereas it was clearly reduced in granulosa cells. Treatment of H rats with estradiol for 3 d partially restored SNURF protein accumulation in granulosa cells. These changes in immunostaining were confirmed by RT-PCR analyses of SNURF mRNA in granulosa cells isolated from hormone-primed H rats. Specifically, SNURF mRNA was expressed to high levels in granulosa cells of intact immature rats but declined markedly (approximately by 80%) after hypophysectomy. Exposure of H rats to estradiol increased SNURF mRNA content approximately 3-fold, close to the levels observed in cells obtained from the intact immature rat. Further treatment of estrogen-primed granulosa cells with FSH and hCG (HEF and HEF/hCG models), which stimulate preovulatory follicle growth, ovulation, and luteinization, slightly augmented SNURF transcript levels by 12 h after treatment. To study the intracellular signaling mechanisms that mediate the regulation of SNURF gene activation, granulosa cells were treated by forskolin and PMA and FSH and testosterone. Under serum-free conditions, SNURF mRNA content was up-



FIG. 3. SNURF mRNA expression in postnatal murine ovary by in situ hybridization at indicated time points. Bright-field (left) and dark-field exposures (right) are shown. NB, Newborn; d, days after birth; GC, granulosa cell; O, primary oocyte.

regulated at 4 h after treatment with 10 ␮m forskolin and 20 nm PMA (Fig. 8C), two agonists known to induce progesterone receptor, early growth response protein-1, activator protein-1 factors, and cyclooxygenase-2 in preovulatory granulosa cells

(27–30). To study the changes in SNURF mRNA levels during granulosa cell differentiation, the cells in culture were further supplemented with FSH (50 ng/ml) and testosterone (10 ng/ ml). Up-regulation of SNURF mRNA accumulation by the LH

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FIG. 4. Localization of SNURF mRNA in mature murine ovary by in situ hybridization. Bright-field exposures are shown on the left and dark-field exposures on the right. A–D, In situ hybridization using SNURF antisense probe; E and F, using SNURF sense probe. G, Expression of SNURF protein in murine ovary detected using anti-SNURF antibody (1:2000 dilution). CL, Corpus luteum; GC, granulosa cell; GF, Graafian follicle; O, oocyte.

agonists forskolin and PMA was transient, in that the increase observed at 4 h was no longer seen at 8 h after hormone exposure. However, at 48 h after the treatment in the differentiated granulosa cells, SNURF mRNA content was slightly elevated by forskolin and PMA over that observed with FSH and testosterone (Fig. 8C).

Discussion

In the present study, we examined the expression pattern of the steroid receptor coregulator SNURF during murine fetal gonad development and in murine postnatal ovaries by in situ hybridization and immunohistochemical methods.



FIG. 5. Accumulation of SNURF mRNA in gonadotropin-stimulated murine ovaries. Immature 3-wk-old female mice received a single ip injection of 2.5 IU PMSG followed by 2.5 IU hCG 48 h later to induce follicular maturation and ovulation. Mice were killed 48 h after PMSG or 5 or 20 h after hCG. The ovaries were harvested, sectioned, and subjected to in situ hybridization using SNURF antisense RNA probe. Unstimulated, Nontreated control mice. In situ hybridization with SNURF sense probe revealed only background signal (indicated as sense). Bright-field exposures are shown on the left and dark-field exposures on the right. GC, Granulosa cell; O, oocyte.

We found that SNURF mRNA is expressed in the embryonic gonads in both sexes from E10.5 dpc onward. Interestingly, steroidogenic factor 1 and the Wilms’ tumor 1 gene product (WT1), both critical for early urogenital development, have also been detected at E10.5 in murine urogenital ridges (31, 32). WT1 was previously identified as one of the potential activators of the murine SNURF promoter in mammalian cells (33), and colocalized expression of WT1 and SNURF during early gonad development supports the possibility that SNURF gene activation is dependent on WT1 signaling in gonads in vivo. The gonads develop initially in a manner that is indepen-

dent of the sex. They are morphologically identical in XX and XY embryos until E12.0 dpc, and in the absence of masculinizing transcription factors and testosterone, the default of the later gonad development is the female ovary (1–2). Sry, the sex-determining gene on Y chromosome, directs the differentiation of pre-Sertoli cells that appear in the male genital ridge at E12.5 dpc (34). By E13.5 dpc, the Sertoli cells have formed testis cords surrounded by peritubular myoid cells and secrete anti-Mu¨ llerian hormone (AMH/MIS) that suppresses the development of the female reproductive tract. In addition, the Sry-related factor Sox9 regulates AMH/MIS gene expression and, AMH/MIS is necessary for testicular

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FIG. 6. Accumulation of SNURF mRNA in estrogen-stimulated murine ovaries detected by in situ hybridization. Three-week-old immature female mice were treated with vehicle (indicated as control), 125 ␮g DES, 50 ␮g of the pure antiestrogen ICI 182,780, or 125 ␮g DES and 50 ␮g ICI 182,780 in combination, respectively, daily for 3 d. Bright-field (left) and dark-field exposures (right) are shown. CL, Corpus luteum; GC, granulosa cell; O, oocyte.

development (34, 35). The development of the male urogenital tract and all secondary sexual characteristics are also dependent on the production of testosterone by fetal Leydig cells. The only steroid receptor reported to be present in male germ cells, ER␤, was detected in murine and human fetal gonocytes at midgestation (36, 37). In agreement with the results of Lemmen et al. (36), we detected ER␤ protein in oogonia and testicular cords at E15.5 dpc using anti-ER␤ antibody. Likewise, SNURF mRNA and protein accumulated to high levels in testicular cords at midgestation (E15.5E17.5 dpc). In addition to AR, SNURF is known to interact with and coactivate ER␣ and progesterone receptor (PR) in mammalian cells (6). It is likely that SNURF also modulates ER␤ function. Environmental or endogenous estrogens affecting the embryonic testis have been implicated in testicular tumorigenesis (37, 38). Our findings suggest that SNURF

may contribute to testicular disease originating from the fetal period. We detected SNURF mRNA and protein in the oocytes and granulosa cells of primary, secondary, and preovulatory follicles and in luteal cells during postnatal ovarian development. The oocytes have entered the long prophase of the first meiotic division perinatally, and they complete the first meiotic division right before ovulation (4, 5). Recently Ret finger protein-like 4, encoding a RING finger-like protein with a B30.2 domain, was found to function as an E3 ubiquitin ligase in the proteasome-mediated degradation of maturationpromoting factor, a heterodimer of p34Cdc2 and cyclin B1, in the regulation of the meiotic cell cycle progression in murine oocytes (39). Also SNURF possesses a RING finger domain typical of several E3 ubiquitin ligases (12), is autoubiquitinylated, and mediates ubiquitinylation (39a), raising



FIG. 7. SNURF mRNA expression in immature and PMSG-treated rat ovary. A, Intact immature rats (aged 23 d) were treated with 10 IU PMSG sc and 10 IU hCG ip to stimulate the growth of preovulatory follicles and luteinization, respectively. With the use of 35S-labeled antisense SNURF RNA probe in the in situ hybridization experiments, an intense signal was detected in the oocytes (designated by arrows) of small and growing follicles in the ovaries from intact and PMSG-treated rats. B, Total RNA (500 ng) isolated from the whole ovaries from rats treated with PMSG and hCG were reverse transcribed using oligo-d(T) primer and AMV reverse transcriptase, and the cDNA was amplified in the presence of [␣-32P]dCTP and primers specific for SNURF and ribosomal protein L19 mRNAs. The amplified DNA was resolved on a 5% polyacrylamide gel, which was dried and exposed to x-ray film.

the possibility that SNURF, expressed abundantly in oocytes, participates in the regulation of oocyte meiosis. SNURF colocalizes with ER␤, AR, and PR in the granulosa cells, and with ER␣ in the oocyte and in the luteal gland. In view of this, it is tempting to suggest that SNURF interacts with steroid receptors and modulates their activity in ovarian cells (40 – 42). The first stages of folliculogenesis can proceed independently of FSH secreted from the anterior pituitary, but the small follicles are responsive to FSH, and the presence of FSH is critical for the follicular development beyond the preantral stage (5). FSH stimulates granulosa cell proliferation and CYP450 aromatase expression in granulosa cells. Aromatase promotes estradiol production by catalyzing aromatization of androgens that diffuse from the neighboring theca cells, and estradiol enhances the effects of FSH on follicle growth (5). In the mouse model, PMSG stimulates the growth of preovulatory follicles that synthesize increased amounts of estrogen. Accumulation of SNURF mRNA was increased in the oocytes of DES-primed ovaries, but exposure to PMSG resulted in somewhat attenuated or unchanged expression of SNURF mRNA in whole rodent ovaries and granulosa cells

by 48 h after treatment, as judged by in situ hybridization and RT-PCR studies, respectively. Nevertheless, the treatment with the antiestrogen ICI 182,780 blocked the stimulation of SNURF mRNA accumulation by DES in the murine oocytes, indicating that enhanced SNURF mRNA expression is regulated by estrogen signaling in murine oocytes. However, SNURF mRNA levels remained relatively constant in mice treated with ICI 182,780 alone, implying that estrogen-dependent signaling is not the only mechanism that regulates SNURF gene expression in oocytes. Interestingly, increased SNURF mRNA accumulation in the oocytes of preantral follicles was concomitant to raising follicular estradiol levels in the unstimulated developing and mature murine ovaries. Furthermore, the expression of SNURF in the oocytes of preantral follicles coincided with the high transcriptional activity at this stage, and SNURF mRNA levels declined in concert with the relative transcriptional inactivity in the preovulatory follicle. These data suggest that SNURF gene expression in maturing follicles is regulated in a stage-dependent fashion. In the H rat model, estradiol is mandatory for the formation of large, antral follicles highly responsive to FSH (43). In

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FIG. 8. Expression of SNURF mRNA and protein in the ovaries derived from H rats. A, Immunohistochemical staining of SNURF protein was analyzed using anti-SNURF antibody (1:500) in the immature intact and H rat ovaries before (H) and after (HE) stimulation with 1.5 mg estradiol sc for 3 d. B, Expression of SNURF mRNA, as analyzed by semiquantitative RT-PCR as described in the legend to Fig. 7B, in whole ovaries derived from intact immature rats and hypophysectomized rats before and after treatment with 1.5 mg estradiol sc for 3 d (HE), with 1 ␮g FSH sc twice daily for 2 d (HEF) and 10 IU hCG ip (HEF/hCG). C, Accumulation of SNURF mRNA in cultured granulosa cells derived from estradiol-primed intact immature rats cultured in serum-free DMEM:F12 medium in the presence of forskolin (F; 10 ␮M) and PMA (P; 20 nM) or with FSH (F; 50 ng/ml) and testosterone (T; 10 ng/ml) for 4, 8, and 48 h. The semiquantitative RT-PCR assays were performed as described in the legend to Fig. 7B.

hypophysectomized rats, SNURF mRNA levels were severely reduced in granulosa cells but remained relatively constant in oocytes after exposure to estradiol, providing further evidence that activation of the SNURF gene in

oocytes is not mediated entirely by estrogen signaling but involves other transcription factors as well. However, a slight increase in SNURF gene expression in response to estradiol in oocytes may not be detectable at the protein


level when immunohistochemical staining is used. To the best of our knowledge, the specific genes induced by estradiol in oocytes have remained elusive, but estrogendependent candidate genes in granulosa cells include cyclin D2 (5), ER␤ (44), Foxo1 (FKHR), Foxo3 (FKHRL1), and IGF1-R␤ (45, 46). The murine SNURF promoter is, however, not responsive to estradiol in mammalian cells of nonovarian origin that express ectopic ER␣ (33), suggesting that the effect of estrogen on SNURF gene transcription is mediated by indirect mechanisms. Granulosa cells of untreated control mice expressed SNURF mRNA at moderate levels, but PMSG or DES treatment did not increase SNURF mRNA accumulation in granulosa cells differentiating in response to these hormones. Furthermore, in cultured granulosa cells, SNURF mRNA content was induced by the LH agonists forskolin and PMA at 4 h after treatment, but this effect vanished by 8 h. However, SNURF levels were again elevated at 48 h in differentiated cells expressing markers of the preovulatory phenotype, such as aromatase and LH receptor. These data suggest that SNURF mRNA induction by LH agonists in granulosa cell cultures is, at least in part, dependent on the differentiation of cells to the preovulatory phenotype. The transient increase in SNURF mRNA accumulation in undifferentiated cells is likely to be mediated by rapid activation of intracellular kinase cascades, the members of which may not be functional during differentiation. Activation of the SNURF gene in differentiated cells could involve transcription factor signaling pathways present only in these cell types. SNURF may, in turn, regulate gene expression in the undifferentiated granulosa cells of growing follicles and in the differentiated granulosa cells in preovulatory follicles. SNURF interacts with PR (6), essential for ovulation, and the results herein imply that SNURF might play a role in granulosa cells during ovulation. The murine progesterone receptor promoter contains cis-elements for binding of Sp1/Sp3 that are critical for basal promoter activity and mediate activation of the promoter by forskolin and PMA in granulosa cells (27). A conserved proximal GC box is also the main regulatory element of the murine SNURF promoter (33), and this element may mediate LH agonist-induced activation of the endogenous SNURF gene. Taken together, SNURF is present in fetal male gonocytes, suggesting a role in the fetal male germ cell development and disease. In addition, SNURF mRNA is expressed to high levels in the oocytes of preantral follicles, implicating SNURF in oocyte maturation. In the immature rat ovary, SNURF is expressed constitutively in the oocytes of growing follicles, whereas SNURF mRNA expression in granulosa cells is dependent, at least in part, on pituitary hormones and estradiol. Further work is required to assess whether SNURF contributes to female fertility and pathogenesis of ovarian disease. Acknowledgments We thank Jorma Toppari for the help with microscopy, Marika Ha¨ kli for recombinant SNURF protein, and Marja-Liisa Ra¨ sa¨ nen for the help with hormone treatments. Received October 3, 2003. Accepted January 9, 2004.


Address all correspondence and requests for reprints to: Dr. Olli A. Ja¨ nne, Biomedicum Helsinki, Institute of Biomedicine (Physiology), University of Helsinki, P.O. Box 63, FIN-00014 Helsinki, Finland. E-mail: [email protected]. This work was supported by grants from Ahokas Foundation, Finnish Cultural Foundation, and Research and Science Foundation of Farmos, Helsinki University Research funds, Academy of Finland, and Biocentrum Helsinki.

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