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Division of Endocrinology (R.S., E.R., H.B.), Department of Physiology and Pharmacology, Göteborg University, SE-40530 Göteborg, Sweden; Department of Physiology (F.-P.Z., J.J.P.), Institute of Biomedicine, University of Helsinki, FIN-00014 Helsinki, Finland; and Department of Physiology (F.-P.Z., I.H.), University of Turku, FIN-20520 Turku, Finland
Address all correspondence and requests for reprints to: Håkan Billig M.D, Ph.D., Division of Endocrinology, Department of Physiology and Pharmacology, Göteborg University, P.O. Box 434, SE-40530 Göteborg, Sweden. E-mail: hakan.billig{at}fysiologi.gu.se.
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Introduction |
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The sequential actions of gonadotropins (FSH and LH) by binding to their respective receptors, FSH receptor and LH receptor, are essential for normal female reproduction, including follicular development, induction of ovulation, resumption of oocyte meiosis, and formation of the corpora lutea (11, 12, 13, 14). Experimental studies have previously shown that FSH receptor is exclusively present in granulosa cells of all healthy follicles throughout follicular development (15), whereas a specific expression of LH receptor in granulosa cells of perovulatory follicles is induced by the synergistic action of FSH and estradiol (16, 17, 18). The expression of LH receptor in granulosa cells is one of the major markers in the process of its differentiation and is essential for the induction of ovulation in response to the preovulatory LH surge (15, 18, 19).
The progesterone receptor (PR) in granulosa cells is considered to be important and an obligatory intermediate for ovulation (12, 13). The intracellular PR is a member of the superfamily of nuclear hormone receptors (that includes androgen, estrogen, glucocorticoid, and mineralocorticoid receptors) that functions as a ligand-activated transcription factor and is known to mainly mediate progesterone actions in target tissues (20, 21). In rodents, PR mRNA and protein are rapidly and transiently expressed in ovarian granulosa cells in response to the preovulatory gonadotropin surge (22, 23, 24), and PR knockout mice fail to ovulate (25), demonstrating that the expression of functional PR in granulosa cells plays a critical role in successful ovulation.
The ubiquitin system and its effects on the posttranslational modification of proteins have been implicated as a key intracellular signaling pathway for regulation of protein function (26). Although the biochemistry and enzymology of the sumoylation pathway have been characterized, a majority of reports have focused on how SUMO-1 recognizes and interacts with target proteins. SUMO-1 modification of protein can regulate protein-protein interaction, transcriptional activity, and subcellular protein translocation in the cells (2, 3, 4). Recently, significant progress has been made in the elucidation of how the nuclear receptors, such as androgen receptor, glucocorticoid receptor, and human PR, interact with SUMO-1 (27, 28, 29, 30). However, the regulation of SUMO-1 expression and functional distribution of SUMO-1 in vivo remain poorly understood.
In the present study, we investigate the LH receptor- mediated regulation of ovarian SUMO-1 expression in both whole ovary and granulosa cells in female mice. To better understand the physiological role of SUMO-1 during ovulation, we examine not only the direct interaction, but also the functional link between SUMO-1 and PR after stimulation of LH receptor in vivo.
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Materials and Methods |
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Animal, tissue, and granulosa cell preparation
Immature female and male mice (C57BL/6) were provided by Taconic M&B, Copenhagen, Denmark. The mice were randomized and housed, five or six per cage, in the temperature-controlled animal room (21 ± 2 C) under a constant 12-h light, 12-h dark cycle. They were fed with standard laboratory chow and water and were conditioned for at least 5 d before the experiments. Different tissues from both female and male mice (26 d old, 13–15 g) were removed, snap-frozen in liquid nitrogen, and stored at -135 C until assayed. All experimental procedures relating to the care and use of animals were approved by the local ethics committee, Göteborg University, Sweden.
In this study, sequential administration of gonadotropins (PMSG and hCG) was used to substitute hormones for effects of the endogenous FSH and LH. LH and hCG recognize the same receptor (LH receptor). Female mice (26 d old, 13–15 g) were killed 48 and 72 h after treatment with PMSG (5 IU ip) to stimulate the development of multiple immature follicles (31). Some of the animals were randomly assigned to receive additional hCG (5 IU ip) 48 h after administration of PMSG to mimic the LH surge and induce ovulation and luteinization (31, 32, 33). These animals were killed 3, 6, 12, 24, and 48 h after hCG treatment. Untreated littermate mice served as controls. The ovaries were dissected out, and adhering tissues and fat were rapidly removed, instantly frozen in liquid nitrogen, and stored at -135 C before processing or fixed in 4% formaldehyde neutral buffered solution (Sigma) for 24 h and processed for paraffin embedding. In addition, the granulosa cells were isolated by puncturing either all follicles from immature mice or only big follicles or early corpora lutea from mice at various times after treatment with PMSG and/or hCG, as described previously (24), and were kept at -135 C until analysis.
Experiment with pentobarbitone sodium in vivo
The effect of blockade of the endogenous LH surge was studied by administration of pentobarbitone sodium in vivo (34). Mice were randomly divided into three groups. After mice were treated with PMSG (5 IU ip) for 52 h, either 0.5–1 mg pentobarbitone sodium in 100 µl saline alone or together with 5 IU hCG, or 100 µl saline by a single ip injection between 1200–1300 h. Mice were killed 72 h after treatment with PMSG. The granulosa cells were isolated and prepared for analysis as described above. To evaluate pentobarbitone sodium inhibition of LH receptor-mediated induction of ovulation (35, 36), ovaries were collected and sectioned, 2 d after pentobarbitone sodium treatment. The number of corpora lutea was used as a measure of successful LH stimulation of preovulatory follicles. Corpora lutea were determined by immunohistochemical staining for CD 31, an endothelial cell marker (37).
Experiments with the PR antagonists in vivo and in vitro
The PR antagonists Org 31710 and RU 486 were dissolved in sesame oil. All ip injections were in a vol of 100 µl. Mice were randomly divided into three groups and given either 2 mg Org 31710 (n = 5) or 1 mg RU 486 (n = 5) in combination with 5 IU hCG after treatment with 5 IU PMSG for 48 h. An equal volume of sesame oil was injected into control mice (n = 10). After 12 h, granulosa cells were isolated from the ovaries and stored at -135 C until assay.
In some experiments, the granulosa cells were isolated by puncturing only the largest hyperemic preovulatory follicles from mice after combined PMSG/hCG treatment as described above. Cells were collected in Earle’s MEM with glutamax-I (MEM, Life Technologies, Carlsbad, CA) supplemented with 0.1% BSA–fraction V (Sigma), 100 U/ml penicillin, and 100 µg/ml streptomycin and pelleted at 200 x g for 5 min at 4 C. Cell viability was always above 70%, as determined by the trypan blue dye exclusion method (Life Technologies). The granulosa cells were placed in tubes (Falcon 12 x 75 mm, Becton Dickinson, Franklin Lakes, NJ) at about 5 x 105/0.5 ml and incubated in the absence or presence of 10 µM Or 31710 or 10 µM RU 486 at 37 C in a humidified atmosphere with 95% air-5% CO2 for 24 h. Org 31710 and RU 486 were solubilized in dimethylsulfoxide or absolute ethanol. Aliquots from each stock solution were added to fresh medium, and each preparation contained less than 0.01% dimethylsulfoxide (vol/vol, final concentration) or ethanol, and served as the control treatment in all experiments. On termination of PR antagonist treatments, granulosa cells were harvested after washing with PBS, and tubes were centrifuged at 200 x g for 5 min at 4 C for Western blot analysis.
Protein extraction and Western blot analysis
The protein extraction and Western blotting were performed as described previously (24). Tissues or granulosa cells were homogenized and pooled, and equal amounts of protein per sample were loaded onto either 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels or 6% Tris-Glycine gels (Novex, San Diego, CA) under reducing conditions, transferred electrophoretically to polyvinyldifluoride membranes (Amersham International, Buckinghamshire, UK), and treated with blocking buffer [0.2% I-Block (TROPIX, Bedford, MA) 0.2% BSA, 5 mM MgCl2, 3 mM NaN3 and 0.3% Tween 20 in PBS, pH 7.4)] for 4 h. The membranes were incubated with the appropriate primary antibody at 1:1,000 dilutions in blocking buffer overnight at 4 C. Binding was detected with alkaline phosphatase-linked secondary antibody (monoclonal secondary antibody at 1:80,000 or polyclonal secondary antibody at 1:40,000 dilutions, respectively) in blocking buffer for 4 h, with gentle shaking, and enhanced using CDP-Star as substrate (Tropix). The second antibodies contributed no nonspecific bands at the concentrations employed. Immunoblotted signals were exposed and developed by ECL-film (Amersham International) and subsequently scanned into a computer. Individual bands were quantified directly from membranes by densitometry using the ImageQuant (version 5.0) software program (Molecular Dynamics, Inc., Sunnyvale, CA). No electronic modifications of the images, such as contrast or brightness adjustment, were performed before quantitation. All steps were carried out at room temperature unless otherwise stated. Blots were reprobed with a mouse monoclonal anti-ß-actin antibody for loading normalization without stripping membranes, as well as confirmed by staining using a colloidal blue staining kit after transfer (Novex).
Immunohistochemical analysis
Embedded tissue was sectioned at 5 µm and mounted onto poly-L-lysine-coated slides, deparaffinized in xylene, and rehydrated through graded series of ethanol. Antigens were retrieved by boiling in 10 mM sodium citrate buffer (pH 6.0) in a microwave oven for 10 min and then cooled down for 20 min. After washing with Tris-buffered saline (TBS, 50 mM Tris, 0.9% NaCl, pH 7.5) three times, endogenous peroxidase activity was abolished by incubating sections in 3% hydrogen peroxide in TBS for 10 min, and nonspecific binding was blocked by incubating sections in 10% normal goat serum for 1 h at room temperature. The primary antibody was diluted 1:250 in TBS containing 1% BSA and incubated overnight at 4 C in a humidified chamber. After washing with TBS, sections were stained using the avidin-biotinylated-peroxidase complex detection system (Mouse on Mouse Immunodetection kits, Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions. Immunostaining was then visualized using 3,3-diaminobenzidine tetrahydrochloride (0.5 mg/ml in PBS and 0.01% H2O2, pH 7.6) for 10 min. Sections were dehydrated and coverslipped using Histo Mounting Medium (Mountex, Histolab, Göteborg, Sweden). The specificity of SUMO-1 primary antibody was monitored in the separate control sections. Additional sections were treated as follows: 1) TBS containing 1% BSA substituted for omission of primary antibody. 2) Normal mouse IgG was used instead of primary antibody. 3) Dilutions of primary antiserum were performed to quench positive staining as a function of specific antibody concentration. For fluorescence immunostaining of PR, the same sections were incubated with PR primary antibody diluted to 1:250 in PBS containing 1% BSA and 3% fat-free milk overnight at 4 C. Immunodetection was accomplished using a biotin-conjugated antirabbit antibody and Cy 3-conjugated streptavidin under the same conditions. The specificity of the PR-positive staining was confirmed by an immunohistochemical absorption study (24). Sections were washed and mounted with either Histo Mounting Medium (Mountex, Histolab) or Fluorescent Mounting Media (Dako, Carpinteria, CA). Slides were viewed under a Nikon E-1000 microscope (supplied by Bergström Instrument AB, Stockholm, Sweden), under either brightfield or epifluorescence optics, and photomicrographed using Easy Image 1 (Bergström Instrument AB).
Coimmunoprecipitation assays
For coimmunoprecipitation experiments, 2 µg of antibodies directed against the PR 538 and SUMO-1 were added to precleaned granulosa cell lysates and incubated for 4 h at room temperature. Nonspecific rabbit IgG and nonspecific mouse IgG were used as controls. Immune complexes were obtained by the addition of 50 µl of Pansorbin cells (Calbiochem, San Diego, CA) and incubated for an additional 2 h at room temperature. The resulting immobilized immune complexes were pelleted by centrifugation at 3000 x g for 5 min at 4 C and washed twice with 1 ml RIRA buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 15 mM MgCl2, 0.5% Nonidet P-40, 0.3% Triton X-100, 0.5% sodium deoxycholate, 1 mM dithiothreitol, 10 mM N-ethylmaleimide, and a cocktail of protease inhibitors, pH 7.8) as described (28). The bound protein was eluted by boiling in 30 µl of 2x SDS loading buffer (125 mM Tris-HCl, 2.5% SDS, 20% glycerol, 5% ß-mercaptoethanol, and 0.01% Coomassie Blue R-250, pH 6.8) for 5 min. Immunoprecipitated complexes were examined by Western blot analysis as described above.
Data analysis and statistics
Each experiment was repeated at least three times unless otherwise stated. Results are expressed as the mean ± SEM. The data were analyzed using Analyze-It program (Analyze-It Software, Ltd., Leeds, UK). One-way ANOVA with post hoc Tukey’s test was used for multiple comparisons. A P value < 0.05 was considered statistically significant.
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Results |
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). Comparing the level of SUMO-1 protein expression between female and male mice, no gender-specific expression pattern was detected in nongonadal tissues. The highest levels of SUMO-1 protein expression occurred in the heart, whereas the lowest levels were detected in the brain in both sexes. In addition, the levels of SUMO-1 protein expression were high in the uterus of female mice and low in the testis of male mice.
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, A and B, top), was detected in all ovarian and granulosa cell samples collected at different times after gonadotropin treatment. Quantitative analysis of the SUMO-1 protein level in the whole ovary detected no change during 48 h of PMSG treatment (Fig. 2A, bottom). The ovaries of PMSG/hCG-stimulated animals showed an initial down-regulation of ovarian SUMO-1 protein at 6 h, with further depression at 12 h, compared with PMSG treatment at 0 h. Complete recovery was observed at 24 h, and the level of SUMO-1 protein was further increased at 48 h (Fig. 2A, bottom). However, the level of SUMO-1 protein in granulosa cells was gradually decreased at 48 h after PMSG treatment and further reduced at 3 and 12 h after hCG treatment (Fig. 2B, bottom). Furthermore, in the ovary with established corpora lutea, i.e. 48 h after hCG treatment, the level of SUMO-1 protein in luteal cells was increased again, although it remained lower than in granulosa cells of untreated immature ovaries (Fig. 2B, bottom).
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). SUMO-1 immunostaining was homogeneously distributed in the nuclear membranes and nuclear bodies of both granulosa and thecal cells (Fig. 3B), consistent with the presence of SUMO-1 within nuclei in a wide range of cell types in a variety of tissues. There was no change on SUMO-1 immunostaining in the granulosa cells from all stages of follicles before 48 h-PMSG treatment. In the ovaries from mice treated with PMSG for 48 h, an immune reaction of SUMO-1 expression was present in the granulosa cells from periovulatory follicles (Fig. 3, C and D). However, a relatively high level of SUMO-1 expression was present in the antral granulosa cells, compared with those adjacent to the basement membrane in the same periovulatory follicles, after treatment with PMSG for 48 h and hCG for an additional 12 h (Fig. 3, E and F). Of note, the amount of SUMO-1 immunostaining in the granulosa cells corresponded to the intensity of the SUMO-1 protein band studied by Western blot analysis. Immunoreactivity in theca cells (Fig. 3, A, C, and E) surrounding the follicles was visible throughout the ovary regardless of the stage of follicular development. Specificity of the immunostaining was demonstrated by replacing the primary antibody with normal mouse IgG at the equivalent titer or omitting the primary antibody. Control sections did not show any evidence of positive labeling (data not shown).
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). To evaluate the specific inhibitory effect of pentobarbitone sodium, LH was replaced by a single injection of hCG (5 IU) in some of those animals. The increased level of SUMO-1 expression, after blocking endogenous LH, was partially reversed by concomitant hCG treatment (Fig. 4).
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), as previously reported (24). Coimmunostaining the same sections with anti-PR 538 antibody (Cy 3 fluorescence, Fig. 5B(-1) and anti-SUMO-1 antibody (peroxidase-diaminobenzidine, Fig. 5B-2) revealed the coexpression of SUMO-1 and PR within the periovulatory granulosa cells, despite the presence of the expected larger numbers of granulosa cells with SUMO-1 protein. Next, to study possible protein interaction between SUMO-1 and PR in vivo, granulosa cell lysates were prepared for either detection of PR expression (Fig. 6A), subjected to Western blot analysis, or coimmunoprecipitation using two different antibodies: mouse monoclonal anti-SUMO-1 or rabbit polyclonal anti-PR 538 antibodies. Western blot analysis was then performed to detect SUMO-1 expression in whole cell and tissue lysates and in both immunoprecipitates (Fig. 6B). We observed that PR proteins were absent in granulosa cells at 48 h after PMSG treatment, whereas they were present in granulosa cells at 6 h after additional hCG treatment using two different rabbit polyclonal anti-PR antibodies. A decrease of SUMO-1 level in granulosa cells after gonadotropin treatment was observed (Fig. 6B, left), consistent with results in SUMO-1 expression analyses (Fig. 2B
). To ensure that SUMO-1 was present in all of the samples tested and that it was immunoprecipitated, the anti-SUMO-1 immunoprecipitates were also immunoblotted with anti-SUMO-1 antibody (Fig. 6B, middle). SUMO-1 was coimmunoprecipitated with PR in granulosa cells at 6 h after additional hCG treatment but not at 48 h after PMSG treatment (Fig. 6B, right). To demonstrate the specificity of the interaction between SUMO-1 and PR, proteins isolated from uterine tissues that express both SUMO-1 and PR as positive controls, and small intestinal tissues that only express SUMO-1 but not PR as negative controls, were studied. Specific interaction was found to exist in mouse uterine tissues as well as ovarian granulosa cells. There was no evidence from coomassie-stained gels or from the immunoreactive normal rabbit IgG and mouse IgG of any nonspecific effects when coimmunoprecipitated with either PR or SUMO-1 (data not shown).
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) and in vitro (Fig. 7B).
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Discussion |
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Our present data, showing a constitutive expression of SUMO-1 in all tissues examined, are consistent with the global role for SUMO-1 in normal tissue physiology (2, 3, 26). This finding is in line with, and extends, previous studies demonstrating SUMO-1 mRNA expression in various human and mouse tissues (7, 8, 9, 10).
During the growth and maturation of ovarian follicles, the expression of the FSH-induced LH receptor in granulosa cells is only detected in preovulatory follicles that are able to respond to the endogenous LH surge, in contrast to the other stage follicles (16, 17, 18). Immunohistochemical analysis revealed that the expression of SUMO-1 protein is dramatically reduced in granulosa cells from periovulatory follicles, but not in granulosa cells from early stages of follicular development, suggesting that the inhibition of SUMO-1 expression is LH receptor dependent in granulosa cells in vivo. In the present study, we present several lines of evidence in support of the LH receptor-mediated regulation of SUMO-1 expression in the mouse ovary: 1) In response to LH receptor stimulation by hCG treatment, both SUMO-1 mRNA (data not shown) and protein expression decreased in both whole ovary and granulosa cells. 2) We detected a significant reduction of SUMO-1 protein in granulosa cells in mice treated with PMSG at the time when the endogenous LH-induced ovulation occurs. In rodents, an endogenous LH surge occurs around 56 h after PMSG treatment and is followed by ovulation 12–14 h later (31, 32, 33). 3) Interruption of the endogenous LH surge by pentobarbitone sodium treatment blocked the decrease of SUMO-1 protein levels in granulosa cells. Furthermore, this effect was reversed when the missing endogenous LH surge was substituted by hCG treatment. Taken together, our in vivo results suggest that the stimulation of LH receptors in granulosa cells is essential for the specific inhibition of SUMO-1 expression, in addition to its well-documented effects on granulosa cell differentiation and induction of ovulation.
The endogenous LH surge is an obligatory signal for the rapid preovulatory rise in ovarian progesterone production under physiological conditions (11, 21). Progesterone acts as a local regulator of ovulation and luteinization mediated by PR in preovulatory follicles (20, 21). Previous studies by us and others (22, 23, 24) have shown that the PR is induced by the stimulation of LH receptor during ovulation in granulosa cells in rodent ovaries. We show that both in vivo and in vitro administration of the PR antagonists induces a significant up-regulation of SUMO-1 protein levels in PR-expressing granulosa cells, suggesting that the effect of LH receptor stimulation on SUMO-1 expression is mediated via progesterone and PR. Indeed, treatment with the PR antagonists suppresses PR mRNA and protein levels in vivo (24, 38). Interestingly, in vitro experiments have recently shown that SUMO-1 interacts with several nuclear receptors (27, 28, 29, 30). Our present finding has shown that SUMO-1 and PR are coexpressed in periovulatory granulosa cells, which express the high level of PR after treatment with PMSG for 48 h and hCG for an additional 6 h. We also demonstrated the interaction between SUMO-1 and PR, using the immunoprecipitation method. SUMO-1 indeed interacts with PR in vivo in ovarian granulosa cells, which expressed both SUMO-1 and PR after gonadotropin treatment, suggesting that the PR is one of the sequential links between the preovulatory LH surge and SUMO-1 expression. At the ovarian level in particular, the inhibition of SUMO-1 by LH receptor stimulation may be a consequence of the induction of PR expression in the mouse ovary. Because SUMO-1 suppresses androgen receptor activity (27), it is reasonable to speculate, from our observations, that SUMO-1, progesterone, and PR belong to a intraovarian negative feedback loop in the mouse ovary: progesterone inhibits SUMO-1 expression, and SUMO-1 interaction with PR suppresses progesterone-PR transcriptional activity. The obligatory function of PR in ovulation has been well delineated using PR gene-deficient mouse ovary (25). Given the fact that the administration of the PR antagonists RU 486 in vivo and Org 31710 in vitro inhibited ovulation in mice (24, 39, 40) and enhanced SUMO-1 expression, our present data suggest that SUMO-1 modulates PR function in the ovulatory process.
The initiation of SUMO-1 activation requires the conversion of its nonfunctional precursor to a mature form by specific proteases (3). It has recently been shown that a specific protease for maturation of SUMO-1 is expressed at high levels in human reproductive organs, including the ovary (41). Furthermore, the mature SUMO-1 conjugates to various target proteins involved in a multienzyme ligase process of the sumoylation pathway which further regulates cellular functions (2, 3, 4). Thus, change of SUMO-1, in particular the mature form of SUMO-1, is of vital importance for sumoylation. Although our data do not distinguish between free SUMO-1 and conjugated SUMO-1 under the experimental conditions used, a minor part of SUMO-1 is free within the cells (42).
In summary, this study provides the first evidence of the regulation and cellular localization of SUMO-1 expression in the mouse ovary. We demonstrated that stimulation of LH receptors in granulosa cells results in down-regulation of SUMO-1 expression with concomitant induction of ovulation. SUMO-1 interacts directly with PR, and the PR antagonists increase SUMO-1 expression both in vivo and in vitro. To our knowledge, this is the first report of physical and functional links between SUMO-1 and PR in the mouse granulosa cell.
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Acknowledgments |
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Footnotes |
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Abbreviations: DAB, Diaminobenzidine; hCG, human chorionic gonadotropin; PMSG, pregnant mare’s serum gonadotropin; PR, progesterone receptor; SDS, sodium dodecyl sulfate; SUMO-1, small ubiquitin-related modifier-1.
Received April 25, 2003.
Accepted for publication September 9, 2003.
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