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BOR - Papers in Press, published online ahead of print January 28, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.025510
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BIOLOGY OF REPRODUCTION 70, 1552–1561 (2004)
DOI: 10.1095/biolreprod.103.025510
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Cell-Specific Expression and Regulation of Soluble Guanylyl Cyclase {alpha}1 and ß1 Subunits in the Rat Ovary1

Fangxiong Shi, Robert L. Stewart, Jr, Emerson Perez, Jean Y.-H. Chen, and Philip S. LaPolt2

Department of Biological Sciences, California State University–Los Angeles, Los Angeles, California 90032


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and carbon monoxide, resulting in cGMP production. Recent studies indicate that NO and cGMP influence ovarian functions. However, little information is available regarding the ovarian expression of sGC. The present study examined sGC {alpha}1 and ß1 subunit protein levels in the ovary during postnatal development, gonadotropin-induced follicle growth, ovulation, and luteinization as well as in cultured rat granulosa cells. In postnatal rats, sGC {alpha}1 subunit immunoreactivity was high in granulosa cells of primordial and primary follicles on Day 5 but low in granulosa cells of larger follicles on Days 10 and 19. Theca cells of developing follicles, but not stromal cells, also demonstrated moderate sGC {alpha}1 immunoreactivity. In gonadotropin- treated immature rats, intense sGC {alpha}1 subunit staining was similarly observed in granulosa cells of primordial and primary follicles, but such staining was low in granulosa cells of small antral follicles and undetectable in granulosa cells of large antral and preovulatory follicles. Following ovulation, corpora lutea expressed moderate sGC {alpha}1 immunoreactivity. Similar ovarian localization and expression patterns were seen for sGC ß1, indicating regulated coexpression of sGC subunits. Immunoblot analysis revealed no change in total ovarian sGC {alpha}1 and ß1 subunit protein levels during gonadotropin treatment. Similarly, no effect of FSH on sGC subunit protein levels was apparent in cultured granulosa cells. These findings indicate regulated, cell- specific patterns of sGC expression in the ovary and are consistent with roles for cGMP in modulating ovarian functions.

cyclic guanosine monophosphate, follicular development, granulosa cells, nitric oxide, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The actions of FSH and LH on ovarian functions are mediated in large part through increased production of the second-messenger cAMP and subsequent activation of downstream signaling pathways [1, 2]. Although the importance of cAMP as a second messenger influencing the ovary is well recognized, other hormones and factors acting through diverse signaling pathways also modulate ovarian functions. In this regard, a large body of evidence indicates potential roles of cGMP as an important cyclic nucleotide regulating ovarian steroidogenesis, gonadotropin-receptor expression, inhibin expression, apoptosis, and ovulation [3– 7]. Thus, understanding the mechanisms regulating cGMP production in the ovary has become increasingly critical.

The synthesis of cGMP from GTP is dependent on the activity of guanylyl cyclase, which exists in both particulate and soluble forms. The particulate, membrane-associated forms serve as receptors, the enzymatic activity of which is stimulated by binding of natriuretic peptides [8, 9]. In contrast, soluble guanylyl cyclase (sGC) is a heme-containing heterodimer consisting of an {alpha} subunit (variously reported as 73–88 kDa) and a smaller ß subunit (70 kDa) [10]. Each subunit has two known isoforms ({alpha}1, {alpha}2, ß1, and ß2), which have slightly differing activities and distributions [11–15]. The {alpha}1 and {alpha}2 isoforms are fairly homologous in their middle and carboxyl terminus portions, but the amino termini differ markedly [11]. The ß1 and ß2 subunits differ markedly at both amino and carboxyl termini, with less than 50% homology in their middle regions [15].

A major activator of sGC is nitric oxide (NO), which binds the heme group of sGC and markedly stimulates activity of this enzyme, increasing cGMP production [16]. Carbon monoxide (CO) also stimulates sGC activity and may play physiological roles in triggering cGMP-dependent signaling pathways [17–19]. In addition to posttranslational activation by NO and CO, sGC clearly is regulated at the message and protein levels by a number of factors, including NO, cGMP, cAMP, and estradiol (E2) [20–24]. Previous studies demonstrate that the sGC subunits are expressed in many organs, including gonadal tissues [14, 25–27]. Recent findings indicate that treatment of granulosa cells with a NO generator or specific activator of sGC increases cGMP accumulation, indicating sGC subunit expression in granulosa cells [28, 29]. Furthermore, our previous studies using an antibody that detects both sGC {alpha} and ß subunits confirm the expression of sGC in rat granulosa cells (unpublished data). Whereas these observations demonstrate sGC expression in granulosa cells, to our knowledge no information is available regarding expression of sGC in other ovarian cell types or concerning regulation of sGC subunit protein levels in the ovary.

Given the growing importance of cGMP as a second messenger in the ovary and the minimal information regarding the control of cGMP production in gonadal tissues, the present study utilized immunohistochemical and immunoblot analysis to examine the cell-specific localization and regulation of sGC {alpha}1 and ß1 subunit protein levels in whole ovaries and cultured granulosa cells. Because the major activator of sGC, NO, is implicated in the inhibition of follicle growth and granulosa cell differentiation [6, 28, 30], we hypothesize that sGC may be down-regulated during these processes as one means of limiting the activity of NO. Furthermore, elucidation of the regulated, cell-specific manner of sGC expression in ovarian cells may provide new insights regarding the control of cGMP production and potential actions of this second messenger in regulating gonadal functions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Hormones and Reagents

Highly purified ovine FSH (oFSH-19-SIAFP; 94 x National Institutes of Health [NIH] FSH S1 U/mg; LH activity, 0.025 x NIH LH S1 U/mg) was obtained through the National Hormone and Pituitary Distribution Program (NIDDK, NIH). Androstenedione, eCG, and hCG were purchased from Sigma Chemical Co. (St. Louis, MO), and McCoy 5A medium (modified, serum-free), streptomycin sulfate, penicillin, and L-glutamine were obtained from Life Technologies (Rockville, MD). All other chemicals were reagent grade and obtained from Fisher Scientific (Pittsburgh, PA) unless otherwise specified.

Animals

Intact, immature Sprague-Dawley rats (age, 21 days; Harlan Sprague- Dawley, Indianapolis, IN) and young (age, 4 mo) rats of the Long-Evans strain (Charles River Laboratories, Wilmington, MA) were obtained from the commercial suppliers indicated. In addition, neonatal and postnatal females were obtained by mating adult Long-Evans males and females. Animals were maintained under a 16L:8D photoperiod with food and water available ad libitum. Animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Institutional Animal Care and Use Committee of California State University–Los Angeles.

Immature rats received a subcutaneous injection of eCG (10 IU) to stimulate follicular development, which was followed 52 h later by an ovulatory dose of hCG (30 IU). Ovaries were obtained at 0, 24, and 52 h after eCG and at 24 and 72 h after hCG (n = 4 rats/time point). One ovary was fixed in 4% paraformaldehyde and processed for immunohistochemical analysis. The remaining ovary was snap-frozen and used for protein extraction and subsequent immunoblot analysis. Similarly, ovaries were collected at approximately 1000 h from rats at Days 5, 10, and 19 of age for immunohistochemical analysis of sGC protein levels (the day of birth was designated Day 1; n = 4 rats/day).

Primary Ovarian Granulosa Cell Culture

To examine the regulation of sGC subunit protein levels in ovarian cells, we used a well-characterized primary granulosa cell culture system as previously described [28]. Ovaries were obtained from immature (age, 25 days) females treated for 5 days with subcutaneous E2 implants to stimulate proliferation of functionally immature granulosa cells. The ovaries were removed aseptically and rinsed in McCoy 5A medium supplemented with streptomycin sulfate (100 U/ml), penicillin (100 U/ml), and L-glutamine (2 mM). Ovarian follicles were then punctured and granulosa cells released with the aid of a 26-guage syringe needle under a stereomicroscope. The cells were harvested, washed, pelleted by centrifugation at low speed, and resuspended in fresh medium. The concentration of viable cells was determined by trypan blue exclusion using a hemocytometer. Approximately 1 000 000 viable granulosa cells were cultured per tube in 12- x 75-mm polypropylene, round-bottomed Falcon culture tubes using McCoy 5A medium supplemented with androstenedione (10–7 M) as substrate for E2 synthesis. Cells were cultured with media alone (control) or with FSH at 37°C in a humidified atmosphere of 95% air and 5% CO2 for the specified times, then harvested for immunoblot analysis of sGC subunit protein levels as described below. Each experiment was repeated five times.

Preparation of Protein Homogenates

Protein was obtained from cultured granulosa cells by disrupting the cell membrane with radioimmunoprecipitation (RIPA) lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.5% NP-40, 20% glycerol, 25 mM benzamidine, 0.5 µg/ml of leupeptin, 0.7 µg/ml of pepstatin A, 2 µg/ml of aprotinin, 10 µg/ml of trypsin inhibitor). The cell lysate was then centrifuged for 20 min at 4°C and 16 000 x g. Similarly, protein was extracted from whole frozen ovaries using RIPA lysis buffer in a Dounce homogenizer (Wheaton, Millville, NJ). After homogenization, samples were incubated for 30 min on ice and centrifuged for 20 min at 16 000 x g. Total protein concentrations were determined by a modification of the Bradford method (Bio-Rad Protein Assay; Bio-Rad Laboratories, Hercules, CA) using BSA standards prepared in an appropriate amount of RIPA buffer. Microplate absorbance readings were obtained at 595 nm. The remaining supernatant was snap-frozen and stored at –80°C.

Immunoblot Analysis

Determination of sGC protein levels was performed by immunoblot analysis. Briefly, the proteins were resolved by 7.5% polyacrylamide-SDS gel electrophoresis under reducing conditions. In each experiment, equal quantities of protein (20 µg for homogenized ovarian samples and 10 µg for granulosa cells) were loaded for each sample. Protein was then transferred electrophoretically from the gel onto nitrocellulose membranes. The blots were blocked with 5% milk-TBST (20 mM Tris-buffered saline, 0.05% Tween 20, pH 7.5) at room temperature for 1 h and incubated overnight at 4°C with diluted antisera specific to sGC {alpha}1 (1:20 000) or ß1 (1:5000) subunits (Sigma). These antisera were raised against regions of {alpha}1 and ß1 subunits not present in {alpha}2 and ß2 isoforms and have been shown to immunoprecipitate the {alpha} and ß type 1, but not type 2, isoforms (unpublished data from commercial supplier). Blots were then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) G, followed by washing and detection of immunoreactivity by chemiluminescence detection methods (Pierce Biotechnology, Rockford, IL). Blots were then used to expose radiographic film to visualize immunoreactive signals.

Analysis of Immunoblot Data

The intensity of immunoblot signals was determined by digital image analysis of radiographs using the ImagePC program (Scion Corp., Frederick, MD). To allow statistical comparison of results from different blots, levels were normalized to the value of the eCG Time 0 group in each blot. Differences in protein levels among treatment groups were determined by one- or two-way analysis of variance, followed by the Tukey post-hoc test. A confidence level of P < 0.05 was considered to be statistically significant.

Immunohistochemistry

After fixation, ovaries were embedded in paraffin, and sections (thickness, 8 µm) were cut and mounted on slides. The sections were then processed for immunohistochemical analysis similar to the method used in our previous report [31]. Briefly, sections were deparaffinized with xylene and rehydrated in graded ethanol before being washed with double- distilled water. To increase epitope exposure, sections were heated for 15 min in sodium citrate buffer (0.01 M, pH 6.0) in a microwave oven. The sections were cooled and washed with 0.01 M PBS (pH 7.2) and then blocked with 5% BSA in TBST for 1 h at room temperature. The sections were incubated overnight at room temperature with polyclonal antibody against either sGC {alpha}1 and ß1 subunit (Sigma), respectively, developed in rabbits. After washing three times with PBS, the sections were incubated with a 1:200 dilution of fluorescence-labeled goat anti-rabbit IgG (Alexa Fluor-488; Molecular Probes, Inc., Eugene, OR) for 1 h at room temperature. The sections were observed using an epifluorescence Nikon Inverted Microscope (Nikon, Tokyo, Japan) equipped with a 496-nm excitation- wavelength filter. Specificity of the antibody was examined using normal rabbit serum (NRS) instead of primary antibody. The sections were counterstained with hematoxylin and mounted with coverslips.

Intensity of immunoreactive staining was scored by two independent observers using the following ratings: –, No staining detected; +, weak; ++, moderate; +++, strong staining. Stages of follicular development were classified as follows: Primordial follicles contained an oocyte surrounded by a single layer of squamous granulosa cells, primary follicles contained an oocyte surrounded by a single layer of cuboidal granulosa cells, preantral follicles contained an oocyte surrounded by multiple layers of granulosa cells but lacking an antrum, antral follicles were those in which the oocyte was contained within multiple layers of granulosa cells in which an antrum was apparent, and preovulatory follicles (diameter >450 µm) exhibited a large antrum and cumulus oophorus that projected into the antrum.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of sGC {alpha}1 and ß1 Subunits in Whole Ovary and Isolated Granulosa Cells

Immunoblot analysis of sGC {alpha}1 and ß1 subunit proteins was performed using protein extracts of whole ovaries and of isolated granulosa cells obtained from immature rats. When blots were incubated with primary antisera directed against the sGC {alpha}1 subunit, an immunoreactive band with an apparent molecular mass of approximately 80 kDa was observed for both whole ovary and isolated granulosa cells (Fig. 1), corresponding to the reported molecular weight of this subunit [10]. Incubation of blots with primary antisera directed against the sGC ß1 subunit yielded a predominant immunoreactive band of approximately 70 kDa (Fig. 1), which is consistent with the smaller molecular mass of this subunit [10]. The apparent molecular weight of the immunoreactive ß1 subunit signal was the same in both whole ovary and isolated granulosa cells.



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  		FIG. 1. Expression of sGC {alpha}1 and ß1 subunit proteins in whole ovary and isolated granulosa cells of 25-day-old rats. Protein homogenates from ovary (20 µg/lane) and isolated granulosa cells (10 µg/lane) were fractionated on SDS-PAGE gels and transferred to nitrocellulose, followed by immunoblot analysis with antisera specific to sGC {alpha}1 and ß1 subunits. For the negative control, immunoblot analysis was performed with omission of primary antisera. The migrating positions of molecular weight standards are shown on the right

Regulation of sGC Subunit Levels During Gonadotropin- Stimulated Follicle Growth, Ovulation, and Luteinization

Potential regulation of sGC subunit levels during follicular development, ovulation, and luteinization was assessed by immunoblot analysis of whole ovaries obtained from immature rats treated with exogenous gonadotropins. Treatment of rats with eCG to stimulate follicular development did not significantly influence whole-ovarian sGC {alpha}1 or ß1 subunit protein levels (Fig. 2). Subsequent treatment at 52 h with hCG to induce ovulation and luteinization also failed to yield statistically significant changes in sGC subunit protein levels in whole-ovarian homogenates. Although levels of both subunits appeared to increase in luteal ovaries 72 h after hCG, this apparent increase was not statistically significant.



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  		FIG. 2. Immunoblot analysis of ovarian sGC {alpha}1 (Top) and ß1 subunit (Bottom) protein levels after treatment with eCG to stimulate follicle growth, followed 52 h later by an ovulatory dose of hCG (n = 4 rats/ group). Ovaries were obtained at indicated times of hormone treatment and processed for immunoblot analysis using antisera specific to sGC {alpha}1 and ß1 subunits

Immunoblot Analysis of sGC Subunit Levels in Cultured Rat Granulosa Cells

Previous reports in other tissues indicate that sGC subunit expression is influenced by E2 and by agents that increase cAMP levels. To examine potential hormonal regulation of sGC subunit protein levels, immature granulosa cells were cultured for 0, 6, 12, 24, and 48 h with medium alone (control) or with FSH (2 ng/ml), followed by immunoblot analysis of sGC {alpha}1 and ß1 subunit proteins. By 48 h of culture, a small but significant decrease was observed in sGC {alpha}1 subunit levels in untreated cells compared with Time 0 (Fig. 3, top). A similar apparent decrease in sGC ß1 subunit levels at 48 h in untreated cells was not statistically significant (Fig. 3, bottom). Immunoblot analysis also revealed that treatment of granulosa cells with FSH did not significantly influence sGC subunit levels at any time point between 6 and 48 h of culture.



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  		FIG. 3. Immunoblot analyses of sGC {alpha}1 subunit (Top) and ß1 subunit (Bottom) protein levels in rat granulosa cells. Cells were treated for the indicated times with medium alone (control; open bars), or FSH (2 ng/ ml; hatched bars), and protein homogenates were then subjected to immunoblot analysis using antisera specific to sGC {alpha}1 and ß1 subunits

Localization of sGC {alpha}1 and ß1 Subunits in the Neonatal and Postnatal Ovary

To determine the cell-specific localization of sGC subunits within the ovary, we also performed immunocytochemical analysis of sGC protein expression. The sGC {alpha}1 subunit reactivity in postnatal rat ovaries was limited primarily to granulosa cells of primordial and small developing follicles and to vascular endothelial cells. In ovaries of 5-day-old rats, the vast majority of follicles were primordial, with some larger follicles containing two layers of granulosa cells and an incomplete layer of theca cells (Fig. 4A). These primordial and primary follicles displayed intense sGC {alpha}1 subunit fluorescence in granulosa cells (Fig. 4B). On Day 10, larger preantral follicles were present with well-developed theca interna (Fig. 4C). However, as follicle size increased, the intensity of sGC staining in granulosa cells decreased (Fig. 4D). On Day 19, follicles were present up to the small antral stage in the ovary (Fig. 4E). Intense sGC immunoreactivity was again evident in primordial and primary follicles, but sGC reactivity was markedly decreased in granulosa cells of small antral follicles (Fig. 4F). No immunofluorescence was observed in sections incubated with NRS (data not shown).



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  		FIG. 4. Immunofluorescent localization of sGC {alpha}1 subunit protein in postnatal rat ovaries. Bright-field phase-contrast photomicrographs (A, C, and E) and corresponding immunofluorescent images (B, D, and F, respectively) of ovarian sections are shown for ovaries obtained from rats at 5 (A and B), 10 (C and D), and 19 (E and F) days of age. Bar = 50 µm

Similar ovarian localization and expression patterns were seen when utilizing an antiserum specific for the sGC ß1 subunit (Fig. 5), with expression limited primarily to granulosa cells of small developing follicles. On Days 5 and 10, sGC ß1 subunit immunoreactivity was strong in granulosa cells of primordial and primary follicles, with markedly lower immunofluorescence in granulosa cells of small antral follicles (Fig. 5, B–D). Whereas sGC ß1 subunit immunoreactivity was occasionally observed in oocytes of primordial and primary follicles, other oocytes in follicles of the same stage did not express sGC ß1 subunit (Fig. 5, D and F). No immunofluorescence was observed in sections incubated with NRS in place of primary antiserum (data not shown).



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  		FIG. 5. Immunofluorescent localization of sGC ß1 subunit in postnatal rat ovaries. Bright-field phase-contrast photomicrographs (A, C, and E) and corresponding immunofluorescent images (B, D, and F, respectively) of ovarian sections are shown for ovaries obtained from rats at 5 (A and B), 10 (C and D), and 19 (E and F) days of age. Bar = 50 µm

Regulation of sGC {alpha}1 and ß1 Subunits During Gonadotropin-Stimulated Follicle Growth, Ovulation,and Luteinization

Immunoblot analysis failed to reveal influences of exogenous gonadotropins on sGC subunit levels in whole- ovarian lysates (Fig. 2), but changes in cell-specific expression or localization of sGC could not be ruled out. Immunohistochemical analysis of ovaries from 25-day-old, immature rats obtained before gonadotropin treatment revealed that sGC {alpha}1 subunit levels were high in ovarian vascular endothelial cells and very intense in granulosa cells of primordial and primary follicles (Table 1 and Fig. 6B). In contrast, sGC {alpha}1 subunit expression was low in the granulosa cells of small antral follicles and undetectable in the granulosa cells of large antral and atretic follicles. Theca cells of developing follicles exhibited moderate sGC {alpha}1 levels. Treatment of rats with eCG stimulated growth of large antral and preovulatory follicles. The sGC {alpha}1 immunoreactivity was undetectable in granulosa cells of such large developing and preovulatory follicles, but it remained high in primordial and primary follicles of the same ovaries (Table 1 and Fig. 6, D and F). In addition, moderate sGC {alpha}1 levels were observed in the thecal layer of preovulatory follicles (Fig. 6F). Treatment with an ovulatory dose of hCG at 52 h after eCG induced ovulation and luteinization of preovulatory follicles, resulting in formation of corpora lutea (Fig. 6, G and I). By 24 and 72 h after hCG, moderate sGC {alpha}1 immunoreactivity was evident in luteal cells (Table 1 and Fig. 6, H and J).


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  		TABLE 1. Relative abundance of sGC {alpha} and ß subunits in the rat ovary during follicular development, ovulation, and luteinization.a



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  		FIG. 6. Immunofluorescent localization of sGC {alpha}1 subunit in ovaries of immature rats following treatment with eCG or eCG followed 52 h later by hCG. Ovaries were obtained from 25-day-old females before eCG treatment (A and B) and at 24 h (C and D) and 52 h (E and F) after eCG. Other females received eCG followed 52 h later by hCG, and ovaries were collected at 24 h (G and H) and 72 h (I and J) after hCG injection. Bright-field micrographs are shown on the left, with corresponding immunofluorescent images shown on the right. cl, Corpora lutea; f, follicle; t, theca. Bar = 50 µm

Similar ovarian localization and expression patterns were observed for the sGC ß1 subunit during gonadotropin-induced follicular development and luteinization. Intense sGC ß1 subunit immunofluorescence was observed in granulosa cells of primordial and primary follicles, decreasing markedly in secondary and antral follicles (Table 1 and Fig. 7, B and D). As observed for sGC {alpha}1, moderate sGC ß1 levels were observed in theca cells of preovulatory follicles (Fig. 7F) and in luteal cells 72 h after hCG (Fig. 7J). Some, but not all, oocytes also exhibited sGC immunoreactivity (Fig. 7, B, D, and H), but this expression did not appear to be associated with stage of follicle growth or the appearance of the oocyte.



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  		FIG. 7. Immunofluorescent localization of sGC ß1 subunit in ovaries of immature rats following treatment with eCG or eCG followed 52 h later by hCG. Ovaries were obtained from 25-day-old females before eCG treatment (A and B) and at 24 h (C and D) and 52 h (E and F) after eCG. Other females received eCG followed 52 h later by hCG, and ovaries were collected at 24 h (G and H) and 72 h (I and J) after hCG injection. Bright-field micrographs are shown on the left, with corresponding immunofluorescent results shown on the right. cl, Corpora lutea; t, theca. Bar = 50 µm

Expression of sGC {alpha}1 and ß1 Subunits in Ovariesof Immature, E2-Treated Rats

The failure to detect changes in sGC {alpha}1 and ß1 subunit levels in granulosa cells following FSH treatment (Fig. 3) may potentially reflect possible down-regulation of sGC expression in these granulosa cells because of in vivo treatment with estrogen implants and/or resulting granulosa cell proliferation. We therefore examined the expression of sGC {alpha}1 and ß1 subunits in ovaries of immature rats treated for 5 days with estrogen implants. Such treatment markedly stimulated granulosa cell proliferation, resulting in development of several large preantral follicles (data not shown). Immunohistochemical analysis of such ovaries revealed that sGC immunoreactivity was high in primordial and small primary follicles, as observed in postnatal and gonadotropin-treated animals. Similarly, sGC subunit expression was decreased in granulosa cells of larger follicles (Fig. 8), confirming down-regulation of sGC {alpha}1 and ß1 levels following estrogen-implant treatment.



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  		FIG. 8. Immunofluorescent localization of sGC {alpha}1 (A and B) and ß1 subunit (C and D) expression in follicles of immature rats treated for 5 days with E2 implants. Bright-field micrographs are shown on the left, with corresponding immunofluorescent results shown on the right. Bar = 50 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study provides new information regarding the cellular localization and regulation of sGC {alpha}1 and ß1 subunit levels in ovarian cells. Given the transient expression of sGC proteins in granulosa cells during follicle growth and the detection of these subunits in theca cells and corpora lutea, our findings suggest that sGC (and the associated second-messenger cGMP) may play significant roles in the process of follicular development and/or survival and in as-yet-undefined thecal and luteal functions.

Our previous reports indicate that treatment of cultured rat granulosa cells with a NO donor or specific activator of sGC results in marked stimulation of cGMP production, which is consistent with expression of sGC in granulosa cells [28, 29]. Similarly, previous immunoblot analysis (using a different primary antisera that detects both sGC {alpha} and ß subunits) also indicated expression of both {alpha} and ß subunits in granulosa cells (unpublished results). The present results confirm and extend these findings. Immunoblot analysis of whole-ovarian lysates did not reveal an effect of gonadotropin treatment, likely reflecting the cell-specific nature of sGC expression. However, immunofluorescence studies suggest an inverse relationship between sGC subunit levels and granulosa cell proliferation in postnatal and gonadotropin-treated rats. Whereas sGC subunit expression is strong in granulosa cells of primordial and primary follicles, a marked decline in sGC levels occurs in granulosa cells at very early stages of follicle growth, with no detectable sGC {alpha}1 or ß1 protein in proliferating granulosa cells of larger developing follicles. Similarly, the absence of sGC down-regulation in cultured granulosa cells in response to FSH may be related to the fact that granulosa cell proliferation is minimal in this serum-free culture system [32, 33]. Together, these findings indicate an inverse relationship between sGC levels and the rate of granulosa cell proliferation. Potentially, inhibition of sGC expression may result in activation of mechanisms facilitating granulosa cell proliferation. In this regard, previous findings demonstrate a role of NO as an inhibitor of cellular proliferation, decreasing activity of cell cycle-regulating factors in a wide variety of cell types [34–37]. Thus, a decline in sGC expression may result in relief from the NO-mediated cytostasis previously proposed to exist in developing follicles [30]. The identities of factors responsible for inhibition of sGC expression during early follicular development remain unknown.

The decrease in sGC {alpha}1 and ß1 expression in maturing granulosa cells of growing and preovulatory follicles is consistent with the reported antagonistic effects of NO and cGMP on the functional maturation of granulosa cells [6, 28, 38]. However, this decrease in sGC does not appear to be a required component of FSH-induced cell maturation, because no change in sGC subunit levels was observed during FSH-induced maturation of cultured granulosa cells. Similarly, FSH-induced increases of E2 production in cultured granulosa cells might also have been predicted to inhibit sGC expression because of the reported inhibitory effects of E2 on sGC expression in the uterus [24]. However, FSH treatment did not significantly affect sGC subunit levels in granulosa cells despite anticipated induction of E2 synthesis. These unexpected results may reflect, in part, the fact that the majority of granulosa cells obtained for culture appear to exhibit down-regulation of sGC subunit levels (Fig. 8), resulting from granulosa cell proliferation and/or E2-implant treatment. Furthermore, the population of granulosa cells used in this culture system may include those from follicles undergoing atresia and, thus, may not completely represent the function of the granulosa cells of healthy follicles. Nevertheless, these findings do indicate that the down-regulation of sGC subunits in granulosa cells of growing follicles is not directly related to FSH-stimulated granulosa cell maturation, because FSH-induced differentiation of cells in this culture system clearly occurred but was not associated with a change in sGC {alpha}1 and ß1 subunit levels.

Our findings may also provide insight regarding the relative importance of NO-derived cGMP as an inhibitor of apoptosis during different stages of follicular development. Previous studies indicate that relatively little apoptosis occurs in granulosa cells of preantral follicles of the ovary and that treatment of cultured preantral follicles with a cGMP analog inhibits programmed cell death [39]. These observations are consistent with the high expression of sGC in such preantral follicles, supporting a potential role for cGMP as an inhibitor of follicular demise. In contrast, studies from the same laboratory also indicate that cGMP effectively inhibits apoptosis in cultured preovulatory follicles [40], which do not appear to express significant levels of sGC {alpha}1 or ß1 subunit. Given our findings of strong sGC expression during early, but not middle and later, stages of folliculogenesis, the role of NO-stimulated cGMP may be as an inhibitor of early follicular demise, with other factors (e.g., FSH) playing roles in survival of follicles at later stages. This proposed importance of NO-derived cGMP as an inhibitor of early follicular demise could explain recent observations in which mice lacking endothelial NO synthase (NOS) expression exhibited decreased numbers of developing antral follicles and smaller ovulation rates [41].

The patterns of sGC regulation observed in the present study are consistent with the reported pattern of NOS expression. Reports of NOS expression in the ovary are not completely consistent, but it has been reported that inducible NOS and endothelial NOS levels decrease during follicular development [30, 42]. This is consistent with the expression of sGC, a target of NO, in early, but not in late, developing follicles. Similarly, our finding that sGC is expressed in corpora lutea is consistent with findings demonstrating luteal NOS expression and action [42–45], although the involvement of sGC in the effects of NO on luteal function is not well established. Similar patterns of expression for NOS and the target receptor for NO are evident, but the molecular mechanisms resulting in such regulated temporal coexpression require further study.

In addition to NO, sGC is also activated by CO, which is generated by heme oxygenases [17–19]. A recent paper demonstrates expression of heme oxygenases in the ovary [46], suggesting a possible role for CO as an activator of sGC in ovarian cells. The observation that E2 increases heme oxygenase levels and cGMP production from human endothelial cells [47] raises further intriguing possibilities regarding CO-mediated feedback mechanisms through which E2 may act on the ovary.

It is important to note that the present study utilizes antibodies that are specific for sGC {alpha}1 and ß1 subunits. Thus, we cannot rule out the possibility that whereas larger developing follicles display low levels of sGC {alpha}1 and ß1 subunits, granulosa cells of such follicles do express the {alpha}2 and ß2 isoforms of sGC. This would allow the possibility that NO and/or CO may act on these target enzymes in larger developing follicles, influencing follicular development and/or steroidogenesis. Future studies utilizing {alpha}2 and ß2 isoform-specific antisera or selective mRNA probes are required to examine the potential expression of these isoforms in the ovary.


   FOOTNOTES
 
1 Supported by NIH SCORE Grant S06 GM008101 to P.S.L. Back

2 Correspondence: Philip S. LaPolt, Department of Biological Sciences, California State University–Los Angeles, 5151 State University Drive, Los Angeles, CA 90032. FAX: 323 343 6451;plapolt{at}exchange.calstatela.edu Back

Received: 13 November 2003.

First decision: 26 November 2003.

Accepted: 19 January 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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