15 Dec

Soluble Guanylyl Cyclase: RESULTS

RESULTS

Expression of sGC a1 and ft] Subunits in Whole Ovary and Isolated Granulosa Cells

Immunoblot analysis of sGC a! and p! 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 a! 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. Incubation of blots with primary antisera directed against the sGC p1 subunit yielded a predominant immunoreactive band of approximately 70 kDa (Fig. 1), which is consistent with the smaller molecular mass of this subunit. The apparent molecular weight of the im-munoreactive p1 subunit signal was the same in both whole ovary and isolated granulosa cells.

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 a1 or pj 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.

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 im-munoblot analysis of sGC a1 and p1 subunit proteins. By 48 h of culture, a small but significant decrease was observed in sGC a1 subunit levels in untreated cells compared with Time 0 (Fig. 3, top). A similar apparent decrease in sGC p1 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.

Localization of sGC a1 and fi1 Subunits in the Neonatal and Postnatal Ovary

To determine the cell-specific localization of sGC subunits within the ovary, we also performed immunocyto-chemical analysis of sGC protein expression. The sGC a1 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 a1 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).

Similar ovarian localization and expression patterns were seen when utilizing an antiserum specific for the sGC (3j subunit (Fig. 5), with expression limited primarily to granulosa cells of small developing follicles. On Days 5 and 10, sGC p1 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 p1 subunit immunoreac-tivity was occasionally observed in oocytes of primordial and primary follicles, other oocytes in follicles of the same stage did not express sGC p1 subunit (Fig. 5, D and F). No immunofluorescence was observed in sections incubated with NRS in place of primary antiserum (data not shown).

Regulation of sGC a1 and j31 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. Im-munohistochemical analysis of ovaries from 25-day-old, immature rats obtained before gonadotropin treatment revealed that sGC a1 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 a1 subunit expression was low in the granulosa cells of small antral follicles and undetectable in the granulosa cells of large antral and atretic follicles. The-ca cells of developing follicles exhibited moderate sGC a1 levels. Treatment of rats with eCG stimulated growth of large antral and preovulatory follicles. The sGC a1 immu-noreactivity 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 a1 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 a! immunoreactivity was evident in luteal cells (Table 1 and Fig. 6, H and J).

Similar ovarian localization and expression patterns were observed for the sGC pi subunit during gonadotropin-induced follicular development and luteinization. Intense sGC p1 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 a1, moderate sGC p1 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.

Expression of sGC a1 and p1 Subunits in Ovaries of Immature, E2-Treated Rats

The failure to detect changes in sGC a1 and p1 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 a1 and p1 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 a1 and p1 levels following estrogen-implant treatment.
Fig1Cell-Specific Expression and-2
FIG. 1. Expression of sGC a-, and p1 subunit proteins in whole ovary and isolated granulosa cells of 25-day-old rats. Protein homogenates from ovary (20 ^g/lane) and isolated granulosa cells (10 ^g/lane) were fractionated on SDS-PAGE gels and transferred to nitrocellulose, followed by immunoblot analysis with antisera specific to sGC a1 and p1 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.

Fig2Cell-Specific Expression and-3
FIG. 2. Immunoblot analysis of ovarian sGC a, (Top) and p, 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 a, and p, subunits.

Fig3Cell-Specific Expression and-1
FIG. 3. Immunoblot analyses of sGC a-, subunit (Top) and p, 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 a-, and p, subunits.

Fig4Cell-Specific Expression and-4
FIG. 4. Immunofluorescent localization of sGC a, 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 |xm.

Fig5Cell-Specific Expression and-5
FIG. 5. Immunofluorescent localization of sGC p, 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 |xm.

TABLE 1. Relative abundance of sGC a and p subunits in the rat ovary during follicular development, ovulation, and luteinization.a
table1Cell-Specific Expression and-6
a Staining intensity: —, No staining detected; +, weak; ++, moderate; + + +, Strong. N/A, Not available.

Ашп6Cell-Specific Expression and-7
FIG. 6. Immunofluorescent localization of sGC a1 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 ^m.

Fig7Cell-Specific Expression and-8
FIG. 7. Immunofluorescent localization of sGC ^ 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 ^m.

Fig8Cell-Specific Expression and-9
FIG. 8. Immunofluorescent localization of sGC a1 (A and B) and p1 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 ^m.

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