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Molecular Endocrinology, doi:10.1210/me.2005-0317
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Molecular Endocrinology 20 (2): 348-361
Copyright © 2006 by The Endocrine Society

Cyclic Guanosine 5'-Monophosphate-Dependent Protein Kinase II Is Induced by Luteinizing Hormone and Progesterone Receptor-Dependent Mechanisms in Granulosa Cells and Cumulus Oocyte Complexes of Ovulating Follicles

Venkataraman Sriraman, Michael D. Rudd, Suzanne M. Lohmann, Sabine M. Mulders and JoAnne S. Richards

Department of Molecular and Cellular Biology (V.S., M.D.R., J.S.R.), Baylor College of Medicine, Houston, Texas 77030; Institute of Clinical Biochemistry and Pathobiochemistry (S.M.L.), University of Wuerzburg, 97080 Wuerzburg, Germany; and N.V. Organon (S.M.M.), 5340 BH Oss, The Netherlands

Address all correspondence and requests for reprints to: JoAnne S. Richards, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: joanner{at}bcm.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cyclic GMP (cGMP)-dependent protein kinase II (Prkg2, cGK II) was identified as a potential target of the progesterone receptor (Nr3c3) in the mouse ovary based on microarray analyses. To document this further, the expression patterns of cGK II and other components of the cGMP signaling pathway were analyzed during follicular development and ovulation using the pregnant mare serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG)-primed immature mice. Levels of cGK II mRNA were low in ovaries of immature mice, increased 4-fold in response to pregnant mare serum gonadotropin and 5-fold more within 12 h after hCG, the time of ovulation. In situ hybridization localized cGK II mRNA to granulosa cells and cumulus oocyte complexes of periovulatory follicles. In progesterone receptor (PR) null mice, cGK II mRNA was reduced significantly at 12 h after hCG in contrast to heterozygous littermates. In primary granulosa cell cultures, cGK II mRNA was induced by phorbol 12-myristate 13-acetate enhanced by adenoviral expression of PR-A and blocked by RU486 and trilostane. PR-A in the absence of phorbol 12-myristate 13-acetate was insufficient to induce cGK II. Expression of cGK I (Prkg1) was restricted to the residual tissue and not regulated by hormones. Guanylate cyclase-A (Npr1; GC-A) mRNA expression increased 6-fold by 4 h after hCG treatment in contrast to pregnant mare serum gonadotropin alone and was localized to granulosa cells of preovulatory follicles. Collectively, these data show for the first time that cGK II (not cGK I) and GC-A are selectively induced in granulosa cells of preovulatory follicles by LH- and PR-dependent mechanisms, thereby providing a pathway for cGMP function during ovulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PITUITARY gonadotropins FSH and LH regulate ovarian follicular development and ovulation by inducing the expression of specific genes in granulosa cells, thecal cells, and cumulus oocyte complexes (COCs) (1). Genes known to be induced by LH during the ovulation process include the progesterone receptor (PR) (Nr3c3) (2), ADAMTS1 (Adamts-1) (3), cyclooxygenase-2 (COX-2) (Ptgs-2) (4), amphiregulin (Areg) (5), and others as recently documented by microarray (6) and differential display RT-PCR (7) technologies. The mechanisms by which LH induces these genes are diverse and involve multiple signaling cascades and specific transcription factors (8). FSH and LH act on ovarian cells by binding their cognate receptors, leading to the activation of adenylyl cyclase and cAMP-mediated signaling events (9). Although cAMP activation of cAMP-dependent protein kinase [protein kinase A (PKA)] is well recognized, many studies indicate the importance of other signaling factors and cross talk between different signaling pathways in modulating ovarian function, including protein kinase C (PKC), phosphatidylinositol 3-kinase, and MAPK pathways (9). In addition, recent studies have shown that components of the cyclic GMP (cGMP) signaling pathway are expressed in ovary and that cGMP can alter granulosa cell function (10). Specifically, cGMP can inhibit cAMP-stimulated LH receptor expression, inhibin A secretion, and estradiol production in cultured rat granulosa cells (10). In an in vitro follicle culture system, cGMP promoted follicular survival by suppression of apoptosis (11). Interestingly, cGMP levels were low when the levels of cAMP were high at the time of proestrus, indicating a functional inverse correlation between their levels that was abolished by blockade of gonadotropin surge by phenobarbital (12).

cGMP is produced by two independent pathways. In one, natriuretic peptides activate membrane bound receptors with intrinsic guanylate cyclase (GC) activity, namely GC-A, GC-B, and GC-C to generate cGMP (13). Three distinct natriuretic peptides are known, atrial (A-type) (ANP), brain (B-type) (BNP), and C-type (CNP). Studies have documented the expression of A- and C-type natriuretic peptides in rat ovary and an increase in their expression after hormone administration (14, 15), However, in mouse ovary only C-type natriuretic peptide expression was detected (16). The natriuretic peptides exhibit differential affinity toward their cognate receptors, ANP and BNP binding preferentially to GC-A, whereas CNP binds to GC-B.

In a second pathway, nitric oxide synthases (NOS) produce NO that activates soluble guanylate cyclases (sGC) to synthesize cGMP. The expression of endothelial and inducible NOS (eNOS and iNOS, respectively) has been documented in rat ovary, and each appears to be hormonally regulated (17). eNOS is expressed in mural granulosa cells and corpus luteum, and its expression increases in response to human chorionic gonadotropin (hCG) (17). iNOS was predominantly localized to the thecal layer and stroma (17). Furthermore, studies in mice null for eNOS revealed longer estrous cycle length with impaired ovulation (18). Expression of the NO target, sGC, occurs preferentially in granulosa cells of primordial and primary follicles of rat ovary (19). Recently, NO signaling has been demonstrated to be essential for optimal meiotic maturation of murine cumulus-oocyte complexes in vitro (20). These results highlight an important role for cGMP signaling in ovary and raise the issue of where and by what mechanisms cGMP might exert its effect on ovulation and follicular maturation.

The effects of cGMP in cells can be mediated by its regulation of cyclic nucleotide gated channels, phosphodiesterase activation (PDE 2 and PDE 5) and inhibition (PDE 3), or stimulation of cGMP-dependent protein kinases (cGK I and cGK II) in a cell-specific manner (13, 21). Furthermore, cGMP can affect PKAs either by direct cross activation in the presence of high cGMP concentrations, or indirectly by activation/inhibition of cAMP hydrolyzing PDEs as mentioned above. Currently, cGK I and cGK II null mice have been characterized with respect to defects in several organ systems; however, ovarian defects have not yet been reported (22, 23). cGK I null mice exhibit a decreased life span with defective relaxation of vascular, visceral, and penile smooth muscle, and impaired platelet activity (13). cGK II has been demonstrated to phosphorylate the cystic fibrosis transmembrane conductance regulator Cl–channel (CFTR) (24) and steroidogenic acute regulatory protein (StAR) (25) to regulate intestinal secretion and adrenal aldosterone release, respectively. Mice null for cGK II exhibit intestinal secretory defects and dwarfism (22). These mice also show defective bone formation due to reduced synthesis of collagen X and ossification that is mediated by C-type natriuretic peptide signaling through cGK II, indicating a role for cGK II in matrix synthesis (26). Many of the effects of cGK I and cGK II and their mechanisms have been recently reviewed (13, 27).

Although previous studies have documented cGK I expression in rat ovary, cGK II expression has not been reported (28). Our preliminary microarray analyses indicated cGK II might impact ovulation because it was induced by hCG (used as an LH-like factor) in preovulatory follicles of pregnant mare serum gonadotropin (PMSG) (used as an FSH-like hormone)-primed mice (data not shown). Furthermore, the microarray data suggested that cGK II might be a target of the nuclear transcription factor PR because expression of cGK II mRNA was reduced in PR knockout (PRKO) mice that do not ovulate and are infertile (29, 30). Of the two isoforms reported for PR, PR-A, and PR-B, the former is predominantly expressed in granulosa cells (2). Based on these considerations, the objectives of present study were to analyze the mechanisms by which LH and PR regulate cGK II expression in mouse ovary. In addition, other components of the cGMP signaling cascade were analyzed to determine the extent to which this pathway might be operational and hormonally regulated during follicular growth and ovulation. To study the regulation of cGK II expression in vivo, immature mice were primed with hormones PMSG and hCG to stimulate follicular growth and ovulation. To analyze the signaling cascades that act downstream of FSH and LH to induce cGK II, granulosa cells and cumulus oocyte complexes were cultured in defined media with hormones with or without selected inhibitors or agonists of specific kinase cascades. Lastly, to define the role of PR in the regulation of cGK II, in vivo studies were performed with hormone-primed PRKO mice and in vitro studies were performed in granulosa cells using an adenovirus that expresses PR-A. Our results show that cGK II is induced by LH- and PR-dependent mechanisms, the former of which involves the PKC and epidermal growth factor (EGF) receptor pathways.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
cGK II mRNA Is Expressed in Granulosa Cells of Ovulatory Follicles
The expression of cGK II mRNA was assessed by semiquantitative RT-PCR in the total RNA isolated from ovaries from mice that were primed with PMSG for 48 h, followed by hCG as described in Materials and Methods. Analyses revealed cGK II was higher in ovaries of PMSG-treated mice than in immature controls. cGK II message was induced further 4 h after hCG, peaked at 12 h and declined progressively thereafter (Fig. 1AGo). cGK II protein was not detected in samples from immature or PMSG-primed mice. However, an intense immunoreactive cGK II band was observed from 4–24 h after hCG and then declined (Fig. 1BGo). In situ hybridization localized cGK II message to granulosa cells of preovulatory and ovulatory follicles. A weak signal observed in granulosa cells at 4 h after hCG increased strongly by 8 h (Fig. 2Go). These results localize cGK II to granulosa cells and confirm the increase between 4 and 8 h. cGK II expression was low in the interstitium, primordial, and primary follicles, as well as in corpora lutea.


Figure 1
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  		Fig. 1. Expression of cGK II mRNA in Ovary Increases during Preovulatory Follicular Development and Ovulation

Hormone (PMSG, hCG), injected as described in Materials and Methods, induced expression of cGK II in the murine ovary was determined by semiquantitative RT-PCR using total ovarian RNA and specific primers for cGK II and the internal standard L19 (in this and subsequent figures) (A), and Western blot analysis, using cell extracts prepared at the indicated intervals (B). Changes in cGK II message are expressed relative to L19 and represent three separate pools of RNA analyzed in duplicate [immature (Imm.) vs. PMSG, hCG 4 h, 8 h, 12 h; ***, P < 0.0001]. Total ovarian protein was extracted by homogenization in urea buffer containing protease inhibitors, and 15 µg protein was resolved by SDS-PAGE and transferred to PVDF membranes. The Western blot shown in B, representative of three determinations, was probed with antibody against cGK II as described in Materials and Methods. Non Sp., A nonspecific band that served conveniently as a loading control.

 

Figure 2
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  		Fig. 2. cGK II mRNA Expression Is Localized to Granulosa Cells of Periovulatory Follicles

In situ hybridization analyses, using a mouse cGK II probe generated by RT-PCR and subcloning, detected cGK II message in granulosa cells of ovarian sections prepared from PMSG-primed mice, as well as those treated with hCG 8 h, but was not detected in ovarian sections of immature mice. Images are shown at x5 (or x10 as indicated) magnification in dark- and light-field illumination. No staining above background levels was observed with the sense riboprobe in these same ovaries (data not shown). PF, Primary follicle; S An, small antral follicle; PO, preovulatory follicle; COC, cumulus oocyte complex; CL, corpus luteum.

 
cGK II mRNA Induction in Granulosa Cells and Cumulus Oocyte Complexes
cGK II mRNA was examined in COCs obtained either from ovaries of PMSG primed mice before or at 8 h after hCG or from oviducts of PMSG-primed mice 24 h after hCG. cGK II mRNA in COCs from mice treated with PMSG was negligible (Fig. 3AGo). However, cGK II expression increased 12- to 14-fold in COCs collected 8 h after hCG treatment and remained detectable even in ovulated COCs collected at 16 and 24 h after hCG treatment (Fig. 3AGo). This increase in cGK II was also apparent in COCs by in situ hybridization 8 h after administration of hCG (Fig. 2Go). Granulosa cells collected from the same ovaries showed a similar pattern of cGK II expression (Fig. 3AGo). cGK II message was induced by FSH in COCs expanded in culture, but not in nonexpanded COCs cultured in media alone (Fig. 3BGo). Thus, cGK II expression increases in granulosa cells and cumulus cells after hCG treatment and in cumulus cells of cultured COCs in response to FSH.


Figure 3
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  		Fig. 3. cGK II mRNA Expression Is High in Expanded COC and Granulosa Cells

A, RT-PCR analysis of cGK II message in RNA prepared from COC collected from ovaries of PMSG, hCG-primed mice. COCs were collected by either ovarian puncture [PMSG (P) and P, hCG 8 and 12 h] or from oviducts (P, hCG 16 and 24 h), and granulosa cells were isolated from the same ovaries. The whole ovary mRNA was analyzed for P, hCG 16 and 24 h. cGK II message levels were highest 8 h after hCG. Changes in cGK II message relative to L19 are shown in the bar graph. (**, P < 0.01; ***, P < 0.001 relative to COCs and granulosa cells from PMSG-primed ovaries to COC and granulosa cells/whole ovary from P, hCG-treated groups, respectively.) B, cGK II message is induced by FSH in conjunction with COC expansion in culture. COCs were isolated by needle puncture of ovaries from PMSG-treated mice and were cultured in defined medium with or without FSH (100 ng) overnight, then examined for expansion and harvested for RNA isolation. Results are representative of three experiments. ***, P < 0.001 relative to control.

 
Reduced Expression of cGK II mRNA and Protein in PRKO Mice
Because preliminary microarray data indicate cGK II might be a PR-regulated gene, the role of PR was analyzed using hormone-primed PRKO mice. The temporal pattern of cGK II expression in ovaries of PRKO mice and their heterozygous (PRHET) littermates treated with PMSG and hCG (Fig. 4AGo) was similar to that of wild-type mice (Fig. 1Go). However, the peak induction of cGK II preceding ovulation (8–12 h after administration of hCG) was reduced 50% (PMSG, hCG 8 h; *, P < 0.01, PMSG, hCG 12 h; ***, P < 0.001) in PRKO mice compared with PRKOHET mice (Fig. 4AGo). Likewise, analyses of cGK II mRNA in granulosa cells isolated from PRKO mice 12 h after treatment with PMSG and hCG also documented a 50% (***, P < 0.0001) decline in cGK II expression (data not shown). Furthermore, levels of cGK II protein in ovaries of PRKO mice treated with PMSG and hCG was reduced approximately 75% at 12 h compared with PRKOHET mice (Fig. 4BGo).


Figure 4
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  		Fig. 4. Expression of cGK II mRNA and Protein Is Reduced in Ovaries of PRKO Mice at the Time of Ovulation

A, RNA was prepared from ovaries of immature, PMSG (P)- and P-hCG primed PRKO (–/–) and PRKOHET (+/–) mice at the indicated time intervals, using ovaries of three individual mice of each genotype analyzed separately. Data are expressed as means ± SD of cGK II mRNA in PRKO and PRKOHET ovaries normalized to L19. Reduced expression of cGK II was evident in PRKO (nonovulating) mice at 8 and 12 h after hCG, the time immediately preceding ovulation in wild-type mice (*, P < 0.05; ***, P < 0.001 relative to heterozygote), but not at 16 h after hCG when PRKOHET, but not PRKO mice, ovulated. B, Western blot analyses and densitometric scanning showing reduced levels of cGK II protein in extracts (20 µg protein) of ovaries from PRKO mice vs. PRKOHET mice at 12 h after P, hCG (***, P < 0.001). More than three samples of each genotype at each time point were analyzed separately. Non Sp., Nonspecific band that serves conveniently as a loading control.

 
Induction of cGK II and PR mRNA by Phorbol 12-Myristate 13-Acetate (PMA) in Cultured Granulosa Cells
To analyze the other cell signaling pathways required for cGK II induction, granulosa cells were isolated from PMSG-primed mice and plated in defined medium. Addition of forskolin (Fo)/PMA (mimics LH signaling by activation of adenylyl cyclase and PKC, respectively) for 4 h to granulosa cells cultured overnight in medium alone induced a marked (10- to 14-fold) increase in cGK II (Fig. 5AGo, lane 2), a response similar to that observed with Fo/PMA in FSH/T-differentiated granulosa cells (Fig. 5AGo, last lane and 5B, fourth column). FSH/T alone did not induce cGK II (Fig. 5AGo, lane 4). Further analyses indicated PMA alone was sufficient to mediate the induction of cGK II suggesting PKC involvement. (Fig. 5BGo, lane 3). This was supported by the reduced expression of cGK II when the PKC inhibitor Calphostin C was added to the cells treated with PMA (Fig. 5CGo). In addition, amphiregulin, an EGF family member that can mediate some actions of LH during ovulation (5) increased the expression of cGK II in cultured granulosa cells, albeit to a lesser degree (Fig. 5BGo). PKC has been known to activate p42/44 and p38 MAPKs in cultured in cultured granulosa cells (31), and the involvement of these kinases in induction of cGK II was confirmed by specific inhibitors. Addition of PD98059 that blocks the activity of p42/44 MAPK reduced the PMA inducibility of cGK II significantly and on the contrary addition of SB203580 that inhibits p38 MAPK did not have any effect of cGK II induction (Fig. 5CGo). Addition of EGF receptor tyrosine kinase inhibitor AG1478 reduced the PMA-mediated induction of cGK II (Fig. 5CGo). These results indicate that induction of cGK II by PMA in granulosa cells in part involves activation of the PKC-p42/44 MAPK pathway and cross talk with the EGF-receptor tyrosine kinases. Interestingly, cGK II induction occurs coordinately with PR induction after Fo/PMA treatment at 4 h (Fig. 5DGo, lane 3).


Figure 5
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  		Fig. 5. cGK II and PR mRNA Expression Increases in Cultured Granulosa Cells after Addition of Either Fo/PMA (to Mimic LH Signaling) or Amphiregulin

Granulosa cells were isolated by needle puncture of PMSG-primed ovaries, washed twice with DMEM-F12, plated in 12-well culture plates, and cultured overnight in DMEM-F12 containing 1% serum. Subsequently, medium was removed and replaced with serum-free medium with or without FSH/testosterone (T) for 48 h, then Fo 10 µM and PMA 20 nM were added for 2 or 4 h (panels A, B, and D). FSH/T was used to promote granulosa cell differentiation, Fo to stimulate acute production of cAMP independent of receptor and PMA to activate diacylglycerol-mediated signaling. In additional cultures (panels B and C), the effects of Fo/PMA were compared with Fo and PMA alone and amphiregulin alone. In additional cultures the effects of PMA were compared as well as to the PKC inhibitor [calphostin C (1 nM)], p42/44 MAPK inhibitor [PD 98059 (18.7 nM)], EGF receptor blocker [AG 1478, (10 nM)] and p38 MAPK inhibitor [SB203580 (20 nM)]. ***, P < 0.0.001; **, P < 0.01 relative to controls). RNA was isolated at the indicated times for RT-PCR analysis of cGK II (panels A–C) and PR (panel D) relative to L19.

 
Because the temporal induction of cGK II in cultured granulosa cells paralleled that of the PR (Fig. 5Go), and because induction of GK II was reduced in PRKO mouse ovaries, the effect of expressing exogenous PR-A in granulosa cells on cGK II induction was assessed. Because the efficiency of plasmid transfection in granulosa cells is relatively low, an adenoviral approach to deliver PR-A-Myc was used. After 14 h of infection, levels of PR-A-Myc protein were elevated (Fig. 6AGo) and at that time agonists (Fo, PMA, and R-5020) were added for 4 h to determine the effect of PR on cGK II expression. PR-A-Myc alone or in the presence of the ligand (R-5020) did not increase cGK II expression (Fig. 6Go B, lane 3 and 6). Fo alone, which does not induce endogenous cGK II mRNA, did exert a small but significant effect on cGK II induction in the presence of PR-A-Myc (*, P < 0.01, lane 4) (Fig. 6BGo). However, addition of PMA alone increased the expression of cGK II and this response to PMA was enhanced approximately 4- to 5-fold in PR-A-Myc-infected cells (***, P < 0.001) when compared with Lac-Z-infected cells exposed to PMA (Fig. 6BGo, lane 5). This enhanced induction of cGK II in the presence of PR-A was blocked by the PR antagonist RU486 (***, P < 0.001) (Fig. 6CGo) and reduced by trilostane a compound that decreases the endogenous progesterone biosynthesis in granulosa cells by inhibiting 3ß-hydroxysteroid dehydrogenase (32). These results indicate PR-A augmented the effects of PMA on cGK II induction but did not induce cGK II by itself.


Figure 6
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  		Fig. 6. Overexpression of PR-A-Myc Increases the PMA-Mediated Induction of cGK II in Granulosa Cells

Cultured granulosa cells were infected PR-A-Myc or Lac-Z adenovirus for 14 h after overnight culture in DMEM-F12. At 14 h after viral infection the expression of PR-A-Myc was determined by Western blot analysis as described employing myc tag polyclonal antibody (A). In additional cultures, fresh medium was added with Fo or PMA or R-5020 (10 nM) and the cells were harvested 4 h later to determine the effects on cGK II expression by RT-PCR analysis. The results are expressed in form of a bar graph as fold induction over lac-Z control normalized to L19 expression obtained from three different experiments (B) (***, P < 0.001 Lac-Z con vs. Lac-Z PMA and PR-A-Myc PMA; *, P < 0.01 Lac-Z con vs. PR-A-Myc Fo; ***, P < 0.0001 Lac-Z PMA vs. PR-A-Myc PMA) and the expression of PR-A-Myc in these cells was also confirmed by RT-PCR (B). The specificity of the PR effects were confirmed employing PR antagonist RU486 (RU, 1 µM) or by blocking ligand synthesis in granulosa cells using trilostane (T, 10 µM) (***, P < 0.001 compared PMA response in PR-A-Myc adenovirus-infected cells) (C).

 
cGK I mRNA and Protein Are Not Hormonally Regulated in the Ovary
The spatio-temporal expression of cGK I mRNA, in relation to that of cGK II in PMSG-hCG primed mice, was determined in additional RT-PCR analyses. Unlike cGK II, ovarian cGK I expression was not altered by administration of PMSG, and declined after hCG (Fig. 7AGo). Western blot analyses using whole ovarian extracts showed that the levels of cGK I protein remained essentially unchanged by PMSG and hCG treatment (Fig. 7BGo). Further analyses of cGK I protein in granulosa cells obtained by needle puncture of PMSG-primed ovaries showed that cGK I expression was negligible in these cells compared with the remaining (residual) tissue that is comprised principally of stromal, interstitial, and vascular cells as well as small follicles (Fig. 7BGo). Likewise, in primary cultures of granulosa cells, cGK I mRNA and protein were undetectable and not induced by Fo/PMA, whereas residual tissue gave a strong cGK I signal (Fig. 7Go, C and D). These results indicate that cGK II rather than cGK I is the major kinase mediator of cGMP signaling in granulosa cells of preovulatory and ovulatory follicles.


Figure 7
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  		Fig. 7. cGK I mRNA and Protein Expression Is Not Affected by Hormonal Treatment and Is Localized to Residual Tissue, Not Granulosa Cells, of Ovulating Follicles

A, Using specific primer sets for cGK I and the internal control L19, together with ovarian RNA as performed in Fig. 1Go, expression of cGK I mRNA was detectable but not hormonally regulated. B, Western blot analyses with ovarian extracts prepared from PMSG and hCG-primed mice document that cGK I protein in the ovary is also not hormonally regulated and is located in residual ovarian tissue (that remaining after removal of granulosa cells) of mice treated with PMSG. Extracts (15 µg protein) were resolved by SDS-PAGE and transferred to PVDF membranes which were probed with antibody against cGK I protein (1:2000) as described in Materials and Methods. Additional RT-PCR (C) and Western blot (D) analyses also demonstrated the absence of cGK I mRNA and protein in cultured granulosa cells.

 
GC-A Is Selectively Induced in Preovulatory Follicles
Having documented cell-specific expression of cGK I and cGK II in the ovary, the expression of upstream components involved in the synthesis of cGMP were assessed. Message encoding GC-A, GC-B, and GC-C were detectable in ovary; however, the expression of GC-C was very low. The expression of GC-A was elevated 6- to 8-fold 4 h after hCG treatment and declined after 16 h (Fig. 8Go, A and B). In situ hybridization analyses localized GC-A message predominantly to granulosa cells of preovulatory and ovulatory follicles after hCG treatment the time at which cGK II was also higher (Fig. 8CGo; P, hCG 8 h). Expression of GC-B showed a subtle increase after PMSG treatment and at 4 h after hCG treatment but was relatively unaffected at other times by hormone treatment (Fig. 8AGo). sGC is a heterodimer comprised of {alpha}- and ß-subunits with isoforms {alpha}1 and ß1 being most relevant physiologically (33). As shown in Fig. 8AGo, message encoding {alpha}1 and ß1-subunits were expressed constitutively in the mouse ovary and not altered by PMSG and hCG treatment. Analyses of the expression of A- and B-natriuretic peptides documented their expression in ovary however they required prolonged exposure times for their detection. The expression of ANP increased after PMSG treatment and administration of hCG elevated ANP expression around the time of ovulation (12–16 h) and again at 48 h (Fig. 8DGo). A similar trend in CNP expression was observed after hormone treatment (Fig. 8DGo).


Figure 8
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  		Fig. 8. GC-A mRNA Is Expressed in Granulosa Cells of Preovulatory Follicles

A and D, Expression of the cGMP generating factors (GC-A,GC-B, GC-C, sGC {alpha} and sGC ß) and natriuretic peptides (ANP and CNP) in total RNA prepared from ovaries of hormone-primed immature mice (as in Fig. 1Go). B, Graphical representation of GC-A expression normalized to L19 expression. C, In situ hybridization analysis showing GC-A localized to granulosa cells of preovulatory (PO) follicles. Images are shown at x5 magnification in dark- and light-field illumination. No staining above background levels was observed with the control, sense GC-A riboprobe in these same ovaries (data not shown). E, RT-PCR analysis demonstrated that Fo/PMA mimics LH signaling by increasing the expression of primarily GC-A in cultured granulosa cells, with little effect on other cyclases that generate cGMP. This increase in expression occurs 4 h after treatment of Fo/PMA treatment and confirms the LH/hCG-mediated increase in GC-A expression in vivo.

 
The LH-mediated increase in GC-A expression in granulosa cells in vivo was mimicked by treating primary cultures of granulosa cells with Fo/PMA. Levels of GC-A mRNA increased 8- to 10-fold by 4 h (Fig. 8EGo). In these same samples, levels of message other cyclases that generate cGMP, namely GC-B and sGC {alpha}1- and ß1-subunits, were relatively unchanged (Fig. 8EGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
These studies provide the first evidence that cGK II is selectively induced by LH/hCG- and PR-dependent mechanisms in granulosa and cumulus cells at the time of ovulation as determined by analyses of hormonally primed wild-type and PRKO mice. The roles of LH and PR were further substantiated by analyses of cGK II induction in granulosa cells and cumulus cells in culture using specific agonist as well as adenoviral delivery of PR-A. Based on the spatio-temporal pattern of cGK II expression during the periovulatory period, and the coexpression of GC-A (presented here) and eNOS (17) in mural granulosa cells of such follicles, one might presume that activation of cGK II by cGMP derived from either GC-A or sGC impacts key steps in the ovulatory process. In other tissues, cGK II has been associated with fluid secretion (22), phosphorylation and activation of CFTR (24, 34), and bone formation (26). Furthermore, cGK II has been shown to phosphorylate StAR, a key regulatory molecule in steroidogenesis, providing a link to ovarian function (25). Mice null for cGK II have been reported to exhibit intestinal secretory defects and are dwarf (22). The dwarf phenotype and the potential impact of cGK II on StAR might lead one to expect suboptimal or defective ovulation if no compensation occurred, although no reports have alluded to this. The fact that cGK II has been identified as a molecular switch coupling the cessation of proliferation to the start of hypertrophic chondrocyte differentiation via attenuation of Sox9 function (35), and that cGK II impacts bone formation and the synthesis of collagen X, suggest (26) that both the production of matrix molecules by expanding COC, and differentiation of granulosa cells, might be regulated or modulated, in part, by cGK II. cGMP has been shown to block cAMP-regulated expression of aromatase, inhibin, and the LH receptor under conditions in which cGK II expression is low to undetectable. Detailed studies in mice null for cGK II (22) and rats harboring a natural mutation of cGK II (KMI) (35) may provide clues to the possible physiological role of cGK II in ovary.

The presence of cGK I in residual tissue but not in granulosa cells of mature follicles suggest that the role of cGK I in ovary is distinct from cGK II. The presence of cGK I in capillary endothelial cells of ovary (36) suggests a putative role in vascular cell function as in other tissues. Although cGK I has been implicated as a regulator of vascular and other smooth muscle function (23), and although changes in vascular function are critical for postovulation and luteinization processes (37), no reproductive alterations have been documented in cGK I null mice, likely due to the fact that 50% of these die before 5 wk of age, and 79% of them die by 2 months of age (23).

Experiments directed toward understanding the signaling mechanisms by which cGK II is induced in response to hormones showed that LH/hCG induction of PR was not essential for the initial induction of this kinase because the level and pattern of cGK II expression was similar in PMSG-primed PRKO and PRKOHET mice treated with hCG for 2 and 4 h (data not shown). However, at 8 and 12 h after hCG, the level of cGK II in the PRKO ovary and granulosa cells of ovulatory follicles was reduced compared with PRHET samples. Because the 12-h peak in cGK II immediately precedes ovulation in wild-type mice, cGK II might mediate certain downstream signaling events critical for PR action leading to ovulation. The synergistic actions of LH and PR appear to involve at least in part the activation of the PKC pathway because expression of PR-A in granulosa cells by adenovirus increased the PKC-mediated induction of cGK II. The induction by PKC involves the activation of p42/44 MAPK and cross talk with EGF receptor tyrosine kinases. The cross talk between PKC and EGF receptor has been documented in other cells including breast cancer and glioblastoma cells (38, 39, 40). The decreased expression of cGK II in PRKO mouse ovaries before ovulation and increased expression of cGK II in adenoviral PR-A-Myc-infected granulosa cells suggests a role for PR in maximal induction of this gene. The promoters of the rat and human cGK II genes do not have a consensus PRE but do have multiple GC-rich regions that bind Sp1/Sp3 (Ref.41 and our unpublished preliminary analysis), suggesting that PR may regulate expression of cGK II via critical Sp1/Sp3 binding sites, as observed for PR regulation of ADAMTS-1 (42) and cathepsin L (43). Alternatively, PR could induce factors that up-regulate cGK II expression through distinct mechanisms.

Amphiregulin, an EGF family member that is believed to mediate some aspects of the LH action in ovulation (5), increased the expression of cGK II, albeit to a lesser extent than PMA. Interestingly, the addition of FSH/T, alone or with Fo, did not induce the expression of cGK II in cultured granulosa cells, but did induce this kinase during in vitro expansion of COCs. These observations suggest that FSH/Fo, via cAMP and PKA (or Epac) or other pathways, may induce factors such as members of the EGF family of growth factors (amphiregulin, epiregulin, and betacellulin) that regulate other signaling cascades that impact COC expansion (5). Alternatively, LH induction of COX-2 and subsequent increased production of prostaglandins may up-regulate the expression of cGK II in COCs; however, this seems less likely because the prostaglandin receptor subtypes expressed in these cells (EP2 and EP4) (44, 45) activate adenylyl cyclase to produce cAMP and therefore should mimic the effects of FSH.

In summary, these studies document for the first time that cGK II is induced markedly by LH/hCG in granulosa cells and COCs of ovulating follicles and that the effects of LH are mediated in part by induction of PR (Fig. 9Go). Not only is the expression of cGK II reduced in anovulatory follicles of PRKO mouse ovaries at 8–12 h after hCG but cGK II induction in granulosa cells by PMA (that mimics the effects of LH) was enhanced by adenoviral expression of PR-A (Fig. 9Go). The sources of cGMP in granulosa cells are GC-A that is induced by LH/hCG in ovulating follicles and sGC that is constitutively expressed in the mouse ovary (Fig. 9Go). Lastly, the data document that cGK I is not expressed in granulosa cells of preovulatory and ovulatory follicles of the ovary. Thus, these studies document the cell-specific expression of cGK II and GC-A in granulosa cells of periovulatory follicles and elucidate the hormonal and signaling pathways by which these components of the cGMP pathway are induced.


Figure 9
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  		Fig. 9. Schematic Model of cGK II Induction and Activation in Granulosa Cells

After the LH surge, cGK II and the GC-A receptor for ANP and CNP are induced in granulosa cells. This induction occurs along with PR and EGF-receptor ligands that collectively increase cGK II induction with LH. Increased cGK II expression is concomitant with high levels of NO, a known activator of sGC. cGMP generated by GC-A and sGC can activate cGK II to phosphorylate various substrates involved in the ovulatory process. Specifically, cGK II increases in ovulatory follicles devoid of cGK I.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
PMSG (Gestyl) was purchased from Professional Compounding Center of America (Houston, TX), hCG (Pregnyl) from Organon Special Chemicals (West Orange, NJ), media and cell culture reagents and materials from Life Technologies, Inc. (Grand Island, NY) and Corning Inc. (Corning, NY), fetal bovine serum from Hyclone Laboratories, Inc. (Logan, UT). Fo and calphostin C, PD 98059, AG 1478, and SB203580 from Calbiochem (San Diego, CA), trypsin, soybean trypsin inhibitor, deoxyribonuclease, PMA, and testosterone from Sigma Chemical Co. (St. Louis, MO), amphiregulin from R&D Systems (Minneapolis, MN), electrophoresis and molecular biology grade reagents from Bio-Rad Laboratories, Inc. (Richmond, CA), and reagents for RT-PCR from Promega Corp. (Madison, WI). Oligonucleotides were synthesized by Sigma-Genosys (Houston, TX), and ovine FSH-16 was a gift from the National Hormone and Pituitary Program (Rockville, MD). Trilostane was kindly provided Prof. L. L. Espey (Trinity University, San Antonio, TX). Rabbit polyclonal antibodies against recombinant human placental cGK I and rat intestine cGK II have been described previously (46). Myc-tag polyclonal antibody was obtained from Cell Signaling (Beverly, MA).

Animals and Hormone Treatments
Immature (23 d old) C57/Bl6 mice obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN) were housed under a 16-h light and 8-h dark schedule in the Center for Comparative Medicine at Baylor College of Medicine and provided food and water ad libitum. Mice were injected with 4 IU PMSG ip to stimulate follicular growth, and after 48 h ip with 5 IU hCG, which like LH induces ovulation and luteinization. Ovulation occurs approximately 12–16 h after hCG administration in this model (47). Ovaries were isolated from these hormone-stimulated mice at selected time intervals for extraction of RNA and protein, or fixed for in situ hybridization. PRKO mice were used in selected experiments because follicles develop normally in response to PMSG but fail to ovulate in response to hCG (3). Ovaries were prepared for RNA and protein analyses as described below. Cumulus-oocyte complexes (COCs) were isolated from preovulatory and ovulating follicles of PMSG- and hCG-stimulated mice, respectively. COCs from preovulatory follicles were also collected for expansion in culture and RNA analyses. All Animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, as approved by the Animal Care and Use Committee at Baylor College of Medicine (Houston, TX).

PR-A-Myc Adenovirus (AdvPR-A-Myc)
A vector for shuttling the cDNA of human PR-A into adenoviral DNA was assembled by ligating the double-stranded product of annealed oligonucleotides 5'-CTAGCGGTACCAGTACTGGATCCGATATCTCTAGAGTAC-3' and 5'-TCTAGAGATATCGGATCCAGTACTGGTACCG-3' into NheI/KpnI digested pShuttle-X (BD Biosciences CLONTECH, Mountain View, CA). The newly created the vector, pShuttle-XR, bore a unique BamHI site in a multiple cloning site otherwise resembling that of pShuttle-X in reverse orientation. Human PR-A (hPR-A) in the PSCT vector (PSCT-PRA) was generously provided by Drs. Neil McKenna and Rainer Lanz (Baylor College of Medicine). To assemble a shuttle construct bearing myc-tagged hPR-A, a BamHI-digested fragment of PSCT-PRA containing the hPR-A cDNA was ligated into pShuttle-XR. A segment from the resulting construct was then amplified by PCR with oligonucleotides 5'-GCTTACTGGCTTATCGAAATTAATACGACTCAC-3' and 5'-GTTGTCCCCGCTCATGGAACAGAAACTCATCTCTGAAGAGGATCTAAGCCGGTCCGTATT-3', which inserted a sequence encoding the myc-tag at the beginning of the hPR-A open reading frame, and the product was substituted into the corresponding RsrII and NheI sites of the shuttle vector containing hPR-A. The PI-Sce-I/I-Ceu-digestion fragment containing myc-tagged hPR-A cDNA from the resulting construct was then ligated into pAdeno-X (BD Biosciences, CLONTECH) as prescribed by the manufacturer, and the resulting pAdeno-X construct was submitted to the Vector Development Laboratory, Baylor College of Medicine for creation of the hPR-A-Myc adenovirus (AdV PR-A-Myc) (see http://vector.bcm.tmc.edu). AdV PR-A-Myc was tested as described by Davis et al. (48) (also see http://vector.bcm.tmc.edu) to verify the absence of replication competent adenovirus, and the titer was determined by plaque assay as described (48). Before use, the efficacy of infection was tested in rat granulosa cells. We determined that a multiplicity of infection of 2:1 produced a 95% infection rate as revealed by immunocytochemical staining with an anti-myc antibody. Under these conditions, expression of PR-A was elevated within 12–14 h and was sufficient to transactivate GRE2-TATA luciferase construct after stimulation with R-5020 (42). A Lac-Z adenovirus obtained from Vector Development Laboratory at Baylor College of Medicine was used as the negative control.

Granulosa Cell Culture and COC Culture
Granulosa cells were harvested by needle puncture from immature mice treated with PMSG (P) on d 23–25 of age as described previously (42). Briefly, cells were cultured in 12-well culture plates in 1% serum-containing medium (DMEM-F12 containing penicillin and streptomycin). On the following day, cells were washed, then cultured for 4 h in fresh, serum-free medium containing Fo (10 µM), which mimics FSH to stimulate cAMP production, PMA (20 nM) to activate diacylglycerol-mediated signaling, or both, and harvested for protein and RNA analysis. Fo and PMA have previously been used to mimic the effects of the LH surge for optimal induction of COX-2 and PR in cultured rat granulosa cells (49, 50). Some cultures were treated with FSH (50 ng/ml) and testosterone (10 ng/ml) for different time intervals (up to 48 h) to stimulate differentiation, followed by Fo/PMA or Amphiregulin (100 nM) [known to mediate some actions of LH (5)] for an additional 4 h. When inhibitors were used [PD 98059 (18.7 nM), AG 1478, (10 nM) and SB203580 (20 nM)], the cultures were pretreated for 45 min with specific inhibitor, and then it was added along with the agonists. Granulosa cells were are also infected with PR-A-Myc and Lac-Z adenovirus after overnight culture for 14 h at a multiplicity of infection of 2:1. RU486 (1 µM) and trilostane (10 µM) were also added to some wells with the virus. After that, fresh medium containing Fo or PMA or R-5020 with or with out RU486 or trilostane was added for 4 h to determine the effect of expression of PR-A on cGK II expression. COCs were cultured in MEM with Earle’s salts supplemented with 25 mM HEPES, 0.25 mM sodium pyruvate, 3 mM L-glutamine, and 1 mg/ml BSA in the presence or absence of FSH as described previously (44). FSH stimulates COC expansion in culture that is complete by 12–16 h (45). RNA and protein were extracted from expanded and unexpanded COCs as described below.

RNA Isolation and RT-PCR
Total RNA was isolated from whole ovaries of immature (d 23) untreated mice, and from PMSG and hCG-treated mice, using TRIzol reagent (Life Technologies, Inc.), and purified as described in the manufacturer’s instructions. Each RNA sample was prepared from ovaries pooled from two or three animals. RNA was similarly prepared from cultured cells. To determine the expression of cGMP signaling components in ovary, 2 µg of RNA were reverse transcribed in a 20 µl reaction containing oligo-deoxythymidine (Amersham Pharmacia Biotech, Newark, NJ) and AMV reverse transcriptase (Promega Corp.) at 42 C for 75 min. cDNA (2 µl) obtained from reverse transcription reaction was subjected to labeled PCR with [P32]deoxy-CTP (ICN, Los Angeles, CA) using Taq polymerase and specific primers for cGMP signaling components (Table 1Go) (47). All PCRs were carried out within the amplification range experimentally determined as linear. The linear range of amplification was determined after PCR amplification of the target gene at different cycles ranging from 22–35 cycles. The product intensities obtained by densitometry were analyzed to determine the cycle number for each amplicon that falls in the linear range of amplification. The ribosomal protein L19 (Rpl19) was employed as an internal control. PCR products were resolved in a 6% PAGE gel that was dried and autoradiographed. The products were quantified using PhosphoImager (Molecular Dynamics, Inc., Sunnyvale, CA). All PCR products were cloned in TOPO vector and their authenticity was confirmed by sequencing at the Baylor College of Medicine Core Facility.


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  		Table 1. List of Primers Employed for RT-PCR and the Expected Size of Amplicon

 
In Situ Hybridization
In situ hybridization was performed as described previously by Wilkensen (51) and as reported by our laboratory (52). The riboprobe in vitro transcription system kit (Promega Corp.) was used to make [S35]uridine triphosphate-labeled antisense and sense probes from mouse cGK II and GC-A cDNA. The cDNA probes were generated by RT-PCR amplification and TOPO TA cloning (Invitrogen, Carlsbad, CA), their identity verified by sequencing and their specificity by basic local alignment and search tool analyses. Ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 7 µm onto Fisher Brand Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Tissue sections were rehydrated, treated with 20 µg/ml proteinase K and 0.1 M triethanolamine/acetic anhydride, dehydrated, and incubated with radiolabeled riboprobe overnight at 55 C. The next day, slides were washed at high stringency, dried, and exposed to X-OMAT film (Kodak, Rochester, NY) overnight to determine the specificity and intensity of the probe. Slides were dipped in photographic NTB-2 emulsion, exposed at 4 C for an appropriate length of time, developed with D-19 developer and fixer (Kodak), and stained with hematoxylin. Tissue histology was observed by light-field illumination, and dark-field illumination was used to visualize the regions of hybridization.

Cell Extracts and Western Blot Analysis
Protein extracts from mouse ovaries were prepared by homogenization in 6 M urea and 0.1% Triton buffers containing protease inhibitor cocktail (Sigma, St. Louis, MO) and EDTA (0.05 M) (53). Protein concentrations were determined by Bradford method (54) and for Western blot analyses of cGK II and cGK I, 15 µg protein was loaded onto gels. Protein was extracted from cultured granulosa cells employing whole cell extract buffer [10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM dithiothreitiol, 400 mM KCl, 10% glycerol, and protease inhibitors], and equal volumes of extract from each treatment group were loaded onto gels (55). Samples were resolved in 10% acrylamide gels under reducing SDS-PAGE conditions and transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp., Bedford, MA). Membranes were blocked with 5% nonfat milk for 2 h followed by overnight incubation with either cGK I or cGK II antibodies at 1:2000 dilution, then washed with TBST [10 mM Tris (pH 7.5), 150 mM NaCl, and 0.1% Tween 20]. Subsequently, blots were incubated with 1:12,500 diluted horseradish peroxidase linked to antirabbit IgG (Amersham Pharmacia Biotech), then washed with TBST. Immunoreactive bands were detected using enhanced chemiluminescence according to the manufacturer’s specifications (Pierce Chemical Co., Rockford, IL). Band intensities were quantitated using a denistometer (Molecular Dynamics, Inc.).

Statistics
Results are presented as means ± SD from at least three different experiments. Significant differences between groups were analyzed by ANOVA followed by the Neuman-Keuls test employing GraphPad Prism software (San Diego, CA). P values less than 0.05 were considered significant.


   ACKNOWLEDGMENTS
 
We thank Dr. L. L. Espey (Trinity University, San Antonio, TX) for providing trilostane.


   FOOTNOTES
 
These studies were supported in part by the Lalor Foundation (to V.S.) and National Institutes of Health Grants HD-16229 and HD-07495; to J.S.R.) and by the Deutscheforschungsgemeinshcaft (SFB 355, to S.L.).

The authors have nothing to declare.

First Published Online October 6, 2005

Abbreviations: ANP, Atrial natriuretic peptide; BNP, brain natriuretic peptide; CFTR, cystic fibrosis transmembrane conductance regulator Cl–channel; cGK I and II, cGMP-dependent protein kinase I and II; cGMP, cyclic GMP; CNP, C-type natriuretic peptide; COC, cumulus oocyte complexes; COX-2, cyclooxygenase-2; EGF, epidermal growth factor; eNOS, endothelial NOS; Fo, forskolin; GC, guanylate cyclase; hCG, human chorionic gonadotropins; hPR-A, human PR-A; iNOS, inducible NOS; NOS, nitric oxide synthase; NP, natriuretic peptide; PDE, phosphodiesterase; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PMSG, pregnant mare serum gonadotropin; PR, progesterone; PRKO, PR knockout; sGC, soluble guanylate cyclases; StAR, steroidogenic acute regulatory protein.

Received for publication August 2, 2005. Accepted for publication September 27, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Richards JS, Russell DL, Ochsner S, Espey LL 2002 Ovulation: new dimensions and new regulators of the inflammatory-like response. Annu Rev Physiol 64:69–92[CrossRef][Medline]
  2. Natraj U, Richards JS 1993 Hormonal regulation, localization and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles. Endocrinology 133:761–769[Abstract/Free Full Text]
  3. Robker RL, Russell DL, Espey LL, Lydon JP, O’Malley BW, Richards JS 2000 Progesterone-regulated genes in the ovulation process: ADAMTS-1 and cathepsin L proteases. Proc Natl Acad Sci USA 97:4689–4694[Abstract/Free Full Text]
  4. Sirois J, Richards JS 1992 Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles. J Biol Chem 267:6382–6388[Abstract/Free Full Text]
  5. Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M 2004 EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 303:682–684[Abstract/Free Full Text]
  6. Jo M, Gieske MC, Payne CE, Wheeler-Price SE, Gieske JB, Ignatius IV, Curry Jr TE, Ko C 2004 Development and application of a rat ovarian gene expression database. Endocrinology 145:5384–5396[Abstract/Free Full Text]
  7. Espey LL, Richards JS 2002 Temporal and spatial patterns of ovarian gene transcription following an ovulatory dose of gonadotropin in the rat. Biol Reprod 67:1662–1670[Abstract/Free Full Text]
  8. Richards JS, Robker RL, Russell D, Yoshioka S, Sharma SC, Lydon JP, O’Malley BW, Espey LL, Richards JS 2000 Ovulation: a multi-gene, multi-step process. Steroids 65:559–570[CrossRef][Medline]
  9. Richards JS 2001 New signaling pathways for hormones and cyclic adenosine 3',5'-monophosphate action in endocrine cells. Mol Endocrinol 15:209–218[Abstract/Free Full Text]
  10. LaPolt PS, Leung K, Ishimaru R, Tafoya MA, You-hsin Chen J 2003 Roles of cyclic GMP in modulating ovarian functions. Reprod Biomed Online 6:15–23[Medline]
  11. McGee E, Spears N, Minami S, Hsu SY, Chun SY, Billig H, Hsueh AJ 1997 Preantral ovarian follicles in serum-free culture: suppression of apoptosis after activation of the cyclic guanosine 3',5'-monophosphate pathway and stimulation of growth and differentiation by follicle-stimulating hormone. Endocrinology 138:2417–2424[Abstract/Free Full Text]
  12. Hubbard CJ 1983 The effect in vivo of alterations in gonadotropins and cyclic nucleotides on oocyte maturation in the hamster. Life Sci 33:1695–1702[CrossRef][Medline]
  13. Feil R, Lohmann SM, de Jonge H, Walter U, Hofmann F 2003 Cyclic GMP-dependent protein kinases and the cardiovascular system: insights from genetically modified mice. Circ Res 93:907–916[Abstract/Free Full Text]
  14. Noubani A, Farookhi R, Gutkowska J 2000 B-type natriuretic peptide receptor expression and activity are hormonally regulated in rat ovarian cells. Endocrinology 141:551–559[Abstract/Free Full Text]
  15. Jankowski M, Reis AM, Mukaddam-Daher S, Dam TV, Farookhi R, Gutkowska J 1997 C-type natriuretic peptide and the guanylyl cyclase receptors in the rat ovary are modulated by the estrous cycle. Biol Reprod 56:59–66[Abstract]
  16. Huang H, Acuff CG, Steinhelper ME 1996 Isolation, mapping, and regulated expression of the gene encoding mouse C-type natriuretic peptide. Am J Physiol 271:H1565–H1575
  17. Jablonka-Shariff A, Olson LM 1997 Hormonal regulation of nitric oxide synthases and their cell-specific expression during follicular development in the rat ovary. Endocrinology 138:460–468[Abstract/Free Full Text]
  18. Jablonka-Shariff A, Ravi S, Beltsos AN, Murphy LL, Olson LM 1999 Abnormal estrous cyclicity after disruption of endothelial and inducible nitric oxide synthase in mice. Biol Reprod 61:171–177[Abstract/Free Full Text]
  19. Shi F, Stewart Jr RL, Perez E, Chen JY, LaPolt PS 2004 Cell-specific expression and regulation of soluble guanylyl cyclase {alpha}1 and ß1 subunits in the rat ovary. Biol Reprod 70:1552–1561[Abstract/Free Full Text]
  20. Jablonka-Shariff A, Olson LM 2000 Nitric oxide is essential for optimal meiotic maturation of murine cumulus-oocyte complexes in vitro. Mol Reprod Dev 55:412–421[CrossRef][Medline]
  21. Pilz RB, Casteel DE 2003 Regulation of gene expression by cyclic GMP. Circ Res 93:1034–1046[Abstract/Free Full Text]
  22. Pfeifer A, Aszodi A, Seidler U, Ruth P, Hofmann F, Fassler R 1996 Intestinal secretory defects and dwarfism in mice lacking cGMP-dependent protein kinase II. Science 274:2082–2086[Abstract/Free Full Text]
  23. Pfeifer A, Klatt P, Massberg S, Ny L, Sausbier M, Hirneiss C, Wang GX, Korth M, Aszodi A, Andersson KE, Krombach F, Mayerhofer A, Ruth P, Fassler R, Hofmann F 1998 Defective smooth muscle regulation in cGMP kinase I-deficient mice. EMBO J 17:3045–3051[CrossRef][Medline]
  24. French PJ, Bijman J, Edixhoven M, Vaandrager AB, Scholte BJ, Lohmann SM, Nairn AC, de Jonge HR 1995 Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II. J Biol Chem 270:26626–26631[Abstract/Free Full Text]
  25. Gambaryan S, Butt E, Marcus K, Glazova M, Palmetshofer A, Guillon G, Smolenski A 2003 cGMP-dependent protein kinase type II regulates basal level of aldosterone production by zona glomerulosa cells without increasing expression of the steroidogenic acute regulatory protein gene. J Biol Chem 278:29640–29648[Abstract/Free Full Text]
  26. Miyazawa T, Ogawa Y, Chusho H, Yasoda A, Tamura N, Komatsu Y, Pfeifer A, Hofmann F, Nakao K 2002 Cyclic GMP-dependent protein kinase II plays a critical role in C-type natriuretic peptide-mediated endochondral ossification. Endocrinology 143:3604–3610[Abstract/Free Full Text]
  27. Munzel T, Feil R, Mulsch A, Lohmann SM, Hofmann F, Walter U 2003 Physiology and pathophysiology of vascular signaling controlled by guanosine 3',5'-cyclic monophosphate-dependent protein kinase. [lsqb Erratum (2003) 108:3165] Circulation 108:2172–2183
  28. Orstavik S, Natarajan V, Tasken K, Jahnsen T, Sandberg M 1997 Characterization of the human gene encoding the type I {alpha} and type I ß cGMP-dependent protein kinase (PRKG1). Genomics 42:311–318[CrossRef][Medline]
  29. Lydon JP, DeMayo F, Funk CR, Mani SK, Hughes AR, Montgomery Jr CA, Shyamala G, Conneely OM, O’Malley BW 1995 Mice lacking progesterone receptor exhibit reproductive abnormalities. Genes Dev 9:2266–2278[Abstract/Free Full Text]
  30. Robker RL, Richards JS 1999 Progesterone: lessons from the progesterone receptor knockout. New York: Springer-Verlag
  31. Salvador LM, Maizels E, Hales DB, Miyamoto E, Yamamoto H, Hunzicker-Dunn M 2002 Acute signaling by the LH receptor is independent of protein kinase C activation. Endocrinology 143:2986–2994[Abstract/Free Full Text]
  32. Espey LL, Yoshioka S, Russell DL, Robker RL, Fujii S, Richards JS 2000 Ovarian expression of a disintegrin metalloproteinase with thrombospondin motifs during ovulation in the gonadotropin-primed immature rat. Biol Reprod 62:1090–1095[Abstract/Free Full Text]
  33. Sharina IG, Krumenacker JS, Martin E, Murad F 2000 Genomic organization of {alpha}1 and ß1 subunits of the mammalian soluble guanylyl cyclase genes. Proc Natl Acad Sci USA 97:10878–10883[Abstract/Free Full Text]
  34. Vaandrager AB, Tilly BC, Smolenski A, Schneider-Rasp S, Bot AG, Edixhoven M, Scholte BJ, Jarchau T, Walter U, Lohmann SM, Poller WC, de Jonge HR 1997 cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl– channels co-expressed with cGMP-dependent protein kinase type II but not type Iß. J Biol Chem 272:4195–4200[Abstract/Free Full Text]
  35. Chikuda H, Kugimiya F, Hoshi K, Ikeda T, Ogasawara T, Shimoaka T, Kawano H, Kamekura S, Tsuchida A, Yokoi N, Nakamura K, Komeda K, Chung UI, Kawaguchi H 2004 Cyclic GMP-dependent protein kinase II is a molecular switch from proliferation to hypertrophic differentiation of chondrocytes. Genes Dev 18:2418–2429[Abstract/Free Full Text]
  36. Perez E, Shi F, Chen J, LaPolt PS 2003 Cell-Specific expression and potential roles of cyclic GMP-dependent protein kinase-I in the rat ovary. Biol Reprod 68(Suppl I):239
  37. Espey LL 1978 Ovarian contractility and its relationship to ovulation: a review. Biol Reprod 19:540–551[CrossRef][Medline]
  38. Chen N, Ma WY, She QB, Wu E, Liu G, Bode AM, Dong Z 2001 Transactivation of the epidermal growth factor receptor is involved in 12-O-tetradecanoylphorbol-13-acetate-induced signal transduction. J Biol Chem 276:46722–46728[Abstract/Free Full Text]
  39. Stewart JR, O’Brian C A 2005 Protein kinase C-{alpha} mediates epidermal growth factor receptor transactivation in human prostate cancer cells. Mol Cancer Ther 4:726–732[Abstract/Free Full Text]
  40. Amos S, Martin PM, Polar GA, Parsons SJ, Hussaini IM 2005 Phorbol 12-myristate 13-acetate induces epidermal growth factor receptor transactivation via protein kinase C{delta}/c-Src pathways in glioblastoma cells. J Biol Chem 280:7729–7738[Abstract/Free Full Text]
  41. Witczak O, Orstavik S, Natarajan V, Frengen E, Jahnsen T, Sandberg M 1998 Characterization of the gene encoding the human type II cGMP-dependent protein kinase. Biochem Biophys Res Commun 245:113–119[CrossRef][Medline]
  42. Doyle KM, Russell DL, Sriraman V, Richards JS 2004 Coordinate transcription of the ADAMTS-1 gene by luteinizing hormone and progesterone receptor. Mol Endocrinol 18:2463–2478[Abstract/Free Full Text]
  43. Sriraman V, Richards JS 2004 Cathepsin L gene expression and promoter activation in rodent granulosa cells. Endocrinology 145:582–591[Abstract/Free Full Text]
  44. Ochsner SA, Russell DL, Day AJ, Breyer RM, Richards JS 2003 Decreased expression of tumor necrosis factor-{alpha}-stimulated gene 6 in cumulus cells of the cyclooxygenase-2 and EP2 null mice. Endocrinology 144:1008–1019[Abstract/Free Full Text]
  45. Ochsner SA, Day AJ, Rugg MS, Breyer RM, Gomer RH, Richards JS 2003 Disrupted function of tumor necrosis factor-{alpha}-stimulated gene 6 blocks cumulus cell-oocyte complex expansion. Endocrinology 144:4376–4384[Abstract/Free Full Text]
  46. Markert T, Vaandrager AB, Gambaryan S, Pohler D, Hausler C, Walter U, De Jonge HR, Jarchau T, Lohmann SM 1995 Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator. J Clin Invest 96:822–830[Medline]
  47. Hsieh M, Johnson MA, Greenberg NM, Richards JS 2002 Regulated expression of Wnts and Frizzleds at specific stages of follicular development in the rodent ovary. Endocrinology 143:898–908[Abstract/Free Full Text]
  48. Davis AR, Wivel NA, Palladino JL, Tao L, Wilson JM 2000 Construction of adenoviral vectors. Methods Mol Biol 135:515–523[Medline]
  49. Morris JK, Richards JS 1993 Hormonal induction of luteinization and prostaglandin endoperoxide synthase-2 involves multiple cellular signaling pathways. Endocrinology 133:770–779[Abstract/Free Full Text]
  50. Sriraman V, Sharma SC, Richards JS 2003 Transactivation of the progesterone receptor gene in granulosa cells: evidence that Sp1/Sp3 binding sites in the proximal promoter play a key role in luteinizing hormone inducibility. Mol Endocrinol 17:436–449[Abstract/Free Full Text]
  51. Wilkensen D 1993 In situ hybridization. In: Stem CD, Holland PWH, eds. Essential developmental biology, a practical approach. New York: Oxford University Press; 258–263
  52. Robker RL, Richards JS 1998 Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27KIP1. Mol Endocrinol 12:924–940[Abstract/Free Full Text]
  53. Russell DL, Doyle KM, Ochsner SA, Sandy JD, Richards JS 2003 Processing and localization of ADAMTS-1 and proteolytic cleavage of versican during cumulus matrix expansion and ovulation. J Biol Chem 278:42330–42339[Abstract/Free Full Text]
  54. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  55. Sharma SC, Clemens JW, Pisarska MD, Richards JS 1999 Expression and function of estrogen receptor subtypes in granulosa cells: regulation by estradiol and forskolin. Endocrinology 140:4320–4334[Abstract/Free Full Text]

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Nuclear Receptors:   PR
Ligands:   Progesterone  |  RU486



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