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Molecular Endocrinology, doi:10.1210/me.2007-0042
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Molecular Endocrinology 21 (10): 2487-2502
Copyright © 2007 by The Endocrine Society

Synaptosomal-Associated Protein 25 Gene Expression Is Hormonally Regulated during Ovulation and Is Involved in Cytokine/Chemokine Exocytosis from Granulosa Cells

Masayuki Shimada, Yoshiari Yanai, Tetsuji Okazaki, Yasuhisa Yamashita, Venkataraman Sriraman, Michael C. Wilson and JoAnne S. Richards

Department of Applied Animal Science (M.S., Y.Yan., T.O., Y.Yam.), Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan; Department of Molecular and Cellular Biology (M.S., J.S.R.), Baylor College of Medicine, Houston, Texas 77030; Department of Internal Medicine (V.S.), University of Texas Medical Branch, Galveston, Texas 77555; and Department of Neurosciences (M.C.W.), University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131

Address all correspondence and requests for reprints to: Masayuki Shimada, Ph.D., Department of Applied Animal Science, Graduate School of Biosphere Science, Hiroshima University, 1-4-4, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan. E-mail: mashimad{at}hiroshima-u.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PITUITARY SURGE of LH acts on granulosa cells of the preovulatory follicles to terminate the follicular program while at the same time stimulating the expression of genes required for ovulation and luteinization (1, 2). Many induced genes regulate prostaglandin biosynthesis, matrix formation and stabilization, as well as progesterone receptor (PGR)-dependent events (3). In addition, DNA microarray and other molecular techniques have identified specific secreted factors that are expressed in granulosa cells and cumulus cells after LH/human (h) chorionic gonadotropin (CG) stimulation. For example, Kawamura et al. (4) reported that expression and secretion of brain-derived neurotrophic factor (BDNF) are induced in mouse granulosa cells and cumulus cells by hCG treatment in vivo. The BDNF receptor, TrkB receptor, is localized on oocyte cytoplasmic membrane, and the treatment of oocytes with BDNF enhances oocyte maturation (4), suggesting that the regulated exocytotic release of this neurotrophic factor also provides a critical step governing ovulation.

The expression of IL family members, including IL-1ß, IL-6, IL-12, IL-17, and IL-18, has been detected in cumulus cells of cumulus oocyte complexes (COCs) by microaaray analysis and cytokine protein array analyses (5, 6). Addition of IL-1ß to LH-containing medium, for example, significantly increases the number of oocytes ovulated from in vitro-perfused ovaries, whereas this is completely suppressed by an IL-1 receptor antagonist (7). It has been demonstrated that IL-6 regulates steroidogenesis in granulosa cells (8, 9). The receptor, GP130, is also expressed in cumulus cells and granulosa cells (10). In Gp130 null mice, oocyte developmental competence after fertilization was reduced presumably due to the lack of IL-6 transduction (10). Moreover, IL-6 is also secreted from cumulus cells of mouse COCs present in the oviduct during the fertilization process (6). Laflamme et al. (11) reported that IL-6 induces sperm capacitation and acrosome reaction. Thus, secretion of cytokines from cumulus cells and granulosa cells, especially IL-1ß or IL-6, impact oocyte maturation, ovulation, and fertilization process.

Transmission electron microscopic analyses have shown that immunoreactive IL-6 accumulates in secretory granules of human mast cells (12), which migrate to the plasma membrane, presumably for secretion from the cell surface (12, 13). Similarly, BDNF accumulates in secretory vesicles and is released from the cells by exocytosis in neuronal cells (14). The process of exocytosis is complex but is known to be controlled by SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) that is composed of SNARE proteins residing in the vesicle membrane (synaptobrevin) and the plasma membrane (syntaxin and SNAP25) (15, 16). SNAP25 is anchored in the plasma membrane by palmitoylation of four cysteines residing in the linker region between the two SNARE motifs (15). After Ca2+-induced SNAP25 proteolysis, the release of vesicle-contained factors is triggered (17, 18). Two isoforms of SNAP25 protein, SNAP25a and SNAP25b, are expressed from a single gene by alternative splicing of exon 5 (19). SNAP25 mutant mice die during embryonic development; however, SNAP25b-specific deficient mice survive for 3–5 wk, suggesting that the expression and functional activities of SNAP25a and SNAP25b isoforms differ (20, 21). Indeed, the SNAP25a isoform is more abundant in the embryonic mouse brain and adult neurosecretory cells, whereas SNAP25b isoform becomes the dominant form in brain after birth during the major period of synaptogenesis (22). During the ovulation process, the expression of Snap25a mRNA is induced transiently in the rat ovary in response to LH/hCG (23, 24), suggesting that the transient expression of SNAP25, a protein in granulosa cells, might contribute to the secretion of numerous factors, such as BDNF and IL family members from granulosa cells. However, there is little information about the molecular and biochemical mechanisms by which soluble proteins are secreted from ovarian cells and whether or not this involves the process of exocytosis. In this study, we analyzed the hormonal regulation and functional activity of SNAP25 in granulosa cells and cumulus cells during the ovulation process by using both in vivo and in vitro models.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Induction of Snap25 mRNA in COCs and Granulosa Cells of Preovulatory Follicles in Vivo by hCG
To analyze the induction of Snap25 mRNA in ovarian cells, COCs as well as granulosa cells were isolated from ovaries of equine (e) CG-primed mice before (0 h) and at 4, 8, and 12 h after hCG as well as from oviducts at 16 and 24 h after hCG (Fig. 1AGo). Semiquantitative RT-PCR analyses of total RNA document induced expression of the gene in COCs within 4 h after hCG, maximum expression of Snap25 mRNA observed at 8 h after hCG, and progressively reduced levels of Snap25 mRNA thereafter. A similar pattern of Snap25 mRNA induction was observed in granulosa cells (Fig. 1AGo).


Figure 1
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  		Fig. 1. The Induction in Vivo of SNAP25 mRNA and Protein in Granulosa Cells and COCs after hCG Stimulation

A, Total RNA was isolated from COCs and granulosa cells harvested from ovaries of mice treated with eCG for 48 h to stimulate growth of preovulatory follicles (0 h) followed by hCG to stimulate ovulation. Ovulated COCs were collected from the oviducts of mice at 16 and 24 h after hCG. The RNA was analyzed by semiquantitative RT-PCR using specific primers as shown in Table 4Go. For reference, the 0-h COC value was set as 1, and the data are presented as fold change. Values are mean ± SEM of three replicates. * and **, The significant differences were observed as compared with that in COCs (*) or granulosa cells (**) before hCG stimulation (0 h). B, SNAP25 protein was analyzed by Western blot using specific anti-SNAP25 monclonal antibody diluted 1:1000. The protein samples were prepared from COCs and granulosa cells harvested from ovaries of mice treated with eCG for 48 h (0 h) followed by hCG. Ovulated COCs were collected from the oviducts of mice at 16 h after hCG. ß-Actin is used as a loading control. Results in each panel are representative of two separate experiments.

 
To measure SNAP25 protein levels, COCs and granulosa cell samples were prepared and subjected to Western blot analyses using a specific anti-SNAP25 antibody and a ß-actin antibody as a control (Fig. 1BGo). An immunoreactive band at 25 kDa was present in low amounts in both COCs and granulosa cells obtained from ovaries of eCG-primed mice before and at 4 h after hCG ovaries. Immunoreactive SNAP25 increased dramatically at 8 h after hCG and declined thereafter, a pattern similar to that observed for Snap25 mRNA in granulosa cells and COCs.

Induction in Vivo by hCG of Snap25 mRNA and SNAP25 Protein in COCs and Granulosa Cells Is Dependent, in Part, on PGR But Not on Prostaglandin Production
Because Ptgs2–/– (COX-2KO) and Pgr–/– [progesterone receptor knockout (PRKO)] mice exhibit impaired ovulation (3, 4), the expression patterns of Snap25 mRNA and SNAP25 protein were analyzed in RNA and protein samples prepared from granulosa cells and cumulus cells of these mutant mouse ovaries and of their respective control wild-type littermates at 8 h after hCG. As shown in Fig. 2AGo, there was no significant difference in Snap25 mRNA levels in granulosa cells prepared from ovaries of Ptgs2+/+, Ptgs2+/–, and Ptgs2–/– mice. However, in Pgr–/– mice, the levels of Snap25 mRNA were significantly lower than those observed in granulosa cells recovered from Pgr+/+ or Pgr+/– mice at 8 h after hCG.


Figure 2
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  		Fig. 2. SNAP25 Expression in Granulosa Cells and COCs Is Down-Regulated in Pgr Null Mice But Not in Ptgs2 Null Mice

A, Snap25mRNA was analyzed in granulosa cells of wild-type, Ptgs2 mutant, and Pgr mutant mice by semiquantitative RT-PCR. Granulosa cells were recovered from ovaries of mice after 8 h hCG. +/+, Wild-type mice, +/–, heterozygous mice; –/–, knockout mice. For reference, the value in +/+ mice was set as 100, and the data are presented as relative amount. Values are mean ± SEM of three replicates. The level of Snap25 mRNA in granulosa cells was similar in Ptgs2 mutant and wild-type mice, whereas Snap25 mRNA levels were reduced in granulosa cells from Pgr null mice compared with cells from Pgr+/+ or Pgr+/– mice (P < 0.01). B, Protein level of SNAP25 in COCs and granulosa cells of Ptgs2+/– and Ptgs2–/– mice, or Pgr+/– and Pgr –/– mice after 8 h hCG stimulation was analyzed by Western blot. C, The localization of SNAP25 in the ovaries of Pgr+/+ (WT) and Pgr –/– (PRKO) mice primed with eCG for 48 h to stimulate preovulatory follicle growth and hCG 8 h (eCG, hCG 8 h) to initiate ovulation. Immunofluorescent images (upper panels) localize SNAP25 (red), and nuclei (4'6-diamidino-2-phenylindole; blue). D, Western blot analyses show the level of SNAP25 protein in whole-ovary protein samples recovered from eCG primed Pgr+/– and Pgr –/– mice at 8 h after hCG. GC, Granulosa cell; WT, wild type.

 
Western blot analyses confirmed that the intensity of immunoreactive SNAP25 band in COCs and granulosa cells of Ptgs2–/– mice was comparable to those of Ptgs2+/– mice at 8 h after hCG stimulation (Fig. 2BGo). A SNAP25 immunoreactive band was also detected in both COCs and granulosa cells prepared from ovaries of Pgr+/– mice. However, levels of SNAP25 protein in Pgr–/– mice were low in both cell types (Fig. 2BGo).

Immunofluorescent (Fig. 2CGo) analysis using the same SNAP25-specific antibody shows intense staining of SNAP25 in granulosa cells and COCs of periovulatory follicles present in ovaries of eCG-primed wild-type mice 8 h after hCG (eCG, hCG 8 h WT) (Fig. 2CGo, left panels). In contrast, positive signals for SNAP25 were detected only in theca/interstitial cells in ovaries of the eCG-primed Pgr–/– mice 8 h after hCG. Little or no SNAP25 signal was observed in either COCs or granulosa cells of large antral follicles present in the Pgr null ovaries (Fig. 2CGo, right panels).

When whole-ovary protein samples were analyzed by Western blots, a weak SNAP25 imunoreactive band was detected in ovaries from eCG-primed Pgr+/– and Pgr–/– mice and in ovaries from Pgr–/– mice 8 h after hCG (Fig. 2DGo). In contrast, an intense SNAP25 signal was observed in ovarian samples from Pgr+/– mice 8 h after hCG. These data document that the induction of Snap25 mRNA and protein expression by hCG in vivo in granulosa cells and cumulus cells is dependent, in part, on the expression and function of PGR.

To analyze the role of PGR in more detail, granulosa cells were harvested from eCG-primed mice and placed in culture in serum-free defined medium. Because primary granulosa cells express negligible amounts of Pgr mRNA in culture unless exposed to the inductive agonists forskolin (For) and phorbol 12-myristate 13-acetate (PMA) (25), the granulosa cells were infected with adenovirus expressing Myc-tagged PGR type A (PGRA) or control (Lac-Z-encoding) adenovirus overnight. The infected cells were stimulated with PMA and/or For for 4 h, a regimen known to potently induce other genes (5, 25). Some granulosa cells were treated with PGR agonist R5020 or antagonist RU486. Total RNA was prepared and analyzed by semiquantitative RT-PCR. As shown in Fig. 3AGo, neither R5020, For, nor PMA alone led to an increase in Snap25 mRNA whereas the treatment with both For and PMA effectively increased gene expression in Lac-Z-infected granulosa cells. Expression of PGRA-Myc alone or in the presence of R5020 (10 nm) or For did not induce Snap25 mRNA. Rather, PGRA-Myc enhanced the effect of PMA on Snap25 induction with or without For. The For/PMA-induced levels of Snap25 mRNA were significantly suppressed by the PGR antagonist RU486 (1 µm) in Lac-Z-infected granulosa cells (Fig. 3BGo). In PGRA-Myc-infected granulosa cells, either RU486 or protein kinase C (PKC) inhibitor Calphostin C significantly suppressed the PMA-induced Snap25 gene expression (Fig. 3BGo). Thus, Snap25 gene expression was modulated by mechanisms involving PGR and PKC.


Figure 3
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  		Fig. 3. The Expression of Snap25 mRNA in PGR-myc Adenovirus-Infected Mouse Granulosa Cells in Culture

A, eCG-primed mouse granulosa cells were infected with PGR-myc or Lac-Z encording adenovirus and then treated with 10 nm R5020, For, and/or PMA for 4 h. Snap25 mRNA levels were significantly up-regulated by For/PMA as compared with that in granulosa cells without any agonist (P < 0.01). When granulosa cells were infected with PGR-myc adenovirus, PMA alone as well as For/PMA increased Snap25 mRNA levels (P < 0.01). For reference, the value of granulosa cells without any antagonists was set as 1, and the data are presented as fold strength. Values are mean ± SEM of three replicates. B, The effects of 1 µm of RU486 (PGR antagonist, Sigma) and 10 nm of Calphostin C [(CalC) PKC inhibitor; Sigma] on the expression of Snap25 mRNA in the PGR-myc- or Lac-Z-infected granulosa cells treated with PMA and/or For. In Lac-Z-infected granulosa cells, the induction of Snap25 mRNA by For/PMA was significantly suppressed by the additional RU486 (P < 0.01). The PMA-induced Snap25 mRNA expression in PGR-myc-infected granulosa cells was significantly suppressed by RU486 (P < 0.05). The PMA-induced Snap25 mRNA expression in PGR-myc-infected cells was also decreased by CalC as compared with that in granulosa cells with PMA alone (P < 0.01). For reference, the value of Snap25 mRNA in Lac-Z-infected granulosa cells without any agonists was set as 1, and the data are presented as fold strength. Values are mean ± SEM of three replicates.

 
Identification of Functional Regions within the Snap25 Promoter
To analyze functional regions within the proximal promoter of the Snap25 gene that confer For, PMA, and PGRA inducibility, granulosa cells from eCG-primed mice were transfected with selected Snap25 promoter-luciferase constructs bearing upstream regions: –1517, –292, –102, –41, +22, or +114 bp (26). These deletional mutants progressively remove regulatory regions for activator protein-1 (AP-1)- and specificity protein-1 (SP-1)-regulatory binding sites, a putative TATA box, and a CRE site as depicted in Fig. 4AGo. For/PMA significantly induced activity of the –1517 kb Snap25-Luc construct (~3-fold; P < 0.05) in the granulosa cells, whereas the more limited –292 bp Snap-25-Luc construct exhibited yet higher activity, indicating that the sequences responsible for induction in granulosa cells reside within the upstream 292-bp sequence proximal to the transcriptional initiation sites. Deletion of the one AP-1 site and three SP-1/SP-3 sites (–41-bp construct) did not alter basal activity (compared with the –1517 construct) but markedly reduced For/PMA inducibility. Importantly, when three SP-1/SP-3 sites were included upstream of the –41 bp Snap25 construction (–102 bp Snap25-Luc), basal activity increased and For/PMA responsiveness was restored. Moreover, mutation of one SP-1/SP-3 binding site resulted in significant reduction of For/PMA responsiveness (supplemental Fig. 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Because the granulosa cell-rich putative SP-1/SP-3 sites bind transcription factors (SP-1 and SP3) that can be activated in granulosa cells (27), and because the transcriptional activities of SP-1 and SP3 have been demonstrated in granulosa cells after hCG stimulation in vivo and in vitro (27, 28), these results indicated that the three SP-1/SP-3 sites contribute to Snap25 promoter activity in granulosa cells in response to agonist stimulation.


Figure 4
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  		Fig. 4. Functional Analyses of the Snap25 Promoter in Granulosa Cells

Panel A, Mouse granulosa cells were transiently transfected with Snap25 promoter-Luciferase constructs: –1517, –292, –102, –41, +22 or +114 bp Snap25-Luc, and stimulated with or without For/PMA for 4 h. For/PMA treatment significantly increased promoter activities as compared with that of nontreated granulosa cells when –1517, –292, or –102 bp Snap25-Luc promoter was transfected (P < 0.05). Firefly luciferase activities were normalized by Renilla luciferase activities. Values are mean ± SEM of three replicates. Panel B, The cotransfection with PGRA significantly enhanced activity of the –292-bp Snap-25-Luc construct in the granulosa cells treated with PMA and/or For (P < 0.05) as compared with that in granulosa cells with empty vector. Values are mean ± SEM of three replicates. Panel C, The positive effects of PGRA cotransfection was not observed in the granulosa cells with –41 bp Snap25-Luc. Values are mean ± SEM of three replicates. Panel D, The PGR antagonist RU486 significantly repressed the activity of –292 bp Snap25-Luc (P < 0.05). Mouse granulosa cells were transiently transfected with –292 bp Snap25-Luc and treated with 1 µm RU486 in the presence of For/PMA for 4 h. Values are mean ± SEM of three replicates. Panel E, The PKC inhibitor, Calphostin C, significantly repressed the activity of –292 bp Snap25-Luc (P < 0.05). Mouse granulosa cells were transiently transfected with –292 bp Snap25-Luc and treated with 10 nm Calphostin C in the presence of For/PMA for 4 h. Values are mean ± SEM of three replicates. C, Control; CalC, calphostin C.

 
Regulation of the Snap25 Promoter by PGR in Granulosa Cells
To analyze the role of PGR in regulating transcription of the Snap25 gene, mouse granulosa cells were cotransfected with either the –292 bp or –41 bp Snap25-Luc construct without or with a PGRA expression vector (Fig. 4Go, B and C). Cotransfection of the –292 bp Snap25-Luc with the PGRA construct showed that PGRA alone did not stimulate Snap25 promoter activity in these granulosa cells (Fig. 4BGo) and that treatment with For alone did not enhance the promoter activity. Interestingly, however, PMA alone or PMA+For enhanced Snap25 promoter activity in the presence of exogenous PGRA as compared with that in cells transfected with the empty vector (Fig. 4BGo). On the other hand, when granulosa cells were transfected with –41 bp Snap25-Luc (which lacks the SP-1/SP3 sites), cotransfection with a PGRA expression vector did not enhance the promoter activity, even if the cells were stimulated with PMA or For/PMA (Fig. 4CGo). Additionally, the single mutation of the SP-1/SP-3 site significantly suppressed the PGR-enhanced promoter activity in the cells treated with For/PMA (supplemental Fig. 1).

To analyze further whether the regulation of Snap25 expression is dependent on functional PGRA and PKC activity, granulosa cells transfected with –292 bp Snap25-Luc reporter constructs were treated with For/PMA in the presence or absence of either the PGR antagonist RU486 or the PKC inhibitor Calphostin C. As shown in Fig. 4DGo, For/PMA significantly increased promoter activity, whereas the addition of RU486 reduced levels of promoter activity as compared with that in the cells without RU486. Snap25 promoter activity was also decreased in the cells treated with Calphostin C (Fig. 4EGo). These results provide evidence that Snap25 gene expression in granulosa cells is regulated by PGRA and PKC via SP-1/SP-3 sites within the Snap25 promoter.

Agonist Induction of Snap25 mRNA and Secreted Cytokines in Mouse COCs and Granulosa Cells
To examine the role of SNAP25 in COCs and granulosa cells during the ovulation process, both COCs and granulosa cell culture systems were used. For these experiments, nonexpanded COCs were isolated from ovaries of eCG-primed immature mice and placed in defined medium containing 1% serum. When both FSH (100 ng/ml) and AREG (250 ng/ml) were added to the COCs, expansion was observed 16 h later, confirming many previous studies (data not shown). Treatment with 10 µm For and 20 nm PMA (For/PMA) also induced gene expression in cumulus cells of COCs. Granulosa cells collected from eCG-primed immature mouse ovaries were cultured with 1 µg/ml LH or For/PMA that mimic LH stimulation, as described above.

In COCs, treatment with FSH/AREG induced a high level of Snap25mRNA within 4 h that declined by 8 h to a level that was nevertheless still higher than that found in COCs before culture (Fig. 5AGo). A similar pattern was observed when COCs were cultured with For/PMA (Fig. 5AGo). In granulosa cells, treatment with LH also induced the Snap25 gene expression, whereas higher levels of Snap25 mRNA were detected after treatment with For/PMA. Therefore, in response to specific agonists, Snap25 mRNA was induced in COCs and granulosa cells within 4 h. The addition of RU486 significantly decreased agonist-induced expression of SNAP25 protein and mRNA in cumulus cells and granulosa cells at 8 h (Fig. 5Go, B and C).


Figure 5
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  		Fig. 5. The Induction of SNAP25 Expression in Cultured COCs and Granulosa Cells Was Dependent on PGR

Panel A, Levels of Snap25 mRNA were determined by real-time RT-PCR. COCs were cultured for 4, 8, or 16 h with FSH/AREG or For/PMA. Granulosa cells (GC) were cultured with LH or For/PMA. * and **, The culture with For/PMA (*) or FSH/AREG (**) significantly increased the expression level of Snap25 mRNA as compared with that in COCs cultured without any agonist at each time point (P < 0.05). # and ##, For/PMA (#) or LH (##) significantly increased Snap25 mRNA at 4 or 8 h as compared with that in granulosa cells cultured in media alone (P < 0.05). For reference, the 0-h COC value was set as 1, and the data are presented as fold strength. Values are mean ± SEM of three replicates. Panels B and C, The levels of SNAP25 in protein (panel B) and mRNA (panel C) mRNA were decreased by RU486. COCs were cultured for 8 h with or without RU486 in the presence of FSH/AREG, or For/PMA. Granulosa cells (GC) were cultured for 8 h with RU486 in the presence of LH or For/PMA. The addition of RU486 to For/PMA- or FSH/AREG-containing medium significantly decreased the level of Snap25 mRNA in COCs (P < 0.05). In granulosa cells (GC), the expression level of Snap25 mRNA was significantly suppressed by the treatment with RU486 (P < 0.05). Values are mean ± SEM of three replicates. ß-Actin is used as a loading control. C, Control; RU, RU486.

 
An analysis of previous microarray data indicated that IL family members, including IL-6, IL-12, IL-17, and IL-18, were expressed in cumulus cells after hCG stimulation (5). Because ILs accumulate in secretory vesicles and are released from the cells via exocytosis (12), we hypothesized that a similar mechanism would control the release of ILs from cumulus cells and granulosa cells. To test this hypothesis, 23 kinds of cytokines and chemokines were analyzed in the medium of COCs or granulosa cells that had been cultured for 12 h with either FSH/AREG or For/PMA, respectively, using the BioPlex Protein Array system.

As shown in Table 1Go, COCs cultured with FSH/AREG secreted increased levels of IL-6, IL-9, and IL-17 compared those of COCs cultured without hormone. Although COCs released keratinocyte-derived chemokine (KC), and regulated upon activation, normal T cell expressed and secreted (RANTES; Ccl5), the levels between control and FSH/AREG-treated samples were not significantly different. When COCs were cultured with For/PMA, the levels of KC as well as those of IL-6, IL-9, and IL-17 were significantly higher than those observed in controls. Moreover, the FSH/AREG-induced secretion of IL-6 and IL-9 as well as For/PMA-induced levels of IL-6, IL-9, IL-17, and KC were significantly suppressed by the treatment with RU486. The secreted levels of IL-1{alpha}, IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-10, IL-12 (p40), IL-12 (p70), IL-13, Eotaxin, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), MIP-1ß, and granulocyte colony-stimulating factor, granulocyte-macrophage colony stimulating factor (GM-CSF), interferon {gamma}, MCP-1, MIP-1{alpha}, MIP-1ß, and TNF{alpha} were below the detection limit.


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  		Table 1. The Levels (pg/ml) of Cytokine and Chemokine Family Members Secreted by Cultured COCs and Detected by BioPlex Protein Array System

 
In granulosa cells, eight different secreted cytokines and chemokines were detected in this system (Table 2Go). A significant increase of IL-1ß, IL-6, IL-9, GM-CSF, KC, MCP-1, and MIP-1ß levels was induced by For/PMA treatment of granulosa cells, whereas no increase in the secretion of RANTES was observed. The treatment of granulosa cells with LH significantly increased the secreted/accumulated levels of IL-1ß, IL-6, IL-9, GM-CSF, and KC as compared with those in the medium when the cells were cultured without any agonist (free). The agonist-induced secretion of cytokines and chemokines was significantly suppressed by the addition of RU486.


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  		Table 2. The Levels (pg/ml) of Cytokine and Chemokine Families Secreted by Granulosa Cells in the Cultured Medium Detected by BioPlex Protein Array System

 
Based on these results and because the elevated levels of IL-6 were observed in agonist-stimulated COCs and granulosa cells, we selected IL-6 to monitor the exocytosis process of cumulus cells and granulosa cells in more detail. As shown in Fig. 6Go, hCG stimulated rapid induction of Il6 mRNA in COCs and granulosa cells in vivo within 4 h that was maximal by 8 h (Fig. 6AGo). Treatment of cultured COCs with FSH/AREG or For/PMA also significantly increased the level of Il6 mRNA at 8 h, which declined by 16 h (Fig. 6BGo). In cultured granulosa cells, Il6 mRNA was induced by For/PMA and to a lesser (but still significant amount) by LH within 4 h and was maximal at 8 h (Fig. 6BGo). Importantly, the expression of Il6 mRNA in cultured COCs and granulosa cells was not altered by the treatment with RU486 (Fig. 6CGo), suggesting that the negative effects of RU486 on the secretion/accumulation of IL-6 protein are mediated by the decrease of SNAP25 protein in the cells.


Figure 6
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  		Fig. 6. Il6 mRNA Is Induced in Vivo and in Cultured COCs and Granulosa Cells by Specific Agonists

Panel A, Time-dependent changes of Il6 mRNA expression in COCs and granulosa cells during the ovulation process in vivo. RNA was analyzed by semiquantitative RT-PCR using specific primers as shown in Table 4Go. For reference, the 0-h COC value was set as 1, and the data are presented as fold strength. Values are mean ± SEM of three replicates. * and #, The significant differences were observed as compared with that in COCs (*) or granulosa cells (#) before hCG stimulation (0 h). Panel B, Levels of Il6 mRNA in cultured COCs and granulosa cells. COCs were cultured for 4, 8, or 16 h with For/PMA or FSH/AREG. Granulosa cells were cultured with For/PMA or LH. * and **, For/PMA (*) or FSH/AREG (**) significantly increased the expression level of Il6 mRNA as compared with that in COCs cultured in media alone (P < 0.05). # and ##, Significantly higher levels were observed in granulosa cells cultured with For/PMA (#) or LH (##) as compared with that in granulosa cells cultured in media alone (P < 0.05). Values are mean ± SEM of three replicates. Panel C, RU486 did not alter agonist-induced expression of Il6 mRNA in COCs or granulosa cells. Values are mean ± SEM of three replicates. C, Control; GC, granulosa cell; RU, RU486.

 
Snap25 Small Interfering RNA (siRNA) Knockdown Affects Granulosa Cell Functions
To identify the role of SNAP25 in exocytosis in more detail, primary cultures of granulosa cells were transfected with Snap25 siRNA. The expression level of Snap25 mRNA and SNAP25 protein and secreted level of cytokine and chemokine were analyzed. As expected, treatment with For/PMA significantly induced Snap25 mRNA and protein levels (Fig. 7Go, A and B), and this induction of Snap25 mRNA and protein was suppressed significantly by the transfection with Snap25 siRNA (P < 0.05) but not by the control siRNA (NC). Interestingly in these same granulosa cells, although the expression of Il6 mRNA was not affected by the Snap25 siRNA (Fig. 7CGo), the levels of IL-6 and other factors (IL-1ß, IL-9, KC, MCP-1, MIP-1ß, and RANTES) in the culture medium were significantly lower in those cells that were transfected with Snap25 siRNA compared with control siRNA (Table 3Go). Thus, the secretion of cytokine and chemokine family members, such as IL-6 from granulosa cells, was dependent on SNAP25 expression.


Figure 7
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  		Fig. 7. The Effects of Snap-25 siRNA on the Levels of SNAP25 in mRNA (A) and Protein Level (B) as Well as Il6 Expression (C) in Granulosa Cells

A and B, Levels of Snap25 mRNA (A) or SNAP25 protein (B) were significantly suppressed by Snap25 siRNA. Granulosa cells were transfected with Snap25 siRNA or scrambled siRNA duplex as a negative control (NC) for 5 h and treated with For/PMA for 4 h (for mRNA) or 12 h (for protein). The level of Snap25 mRNA was determined by real-time RT-PCR. The protein level was analyzed by Western blot. Significantly lower levels of Snap25 mRNA and SNAP25 protein were detected in granulosa cells transfected with specific siRNA as compared with that in granulosa cells with scrambled siRNA (NC) (P < 0.05). Values are mean ± SEM of three replicates. ß-Actin is used as a loading control. C, The level of Il6 mRNA was determined by real-time RT-PCR. Granulosa cells transfected with Snap25 siRNA or scrambled siRNA duplex as a negative control (NC) for 5 h and treated with For/PMA for 4 h.

 

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  		Table 3. The Effect of Snap25 siRNA on the Secretion of Cytokines and Chemokines by Cultured Granulosa Cells Detected by BioPlex Protein Array System

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Induction of PGR by LH plays an integral role in the process of ovulation, as shown by ovulation failure in Pgr null mice (29, 30). Although the specific targets of PGR that control ovulation are not yet entirely clear, our laboratory has demonstrated that induction of ADAMTS-1 (29), cathepsin L (29, 31), cGMP-dependent protein kinase II (Prkg2) (25), amphiregulin (AREG) (32), and epiregulin (32) are reduced in these mice. Doyle et al. (33) showed that CCAAT enhancer binding protein-ß, GA binding protein (GABP), nuclear factor-1-like factor, early growth response 1, and SP-1/SP-3 bound the Adamts1 proximal promoter region, whereas this gene lacks a distinguishable consensus PGR response element. Nevertheless, cotransfection of a PGRA expression vector enhanced Adamts1 promoter activity via granulose cell-rich SP-1/SP-3 binding sites (34). Importantly, other genes regulated by PGR in granulosa cells appear to have at least one SP-1/SP-3 site (25, 31, 34, 35). Moreover, ligand-activated PGR stimulates glycodelin-A gene expression in endometrial cells through functional SP-1 sites (36), and SP-1 protein coprecipitates with either ligand-activated human PGRA or PGRB in the decidual cell nuclear extract (37). Thus, the function of PGR in granulosa cells is mediated, at least in part, by SP-1/SP-3 factors and essential granulose cell-rich SP-1/SP-3 sites.

We show herein that SNAP25 is a new member of PGR-target genes in the ovary because induced expression of Snap25 mRNA was repressed in granulosa cells and cumulus cells from Pgr–/– mice as compared with Pgr+/– or wild-type mice at 8 h after hCG, the time of maximal induction. Although the proximal Snap25 promoter region has three recognizable AP-1 sites, a TATA box and a cAMP-responsible element (CRE) site (26), we document that the GC-rich region containing three SP-1/SP-3 sites was critical in granulosa cells. These same SP-1/SP-3 sites were also essential for high basal expression of the Snap25 promoter in neuronal PC12 cells (26). SP-1 family members are widely known to participate in the regulation of a variety of genes (38). Because SP-1 is constitutively expressed in both granulosa cells (27) and PC12 cells (39), and because the results described herein show that the deletion of SP-1/SP-3 sites reduces the activity of Snap25 promoter, this provides an explanation for the high level of Snap25 promoter activity in transfections assays and induction of Snap25 expression in both granulosa cells and PC12 cells.

Although the Snap25 gene is expressed in both granulosa cells and PC12 cells, the regulatory mechanisms that activate the SP-1/SP-3 promoter sites appear to be completely different. In PC12 cells, but not granulosa cells, treatment with For alone stimulated Snap25 promoter activity even if the SP-1/SP-3 sites were deleted (supplemental Fig. 2). Sanberg and Low (40) reported that Snap25 expression was increased in a cAMP-dependent manner in neuronal cells, suggesting that in PC12 cells Snap25 gene expression is regulated not only via activation of SP-1/SP-3 but also by cAMP activation of the CRE region. In contrast, cotransfection with a PGRA expression vector enhanced PMA-stimulated promoter activity in granulosa cells, but not in PC12 cells (supplemental Fig. 2). The importance of PGRA in granulosa cells was documented further by the ability of adenoviral-expressed PGR to enhance PMA induction of Snap25 mRNA. Although the mechanisms by which PMA up-regulates Snap25 mRNA expression and modulates PGR function are unclear, we have previously shown that induction of Areg and Prkg2 mRNA expression in granulosa cells was also enhanced by overexpression of exogenous PGR, but only in the presence of PMA (32). Additionally, Biggs et al. (41) reported that the transcriptional activity of SP-1/SP-3 was modified by a PKC-dependent pathway. Moreover, SP-1 protein bound either ligand-activated human PGRA or PGRB in the decidual cell nuclear extract, and the interaction increased the transcriptional activity (37). Based on these results and those in the present study, we hypothesized that the Snap25 gene is transiently expressed in granulosa cells and cumulus cells during the ovulation process through an ovarian-specific mechanism that involves both PGR and SP-1/SP-3 and the modification of their functional activities by PKC.

SNAP25 is one plasma membrane protein of the SNARE complex (15, 16) that is abundantly expressed in neuronal and neuroendocrine cells. SNAP25 and the SNARE complex have specialized functions in fast-regulated secretion pathways, such as synaptic vesicle exocytosis (16). Recently, SNAP25 expression has been observed in nonneuronal cells, such as granulosa cells (23), pancreatic ß-cells (42), chromaffin cells (43, 44), and sperm (45). In chromaffin cells, the Ca2+-triggereed exocytosis burst responsible for regulated catecholamine secretion requires SNAP25 and associated SNARE proteins (44), which suggests that SNAP25 participates in regulated stimulus-driven secretory processes exercised in these nonneuronal cells. Because the present study showed that SNAP25 is induced markedly in granulosa cells and cumulus cells of ovulating follicles and other SNARE components, syntaxin and synaptobrevin are expressed in both cells, and these expressions were not regulated by PGR (data not shown), the regulated exocytosis might be required for these cells to release specific vesicle-contained factors, such as cytokines and chemokines, known to be present in human follicular fluid IL-1ß, IL-6, GM-CSF, KC, MCP-1, MIP-1ß, and RANTES (46, 47, 48, 49, 50, 51, 52, 53). Importantly, we have shown previously by microarray data that cumulus cells express IL-6, IL-12, IL-17, and IL-18 (5). The release and accumulation of IL-6, IL-9, and IL-17 protein from cumulus cells and granulosa cells was confirmed using BioPlex Protein Array system. Moreover, because IL-6 can be induced in granulosa cells and cumulus cells recovered from the ovaries of mice after 8 h hCG (54) and because IL-6 has been shown to accumulate in vesicles that appear to migrate to the plasma membrane before fusing at the cell surface (12), we chose this cytokine to assay secretion in these studies.

In in vitro cultures agonist-induced expression of SNAP25 was significantly suppressed by RU486 as compared with that in the cells cultured without the antagonist. The treatment with RU486 did not alter the levels of Il6 mRNA but did reduce significantly the release/accumulation of IL-6 as well as other cytokine family members in the cultured medium. Moreover, when granulosa cells were transfected with Snap25 siRNA, the expression level of Snap25 mRNA was less than half of that observed in the cells transfected with scrambled siRNA duplex, and the SNAP25 protein level decreased to about 25% with the Snap25 siRNA. In the same experiments, levels of Il6 mRNA were not affected by the Snap25 siRNA whereas the secretion/accumulation of IL-6 in the media of treated cells was significantly suppressed by the Snap25 siRNA transfection. The secretion of other cytokines and chemokines was also repressed when granulosa cells were transfected with Snap25 siRNA. Thus, IL-6, as well as other factors, appears to be secreted from granulosa cells by SNAP25-dependent exocytosis.

Although multiple cytokines are present in human follicular fluid (46, 47, 48, 49, 50, 51, 52, 53) and can be released from mouse granulosa cells and COCs in culture, the functional roles of these secreted factors and the triggers regulating their exocytotic release remain to be determined. For example, IL-1ß and its receptor are expressed in granulosa cells after hCG injection, and secretion of this cytokine can mediate, by autocrine or paracrine mechanisms, the increased progesterone production, prostaglandin production, and glucose uptake by granulosa cells during the ovulation process (51). Moreover, IL-6 can also regulate progesterone production in granulosa cells (8, 9). Despite the current lack of known functions for other cytokines, levels of these factors in follicular fluid could be useful predictive parameters of successful pregnancy or infertility in in vitro fertilization patients (55, 56, 57, 58).

In summary, the present study documents that the expression of Snap25 mRNA is induced by hCG- and PGR-dependent mechanisms in granulosa cells and COCs of periovulatory follicles. Promoter analyses indicated that PGR and PMA acted synergistically to activate the proximal Snap25 promoter in granulosa cells via a GC-rich region containing three SP-1/SP-3 sites. Finally, using Snap25 siRNA we show that reduction of Snap25 mRNA and protein is associated with a marked decrease in the levels of IL-6 released/accumulated in the cultured medium but not to a decrease in Il6 mRNA. From these results, we concluded that progesterone-PGR-mediated SNAP25 expression in COC and granulosa cells contribute to release secreted factors including cytokine and chemokine family via the exocytosis system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
eCG was purchased from Calbiochem (San Diego, CA) or Asuka Seiyaku (Tokyo, Japan). hCG was purchased from St. Luke Episcopal Hospital Pharmacy (Houston, TX) or Asuka Seiyaku. FSH (oFSH-16) and LH (pLH) were a gift from the National Hormone and Pituitary Program (Rockville, MD). For and PMA were purchased from Calbiochem. R5020 was gift from Dr. Nancy Weigel (Baylor College of Medicine, Houston, TX). DMEM:F12 medium and penicillin-streptomycin were from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from Hyclone Laboratories, Inc. (Logan, UT) or Life Technologies, Inc. (Gaithersburg, MD). Oligonucleotide poly-(dT) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ), and avian myeloblastoma virus reverse transcriptase and Taq polymerase were from Promega Corp. (Madison, WI). Radiolabeled [p32]dCTP was purchased from ICN (Los Angeles, CA). Routine chemicals and reagents were obtained from Fisher Scientific (Pittsburgh, PA), Nakarai Chemical Co. (Oosaka, Japan), or Sigma Chemical Co. (St. Louis, MO).

Animals
Immature female C57BL/6 mice were obtained from Harlan, Inc. (Indianapolis, IN) or Clea Japan (Tokyo, Japan). On d 23 of age, female mice were injected ip with 4 IU of eCG to stimulate follicular growth followed 48 h later with 5 IU hCG to stimulate ovulation and luteinization (29, 59). PGR null (Pgr; PRKO) mice and prostaglandin synthase 2 (Ptgs2) null mice were used in selected experiments because follicles develop normally in response to eCG but fail to ovulate in response to hCG (60, 61). PRKO mice were obtained originally from John Lydon, at Baylor College of Medicine (30); Ptgs2 null mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were housed under a 16-h light, 8-h dark schedule in the Center for Comparative Medicine at Baylor College of Medicine or Experiment Animal Center at Hiroshima University and provided food and water ad libitum. Animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals, as approved by the Animal Care and Use Committee at Baylor College of Medicine or at Hiroshima University.

Granulosa Cell Culture
Granulosa cells were harvested by needle puncture from immature mice treated with eCG on d 23–25 of age as described previously (25). Briefly, 1 x 106 cells were cultured in 12-well culture plates in 1% serum-containing medium (DMEM:F12 containing penicillin and streptomycin). After 8 h culture, cells were washed and then cultured for 4, 8, or 16 h in fresh, serum-free medium containing 1 µg/ml of LH or For (10 µm), which mimics LH stimulation of cAMP production, PMA (20 nm), which activates diacylglycerol-mediated signaling, or both, and harvested for protein and RNA analysis. For and PMA have previously been used to mimic the effects of the LH surge for optimal induction of Ptgs2 and Pgr mRNA in cultured rat granulosa cells (28, 62). In other experiments, granulosa cells were infected with PGRA-Myc and Lac-Z-expressing adenoviral vectors for 14 h (overnight) at a multiplicity of infection of 2:1 before the addition of agonists (25).

COC Isolation and Culture
Ovaries of immature mice primed with eCG for 48 h contain multiple preovulatory follicles. COCs were isolated from these follicles by needle puncture and collected by pipette. Nonexpanded COCs were selected and 100 COCs were cultured in separate wells of a Falcon 96-well plate (Becton Dickinson, Franklin Lakes, NJ) in 150 µl of defined medium (61) containing 1% FBS with FSH (100 ng/ml) and AREG (250 ng/ml; R&D Systems, Minneapolis, MN), or 10 µm For and 20 nm PMA. After 4, 8, or 16 h, the COCs total RNA or protein was extracted (see below).

siRNA Treatment Procedure in Cultured Mouse Granulosa Cells
Snap25 Silencer Pre-designed siRNA was purchased from Ambion, Inc. (Austin, TX). The sequences were: sense, GCAAAUAUAUGUUUHHCUGtt; antisense, CAGCCAAACAUAUAUUUGCtt. Scrambled siRNA duplex (Ambion) was used as a negative control. Mouse granulosa cells (1 x 106 cells per well) recovered from eCG-primed mice were plated in 12-well culture plates for 3 h before transfection. Transfection of siRNA (25 nm) was accomplished with HVJ envelope vector kit GenomONE neo (Ishihara Sangyo, Tokyo, Japan) according to the manufacturer’s instructions. Cells were incubated at 37 C in a CO2 incubator, and the culture medium was replaced 5 h after transfection. After transfection, granulosa cells were cultured with For (10 µm) and PMA (20 nm) for 4 h (collected for RNA isolation) or 12 h (collected for protein). The cultured media was kept under –80 C until BioPlex protein array analysis.

RT-PCR Analyses
Total RNA was obtained from COCs or granulosa cells using the RNAeasy mini kit (QIAGEN Sciences, Germantown, MD) according to the manufacturer’s instructions, and semiquantitative RT-PCR analyses were performed as previously described (63). Briefly, total RNA was reverse transcribed using 500 ng poly-dT (Amersham Pharmacia Biotech) and 0.25 U avian myeloblastosis virus-reverse transcriptase (Promega Corp.) at 42 C for 75 min and 95 C for 5 min. For the amplification of the cDNA products, specific primers pairs were selected and analyzed as indicated in Table 4Go. All PCRs contained [32P]dCTP (ICN, Los Angeles, CA), Taq polymerase, and Thermocycle buffer (Promega Corp.). cDNA products were resolved on 5% polyacrylamide gels that were dried and exposed to film. The radioactive PCR product bands were quantified using a Storm 860 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). The authenticity of the PCR products was verified by sequencing.


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  		Table 4. List of Primers Employed for RT-PCR and the Expected Size of the Amplified Product

 
Real-Time PCR Analyses
cDNAs produced by the above method were also used for real-time PCR analysis. cDNA and primers were added to 15-µl reaction mixture (Power SYBR Green PCR Master Mix; Applied Biosystems, Foster City, CA). PCR were then performed using the iCycler thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA). Conditions were set to the following parameters: 10 min at 95 C followed by 45 cycles each of 15 sec at 95 C and 1 min at 62 or 64 C. Specific primer pairs were selected and analyzed as indicated in Table 4Go.

Western Blot Analyses
Protein samples from granulosa cells or cumulus cells were prepared by homogenization in whole-cell extract buffer and then diluted by the same volume of 2x SDS sample buffer (5). Extracts (20 µg protein) were resolved by SDS-PAGE (15%) and transferred to Immobilon-P nylon membranes (Millipore Corp, Bedford, MA). Membranes were blocked in Tris-buffered saline and Tween 20 [TBST; 10 mm Tris (pH 7.5), 150 mm NaCl and 0.05% Tween 20] containing 5% nonfat Carnation instant milk (Nestle Co., Solon, OH). Blots were incubated with primary antibody [1:1000 dilution of anti-SNAP25 monoclonal antibody SMI 81 (Abcam, Cambridge, UK) or 1:10,000 dilution of anti-ß-actin antibody, AC74 (Sigma)] overnight at 4 C. After washing in TBST, enhanced chemiluminescence (ECL) detection was performed by using Pierce Super Signal according to the manufacturer’s specifications (Pierce Chemical Co.) and appropriate exposure of the blots to Kodak x-ray film (Eastman Kodak, Rochester, NY). Specific bands were quantified by densitometric analyses using a Molecular Dynamics Personal Densitometer.

Immunofluorescence
Ovaries were embedded in ornithine carboxytransferase compound (Miles, Inc., Elkhart, IN) and stored at –70 C before the preparation of 5-µm sections, which were fixed overnight in PBS-buffered 4% paraformaldehyde at 4 C. Sections were then sequentially probed with primary anti-SNAP25 antibody and secondary Alexa Fluor 594- or 488-conjugated goat antimouse IgG antibodies (Molecular Probes). Slides were mounted using VectaShield with 4'6-diamidino-2-phenylindole (Vector Laboratories, Inc., Burlingame, CA).

Transient Transfection and Luciferase Reporter Assay
Granulosa cells from eCG-primed mice or PC 12 cells were cultured as described above. Transfections with specific expression and reporter constructs were done 3 h after plating cells using the Fugene transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions. Cells were transfected with 0.5 µg of the indicated Snap25 promoter-reporter constructs (no. 26, shown in Fig. 4AGo) and 10 ng of pRL Renilla luciferase control vector (Promega). Cotransfections with either empty vector or a human PGRA expression vector (gifted by Drs. Neil McKenna and Rainer Lanz, Baylor College of Medicine) used 10 ng of the plasmid DNA. After overnight culture in DMEM:F12 containing 5% fetal bovine serum, cells were washed with serum-free medium and then placed in the same media containing agonist and/or antagonist as indicated. After 4 h of agonist treatment, cells were harvested in lysis buffer [0.2 m Tris (pH 8.0) containing 0.1% Triton X-100]. Luciferase activity was analyzed according to a standard protocol. In brief, a 40-µl aliquot of the cell lysate was mixed with 100 µl of the firefly luciferase substrate (Promega) or Renilla luciferase substrate (Promega), and each reaction was monitored by a Dynex Technologies, Inc. (Chantilly, VA) MLX Luminometer. Firefly luciferase activities were normalized by Renilla luciferase activities. Each experiment was performed in triplicate at least three times.

BioPlex Protein Array System
Granulosa cells were transfected with Snap25 siRNA for 5 h and treated with or without For/PMA for 12 h. At that time, media samples were collected and cytokines present in the media were analyzed with the BioPlex Protein Array system (Bio-Rad) using BioPlex Mouse Cytokine 23-Plex Panel including antibodies for IL family members [IL-1{alpha}, IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17], Eotaxin, granulocyte colony-stimulating factor, GM-CSF, interferon {gamma}, keratinocyte-derived chemokine (KC), MCP-1, MIP-1{alpha}, MIP-1ß, RANTES (regulated upon activation, normal T cell expressed and secreted; Ccl5), and TNF{alpha}, according to the manufacturer’s instructions.

Statistics
Statistical analyses of all data from three or four replicates for comparison were carried out by one-way ANOVA followed by Duncan’s multiple-range test (Statview; Abacus Concepts, Inc., Berkeley, CA).


   ACKNOWLEDGMENTS
 
We thank Dr. Derek Boerboom (Université de Montréal) and Dr. Michael D. Rudd (Baylor College of Medicine) for technical suggestions to modify Snap25 promoter-reporter constructs; Dr. Syunichi Tanabe (Hiroshima University) for technical advice on the use of BioPlex protein array system; and Dr. Sabine M. Mulders (Organon, Oss, The Netherlands) for early discussions on the expression of Snap25 mRNA expression in mouse ovaries.


   FOOTNOTES
 
This work was supported, in part, by an Overseas Advanced Educational Research Practice Support Program (16-311) and JSPS-18688016 (to M.S.); National Institutes of Health (NIH)-HD-16229 and HD-07495 (Project III, Specialized Cooperative Program in Reproductive Research, SCPRR) (to J.S.R.); American Society for Reproductive Medicine/Organon grant and BIRCWH program (HD052023) (to V.S.); and NIH-MH-48989 (to M.C.W.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 26, 2007

Abbreviations: AREG, Amphiregulin; AP-1, activator protein-1; BDNF, brain-derived neurotrophic factor; CG, chorionic gonadotropin; COC, cumulus oocyte complex; CRE, cAMP-responsible element; For, forskolin; GM-CSF, granulocyte-macrophage colony stimulating factor; KC, keratinocyte-derived chemokine; MCP-1, monocyte chemotactic protein-1; MIP-1{alpha}, macrophage-inflammatory protein-1{alpha}; PGR, progesterone receptor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PRKO, progesterone receptor knockout; RANTES, regulated upon activation, normal T cell expressed and secreted; siRNA, small interfering RNA; SNAP, synaptosomal-associated protein; SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor; SP-1 or -3, specificity protein-1 or -3.

Received for publication January 22, 2007. Accepted for publication June 21, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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