Modulatory Action of Nitric Oxide on the Expression of Transcription Factor Genes, c-fos and c-jun, in Developing Porcine Granulosa Cells In Vitro.

Abstract

Abstract. The present study was performed to clarify the synthesis of nitric oxide (NO) and its effect on the expression of transcription factor genes (c-fos, c-jun and ATF-4) during the differentiation of granulosa cells. Granulosa cells prepared from porcine ovarian follicles (1-4 mm diameter) were matured with FSH for 48 h. From 40 h to 48 h of ovine FSH stimulation, nitrite and nitrate increased by 2 folds, and this was accompanied by an increase of cyclic GMP. The cells were exposed to either NO scavenger (carboxy-PTIO: 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide) or NO donor (NOC18: 2,2'-(hydroxynitrosohydrazino)bisethanamine) before or after NO generation, and further stimulated with ovine LH until 48 h. Removal of endogenous NO (NO scavenger) induced a serious impairment in the LH-induced synthesis of progesterone, whereas NO donor had no significant effect on the synthesis. In the semi-quantitative reverse transcriptase-PCR of the transcription factor mRNAs, removal of endogenous NO resulted in a reduction of c-fos expression, an increase of c-jun and no changes in ATF-4 expression. In contrast, NO donor had no significant effects on the expression of these transcription factor genes. Consequently, the transient generation of NO may have critical roles in the LH-induced expression of transcription factor genes and thereafter transformation of the granulosa cell to the luteal cell. Key words: Nitric oxide, Transcription factor genes, Granulosa cell differentiation.(J. Reprod. Dev. 46: [167][168][169][170][171][172][173][174][175] 2000) The expression of mRNAs and proteins for eNOS and iNOS in the rat ovary has been shown during follicular development, ovulation and pseudopregnancy [8,9]. In rat and human ovarian cells, NO has been shown to be involved in folliculogenesis, ovulation and steroidogenesis [10][11][12][13][14][15]. In our previous study using rat granulosa cells [16], NO was proposed as functioning as a modulator in cell differentiation, because removal of NO during cell differentiation suppressed itric oxide (NO) is a short-lived messenger molecule that mediates a variety of cellular functions. NO is generated from molecular O2 and L-arginine by nitric oxide synthase (NOS) [1][2][3][4]. Constitutive isoforms such as endothelial (eNOS) and neural NO (nNOS) synthases [5,6], and an inducible isoform (iNOS) [7] have been identified.

Expression of endothelial nitric oxide synthase gene in cultured porcine granulosa cells after FSH stimulation

“…A similar finding for LH receptor mRNA expression was also reported using Northern blots and receptor binding studies (Goxe et al 1992). As NO may be involved in ovulation (Shukovski & Tsafriri 1995, Bonello et al 1996, Yamauchi et al 1997) and differentiation of the granulosa cells (Hattori et al 1996, Nishida et al 2000, it is reasonable to propose that expression of eNOS mRNA preceded maximal expression of LH receptor mRNA in cultured granulosa cells. eNOS has been considered to be a constitutively expressed protein, but there are many reports of regulation of the expression of its gene , Bryant et al 1995, Van Voorhis et al 1995, Arnet et al 1996, JablonkaShariff & Olson 1997, El Dwairi et al 1998, Vega et al 1998, Hangai et al 1999, Srivastava et al 1999, Venkov et al 1999.…”

“…The concentrations of nitrite and nitrate remained relatively constant until 42 h of stimulation, after which they increased significantly up to twofold at 48 h (P<0·05). Production of cGMP stimulated by an NO donor was observed at the initial periods of the cell development (Nishida et al 2000). These results indicate that NO synthesis is transiently promoted after 42-48 h of FSH stimulation, and that NO in turn stimulates guanylate cyclase.…”

“…Nitrite, an NO metabolite, is accumulated in luteinized ovarian cells of the rat (Olson et al 1996), suggesting an important function of NO during the periovulation period. In our previous study (Nishida et al 2000), we found that NO was synthesized by cultured porcine granulosa cells. Northern blot analysis has shown that iNOS and eNOS are expressed in the rat ovary (Van Voorhis et al 1995).…”

Upregulation of P-Glycoprotein Activity in Porcine Oocytes and Granulosa Cells During In Vitro Maturation

“…Porcine granulosa cells were prepared from prepubertal gilts as previously described [19,20] with minor modifications. Briefly, medium sized (3-6 mm diameter) follicles were removed with forceps and scissors under microscopy, and granulosa cell layers were isolated from the follicles.…”

Stimulation of Nitric Oxide Synthesis in Porcine Granulosa Cells by Phorbol Ester.

“…The cells were treated with 50 µg/ ml DNase I (Boehringer Mannheim, Mannheim, Germany) for 5 min, washed and suspended in Dulbecco's Modified Eagle's Medium (DMEM)/ Ham's F10 (1:1) (GIBCO Laboratories, Grand Island, NY, USA) supplemented with 10 mM Hepes, 50 µg/ml gentamycin (GIBCO), and 20 IU/ ml nystatin (GIBCO) [10]. Viable cells were seeded in 48-well plates (2.5 × 10 5 cells/0.3 ml/well) or 35 mm dishes (2 × 10 6 cells/2.5 ml/dish) coated with human fibronectin (Becton-Dickinson, Oxnard, CA, U S A ) a n d c u l t u r e d f o r 2 4 h w i t h 1 0 0 n M androstenedion, 100 nM hydrocortisone, 1 µg/ml insulin, 5 µg/ml transferrin and 0.1% bovine serum albumin (BSA) (Sigma, St. Louis, MS, USA) at 39 C in humidified 95% air-5% CO2 [11]. The cells were then cultured for 48 h with 10 ng/ml ovine FSH (oFSH) (NIDDK-oFSH-20).…”