Gap Junction Communication and Connexin 43 Gene Expression in a Rat Granulosa Cell Line: Regulation by Follicle-Stimulating Hormone1

Abstract

Follicle-stimulating hormone is the major regulator of growth and development of antral follicles in the ovary. Granulosa cells (GCs) in these follicles are coupled via gap junctions (GJs) consisting of connexin 43 (Cx 43). Because we and others have found that Cx 43 and GJs, respectively, are more abundant in large antral follicles compared with small antral and preantral follicles, we hypothesized that FSH may control Cx 43 gene expression, GJ formation, and intercellular communication. To directly address these points, we chose a rat GC line (GFSHR-17) expressing the FSH receptor and the Cx 43 gene. The functionality of FSH receptors was shown by the effects of porcine FSH, namely cell rounding, reduced cellular proliferation, and stimulation of progesterone production of GFSHR-17 cells, which are effects that were detectable within hours. Treatment with FSH also statistically significantly increased Cx 43 mRNA levels, as shown after 6 to 9 h in Northern blots. These effects were antedated by altered GJ communication, which was observed within seconds. Using a single-cell/whole-cell patch clamp technique, we showed that FSH rapidly and reversibly enhanced electrical cell coupling of GFSHR-17 cells. Increased GJ communication was associated with statistically significantly decreased phosphorylation of Cx 43, which was observed within 10 min after FSH addition, during immunoprecipitation experiments. Our results demonstrate, to our knowledge for the first time, that the gonadotropin FSH acutely and directly stimulates intercellular communication of GFSHR-17 cells through existing GJs. Moreover, FSH also increases levels of Cx 43 mRNA. These changes are associated with reduced proliferation and enhanced differentiation of GFSHR-17 cells. In vivo factors in addition to FSH may be involved in the regulation of GJ/GJ communication between GCs in the follicle, but our results suggest that improved cell-to-cell coupling, enhanced Cx 43 gene expression, and possibly, formation of new GJs are direct consequences of FSH receptor activation and may antedate and/or initiate the pivotal effects of FSH on GCs.

Changes in Expression and Localization of Connexin 43 mRNA and Protein in Porcine Ovary Granulosa Cells during Follicular Atresia

“…healthy follicles of porcine ovaries is similar to that in ovine and bovine follicles but not in rodent follicles [18][19][20]. In atretic tertiary follicles, less Cx43 mRNA was noted, and a rapid disappearance of phosphorylated and native Cx43 proteins was observed.…”

“…Gap junctions are essential for maintaining interactions between cells within a tissue and their homeostasis, and regulate growth/proliferation and death [24,25]. In ovarian follicles, gap junctions are considered to play i m p o r t a n t r o l e s i n f o l l i c u l a r g r o w t h a n d development and in the prevention of follicular atresia in developing preantral secondary follicles [13][14][15][16][17][18][19][20][21][22][23]. Although the role of gap junctions in mammalian follicles has been investigated, there is insufficient information on whether or not Cx43, which is a dominant gap junction protein in porcine follicles [16,17] and has multiple phosphorylated/ activated and non phosphorylated/native forms [30], is involved in granulosa cell apoptosis during atresia in porcine ovaries.…”

Expression of connexin 43 and gap junctional intercellular communication in the cumulus–oocyte complex in sheep

“…The stimulatory effects of FSH on GJIC have been demonstrated for bovine granulosa cells (Johnson et al 2002) and for a rat granulosa cell line (Sommerburg et al 2000). Stimulation by FSH is followed by an elevation of cAMP in granulosa/cumulus cells (Salustri et al 1985, Webb et al 2002.…”

“…The expression of Cx43 in the ovarian follicle has been associated with the profile of serum concentrations of FSH and LH (Wiesen & Midgley 1993, Granot & Dekel 1997. Numerous studies have demonstrated the specific effects of FSH and LH on gap junction presence, Cx43 expression and GJIC in the ovarian follicle (Burghardt & Matheson 1982, Mayerhofer & Garfield 1995, Grazul-Bilska et al 1996, Granot & Dekel 1997, Sommerburg et al 2000. Both FSH and LH affect the function of granulosa/cumulus cells directly by binding to specific receptors (Greenwald & Roy 1994).…”

Proliferative action of mast-cell tryptase is mediated by PAR2, COX2, prostaglandins, and PPARγ: Possible relevance to human fibrotic disorders

“…HFFF2 cells per well were plated on 96-well plates (Nunc) and incubated for 24 h in the presence or absence of tryptase, HMC-1-conditioned medium, meloxicam, PAR2 agonist peptide, PGs, and BADGE. Cell proliferation was determined by using the CellTiter 96 AQueous One Solution cell-proliferation assay (Promega) (25). The specificity and sensibility of this method was evaluated previously in our lab by comparison with a [ Archival human testicular biopsies had been fixed in Bouin's fixative and were embedded in paraffin.…”