Lindane, a gap junction blocker, suppresses FSH and transforming growth factor β1-induced connexin43 gap junction formation and steroidogenesis in rat granulosa cells

Abstract

The present study was designed to explore the role of gap junctions in follicle-stimulating hormone (FSH) and transforming growth factor beta1 (TGF beta1)-stimulated steroidogenesis in ovarian granulosa cells of gonadotropin-primed immature rats. There were three specific aims. First, we investigated the effect of FSH and TGF beta1 as well as lindane (a general gap junction blocker) on the level of connexin43 (Cx43), the major gap junction constituent in granulosa cells, and on gap junction function. The second aim was to determine the effect of lindane on FSH and TGF beta1-stimulated progesterone production and the levels of two critical players, cytochrome P450 side-chain cleavage (P450scc) enzyme and steroidogenic acute regulatory (StAR) protein. The third aim was to further investigate the specific involvement of Cx43 gap junctions in FSH and TGF beta1-stimulated steroidogenesis using a Cx43 mimetic peptide blocker. Immunoblotting analysis showed that FSH plus TGF beta1 dramatically increased the levels of phosphorylated Cx43 without significantly influencing the level of nonphosphorylated Cx43, and this stimulatory effect was completely suppressed by lindane. Also, immunofluorescence analysis showed that Cx43 immuno-reactivity increased in the FSH plus TGF beta1-treated group and predominantly appeared in a punctate pattern at cell-cell contact sites, and lindane reduced such cell periphery immunostaining. Furthermore, TGF beta1 enhanced the FSH-induced gap junction intercellular communication and lindane completely suppressed this effect. In addition, lindane suppressed the FSH and TGF beta1-stimulated increases in progesterone production and the levels of P450scc enzyme and StAR protein. This study demonstrates a clear temporal association between the Cx43 protein level/gap junction communication and progesterone production in rat ovarian granulosa cells in response to FSH and TGF beta1 as well as lindane. Furthermore, a specific Cx43 gap junction blocker suppressed FSH plus TGF beta1-stimulated progesterone production. In conclusion, this study suggests that Cx43 gap junctions may play a critical role in FSH plus TGF beta1-stimulated progesterone production in rat ovarian granulosa cells.