PHOSPHORYLATION AND EXPRESSION OF CONNEXIN-43 OVARIAN GAP JUNCTION PROTEIN ARE REGULATED BY LUTEINIZING-HORMONE

One important role of the junctional communication in the ovarian foll icle is to mediate transmission of cAMP, the regulatory signal that ma intains the oocyte in meiotic arrest. Luteinizing hormone (LH) interru pts cell to cell communication within the ovarian follicle, leading to a decrease in intraoocyte concentrations of cAMP followed by resumpti on of meiosis. Our experiments were directed at exploration of mechani sms involved in the LH-induced communication breakdown in the preovula tory ovarian follicle. Immunofluorescence and Western blot analysis, u sing highly specific antibodies, showed that connexin-43 (Cx43), the o varian gap junction protein, is present in the cytoplasmic mem branes of the follicular cells in multiple phosphorylated forms. The relative amounts of the different forms of Cx43 vary in response to LH: short time exposure (10 min) stimulated phosphorylation of Cx43 followed by immediate dephosphorylation, while longer incubations (8 and 24 h) wit h this hormone resulted in elimination of the protein. Forskolin mimic ked the LH-induced phosphorylation/dephosphorylation, as well as the d ecrease of Cx43 protein level. A gonadotropin-releasing hormone analog (GnRHa) also induced an immediate phosphorylation/dephosphorylation o f Cx43 and a later reduction of the amount of Cx43. The direct PKC act ivator, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced phosphoryl ation of Cx43 that was completely blocked by the protein kinase C inhi bitor, staurosporine. This kinase inhibitor partially interfered with LH, but not forskolin-induced phosphorylation of Cx43. Analysis of the effect of LH on Cx43 gene expression revealed a significant decrease (45%) in Cx43 mRNA level at 24 h of incubation. A drop of Cx43 mRNA wa s also induced by GnRHa. Our results suggest that the LH-induced gatin g mechanism of the gap junctions in rat ovarian follicles is comprised of two steps: the immediate response is represented by a change in th e phosphorylation state of the Cx43 protein, and the later response is manifested by a reduction of Cx43 protein level, due to attenuation o f its gene expression. Phosphorylation of Cx43 may occur through PKA-, as well as PKC-dependent pathways.