Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone.

I Granot,N Dekel

doi:10.1016/s0021-9258(18)43842-2

Abstract

One important role of the junctional communication in the ovarian follicle is to mediate transmission of cAMP, the regulatory signal that maintains the oocyte in meiotic arrest. Luteinizing hormone (LH) interrupts cell-to-cell communication within the ovarian follicle, leading to a decrease in intraoocyte concentrations of cAMP followed by resumption of meiosis. Our experiments were directed at exploration of mechanisms involved in the LH-induced communication breakdown in the preovulatory ovarian follicle. Immunofluorescence and Western blot analysis, using highly specific antibodies, showed that connexin-43 (Cx43), the ovarian gap junction protein, is present in the cytoplasmic membranes 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) with this hormone resulted in elimination of the protein. Forskolin mimicked the LH-induced phosphorylation/dephosphorylation, as well as the decrease of Cx43 protein level. A gonadotropin-releasing hormone analog (GnRHa) also induced an immediate phosphorylation/dephosphorylation of Cx43 and a later reduction of the amount of Cx43. The direct PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced phosphorylation of Cx43 that was completely blocked by the protein kinase C inhibitor, 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 was also induced by GnRHa. Our results suggest that the LH-induced gating mechanism of the gap junctions in rat ovarian follicles is comprised of two steps: the immediate response is represented by a change in the phosphorylation state of the Cx43 protein, and the later response is manifested by a reduction of Cx43 protein level, due to attenuation of its gene expression. Phosphorylation of Cx43 may occur through PKA-, as well as PKC-dependent pathways.

Highlights

Irit Granot and Nava Dekel From the Department of Hormone Research, The Weizmann Instituteof Science, 76100Rehouot, Israel
In the ovarian follicle is to mediatetransmission of It was previously shownthatintraoocyteconcentrations of CAMP,the regulatory signal that maintains the oocyte in CAMPnegatively regulate themeiotic status of the oocyte, and meiotic arrest
Our results suggest that the LHinduced gating mechanism of the gap junctions in rat in intraoocyte concentrationosf CAMPfollowed by resumption of meiosis (reviewed by Dekel (1988b))

Summary

RESULTS

Ribosomal RNA extracted from hormone-treated rat ovarian follicles, using the T7R3 a19 cDNA probe which encodes Cx43 (Risek et al, 1990). Simultaneous diated by the CAMP-dependent PKA biochemical pathway was exposure of follicles to TPA and staurosporine totallyinhibited investigated by incubating isolated follicles with forskolin, a the TPA-induced phosphorylation of Cx43 (Fig. 7). 1991).a net reduction in the amount of gap the decrease in thelevel of the Cx43 transcript, junction membrane a t 12 h, but not immediately after human observed in the extractsof crude ribosomal RNA a t 24 h after chorionic gonadotropin administration, was reported for LH (43%, Fig. 9), was nodtifferent from that observed in prepa- rabbit granulosacells (Larsen et al, 1981).Along with thisline, rations of total RNA, suggesting thatmost of the Cx43 mRNA immunocytochemistry of rat ovaries revealed a decreasein the is present in the crudriebosomal fraction. Exposure of follicles Cx43 gapjunctionproteininthe preovulatory follicles that to GnRHa for 24 h resulted in a decrease (50%)in Cx43 could not be detected before 23:OO h on the night of proestrus, mRNA level (Fig. 10)

DISCUSSION

ToRtaNl A

Findings

LH modulation of the ovarianfollicle gap junctionis not only