PARENT SESSION
Follicular Development

EFFECTS OF FSH ON CX43 IN GAP JUNCTION FORMATION.

Yogo, Keiichiro¹, Ogawa, Takuya¹, Ishida, Norihiro¹, Takeya, Tatsuo¹, ¹

ABSTRACT- Gap junctions are specialized plasma membrane structures that allow the passage of small molecules between neighboring cells. In mammalian ovaries, it is known that six different connexin genes are expressed and these connexins play their respective roles in oogenesis as well as folliculogenesis. In this study, we focused on connexin 43 protein (Cx43) in rat primary granulosa cells and examined the effects of FSH on its phosphorylation profile, trafficking and gap junctional plaque formation. Various studies including use of gene targeting mice have shown that Cx43 is essential for follicular development. First, FSH was found to increase the phosphorylation level of Cx43, concurrent with the protein level. The phosphorylation was observed even in the presence of cycloheximide. Then, detergent solubility assay revealed that phosphorylated forms of Cx43 were accumulated in 1% Triton X-100 insoluble fraction, suggesting that FSH promote the gap junctional plaque formation. When intracellular trafficking was blocked with brefeldin A (BFA) for 6 hrs, the phosphorylated forms disappeared. After the removal of BFA, phosphorylation and acquisition of Triton insolubility recovered at 2 hrs after in FSH treated cells whereas at 8 hrs in control cells. Phosphoamino acid analysis and two-dimensional tryptic peptide mapping showed that serine residues were exclusively phosphorylated and that several spots became prominent after FSH treatment. These phosphopeptide spots could also be observed when a recombinant protein of Cx43 cytoplasmic tail (Cx43-CT) was subject to in vitro phosphorylation, and Ser-365 and Ser-373 residues were identified as major FSH induced phosphorylation sites by introducing the mutations into the Cx43-CT. These results indicate that FSH promote trafficking and/or gap junctional plaque formation of Cx43 and that phosphorylation of Ser-365 and Ser-373 could play important roles for the processes.

KEY WORDS: gap junction, connexin, FSH, phosphorylation