Expression of ectodermal neural cortex 1 and its association with actin during the ovulatory process in the rat

Ectodermal neural cortex (ENC) 1, a member of the kelch family of genes, is an actin-binding protein and plays a pivotal role in neuronal and adipocyte differentiation. The present study was designed to examine the gonadotropin regulation and action of ENC1 during the ovulatory process in immature rats. The levels of ENC1 mRNA and protein were stimulated by LH/human chorionic gonadotropin (hCG) within 3 h both in vivo and in vitro. In situ hybridization analysis revealed that ENC1 mRNA was localized not only in theca/interstitial cells but also in granulosa cells of preovulatory follicles but not of growing follicles in pregnant mare's serum gonadotropin/hCG-treated ovaries. LH-induced ENC1 expression was suppressed by a high dose of protein kinase C inhibitor RO 31-8220 (10 microM) but not by low doses of RO 31-8220 (0.1-1.0 microM), suggesting the involvement of atypical protein kinase C. ENC1 was detected in both nucleus and cytoplasm that was increased by LH/hCG treatment. Both biochemical and morphological analysis revealed that LH/hCG treatment increased actin polymerization within 3 h in granulosa cells. Interestingly, ENC1 physically associated with actin and treatment with cytochalasin D, an actin-depolymerizing agent, abolished this association. Confocal microscopy further demonstrated the colocalization of ENC1 with filamentous actin (F-actin). The present study demonstrates that LH/hCG stimulates ENC1 expression and increases F-actin formation in granulosa cells. The present study further shows the physical association of ENC1 and F-actin, implicating the role of ENC1 in cytoskeletal reorganization during the differentiation of granulosa cells.