Chicken epiregulin (ER) gene: cDNA cloning, genomic organization, and regulation of its mRNA expression in ovarian granulosa cells

Abstract

Growing evidence suggests that epiregulin (ER), a ligand of epidermal growth factor receptor (EGFR), is involved in controlling follicular development in mammals. However, there is no information on its expression in the ovary of non-mammalian species including chicken. In this study, we first cloned the full-length cDNA of chicken epiregulin gene from ovary. The cloned cDNA is 948 bp in length and encodes a membrane-anchored precursor of 153 amino aicds, which shares high sequence identity (~59%) with its mammalian counterpart. Genomic structural analysis shows that chicken epiregulin gene spans approximately 8 kb on chromosome 4 and consists of 5 exons. Using RT-PCR assay, ER mRNA was detected to be expressed in embryonic (embryonic day 20), sexually immature (3 week), and mature ovaries (6 chickens used at each stage), suggesting a role of ER in ovarian development. Compared with the other six EGFR ligands, epiregulin appeared to be expressed at a low level. However, using semiquantitative RT-PCR assay, epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) have been demonstrated to be capable of inducing ER mRNA expression significantly in cultured ovarian granulosa cells from preovulatory follicle (F1) in a timedependent manner. The expression of epiregulin reached the maximum at 4 h and was sustained at 24 h post-treatment. Moreover, phorbol 12-myristate 13-acetate (PMA), a potent protein kinase C (PKC) activator, could also significantly induce epiregulin mRNA expression in a dose-dependent manner, suggesting that PKC (or PKC-activated) signaling pathway may be involved in controlling ER expression in the chicken ovary. These findings suggest that ER is a potential paracrine/autocrine factor involved in controlling ovarian development and its expression is regulated by local ovarian factors including EGFR ligands from chicken ovary.