PARENT SESSION

(49) LOCALIZATION, REGULATION, FUNCTION OF RUNX1 IN RAT PERIOVULATORY OVARIES.

Jo, Misung¹, Curry, Thomas ¹, ¹ University of Kentucky, Lexington, KY

ABSTRACT- The LH surge induces specific transcription factors that regulate the expression of a myriad of genes in periovulatory follicles to bring about ovulation and luteinization. We recently identified the dramatic, transient induction of mRNA for Runx1, a nuclear transcription factor, in periovulatory follicles of ovaries from eCG/hCG-primed immature rats. Therefore, in the present study we investigated 1) the localization of Runx1, 2) the regulation of Runx1 mRNA expression, and 3) its potential function in rat ovaries. Immunohistochemical analysis revealed the induction of Runx1 protein in granulosa cells of preovulatory follicles after hCG injection in the eCG-primed model. In cycling rats, Runx1 mRNA and protein were also detected in preovulatory follicles only after the LH surge by in situ hybridization and immunohistochemical analyses, confirming the periovulatory expression of Runx1 in the natural setting. Next, we investigated regulatory mechanisms involved in Runx1 mRNA expression in vitro using preovulatory granulosa cells isolated from eCG-primed immature rat ovaries. Treatments with hCG, forskolin (PKA agonist), or PMA (PKC agonist) stimulated Runx1 mRNA expression. The stimulatory effect of hCG was reduced by inhibitors for PKA (H89), MEK (PD98059), and p38 kinase (SB2035850), demonstrating that the Runx1 expression is mediated by the LH-induced activation of these signaling mediators. In addition, cyclohexamide blocked the hCG-induced Runx1 mRNA expression, indicating the requirement of new protein synthesis for Runx1 expression. Furthermore, Runx1 mRNA expression is dependent on progesterone action and EGF receptor activation by EGF-like peptides such as amphiregulin. For example, the hCG-induced Runx1 mRNA expression was inhibited by a progesterone receptor antagonist (ZK98299) and an EGF receptor tyrosine kinase inhibitor (AG1478), whereas amphiregulin stimulated Runx1 mRNA expression. Finally, knockdown of Runx1 mRNA by Runx1 siRNA resulted in decreased progesterone accumulation in granulosa cell cultures. In conclusion, the transient, hormonally-regulated expression of Runx1 in periovulatory follicles as well as its involvement in progesterone production suggest an important role of Runx1 in the processes of ovulation and luteinization. Supported by NIH P20 RR15592.

KEY WORDS: runx1, ovary, lh, progesterone