MON-227 Roles of Clock Expression Modulated by BMP-7 in Ovarian Steroidogenesis by Human Granulosa Cells

In mammals, the circadian pacemaker is known to control and integrate physiological rhythms via the coordination of oscillating peripheral clocks that reside in various organs, through the biologic actions of various neurochemical and/or hormonal transmitters. The expression of clock genes has been demonstrated in tissues composing the axis of the hypothalamic-pituitary-gonadal system. The roles of hypothalamic oscillators to produce the biological timings and rhythms in the reproductive axis have been recognized. Results of recent studies have shown that clock genes are expressed in the ovary, and accumulated findings have demonstrated a significant interrelationship between clock gene expression and follicular development. It has been reported that the rhythms of clock gene expression are undetectable in primordial and preantral follicles but can be detected at the timing of early antral stages of follicles and then become clearer at the late antral stages to preovulatory follicles. In the ovarian follicles, clock genes are expressed in granulosa cells, theca cells, oocytes and stromal cells composing the developing follicles. Dysfunction of clock gene expression in the ovary and/or desynchronicity between ovarian clocks and extra-ovarian circadian oscillators may cause various reproductive abnormalities. However, the regulatory mechanism and the physiological roles of the intrinsic clock activity in the ovary have remained uncertain. In the preset study, functional link between clock gene expression and ovarian steroidogenesis was studied using human granulosa KGN cells. Similarities between the oscillating patterns of mRNA and protein expression of Bmal1 and Clock and that of Per2 and Cry1 were demonstrated during 24-h culture of KGN cells in the presence of forskolin. Among the interrelationships between the expression levels of clock and steroidogenic factors, Clock mRNA had a strongly positive correlation with P450arom and a negative correlation with 3βHSD. Knockdown of Clock gene by siRNA resulted in a significant reduction of estradiol production by inhibiting P450arom expression, while it induced a significant increase of progesterone production by upregulating 3βHSD in KGN cells treated with forskolin. Moreover, BMP-7 had an enhancing effect on the expression of Clock mRNA and protein in KGN cells. Collectively, the expression levels of Clock, being upregulated by forskolin and BMP-7, were functionally linked to estradiol production and progesterone suppression by human granulosa cells.