May 6, 2005

Expression and Potential Function of the Cyclic Nucleotide Gated Channel CNGA3
in Rat Granulosa Cells

Marianela Arias
California State University, Los Angeles
MBRS-RISE Graduate Student

Abstract: In females, the production of estradiol (E2) is essential for reproductive function. Previous studies suggest that increased intracellular calcium levels contribute to the stimulatory effects of follicle-stimulating hormone (FSH) on E2 production. Cyclic nucleotide gated channels (CNGs) are cation channels which mediate calcium influx into cells. To determine the role of CNG-mediated calcium flux on E2 production, this study examined the expression of CNG channels in the ovary, and determined the effects of EGTA (a calcium chelator) and LCD (a CNG blocker) on FSH-stimulated E2 levels. The role of CNGs in E2 production was examined using cultured rat granulosa cells. While untreated cells displayed low E2 production, incubation with FSH markedly increased E2 levels. Removal of extracellular calcium by incubation with EGTA markedly inhibited FSH-stimulated E2 levels in a dose-dependent manner, indicating that calcium influx into cells contributes to the stimulation of E2 synthesis. Similarly, blockage of CNG channels with LCD abolished FSH-stimulated E2 production in a dose dependent manner, indicating a role of CNG channels in calcium influx. Cell viability studies demonstrated increase in cell death with increasing treatment concentration. RT-PCR of ovarian RNA using specific primers for CNG3 resulted in the amplification of a product of expected size, suggesting the presence of CNG3 in the ovary. However, using Northern analysis to determine whether CNG3 mRNA was regulated in granulosa cells or whole ovary, no CNG3 transcript was detected. Similarly, using Immunoflourescence analysis to determine the presence and cellular localization of the CNG3 protein, expression was detected in the oocyte and corpus luteum but not in granulosa cells as expected. Collectively, these findings suggest that CNG3 expression may play a role in oocyte maturation and luteal steriodgenesis but other CNG isoforms may be important for granulosa cell functions since they play a permissive role in E2 production and cell viability.



The Preparation and Characterization of Silica Gel Supported Chiral Zirconium Complexes

Marisa Monreal
California State University, Los Angeles
MBRS-RISE Graduate Student