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.
and
The Preparation and Characterization of Silica Gel Supported
Chiral Zirconium Complexes
Marisa Monreal
California State University, Los Angeles
MBRS-RISE Graduate Student