Dose-Responsive Relationship between Steroids and the Expression of Cell Death Genes BCL-2 and FAS in Granulosa Cells
Onafuwa, Obayemi B.
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Apoptosis, or programmed cell death, has recently been identified as the cause of follicular atresia. Although the genetic mechanisms that underlie apoptosis are yet to be fully understood, several genes have been linked with the process. Primary among these are the proto-oncogene bcl-2 and the tumor necrosis factor receptor Fas. To study the steroid dosage dependence of the expression of bcl-2 and Fas, rat ovarian granulosa cells were cultured in DHEA of varying concentrations (10 -7, 10 -6, 10 -5 M) either with 50 ng/mI FSH or no FSH at all for a 24 hour period. Fresh granulosa cells were used as controls. RNA was extracted.from the granulosa cells after 24 hours using the RNAzol technique. Rat 𝛽-actin, bcl-2 and Fas cDNA clones were transcribed from RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) technique. The mRNA expression levels of bcl-2 and Fas were then measured by riboprobe synthesis, ribonuclease protection assay (RPA) and PhosphoImager, normalized relative to 𝛽-actin levels. In a similar experiment by Laohaprasitiporn et al. (1995), the assay analysis indicated no statistically significant change in the expression of either bcl-2 or FaS in a dose response 24 hour study. The presence of extraneous bands in both the 𝛽-actin and the triple hybridization phosphoimage did raise some questions as to the accuracy of the readings. It was hypothesized that the use of a smaller base pair fragment of of 𝛽-actin would free up the larger base pair regions for a clearer triple hybridization phosphoimage, allowing a fuller comparison of bcl-2 and Fas levels with 𝛽-actin.