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Follicular atresia is characterized by the initial rapid loss of granulosa cells by apoptosis, followed by the loss of thecal cells at a slower rate. We have previously shown that treatment of subconfluent cultures of thecal/interstitial cells (T/I) with transforming growth factor (TGF) alpha plus TGF beta caused chromatin condensation and internucleosomal fragmentation characteristic of apoptosis, whereas in the presence of either TGF alpha or TGF beta alone the cells remained healthy. In this study we have examined the effect of TGF alpha and TGF beta alone and in combination on the levels of mRNA encoding bcl-2 and interleukin-1 beta-converting enzyme (ICE) in T/I cells using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Bcl-2, a cell survival gene, has been implicated in regulating the balance between cell proliferation and cell death in physiological processes. ICE, the homolog of the C. elegans cell death gene, ced-3, is also involved in apoptotic signal transduction. The levels of mRNA encoding specific PCR products for bcl-2 (430 bp) and ICE (453 bp) were amplified from T/I cell cDNA. Untreated T/I cells and TGF alpha- or TGF beta-treated cells contained comparable levels of bcl-2 mRNA. Treatment of T/I cells with TGF alpha plus TGF beta significantly decreased the levels of bcl-2 mRNA expression. TGF alpha plus TGF beta caused a significant decrease in bcl-2 mRNA levels within 3 h of treatment of T/I cells, followed by a progressive decline to 10% of control levels after 24 h of treatment. In contrast, in control T/I cells, the levels of ICE mRNA were low. TGF alpha plus TGF beta caused a progressive increase in ICE mRNA, reaching levels 2- and 3-fold higher than control cells after 5 and 7 h respectively. DNA analysis showed that DNA fragmentation, indicative of apoptosis, occurred after 10 h of treatment with TGF alpha plus TGF beta. These studies demonstrated that treatment of T/I cells with TGF alpha plus TGF beta influenced gene expression of bcl-2 and ICE prior to the time at which DNA fragmentation was observed. We propose that the gene products of bcl-2 and ICE are involved in the apoptotic signal transduction pathway induced by TGF alpha plus TGF beta in T/I cells.

journal of endocrinology/journal of endocrinology

Induction of apoptosis in thecal/interstitial cells: action of transforming growth factor (TGF) alpha plus TGF beta on bcl-2 and interleukin-1 beta-converting enzyme