INTERLEUKIN-1-BETA-CONVERTING ENZYME-RELATED PROTEASES (IRPS) AND MAMMALIAN-CELL DEATH - DISSOCIATION OF IRP-INDUCED OLIGONUCLEOSOMAL ENDONUCLEASE ACTIVITY FROM MORPHOLOGICAL APOPTOSIS IN GRANULOSA-CELLS OF THE OVARIAN FOLLICLE

The Caenorhabditis elegans death susceptibility gene, ced-3, has a num ber of homologs in vertebrate species, including interleukin-1 beta (I L-1 beta)-converting enzyme (ICE), Ich-1(long), and CPP32. These genes , which encode a family of related proteases, have been shown to induc e apoptosis when transfected into eukaryotic cells. However, it remain s to be determined whether these proteases are involved in apoptotic c ell death under physiological conditions. The purpose of these studies was to examine the role of ICE-related proteases (IRPs) in apoptosis using a physiologically relevant model system, the ovarian follicle. S omatic granulosa cells within ovarian follicles undergo apoptosis duri ng follicular atresia, a process responsible for the depletion of grea ter than 95% of the follicles established in the postnatal ovary. To a ccomplish these studies, me cloned partial rat complementary DNAs enco ding ICE, Ich-1, and CPP32 and used these complementary DNAs to examin e the gonadotropin regulation of ICE, Ich-1, and CPP32 gene expression in the immature rat ovary. We also examined levels of ICE activity in healthy and atretic rat follicles by monitoring the conversion of exo genous pro-IL-1 beta to the active cytokine, and then evaluated the ac tions of recombinant IL-1 beta on apoptosis in follicles incubated in vitro. Finally, we tested the requirement for IRP activity in granulos a cell apoptosis and follicular atresia by incubating follicles withou t and with IRP inhibitors. Northern blot analysis of total RNA samples indicated that gonadotropin-promoted follicular survival was associat ed with reduced ovarian expression of messenger RNAs encoding Ich-1 an d CPP32. In contrast, ICE messenger RNA levels were extremely low and were not affected by gonadotropin treatment. We were also unable to de tect ICE activity in proteins extracted from either healthy or atretic rat follicles, collectively suggesting that ICE per se may not functi on in granulosa cell death. As another approach to determine whether I CE is involved in atresia, healthy antral follicles were isolated from ovaries of gonadotropin-primed immature rats and incubated for 24 h i n the absence or presence of 100 ng/ml transforming growth factor-alph a (TGF alpha) without and with 100 ng/ml IL-1 beta. Granulosa cells wi thin follicles incubated in medium alone exhibited extensive levels of apoptosis, and this onset of apoptosis was prevented by the inclusion of TGF alpha. Addition of IL-1 beta did not alter basal levels of apo ptosis nor did the cytokine antagonize TGF-alpha-promoted follicle sur vival, providing additional evidence that ICE activity is not required for atresia to occur. To further evaluate whether IRPs are involved i n atresia, we analyzed the effects of several protease inhibitors on a poptosis in follicles incubated in vitro. Treatment of hormone-deprive d follicles with either of two inhibitors of IRP activity, sodium auro thiomalate (0.01-1 mM) or iodoacetic acid (10 mu M), effectively suppr essed internucleosomal DNA cleavage associated with apoptosis. However , histological analysis of follicles treated with these inhibitors rev ealed extensive cellular degeneration, dramatically contrasting with t he biochemical results obtained through DNA analysis. In summary, thes e data have provided the first evidence that the expression of IRPs, b ut not ICE per se, is down-regulated during gonadotropin-promoted foll icular survival. Furthermore, the activity of IRPs may be involved in activation of oligonucleosomal endonucleases, an event that can be dis sociated from morphological indexes of apoptosis, in granulosa cells d uring atresia.