A growing body of evidence suggests that intraovarian interleukin-1 beta (IL-1 beta) may play an intermediary role in the ovulatory process. Furthermore, induction of nitric oxide (NO) by IL-1 beta has been reported in a wide variety of tissues. As the majority of ovarian follicles undergo an atretic degeneration process involving apoptotic cell death, we set out to determine whether IL-1 beta rescues follicles from apoptosis and the possible involvement of NO. Preovulatory follicles obtained from PMSG-primed rats were cultured for 24 h in serum-free medium with or without hormone treatments. After culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends with [32P]dideoxy-ATP. Follicular NO production was also determined by a colorimetric method. Treatment with IL-1 beta dose-dependently suppressed the spontaneous onset of apoptosis in cultured follicles, but stimulated NO production. In contrast, the addition of IL-1 receptor antagonist eliminated both effects of IL-1 beta, confirming receptor mediation. Follicles treated with sodium nitroprusside, a NO generator or an analog of cGMP, the second messenger for NO, also showed decreased follicle apoptosis. Moreover, the addition of NG-monomethyl-L-arginine, a NO synthase inhibitor, reversed both IL-1 beta stimulation of NO production and suppression of apoptosis, suggesting a mediatory role of NO in these IL-1 beta effects. Gonadotropins also prevent follicle apoptosis. Of interest, treatment with hCG stimulated NO production, and the hCG suppression of follicle apoptosis and stimulation of NO production were partially blocked by cotreatment with IL-1 receptor antagonist, indicating the mediation of endogenous IL-1 beta. Treatment with IL-1 beta also stimulated a small increase in the production of cAMP, estrogen, and progesterone. Taken together, these findings suggest that IL-1 beta is a survival factor for ovarian follicles, and its action is partially mediated via NO and cGMP generation. Moreover, part of the suppressive action of gonadotropins on follicle apoptosis is mediated by endogenously produced IL-1 beta.

Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of apoptotic DNA fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of apoptotic DNA fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of apoptotic DNA fragmentation which was prevented by treatment with FSH in a dose-dependent manner (60% maximal suppression and apparent ED50 of 30 ng/ml). Similarly, treatment with (Bu)2cAMP also suppressed follicle apoptosis. Treatment with LH or human CG, however, minimally suppressed apoptotic DNA fragmentation (35% maximal suppression). Insulin-like growth factor-I (IGF-I) also suppressed apoptosis by 45%. Moreover, the suppressive effect of FSH on apoptosis was partially reversed by coincubation with IGF-binding protein-3, suggesting a potential mediatory role of endogenous IGF-I. However, recombinant bovine GH had no effect on follicle apoptosis despite its ability to stimulate IGF-I messenger RNA (mRNA) levels. Incubation of follicles with epidermal growth factor (EGF) and basic fibroblast growth factor maximally suppressed follicle apoptosis by only 32% and 42%, respectively. Ligand binding analysis indicated the minimal effectiveness of EGF on apoptosis in early antral follicles, as compared with its potent action in preovulatory follicles reported earlier, may be due to a 3.5 fold increase in EGF receptor concentration in the mature follicles. High doses (150 or 500 ng/ml) of interleukin-1beta also suppressed apoptosis by 48% whereas treatment with an NO generator, sodium nitroprusside, or a cyclic GMP analog suppressed apoptosis as effectively as that of FSH. Furthermore, treatment with activin resulted in a dose-related suppression of follicle apoptosis, reaching a maximal 40% suppression. In contrast, cotreatment of activin with its binding protein, follistatin, abolished this effect. Collectively, these data demonstrated a stage-dependent difference in the hormonal regulation of follicle apoptosis. Although FSH, LH/human CG, GH, IGF-I, EGF, basic fibroblast growth factor, and interleukin-1beta are all effective survival factors for preovulatory follicles, FSH is a major survival factor for early antral follicles, the stage during which a majority of follicle undergo atresia under physiological conditions.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA at the ends of eukaryotic chromosomes. It has been hypothesized that telomerase activity is necessary for cellular immortalization and that telomerase activity is present in cells of germline origin. The objective of the present study was to determine the level of telomerase activity in the following rat cells: 1) oocytes from follicles at different stages of development, 2) spermatogenic cells, and 3) early embryos. Telomerase activity was quantitated using a recently developed, sensitive polymerase chain reaction-based assay and a human kidney cell line (293) as a standard. Telomerase activity was found in oocytes from early antral and preovulatory follicles, as well as in ovulated oocytes. The level of enzyme activity in early antral and preovulatory follicles was comparable to that of the 293 cells, while levels in ovulated oocytes were 50-fold lower. Telomerase activity was present in even lower levels in pachytene spermatocytes and round spermatids, and no telomerase activity was detected in spermatozoa from either the caput or the cauda epididymis. After fertilization, telomerase activity was present in 4-cell embryos. Telomerase activity was also detected in several rat somatic tissues. These data demonstrate that telomerase activity is present in germ cells at several stages of differentiation, with the exception of spermatozoa, and suggest that telomerase activity may be important during meiosis. The high levels of telomerase activity in individual oocytes may serve as a marker for monitoring the effects of hormonal agents, aging, and toxins on oocyte quality.

This is the second publication of Clinical Development Plans from the National Cancer Institute, Division of Cancer Prevention and Control, Chemoprevention Branch and Agent Development Committee. The Clinical Development Plans summarize the status of promising chemopreventive agents regarding evidence for safety and chemopreventive efficacy in preclinical and clinical studies. They also contain the strategy for further development of these drugs, addressing pharmacodynamics, drug effect measurements, intermediate biomarkers for monitoring efficacy, toxicity, supply and formulation, regulatory approval, and proposed clinical trials. Sixteen new Clinical Development Plans are presented here: curcumin, dehydroepiandrosterone, folic acid, genistein, indole‐3‐carbinol, perillyl alcohol, phenethyl isothiocyanate, 9‐cis‐retinoic acid, 13‐cis‐retinoic acid, l‐selenomethionine and 1,4‐phenylenebis(methylene)selenocyanate, sulindac sulfone, tea, ursodiol, vitamin A, and (+)‐vorozole. The objective of publishing these plans is to stimulate interest and thinking among the scientific community on the prospects for developing these and future generations of chemopreventive drugs. © 1997 Wiley‐Liss, Inc.