In order to investigate the action of leptin on early follicular growth, preantral follicles, 95-115 microm in diameter were mechanically isolated from the ovaries of BDF1 hybrid immature (11-day-old) and adult (8-wk-old) mice, and cultured for 4 days in vitro. Follicular growth was assessed by daily changes in follicular diameter and by the amount of estradiol and immunoreactive (IR)-inhibin released into the culture medium at Day 4. Preantral follicles from immature mice showed a significant development in follicular growth as a result of stimulation by GH (1 mIU/ml), insulin-like growth factor (IGF)-I (100 ng/ml) + FSH (100 mIU/ml), and GH (1 mIU/ml) + FSH (100 mIU/ml). Although leptin at concentrations of 1-1000 ng/ml did not have any significant effect on follicular growth stimulated by IGF-I or GH, it significantly inhibited follicular growth in a dose-related manner when follicles were stimulated by IGF-I + FSH and GH + FSH, respectively, suggesting that leptin attenuated the additive effect of FSH. On the other hand, preantral follicles from adult mice were cultured in the presence of FSH, and FSH-dependent follicular growth was inhibited by leptin in a dose-related manner. Because FSH stimulates cAMP production, we investigated the involvement of cAMP in the inhibitory mechanisms of leptin. Preantral follicles from immature and adult mice were cultured in the presence of either 8-Br-cAMP or forskolin. Both 8-Br-cAMP and forskolin significantly increased follicular diameter and hormone secretion in both immature and adult mice. However, 8-Br-cAMP and forskolin-stimulated follicle growth and hormone secretion were significantly inhibited in immature mice by coadministration of leptin, whereas growth of preantral follicles from adult mice was not inhibited by addition of leptin to cultures. These results indicate that leptin causes an inhibitory effect on the early follicular development of both immature and adult mice, but the inhibitory mechanisms of leptin are different.

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The present study investigates the physiological significance of dehydroepiandrosterone, dehydroepiandrosterone sulfate, T, androstenedione (Delta(4)), dihydrotestosterone (DHT), estrone (E1), and E2 on recombinant human FSH- (rhFSH) resistant type 4 follicles obtained from immature mice. Type 4 follicles of a diameter of 100-120 microm with one or two granulosa cell layers around the oocyte and an intact basal lamina with theca cells were isolated from the ovaries of 11-d-old BDF-1 mice and cultured with medium alone (control) or with dehydroepiandrosterone, dehydroepiandrosterone sulfate, T, Delta(4), DHT, E1, or E2 at concentrations ranging from 1 x 10(-11) to 1 x 10(-7) M for 4 d. We examined the mean diameters of type 4 follicles, levels of immunoreactive (IR)-inhibin, and E2 and progesterone in the culture media on day 4. In addition, we evaluated follicular cell proliferation by immunofluorescence staining with 5-bromo-2'-deoxyuridine. All tested androgens significantly increased the diameter of type 4 follicles in a dose-dependent manner without the production of IR-inhibin and E2. The nuclei of granulosa cells in type 4 follicles cultured with all tested androgens exhibited intense 5-bromo-2'-deoxyuridine-positive staining, compared with those of controls. In contrast, neither E1 nor E2 had any stimulatory effects. The stimulatory effects of T, Delta(4), or DHT were inhibited by an AR antagonist in a dose-related fashion but not by an aromatase inhibitor. Furthermore, all tested androgens had a synergistic effect with rhFSH on follicular growth and the production of IR-inhibin and E2. These results demonstrated that neither adrenal nor ovarian androgens are arteriogenic but that they stimulate type 4 follicles unresponsive to rhFSH and augment the responsiveness of these follicles to rhFSH.

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To clarify the role of leptin in women with polycystic ovary syndrome (PCOS), we analyzed whether serum leptin levels correlate with other hormonal parameters in obese and non-obese women with PCOS. We studied 20 obese (body mass index, BM ≥25 kg/m) and 20 non-obese (BMI <25 kg/m) women with PCOS diagnosed by the existence of menstrual disturbance, elevated serum level of luteinizing hormone (LH) with normal follicle-stimulating hormone (FSH) and the characteristic polycystic appearance of the ovaries on transvaginal ultrasound images. Blood samples for LH, FSH, estradiol, testosterone (T), androstenedione (Δ) and leptin were obtained, and the relationships between variables were examined by calculating Spearman correlation coefficients. Mean levels of leptin, T and Δ in obese PCOS women were significantly higher than those in non-obese PCOS women, but this was not the case for BMI, bodyweight and waist to hip ratio. In all the 40 PCOS women considered together, there were significant positive correlations of leptin with BMI, waist to hip ratio, and Δ levels. However, in each group separately, serum leptin levels in obese PCOS women correlated only with BMI and bodyweight, whereas serum leptin levels in non-obese PCOS women correlated with serum A4 levels. Although further study is needed to assess the role of leptin on ovarian function in non-obese women with PCOS, present findings do not support the fact that leptin is involved in the development of hormonal abnormalities in obese women with PCOS. (Reprod Med Biol 2002;: 49-54).