No abstract

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.

No abstract

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.

The aim of this study was to investigate whether GH and insulin-like growth factor I (IGF-I) are involved in preantral folliculogenesis and, if so, to clarify the relationship between GH/IGF-I and activin/follistatin (FS) systems in immature female mice. Ovaries were obtained from 11-day-old mice, and preantral follicles, 100-105 microm in diameter, were mechanically isolated and selected for culture. Ten preantral follicles per well were cultured with different quantities and combinations of additives as follows: no additives (control), recombinant human FSH (rhFSH), IGF-I, recombinant human GH (rhGH), activin A, and recombinant human FS (rhFS). Mean diameters of the follicles were measured daily, and estradiol and immunoreactive inhibin levels in the cultured medium were assayed by RIA on day 4. rhGH showed stimulatory effects on the follicular diameter and the secretion of estradiol and immunoreactive inhibin. These effects were augmented by the presence of IGF-I and activin A. IGF-I alone did not show any stimulatory effect. The addition of rhFSH to activin A or to rhGH and activin A promoted preantral follicular growth and hormone production. On the other hand, GH- or activin-stimulated follicular growth was suppressed by rhFS in a dose-dependent manner. These results indicate that activin A and rhGH may play an important role in controlling earlier phases of follicular development during the infantile period, which is considered to be gonadotropin independent.