Control of peptides regulating mitosis of granulosa cells in immature rat ovary by oestrogen and gonadotrophin

in Reproduction

Previous studies in this laboratory have demonstrated the presence of a peptide that inhibits granulosa cell proliferation in medium sized follicles. This peptide was produced after 72–96 h of exposure to diethylstilboestrol (DES). This study analyses oestrogen and gonadotrophin modulation of this and another stimulatory peptide found in large follicles. Intact, immature, female rats were assigned to the following study groups: (i) one to four injections of DES (2 mg per rat) given at intervals of 24 h, animals killed 24 h after the last injection; (ii) DES at 0 and 24 h, animals killed 24 or 48 h after the last injection; and (iii) DES plus pentobarbital (37 mg kg−1 body weight) at 30 h, animals killed 48 h after the last injection. Small, medium and large follicles (diameters of <200, 200–400 and > 400 μm, respectively) were isolated from ovaries, granulosa cells were harvested and follicular fluid supernatant (FFS) was collected. FFS proteins were tested for their effects on incorporation of [3H]thymidine into granulosa cell DNA. Results showed that unfractionated FFS protein (150 μg) from medium follicles in groups (i) (three and four injections only), (ii) and (iii) inhibited [3H]thymidine incorporation. Pentobarbital blockage of gonadotrophin secretion had no effect on the inhibitory peptide activity and two injections of diethylstilboestrol were enough to stimulate synthesis of the inhibitory peptide, provided sufficient time was allowed; FFS protein (150 μg) from large follicles was stimulatory, but only when it was collected 24 h after the second injection, and the effect was abolished with pentobarbital treatment. Fractionation of pooled FFS into proteins with three molecular mass ranges (< 10 kDa, 10–30 kDa and > 30 kDa) showed that the inhibitory activity was in the < 10 kDa fraction while the >30 kDa fraction stimulated thymidine incorporation. The number of medium and large follicles increased 24 h after the second DES injection, but the number of granulosa cells in the large follicles was significantly reduced after pentobarbital blockage of gonadotrophins. Taken together, these findings show that (i) the inhibitory peptide is induced in effective amounts in the medium follicles 48 h after the second DES injection and induction is not modulated by gonadotrophins; and (ii) stimulatory activity seen in the large follicles is transient and under gonadotrophic control, since blockage of follicle-stimulating hormone (FSH) secretion led to loss of stimulatory activity and a resulting reduction in number of granulosa cells in large follicles. We, therefore, propose that, besides recruiting small follicles, FSH boosts large follicle growth in the penultimate stages of follicle development via the synthesis of a stimulatory peptide of molecular mass > 30 kDa. Medium follicle growth, however, is regulated more by the oestrogen-induced inhibitory peptide.

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