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A. Chakravorty, V. B. Mahesh and T. M. Mills

Summary. Intact immature female rats were treated with 1, 2, 3 or 4 subcutaneous injections of 2 mg diethylstilboestrol (DES)/rat at intervals of 24 h and then killed. Ovaries were collected, cleaned, enzymically digested and serially filtered through Teflon sieves to yield follicles of diameter <200 μm (small), 200–400 μm (medium) and >400μm (large). Follicular supernatant was collected and granulosa cells were extracted from these isolated follicles. There was a general increase in [3H]thymidine incorporation in all sizes of follicles after 1 or 2 DES injections, the increase in the medium and large follicles being significant after 2 doses. With 3 and 4 injections of DES, there was a sudden decrease in the rates of [3H]thymidine incorporation, particularly in the medium-sized follicles, which also had higher concentrations of follicular supernatant protein. Protein contents in small and large follicles did not change significantly. The follicular supernatant protein had a specific and dose-dependent inhibitory effect on [3H]thymidine incorporation when added to cultures of rapidly dividing granulosa cells. Addition of the same amounts of bovine serum albumin (BSA) to the cultures had no effect. Heat-denaturing did not abolish the inhibition by the protein. Removal of the protein from the cultures after the first 48 h resulted in a rebound increase in [3H]thymidine incorporation during the following 48 h, showing that the inhibitory effects were reversible. Though aromatase activity after 1 or 2 DES injections abruptly decreased after 3 and 4 injections, follicular supernatant protein had no effect on steroidogenesis in cultured granulosa cells.

Taken together, these findings suggest that oestrogen can inhibit follicular development, depending on the duration of exposure. We propose that the inhibitory effects of DES on cell proliferation are mediated via the synthesis of a specific peptide factor which is produced in high amounts in the medium-sized follicles only, on prolonged exposure to the oestrogen. This factor may be autocrine or paracrine, serving as an in-built autoregulatory control mechanism for follicle development, particularly at pro-oestrus, when oestrogen concentations are highest.

Keywords: diethylstilboestrol; follicle; thymidine; protein; inhibition; rat

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A. Chakravorty, V. B. Mahesh and T. M. Mills

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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A. Chakravorty, V. B. Mahesh and T. M. Mills

Summary. Immature female rats received either one injection of 2 mg diethylstilboestrol (DES)/rat subcutaneously and were killed 12 h later or received two injections of DES at 0 and 24 h and were killed at 24, 36 and 48 h after the initial injection. The ovarian follicles were released by enzymic digestion with collagenase and separated into those of small, medium and large diameter (<200 μm, 200-400 μm and >400μm) by filtration through graded Teflon sieves and granulosa cells were extracted from these follicles. The ovaries of immature rats treated with pregnant mares' serum gonadotrophin (PMSG) were used for comparative purposes.

Incorporation of [3H]thymidine into granulosa cell DNA was augmented by DES and by PMSG. Small follicles were more strongly stimulated by DES at 12 h than those of other sizes, but rates increased significantly in medium and large follicles at 48 h. Aromatase activity in the DES-treated group was low at all times and in all follicles. Rates of oestrogen and progesterone production in response to 36 h of exposure to follicle-stimulating hormone (FSH) in vitro were significantly lower than in the PMSG-treated group. FSH-stimulated steroid production in the DES group at 36–48 h was lower, particularly in the medium follicles. A significant rise in serum FSH, luteinizing hormone (LH) and progesterone concentrations was noted only at 36 h after DES treatment, while serum and follicular fluid oestrogen values remained unchanged. When these changes were compared with those in PMSG-treated rats, there were obvious differences. The pattern of thymidine incorporation and aromatase activity differed with time and follicle size. Serum FSH and LH values were not affected by PMSG treatment, but serum and follicular fluid oestradiol values increased with time. The PMSG-treated animals ovulated in response to human chorionic gonadotrophin, but the DES-treated rats did not ovulate in spite of the presence of some large antral follicles in the ovaries.

These findings show that initial exposure of follicles to high concentrations of oestrogen results in follicles which fail to respond to subsequent gonadotrophin surges and are thereby restricted in their ability to differentiate fully.

Keywords: diethylstilboestrol; follicle; aromatase; FSH; thymidine; rat