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  • Author: G. S. GREENWALD x
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G. S. Greenwald

Summary. Follicles failed to respond to the ovulating hormone stimulus (5 or 25 μg LH) when this was given 8 h after hypophysectomy. Shorter delays resulted in full or partial (<6 ova) ovulation.

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G. S. Greenwald

Summary. Treatment of hamsters with 30 i.u. PMSG induced ovulation of 46 ova/animal, 90% of which were fertilized. CL growth was normal. About 27 zygotes/animal implanted and at term (Day 16) the litter size of the PMSG-treated hamsters was 21 compared to the normal number of 10. Parturition occurred at the normal time. Average weight at birth was about 500 mg less for young of PMSG-treated mothers than for controls.

Differences were found in the serum progesterone and oestradiol concentrations of the treated and control hamsters, but serum FSH and LH levels were similar.

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K. Taya and G. S. Greenwald

Summary. Hamsters hypophysectomized (hypox.) and concurrently injected i.p. with 2·5 μg ovine LH or 25 μg ovine FSH at 13:00 h on the day of pro-oestrus ovulated the normal complement of ova the next morning (Day 1). At 09:00 h on Day 1, serum levels of progesterone were comparable between sham-hypox. and LH-treated hypox. hamsters. In contrast, there were no differences in serum progesterone between FSH-treated or saline-treated hypox. hamsters; a slight but significant increase in serum progesterone occurred at 15:00 h in the FSH-treated animals and this was continued on Day 2, when values were comparable to those in the LH-treated and sham-hypox. hamsters.

The CL induced by LH or FSH produced significant amounts of progesterone in vitro on Day 1 at 09:00 and 15:00 h. On Day 2, there was a significant increase in production of progesterone by the CL of both groups. Production rates of progesterone by CL in vitro were the same in the LH- and FSH-treated animals on Days 1 and 2. Therefore, once ovulation has occurred in the hamster the newly developed CL gain the ability to synthesize, store and secrete progesterone independently of pituitary hormones for 2 days, although at a delayed rate after FSH treatment.

The results of incubations of the non-luteal (residual) ovarian tissues showed that large quantities of progesterone (about 7 ng/mg/h) were produced on Day 1 by tissue from FSH-treated animals while that of LH-treated hamsters lost progesterone at the rate of about 1 ng/mg/h. In saline-treated hypox. hamsters the residual tissue produced large amounts of progesterone and small amounts of oestradiol-17β and testosterone in vitro for at least 2 days after hypox. Little oestradiol-17β was produced by the CL of hamsters treated with LH, FSH or sham-hypox. By Day 2 the CL of all 3 groups produced appreciable amounts of testosterone in vitro, whereas on Day 1 only the CL induced by LH secreted testosterone.

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Chandrima Shaha and G. S. Greenwald

Summary. Immediately after hypophysectomy, 30 i.u. PMSG were injected s.c. and 3 days later an antiserum to PMSG was injected i.p. Groups of hamsters were killed at 0, 24, 48 and 72 h after PMSG antiserum. The ovaries were prepared for topical autoradiography and the numbers of silver grains in different ovarian compartments were counted. The numbers of binding sites for 125I-labelled FSH in the granulosa cells of the antral follicles dropped sharply to 33, 14 and 5% of that at 0 h at 24, 48 and 72 h respectively. Binding of 125I-labelled hCG to granulosa cells declined more slowly, being 47, 27 and 24%, respectively. Binding of 125I-labelled hCG to thecal and interstitial cells was unaffected. Compared to other models of atresia, the changes in gonadotrophin binding observed in this model occur at an accelerated rate because of the acute deprivation of PMSG.

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Yuichi Wada and G. S. Greenwald

Summary. Immature rats and adult hamsters were killed on Days 2,4 or 8 of pregnancy (Day 1 = sperm positive vaginal smear). Dispersed luteal cells (5 × 104 cells) were incubated for 2 h in the absence or presence of graded doses of ovine LH. In the absence of LH, incubation of rat luteal cells compared to hamster cells produced about 3–6-fold as much progesterone, 26–66 times as much 20α-dihydroprogesterone and about the same amounts of 17α-hydroxyprogesterone. For the rat, 1 ng LH was the minimal dose which stimulated synthesis of progesterone and 17α-hydroxyprogesterone by luteal cells on Days 2 and 4 whereas 10 ng LH stimulated maximal production of progesterone by Day-8 luteal cells. As pregnancy progressed from Day 2 to Day 8, there was an inverse relationship between the levels of progesterone and 20α-dihydroprogesterone accumulated by rat luteal cells. For the hamster, 1 ng LH significantly stimulated accumulation of progesterone and 17α-hydroxyprogesterone by Day-2 luteal cells but not by Day-4 or Day-8 cells. Hamster luteal cells on Day 4 produced the highest levels of progesterone in response to 10 or 100 ng LH, with a maximal rate of accumulation by Day-8 cells with 10 ng LH.

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S. K. Roy and G. S. Greenwald

Summary. To assess the roles of FSH and LH on follicular growth, after various experimental manipulations, hamster follicles were sorted into 10 stages and incubated for 4 h with [3H]thymidine. Stages 1–4 correspond to follicles with 1–4 layers of granulosa cells, respectively; Stage 5 = 5 or 6 layers of granulosa cells plus theca; Stage 6 = 7–8 layers of granulosa cells plus theca; Stage 7 = early formation of the antrum; Stages 8–10 = small, intermediate and large antral follicles, respectively.

Phenobarbitone sodium injected at 13:00 h on pro-oestrus blocked the normal rise of blood FSH and LH concentrations at 15:00 h and prevented the increase of [3H]thymidine incorporation into follicles of Stages 1–9. The optimal treatment to reverse the effects of phenobarbitone was 1 μg FSH and 2 pg LH injected i.p. at 13:00 h which restored DNA replication to follicles of Stages 2–10: FSH acted primarily on Stages 2–5 and LH on Stages 5–10. Injection of phenobarbitone at 13:00 h on prooestrus followed by 2·5 μg FSH at 22:00 h restored DNA synthesis by the next morning to follicles at Stages 1–8. In hamsters hypophysectomized at 09:00 h on the day of oestrus (Day 1), injection on Day 4 of 2·5 μg FSH restored DNA synthesis 6 h later to Stage 2–6 follicles. Unilateral ovariectomy on Day 3 resulted 6 h later in an acute rise in FSH and LH and change of follicles from Stage 4 to Stage 5 but, paradoxically, there was decreased synthesis of DNA in follicles of Stages 5–10. The incorporation of [3H]thymidine was at normal low levels for Stage 5–10 follicles by 24 h after unilateral ovariectomy with significant increases in follicles of Stages 1–4. We conclude that small preantral follicles are already dependent on FSH for DNA synthesis and LH action is manifested when the follicles acquire a theca (Stage 5 onwards) and when LH receptors appear in granulosa cells.

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S. L. Silavin and G. S. Greenwald

Summary. Hypophysectomized PMSG-primed hamsters were injected with PMSG antiserum and the theca and granulosa cells of the resulting atretic follicles were incubated in vitro. In the absence of added hormone, 17α-hydroxyprogesterone and oestradiol production was not detectable in granulosa cells collected and incubated at 0, 12 and 24 h after antiserum. Progesterone production was not detected in control incubations at 0 h but was measurable with cells collected at 12 h after PMSG antiserum. When incubated with androstenedione or pregnenolone (10 ng/ml for each) 17α-hydroxyprogesterone and progesterone production by granulosa cells were significantly increased at 0, 12 and 24 h after antiserum. Granulosa cells were capable of aromatizing androstenedione to oestradiol at all times examined. At 0 and 12 h after antiserum to PMSG, isolated thecal shells produced androstenedione. LH stimulation caused increased androstenedione production in all thecae at 0 h, in 50% of the thecae at 12 h and in none at 24 h after antiserum. Thecal shells produced 17α-hydroxyprogesterone in response to LH at 0,12 and 24 h after antiserum, and produced progesterone at all times examined. Thecae also responded to LH with increased progesterone production up to 72 h after antiserum. These experiments demonstrate that one important steroidogenic event in atresia may be the loss of activity of C 17,20 lyase in the theca leading to loss of substrate (androstenedione) for granulosa cell aromatization, although aromatase activity is present until at least 24 h after the induction of atresia.

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S. K. Roy and G. S. Greenwald

Summary. Follicles were isolated from hamster ovaries at 09:00 h and 15:00 h on each of the 4 days of the oestrous cycle (Day 1 = oestrus; Day 4 = pro-oestrus) by microdissection and by a mixture of enzymes and classified into 10 stages with pre-calibrated pipettes (stage 1 = preantral follicles with 1 layer of granulosa cells; stage 10 = preovulatory antral follicles). The follicles at each stage were incubated for 4 h with [3H]thymidine with incorporation expressed per μg follicular DNA or per follicle. A significant increase in thymidine per follicle occurred at 15:00 h on Days 1 and 3 of the cycle from stage 2 (bilaminar follicle) to stage 6 (7–8 layers granulosa cells plus theca). When expressed as thymidine per follicle or μg DNA, there was a significant increase in incorporation for stages 1–4 (4 layers granulosa cells) on Day 4 at 15:00 h compared to 09:00 h, presumably as a consequence of the preovulatory increase in gonadotrophins. Follicles in stages 5 to 8 (preantral follicles with 5 or more layers of granulosa cells to small antral follicles), from which the next set of ovulatory follicles will be selected, did not show a significant peak in incorporation per μg DNA until Day 1 at 09:00 and 15:00 h when the second increase in FSH is in progress. DNA synthesis was similarly sustained throughout Day 1 for stage 1–4 follicles. These results suggest that periovulatory changes in FSH and LH, directly or indirectly, are not only responsible for ovulation and the recruitment of the next set of follicles destined to ovulate but also stimulate DNA replication in smaller follicles which develop over the course of several cycles before they ovulate or become atretic.

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S. K. Saidapur and G. S. Greenwald

Summary. Injection (s.c.) of 2 mg cycloheximide at 14:00 h on the day of pro-oestrus prevented the normal rise in serum progesterone and significantly lowered progesterone levels at 15:00 h. Values then rose but only to approximately half of the control values between 16:00 h and 19:00 h. Oestradiol levels also decreased drastically by 15:00 h but were significantly higher in cycloheximide-treated animals until 19:00 h. FSH and LH concentrations were not affected when cycloheximide was given at 14:00 h but treatment at 10:00 h resulted in generally lower values. Animals treated with cycloheximide at 14:00 h failed to ovulate (N = 9), but simultaneous injection of 50 μg progesterone restored ovulation in 50% of the treated animals. In contrast, hamsters injected with cycloheximide at 10:00 h ovulated the next morning, suggesting that protein synthesis essential for ovulation is limited to the first 4–5 h after the release of LH.

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S. K. Roy and G. S. Greenwald

Summary. Preantral follicles from pro-oestrous and oestrous hamsters were isolated enzymically (Stages 1–5) and by microdissection (Stage 6) and cultured for up to 168 h in the absence or presence of 100 ng ovine FSH or LH separately or combined or 1 or 10 μg progesterone or oestradiol-17β in serum-free defined medium and exposed to 1 μCi [3H]thymidine for 24 h before termination. In the presence of insulin and hydrocortisone but not gonadotrophins, the morphology of follicles from pro-oestrous animals at Stages 1–4 (1–4 layers granulosa cells; no theca) were unaffected for up to 48 h whereas for Stages 5 (5–6 layers granulosa cells and developing theca) and 6 (7–8 layers granulosa cells and theca), atresia was prominent by 24 h. FSH significantly reduced the percentage of atretic follicles in Stages 1–5 throughout the culture period; but was effective only up to 96 h for Stage-6 follicles. LH was also effective, albeit to a lesser extent. FSH increased follicular labelling indexes during every 24-h labelling period and, during a pulse-chase period, follicular DNA content and granulosa cell numbers. FSH, but not LH, induced differentiation by 96 h of preantral follicles at Stage 6 into small antral stages (Stages 7–8). FSH and LH together induced almost the same effect as FSH alone. However, neither progesterone nor oestradiol had any significant long-term effects on DNA synthesis and oestradiol induced atresia beyond 24 h. Both FSH and LH induced follicular maturation in vitro as evident from increases in progesterone, androstenedione and oestradiol production. Follicles (Stages 1–4) collected from oestrous hamsters responded to FSH to a lesser extent than did those from pro-oestrous animals, possibly because of in-vivo exposure to periovulatory changes in gonadotrophins; however, an antrum formed in Stage-6 follicles by 72 h.

Keywords: preantral follicle; hamster; long-term culture; DNA synthesis; gonadotrophins