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.
S. L. Silavin and G. S. Greenwald
I. Kim, Chandrima Shaha and G. S. Greenwald
Summary. Intact or hypophysectomized 23-day-old hamsters and rats were injected s.c. with 2 mg diethylstilboestrol (DES) or 1 mg oestradiol cyclopentylpropionate (OECP) on Days 23–25 and killed on Day 26. Although serum oestradiol was elevated to the same high levels by OECP, ovarian and uterine weights were increased in the rat by OECP or DES whereas only the uterus responded in the hamster. This correlated with the ability of the oestrogens to increase significantly the number of large preantral and antral follicles in the intact rat but only the number of follicles with 2–3 layers of granulosa cells in the immature hamster. Qualitative study revealed that DES and OECP increased the number of large preantral follicles in the adult hypophysectomized rat but were ineffective in the adult hamster. It is concluded that for the immature and adult hamster oestrogens do not play a major role in the recruitment of large preantral follicles.
P. F. Terranova and G. S. Greenwald
Summary. Cyclic guinea-pigs were injected s.c. on Day 12 (Day 1 = day of ovulation) of the cycle with 0·4 or 0·8 ml equine antiserum to bovine LH or with 0·4 ml normal horse serum (NHS). Treatment with 0·4 or 0·8 ml anti-LH delayed vaginal opening by 3 days and prolonged the oestrous cycle to 20 days compared with 16 days in controls; ovulation rates for these treatments were 5·6 ± 0·6 ova (P < 0·01), 4·5 ± 0·9 ova and 2·9 ± 0·3 ova respectively. Serum concentrations of oestradiol and progesterone were not affected but androgen values were decreased by treatment with LH antiserum. The number of antral follicles (>520 μm diam./ovary 48 h after 0·4 ml anti-LH (Day 14) was similar to that observed in controls, but on Day 18 6·0 ± 1·4 follicles/ovary were present in antiserum-treated guinea-pigs, whereas in controls on Day 16 there were only 2·4 ± 0·5 antral follicles. The results indicate that the increased ovulation rate induced in the guinea-pig by LH antiserum is not associated with recruitment of increased numbers of antral follicles but may be related to reduced atresia at the end of the cycle.
D. D. Chiras and G. S. Greenwald
Summary. Hamsters were hemispayed at 09:00 h on Day 3 of the cycle (Day 1 = ovulation) and were killed 1 h after injection of [3H]thymidine at 09:00, 12:00, 17:00 or 22:00 h. Unilateral ovariectomy had no effect on the number of Stage 1 or Stage 2 follicles, but there were significantly fewer Stage 3 follicles between 10:00 and 13:00 h. This decrease was not encountered in intact hamsters and was reflected in an increase in the number of Stage 6 (antral) follicles. At 13:00 h there was no difference in the number of atretic follicles between the experimental and control groups. It is concluded that preantral follicles with 6–7 layers of granulosa cells were recruited within 4 h after unilateral ovariectomy and transformed into antral follicles.
J. B. CHOUDARY and G. S. GREENWALD
To determine the mechanism by which oestrogen induces luteolysis, cyclic guinea-pigs were given a single subcutaneous injection of 10μg of oestradiol cyclopentylpropionate (OECP) on Day 1 and daily subcutaneous injections of various gonadotrophins on Days 1 to 9. The mean diameter of the corpora lutea and their histology were used as end points in assessing luteal status.
Ovine lh, prolactin, or a combination of both did not reverse the effect of OECP. Daily treatment with 200 μg of ovine fsh in normal saline partially counteracted the luteolytic effect of OECP on the size and morphology of the corpora lutea. A combination of 100 μg of fsh and 1 mg of prolactin or 10 μg of lh was less effective than 200 μg of fsh. Treatment with 10 or 25 i.u. of pmsg in saline or 200 μg of fsh in 5% beeswax-sesame oil suspension counteracted the luteolytic effect of OECP in the majority of animals. The corpora lutea regressed when the initial injection of pmsg was deferred until Day 4.
The results indicate that oestrogen induces luteal regression in the cyclic guinea-pig by inhibiting the pituitary secretion of fsh which is apparently necessary—at least in part—for the normal maintenance of corpora lutea.
J. T. McCORMACK and G. S. GREENWALD
Measured by radioimmunoassay, the peripheral plasma oestradiol concentration reached a peak (21·2±1 pg/ml) between 10.00 to 12.00 hours on Day 4 of pregnancy in mice. These values were significantly different from all others measured between 07.00 and 18.00 hours. To determine the physiological significance of this peak, ovariectomies were performed at selected intervals. Ovariectomy at 08.00 hours or earlier usually prevented implantation. The period from 10.00 to 12.00 hours on Day 4 was found to represent a critical time period before which insufficient oestrogen is secreted, in most instances, to initiate implantation.
PAMELA J. MOORE and G. S. GREENWALD
Several studies from this laboratory have used PMSG to induce superovulation in the cycling hamster (for references see Greenwald, 1973). The same dose of PMSG given at different times of the year resulted in different ovulation frequencies. This raised the question of whether there is a seasonal variation in ovarian responsiveness to exogenous gonadotrophins.
Aliquots of 5, 15 or 30 i.u. PMSG (concentration/0·1 ml), sufficient to last for 1 year, were prepared and frozen at the beginning of the experiment. Individual vials were then unfrozen and used only once. For 1 year, beginning in May 1972, on the 14th or 15th of each month, three groups of adult golden hamsters (five per group) were injected subcutaneously with 5, 15 or 30 i.u. of PMSG on the morning of Day
H. M. WEITLAUF and G. S. GREENWALD
Mature mice were ovariectomized on the 4th day post coitum (Day 1 = vaginal plug) and given no further treatment. Unimplanted (delayed) blastocysts were recovered from these females between 5 and 40 days post coitum. They were tested for viability by determining the proportion that developed into normal foetuses following transfer to Day-4 pseudopregnant recipients.
It was found that the proportion of transferred blastocysts developing into foetuses was similar after delays of from 0 to 10 days. After delays of more than 10 days, the proportion of blastocysts that were capable of developing into normal foetuses decreased; 30 to 32% developed following transfer on Days 5 to 15, 18% with transfer on Day 20, and 3% with transfer on Day 30. The finding that blastocysts can be delayed in the absence of ovarian hormones and yet remain capable of developing into normal foetuses demonstrates that progesterone is not essential for blastocyst survival in mice.
G. S Greenwald and P. F. Terranova
Summary. Hamsters injected s.c. on the day of ovulation (Day 1) with 100 μl equine anti-bovine LH serum ovulated 28 eggs at the end of a 5-day cycle. When a second injection of anti-LH serum was administered 4–93 days later, the animals did not superovulate and had normal 4-day cycles. Injection of 100 μl normal rabbit serum (NRS) on Day 1 followed 14 days later by anti-LH serum resulted in the ovulation of 32 ova whereas a priming injection of 100 μl normal horse serum (NHS) followed by anti-LH serum resulted in the ovulation of only 18 ova. When hamsters were injected on Day 1 with anti-LH serum, NHS or NRS and then with anti-LH serum in the 4th cycle, high titres of free antibodies to LH were present on Days 2–4 only in the animals treated with NRS; these hamsters ovulated a mean of 35 ova.
These experiments suggest that the hamster rapidly forms antibodies to equine immunoglobulins, thus preventing a second injection of anti-LH serum from inducing superovulation.
C. J. Hubbard and G. S. Greenwald
Summary. Atresia was induced in antral follicles of hypophysectomized PMSG (30 i.u.)-treated hamsters by an i.p. injection of PMSG antiserum. Mature antral follicles were isolated and incubated for 1 h. When expressed per follicle, cAMP increased 108% above control levels at 2 h, while cGMP rose 117% at 4 h; both nucleotides then declined (12–72 h). DNA per follicle fell below control values by 24 h and reached its lowest point at 48 h. Serum concentrations of oestradiol-17β and testosterone were 50% lower by 1 h after injections of PMSG antiserum. Oestradiol and testosterone in serum, incubation medium and tissue dropped rapidly to reach basal values by 12 h. Serum progesterone showed a sharp increase at 4 h then dropped below control levels by 12 h and remained constant thereafter. In tissue and medium, progesterone rose to peak values at 8 h, then dropped to control levels with no significant changes for the remaining 64 h. The precipitous decreases in both oestradiol and testosterone in the serum, medium and tissue indicate inhibition of steroid synthesis at some point after progesterone formation.
These results demonstrate the usefulness of this model for following atretic changes in a synchronized population of Graafian follicles.