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F. Garza and P. F. Terranova

Summary. Implants of progesterone on the day of dioestrus II in the hamster induced on the following day an increase in circulating levels of progesterone (6·0 ± 0·7 ng/ml, N = 8; sesame oil controls, <0.5 ng/ml, N = 6) and a decline in serum levels of LH (5·3 ± 0·4 ng/ml; controls 12 ± 2 ng/ml) and oestradiol (10 ± 2 pg/ml; controls 69 ± 5 pg/ml). The production of androstenedione and oestradiol by antral follicles in vitro was reduced in progesterone-treated hamsters when compared with controls, but progesterone production was not affected. Aromatizing activities of antral follicles were the same in progesterone-treated and sesame oil-treated hamsters. Androstenedione production by theca was significantly less in progesterone-treated hamsters than in controls. On dioestrus II, LH replacement therapy (200 μg ovine LH by osmotic minipump inserted s.c.) prevented the decline in follicular androstenedione and oestradiol production induced by progesterone alone, and also prevented the decline in thecal androstenedione production in vitro. The results indicate that exogenous progesterone on dioestrus II lowers circulating levels of LH by the following day, inhibits thecal androstenedione production and thus reduces follicular oestradiol production without alteration in aromatizing ability.

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S. Rousey and P. F. Terranova

Summary. Insertion of osmotic minipumps containing 1 mg ovine LH on Day 1 (oestrus) elevated circulating serum concentrations of LH, progesterone and androstenedione when compared with values at pro-oestrus. Ovulation was blocked for at least 2 days at which time there were twice the normal numbers of preovulatory follicles. Follicular and thecal progesterone production in vitro was elevated when compared with that in pro-oestrous controls. Follicular and thecal androstenedione production in vitro was lower than in controls even though serum concentrations of androstenedione were elevated; the higher androstenedione values may be due to the increase in number of preovulatory follicles when compared with pro-oestrous controls. Follicles from LH-treated hamsters aromatized androstenedione to oestradiol and follicular production of oestradiol was similar to that in pro-oestrous follicles despite low follicular androstenedione production in the LH-treated group. Treatment with 20 i.u. hCG on Days 4 or 6 after insertion of an LH osmotic minipump on Day 1 induced ovulation of ~30 ova, indicating that the blockade of ovulation was not due to atresia of the preovulatory follicles. Serum progesterone concentrations on Days 2, 4 and 6 in LH-treated hamsters were > 17 nmol/l, suggesting that the blockade of ovulation might have been due to prevention of the LH surge by high serum progesterone concentrations.

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K. F. Roby and P. F. Terranova

Summary. An inhibitor of endothelial cell thymidine incorporation in vitro was partly purified from cow ovaries using ammonium sulphate (AS) precipitation. Supernatant fluid from the 100 000 g pellet of freshly homogenized ovaries was subjected to stepwise AS precipitation. Precipitates were collected sequentially at 40%, 60%, 80% and 95% saturation, and then each was dissolved, dialysed (M r 8000 cutoff) and examined in tissue culture for effects on cellular thymidine incorporation by cow pulmonary artery endothelial cells (CPAE) and mouse fibroblasts (L929 and 3T3). The 80% AS precipitate (ppt.) inhibited the in-vitro uptake of [3H]thymidine by CPAE and L929 cells, but not 3T3 cells. Heparin–Sepharose (HS) chromatography of the 80% AS ppt. revealed that the inhibitory activity on CPAE and L929 cells did not bind to HS; the inhibitory fraction was found in the HS column breakthrough (80% BT). The 80% BT fraction reduced CPAE [3H]thymidine uptake as determined by autoradiography and increased cellular uptake of trypan blue. Serial fractions from Sephacryl S-200 exclusion chromatography of the 80% BT contained CPAE inhibitory activity in the M r range 30 000– 50 000. The inhibitory activity on endothelial cells and L929 fibroblasts and the nonreduced molecular weight range of that fraction are similar to those of tumour necrosis factor alpha (TNFα). The results indicate that the cow ovary contains a fraction that inhibits endothelial cell growth in vitro and may have important roles in follicular atresia and luteal regression.

Keywords: endothelial cell; fibroblast; growth factor; ovary; tumour necrosis factor

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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.

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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.

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J. D. Brannian and P. F. Terranova

Summary. In cyclic hamsters, exogenous progesterone (100 μg) administered s.c. at 09:00 h on the day of dioestrus II reduced prostaglandin (PG) E and 6-keto PGF-1α but not PGF concentrations in preovulatory follicles measured at 09:00 h of prooestrus. The injection of 10 μg ovine LH (NIADDK-oLH-25) concurrently with 100 μg progesterone on dioestrus II prevented the decline in follicular PGE and 6-keto PGF-1α values. Administration of LH alone did not significantly alter follicular PG concentrations. Inhibition of follicular PGE accumulation by progesterone was due to a decline in granulosa PGE concentration and not thecal PGE. Progesterone administration also reduced follicular oestradiol concentrations. Administration of oestradiol-17-cyclopentanepropionate (ECP) (10 μg) with progesterone did not prevent the decline in follicular PGE and 6-keto PGF-1α but did increase follicular PGF concentrations. However, ECP given alone on dioestrus II reduced follicular PGE and increased PGF concentrations in preovulatory follicles on pro-oestrus. It is concluded that exogenous progesterone administered on dioestrus II inhibits granulosa PGE and 6-keto PGF-1α accumulation in preovulatory follicles, probably by reducing serum LH concentrations, and that the granulosa cells, which are LH-dependent, are a major source of follicular PGE.

Keywords: prostaglandins; progesterone; ovary; follicles; hamster

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J. D. Brannian, F. Griffin, H. Papkoff, and P. F. Terranova

Summary. Serum samples were collected from 3 mature female African elephants once each week for 15–18 months. Circulating concentrations of progesterone, oestradiol and LH were determined by radioimmunoassay (RIA). The LH RIA was validated by demonstrating parallel cross-reaction with partly purified elephant LH pituitary fractions. Changing serum progesterone concentrations indicated an oestrous cycle length of 13·3 ± 1·3 weeks (n = 11). The presumed luteal phase, characterized by elevated serum progesterone values, was 9·1 ± 1·1 weeks (n = 11). Two abbreviated phases of progesterone in serum lasting 2–3 weeks were observed in 2 elephants, indicating short luteal phases. Oestradiol concentrations in serum were variable, with no clear pattern of secretion. More frequent blood samples were collected during periovulatory periods and 9 distinct LH peaks were detected; all were followed by rises in serum progesterone concentrations. Periovulatory changes in progesterone and LH in sera correlated with external signs of oestrus and mating behaviour.

Keywords: elephant; Loxodonta; oestrous cycle; progesterone; LH

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A. Krishna, P. F. Terranova, R. L. Matteri, and H. Papkoff

Summary. Constant infusion of LH (400 μg NIH-S24) through an osmotic minipump inserted on Day 1 (oestrus) of the cycle in the hamster resulted in spontaneous superovulation (≃ 29 ova) at the next expected oestrus, increased blood flow (P<0·001) to the ovary on Day 3, and slight depletion (0·1 >P>0·05) of histamine in the ovary. Treatment with antihistamine (alpha-fluoromethylhistidine, an irreversible inhibitor of histidine decarboxylase, or cimetidine, an H2 blocker) by injections or by infusion using separate osmotic minipumps significantly (P<0·01) reduced the number of ova shed in the LH-treated hamsters. Infusion of LH with alpha-fluoromethylhistidine in the same osmotic minipump reduced the bioactivity of the LH. Infusion of antihistamine alone did not alter the normal number of ova shed. The results suggest that the LH-induced superovulation involves stimulation of histamine release; the histamine then may increase ovarian blood flow thus allowing more gonadotrophins to reach the ovary.

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V. Montgomery Rice, S. D. Limback, K. F. Roby, and P. F. Terranova

Tumour necrosis factor α (TNF-α) concentrations were measured during periods of controlled and natural follicular development and ovulation in rat ovaries. Concentrations of bioactive TNF-α were determined in the ovaries and sera of rats during puberty (the period of vaginal opening and the first ovulation) and in immature rats after gonadotrophin treatment. Ovaries and sera were collected from 33-, 35-, 37-, 39-, 41- and 43-day-old rats (n = 6 or 7 per group); vaginal opening occurs on day 35. The presence of ovarian follicles and corpora lutea or ova in the oviducts was assessed. For gonadotrophin treatment, a single subcutaneous injection of 5 iu equine chorionic gonadotrophin (eCG) was administered at 08:00 h to 28-day-old rats to stimulate follicular development. A single subcutaneous injection of 10 iu human chorionic gonadotrophin (hCG) was administered 48 h later to induce ovulation. Ovaries and sera from three to six animals per group were collected 0, 3, 24, 48, 51, 54, 57, 60 and 72 h after injection of eCG. At puberty, ovarian concentrations of TNF-α were highest (approximately 1.1 fg μg−1 ovarian protein) before vaginal opening and the first ovulation. After vaginal opening and ovulation at day 37, ovarian concentrations of TNF-α were markedly reduced (0.091 fg μg−1 ovarian protein) and remained low up to day 43. Serum concentrations of TNF-α remained low throughout the period of vaginal opening and the first ovulation (8–32 pg ml−1). In 43-day-old rats serum concentrations of TNF-α increased (105 pg ml−1). In the immature ovaries of 28-day-old rats TNF-α concentrations were highest before injection of eCG (approximately 1.2 fg μg−1 ovarian protein) and decreased to approximately 0.4 fg μg−1 protein 3 h after injection. TNF-α concentrations decreased further 24 h after eCG injection (< 0.1 fg μg−1 protein) and remained low until 48 h after eCG injection. Serum concentrations of TNF-α did not change during the 48 h period after injection of eCG. hCG was administered 48 h after eCG, and ovarian and serum TNF-α concentrations increased transiently. Serum TNF-α concentrations increased 3 h after hCG and remained elevated until 9 h after injection, after which concentrations decreased. Ovarian concentrations of TNF-α increased 6 h after hCG, peaked (approximately 0.5 fg μg−1 protein), and then declined. These results indicate that during puberty and the first ovulation, circulating and ovarian TNF-α concentrations change. In addition, exogenous gonadotrophins alter circulating and ovarian TNF-α concentrations. These data suggest that TNF-α has a role in follicular development and ovulation during puberty and in immature rats treated with gonadotrophins to induce ovulation.