Serial injections of a mixture of prostaglandin (PG) E₂ and F_2α 0, 2, 4, and 6 h after simultaneous injection of human chorionic gonadotrophin (hCG) and indomethacin incompletely restored the ovulation that would have been blocked by indomethacin in immature rats treated with pregnant mare serum gonadotrophin followed by hCG. Serial injections of another mixture of PGE₂ and PGF_2α 6, 8, 10 and 12 h after simultaneous injection of hCG and indomethacin similarly reversed, in part, the inhibitory effects of indomethacin on hCG-induced ovulation. In contrast, serial injections of the mixtures of PGE₂ and PGF_2α 0, 2, 4, 6, 8, 10 and 12 h after simultaneous injection of hCG and indomethacin completely restored the indomethacin-blocked ovulation, suggesting that the prostaglandins mediate the action of hCG on ovulation both in the earlier and later stages of the preovulatory process.

Six hours after simultaneous injection of hCG and indomethacin serial injections of a mixture of PGE₂ and PGF_2α reproduced the acute and temporary increase in concentrations of progesterone and testosterone in plasma which would have been abolished by indomethacin. Progesterone given concurrently with hCG and indomethacin partially antagonized the inhibitory action of indomethacin on ovulation. Serial injections of a' mixture of PGE₂ and PGF_2α 6, 8, 10 and 12 h after concurrent administration of progesterone with hCG and indomethacin completely restored the indomethacin-blocked ovulation, suggesting that progesterone can substitute the action of prostaglandins injected serially in the first half of the preovulatory process.

It was concluded that the co-operation of progesterone in the earlier stage and of prostaglandins in the later stage of the preovulatory interval is required to mediate the action of hCG on ovulation.

ABSTRACT

To study the effects of immunoneutralization of endogenous inhibin on gonadotrophin secretion and ovarian function, prepubertal heifers (n = 6) were actively immunized against a synthetic peptide replica of the N-terminal sequence of bovine inhibin α subunit bIα(1–29)Tyr³⁰) coupled to ovalbumin. In contrast to ovalbumin-immunized controls (n=6), bIα(1–29)Tyr³⁰-immunized heifers had detectable inhibin antibody titres (% binding to ¹²⁵I-labelled bovine inhibin at 1:2000 dilution of plasma) of 17 ± 3% (s.e.m.) at puberty, rising to 31 ± 5% by the end of the study period 7 months later. Neither age (immunized: 295 ± 8 days; controls: 300 ± 5 days) nor body weight (immunized: 254 ± 13 kg; controls 251 ± 9 kg) at onset of puberty differed between groups. Although the difference did not reach statistical significance, mean plasma FSH concentrations recorded in inhibin-immunized heifers remained 35–40% higher than in controls throughout the 12-week period leading up to puberty (P = 0·14) and during nine successive oestrous cycles studied after puberty (P=0·10). Plasma LH concentrations did not differ between groups at any time during the study. Inhibin immunization had no effect on oestrous cycle length (immunized: 19·8±0·5 days; controls: 19·9±0·5 days). However, in comparison with controls, inhibinimmunized heifers had more medium sized (≥0·5 to <1 cm diameter) follicles during both the preovulatory (95%, P<0·001) and post-ovulatory (110%, P < 0·05 waves of follicular growth and more large (>1 cm diameter) follicles during the preovulatory wave (49%, P<0·05). In addition, the number of corpora lutea observed during the post-ovulatory phase of each cycle was significantly greater in the inhibin-immunized group (43%, P<0·01), as was the recorded incidence of cycles with multiple ovulations (19/56 in the inhibin-immunized group compared with 0/54 in controls; P<0·001). All six inhibinimmunized heifers had at least one cycle with multiple ovulation whereas none of the control heifers did so.

These results support the conclusion that immunoneutralization of endogenous inhibin using a synthetic peptide-based vaccine can enhance ovarian follicular development and ovulation rate in heifers. Whether this ovarian response is dependent upon the expected increase in secretion of FSH remains to be established.

Journal of Endocrinology (1992) 134, 11–18

ABSTRACT

Mating induces a surge of both LH and FSH in the blood of female rabbits, followed 10–12 h later by a surge of FSH only, which begins at the time of ovulation. We have studied the effect of suppression of ovulation on the post-ovulatory surge of FSH. In the first experiment, follicular fluid and oocytes were withdrawn from the largest follicles 8 h after coitus. In the second experiment, ovulation was inhibited by injecting the rabbits with 25 mg indomethacin/kg body weight 7·5 h after mating. Levels of serum FSH and LH were measured for 24-48 h after mating. Control rabbits ovulated normally in both experiments. The treatments did not significantly affect the levels of serum FSH in either experiment, although the second surge of FSH was slightly higher after fluid had been aspirated from the preovulatory follicles. These observations show that the post-ovulatory surge of serum FSH is not dependent upon the completion of ovulation and that it is programmed before 7·5–8 h post coitum.

J. Endocr. (1987) 112, 57–61

Abstract

The positive relationship between nutrition and ovulation rate was investigated in sheep infused intravenously with glucose. Ovulation rate increased (2·0±0·0 vs 2·4 ± 0·3) when ewes were given an infusion of glucose (60–65 mm/h) for five days in the late luteal phase of the oestrous cycle. The effect of glucose was obtained without any significant change in LH secretion. The concentration of FSH in glucose-infused ewes was lower during the infusion (luteal phase) but higher during the early follicular phase. These data suggest that the change in ovulation rate occurred without increased gonadotrophin support to the follicle during the late luteal phase, which is the period of the sheep oestrous cycle during which improved nutrition increases ovulation rate. There were no changes in GH or prolactin, but changes in circulating glucose and insulin levels were detected. We conclude that insulin, because of its role in cell growth and metabolism, is involved in mediating ovulation responses to nutritional stimuli, either directly or more likely by the stimulation of insulin-mediated glucose uptake.

Journal of Endocrinology (1995) 146, 403–410

ABSTRACT

This study in ewes examined the effects on ovarian function of a pulsatile regimen of ovine FSH (NIADDK-oFSH-17) administered over a 24- to 28-day period beginning on day 1 of the oestrous cycle (day 0 = oestrus). The FSH (1·66 μg or 5·00 μg) was administered i.v. over a 1-min interval once every hour throughout the treatment period. In other ewes ovine LH (NIDDK-oLH-23) was administered (10 μg once every 2 h) for 24–28 days together with oFSH (1·66 μg/h).

Compared with untreated controls (n = 19 ewes), FSH alone at both doses (n = 19 ewes/dose) as well as the FSH +LH treatment (n=10) led to significant increases in the plasma concentrations of FSH (P <0·01), ovarian weight (P <0·05) and ovulation rate (P <0·01) but there was no change in the mean weight of individual corpora lutea (CL). Exogenous FSH at the high but not the low dose alone or with LH stimulated a significant overall increase in plasma inhibin concentrations (P <0·05). The geometric mean (and 95% confidence limits) ovulation rates in the high FSH (i.e. 5·00 μg/h), low FSH (i.e. 1·66 μg/h), low FSH+LH, and control treatment groups were 15·3 (9·3, 24·8), 3·7 (2·1, 6·0), 3·7 (2·5, 5·8) and 1·4 (1·2, 1·7) respectively. The FSH or FSH+LH treatments did not alter the total numbers of antral follicles (≥1 mm diameter). However, the high but not the low FSH or low FSH + LH treatment led to significant increases in the mean numbers of large follicles (i.e. >4·5 mm diameter; P<0·01) and a higher proportion of non-atretic antral follicles.

Highly significant linear relationships were found between the mean plasma concentrations of FSH or inhibin and the ovulation rate (FSH: r=0·74, P<0·0001; inhibin: r=0·93, P<0·0001). Highly significant linear relationships were also found between the plasma concentrations of FSH or inhibin and the number of large follicles (i.e. >4·5 mm diameter; FSH, r=0·78, P<0·0001; inhibin, r=0·80, P<0·0001) and between the plasma concentrations of inhibin and the number of granulosa cells in large follicles (r=0·78, P<0·0001). After the high FSH but not the low FSH treatment there were significant increases in both FSH- and LH-induced responsiveness in granulosa cells with respect to cyclic AMP synthesis in vitro. In the high FSH treatment group, granulosa cells from 1–2·5 mm diameter follicles were responsive to LH whereas, in the low FSH or FSH + LH treatment groups and the controls, granulosa cells were not responsive to LH until the follicles were >4·5 mm in diameter. FSH or FSH+ LH treatment did not lead to increases in aromatase activity in granulosa cells (i.e. when expressed on a per cell basis) or to increases in oestradiol in follicular fluid.

Collectively these results show that chronic increases in plasma FSH concentrations influence, in a dose–responsive manner, the size distribution of antral follicles, the proportion of non-atretic follicles, the number of follicles with peak aromatase activity and the ovulation rate, without altering the total number of antral follicles, the granulosa cell composition of individual follicles or the sizes of individual CL. Exogenous FSH treatment at high but not low doses enhanced the sensitivities of granulosa cells to both FSH and LH in vitro. Increases in plasma FSH also led to higher concentrations of plasma inhibin as a consequence of an increase in the number of large follicles and thus the number of granulosa cells.

Journal of Endocrinology (1993) 138, 315–325

ABSTRACT

Ovulation rate, follicle growth, serum FSH and oestradiol concentrations were studied after a single intraperitoneal injection of inhibin antiserum in 5-day-cyclic rats. Control rats received (non-immune) serum from castrated sheep or saline. Rats were injected at 10.00 h on dioestrus-1 (D1), i.e. the day following the day of oestrus, or at 17.00 h on dioestrus-2 (D2). The ovaries were excised at necropsy 48 h after injection, or at first or second oestrus after injection. After routine histology fresh corpora lutea were counted and/or differential follicle counts were made.

Results from rats injected with either (non-immune) serum from castrated sheep or with saline were not different and were therefore combined to form the control group. The activity of inhibin-neutralizing antibodies in the circulation of antiserum-treated rats was reduced by approximately 39% between 8 h and second oestrus after injection, as determined by the binding of purified bioactive radioiodinated 31 kDa bovine inhibin.

Rats were injected on D1 and killed at first oestrus. The number of fresh corpora lutea was significantly higher in antiserum-treated rats than in controls (13·9±0·4 vs 11·8±0·4; P < 0·05). Other rats injected on D1 were killed either 48 h or at the second oestrus after injection. Blood was collected 8, 16, 24 and 48 h and at first and second oestrus after injection. At 48 h after injection differential follicle counts showed that the ovaries of antiserum-treated rats contained approximately 32 more healthy follicles and 11 fewer atretic follicles than controls (both P < 0·05 vs control; data for follicles with volume > 100 × 10⁵μm³ and diameter > 260 μm). The ovaries of the antiserum-treated group collected at second oestrus contained more corpora lutea than controls (17·5±0·5 vs 13·6±0·4; P < 0·001). Serum FSH levels at 8, 16, 24 and 48 h after antiserum injection were elevated (P < 0·05). Overall oestradiol levels in antiserum-treated rats were increased from 8 to 24 h and at first oestrus (P < 0·05) as compared with control rats. Further rats were injected on D2 and necropsied at first or second oestrus which caused ovulation rate to almost double at first oestrus (antiserum 23·7±1·4 vs control 12·4±0·4; P < 0·01), while at second oestrus there was no difference between antiserum-treated and control rats.

The rise in FSH level after injection of antiserum on D1 caused follicle recruitment in addition to that normally occurring on the morning of oestrus (36 h earlier) and reduced atresia, resulting in a moderately increased ovulation rate on the first and second oestrus after injection. If the interval between antiserum injection and the next oestrus was shortened (injection on D2), ovulation rate was doubled, while on the next oestrus (second) there was no difference compared with controls. It is concluded that inhibition is progressively involved in the control of follicle growth and ovulation rate via its effect on serum FSH levels during the oestrus cycle of the rat.

Journal of Endocrinology (1991) 130, 297–303

Department of Anatomy and Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90024, U.S.A.

(Received 21 July 1977)

The existence of a 'critical period' in the ovulatory surge of pituitary gonadotrophin during the afternoon of pro-oestrus was demonstrated by Everett, Sawyer & Markee (1949). It was assumed that during this period, between 14.00 and 16.00 h, an amount of gonadotrophin sufficient to cause ovulation was released from the anterior pituitary gland. Administration of a number of neuropharmacologically active drugs before or during the 'critical period' was shown to inhibit the ovulatory response (Everett, 1961). Recently, Blake (1974) described the existence of an 'activation period' as well as a 'potential activation period' for the preovulatory release of luteinizing hormone (LH) in female rats. The 'activation period' is an extended 'critical period', from 14.00 to 17.00 h, during which time a neurohumoral signal continues to stimulate the anterior pituitary gland

The desamino analogues of arginine-vasotocin (dAVT) and arginine-vasopressin (dAVP), when administered on the afternoon of pro-oestrus to immature female rats primed with pregnant mare serum, blocked the preovulatory progesterone surge and inhibited ovulation. Statistical analysis revealed no difference in the potency of the two compounds. At a dosage of 2·0 μg, dAVT and dAVP yielded a 70 and 87% block of ovulation respectively. Simultaneous administration of dAVT and the antipressor compound [1-deaminopenicillamine-4-valine-8-d-arginine]-vasopressin had no statistically significant inhibitory effect when compared with saline-treated controls.

These data suggest that the inhibitory effects of dAVT and dAVP on the progesterone surge and subsequent ovulation are mediated through receptor sites similar to those mediating the pressor actions of the parent hormones.

ABSTRACT

The effects were analysed of a unilateral implant of atropine on ovulation in intact and hemiovariectomized adult rats, together with the response of the atropine-implanted rats to hormone replacement. An outer cannula directed to the left or right preoptic (POA)-anterior-hypothalamic area (AHA) was implanted into cyclic adult rats. A group of animals in oestrus was hemiovariectomized and some were also implanted with a cannula. After two consecutive 4-day cycles, the hemiovariectomized animals were implanted with atropine (23 ± 4 μg) or cholesterol (25 ± 2 μg) on the day of oestrus. Atropine implanted into the left side of the POA-AHA blocked ovulation and compensatory ovarian hypertrophy, whilst implants in the right side had no effects. Administration of gonadotrophin-releasing hormone (GnRH; 3·7 μg/kg) at 13.00 h on the expected day of pro-oestrus induced ovulation in six out of seven treated animals. Of 19 rats with an implant of atropine in the left side of the POA-AHA, one ovulated after treatment with pregnant mare serum gonadotrophin (PMSG) on oestrus, or oestradiol benzoate or human chorionic gonadotrophin (hCG) on day 2 of dioestrus. The effects on ovulation of a unilateral implant of atropine into the POA-AHA of cyclic adult rats and the responses of such rats to GnRH, PMSG, hCG and oestradiol benzoate replacement were also studied. Ovulation was induced in rats with a unilateral implant of atropine and which had been treated with GnRH or hCG at 13.00 h on the expected day of pro-oestrus after the implant. In rats with an atropine implant, treatment with PMSG on the day of implantation or with oestradiol benzoate on day 2 of dioestrus restored ovulation in those with the implant in the left side of the hypothalamus, but was ineffective in those with the implant in the right side. The results suggested that in the adult rat the muscarinic mechanisms regulating preovulatory GnRH release, as well as the stimulatory effects of oestrogen, are lateralized. As the results observed in intact and hemiovariectomized animals subjected to the same treatments were different, it was concluded that the cholinergic neuroendocrine mechanisms regulating ovulation are related to the neural information arising from the ovaries and reaching the POA-AHA.

Journal of Endocrinology (1992) 133, 205–210

ABSTRACT

Rats with a 4-day oestrous cycle were injected with 2·5 mg haloperidol/kg, a dopaminergic blocker, or with 2·0 mg propranolol/kg, a β₁- and β₂-receptor blocker, at 13.00 h on oestrus, dioestrous day 1, diestrous day 2 or pro-oestrus. Animals were autopsied on the next expected day of oestrus. Haloperidol blocked ovulation when injected on oestrus, dioestrous day 1 or pro-oestrus and was less effective when injected on dioestrous day 2. Propranolol caused a decrease in the number of ova shed when injected on dioestrous day 2 or pro-oestrus. Serum concentrations of FSH at oestrus were below the control values in those animals in which ovulation was blocked by haloperidol. No significant changes in serum concentrations of LH were observed. The normal gonadotrophin peak which occurs during the afternoon of pro-oestrus was blocked by administration of haloperidol on oestrus or dioestrous day 1. Administration of gonadotrophin-releasing hormone (GnRH) to haloperidol-treated animals on oestrus or dioestrous day 1 did not restore ovulation or increase serum FSH levels. When the same dose of GnRH was given to rats treated with haloperidol on pro-oestrus, they all ovulated and their FSH levels rose normally. Treatment with both FSH and LH of rats given haloperidol at oestrus restored ovulation in 50% of the animals, whereas it was ineffective in animals treated on dioestrous day 1. Fifty per cent of the animals treated with haloperidol on oestrus or dioestrous day 1 ovulated when oestradiol benzoate was injected on dioestrous day 2.

These results suggest that the dopaminergic and adrenergic systems have different roles in the regulation of the oestrous cycle and ovulation. The decrease in the number of ova shed induced by propranolol may be related to the blockade of receptors at the level of the ovary. The sensitivity of the dopaminergic systems involved in gonadotrophin control seems to vary depending on the day of the oestrous cycle. It is possible that when the dopaminergic system is blocked at the beginning of the cycle, an alteration in oestrogen secretion might be produced, initiating a chain of events at the hypothalamic-pituitary-ovarian axis which will ultimately inhibit ovulation. Since dopaminergic blockade induces an increase in prolactin secretion, and prolactin enhances dopamine turnover at the median eminence and blocks oestrogen secretion at the level of the ovary, the present results might also reflect the effects of haloperidol at two different levels, i.e. increased dopamine turnover at the median eminence and decreased secretion of oestrogens.

J. Endocr. (1987) 113, 37–44