ABSTRACT

Seven Merino–Border Leicester cross–bred ewes were immunized with a purified fusion protein, produced by recombinant DNA methods, of the a subunit of bovine inhibin. Four animals were immunized with the fusion protein alone and three with a conjugate made by coupling the fusion protein to keyhole limpet haemocyanin (KLH) using glutaraldehyde. Each animal received four injections of the fusion protein over 93 days. The animals were synchronized using progestagen sponges and subjected to laparoscopy for the determination of ovulation rates in two consecutive cycles (days 115 and 135). The immunized animals had overall mean ovulation rates for each cycle of 3.4 and 3.4 which was significantly (P < 0.001) above the rates of 1.1 and 1.4 determined for the controls, which had either received no treatment (n=5) or had been immunized with 300 μg KLH (n=4). Analysis of antisera taken on day 115 showed significant fusion protein antibodies and iodinated inhibin–binding capacity in the test but not control groups. Furthermore, antisera to the fusion protein in four out of seven ewes neutralized the inhibin bioactivity of ovine follicular fluid in an in–vitro bioassay. These data demonstrate that neutralization of inhibin can be effected by immunization with bovine inhibin a subunit and that such immunization results in increased ovulation rates as predicted from the biological role of inhibin as a suppressor of FSH.

The role of oestradiol in the regulation of LH release in the hen was studied by use of the anti-oestrogen, tamoxifen (ICI 46,474).

Intramuscular injection of laying hens with 2 or 4 mg tamoxifen on 2 successive days delayed or prevented the occurrence of the preovulatory release of LH and ovulation expected on day 3. Ovulation could be restored by i.v. injection of 20 pg LH releasing hormone (LH-RH). Tamoxifen at a dose of 1 mg affected neither the timing of the preovulatory release of LH nor ovulation. Treatment with 2 or 4 mg tamoxifen on 2 successive days reduced the effectiveness of an i.m. injection of progesterone to stimulate a release of LH. Injection of 1, 2 or 4 mg tamoxifen on 2 successive days significantly raised basal levels of LH in the blood at 24 h after the last injection. This was associated with an increase in the capacity of the pituitary gland to respond to an injection of synthetic LH-RH by a release of LH.

These studies suggest that oestradiol has at least two roles in the regulation of LH release in the hen. First, it maintains a low basal level of LH in the blood by reducing the responsiveness of the pituitary gland to LH-RH. Secondly, oestradiol has a facilitative role in the mechanism by which progesterone stimulates the preovulatory release of LH.

ABSTRACT

The noradrenergic innervation of the ovary of prepubertal rats causes an inhibitory response of the follicles to gonadotrophins, leading to ovulation. We investigated the possibility that noradrenergic peripheral denervation at birth, produced by treatment with guanethidine, modifies the positive feedback effects of gonadotrophins and oestradiol in prepubertal rats, and also the possibility that peripheral denervation can modify the anovulatory syndrome induced by androgenization at birth.

Noradrenergic peripheral denervated rats of 18 days of age treated with pregnant mare serum gonadotrophin (PMSG) ovulated 96 h later, while normal animals did not ovulate (4/9 vs 0/12, P < 0·05) and the number of ova shed was lower than in rats which ovulated spontaneously at first vaginal oestrus (3·5 ± 0·6 vs 8·3 ± 0·4 (s.e.m.), P < 0·01). Oestradiol benzoate (10 μg) did not induce ovulation in either normal or denervated animals (0/11 and 0/11). The anovulatory syndrome induced by the administration of testosterone propionate (75 μg) at birth was partially blocked by noradrenergic peripheral denervation (4/7 ovulated vs 0/10).

The results suggest that some neural information arising from the ovary modulates, in an inhibitory way, the stimulatory feedback mechanisms required to induce ovulation. Partial inhibition of the anovulatory syndrome resulting from androgenization caused by peripheral noradrenergic denervation suggests that noradrenergic neural information sent by the ovary to the hypothalamus results in a decreased concentration of noradrenaline in the hypothalamus and in the aromatization of androgens to oestrogens.

Journal of Endocrinology (1992) 135, 415–420

ABSTRACT

The physiological role of activated hypothalamic N-methyl-d-aspartate (NMDA) receptors during the final phase of female sexual maturation was explored in the rat. The effects of administration of the specific non-competitive receptor antagonist MK-801 on the occurrence of first ovulation and on LH secretion were studied. Injections of MK-801 (0·1–0·2 mg/kg body wt, s.c.) were given once or twice daily, starting at 28 or 35 days of age and continuing up to the day of first ovulation, resulted in a significant delay of this ovulation. Rats that were treated daily with 0·2 mg MK-801/kg, starting on days 30 or 34 and continuing up to day 38, but not including the day of first pro-oestrus, also showed retarded first ovulation. No decrease in serum LH concentration, compared with control rats, could be detected in these rats.

Acute treatment with MK-801 (one or two injections of 0·2, or one injection of 0·5 mg/kg) given at 11.30 h (and 16.00 h) on the day of first pro-oestrus produced partial (1 × 0·2 mg/kg) or complete (2×0·2 and 1 × 0·5 mg/kg) blockade of first ovulation; blocked rats ovulated 1 day later. Serum LH concentrations at 16.00 h on the day of pro-oestrus were significantly decreased in all MK-801-treated groups compared with saline-injected control rats. At 19.00 and 22.00 h LH concentrations remained low in all non-ovulating MK-801-treated rats, but increased in the MK-801-treated rats that ovulated.

Thus chronic blockade of the NMDA receptors by the antagonist MK-801 delays but does not prevent first ovulation, whereas acute treatment blocks the pro-oestrous LH peak.

It was concluded that activation of NMDA receptors plays an important role both in tonic and preovulatory LH secretion during the onset of puberty in the female rat.

Journal of Endocrinology (1991) 131, 435–441

The ability of 20α-hydroxy-4-pregnen-3-one (20α-dihydroprogesterone, 20α-DHP) and its 5α-reduced metabolites, 20α-hydroxy-5α-pregnan-3-one and 5α-pregnane-3α,20α-diol, to facilitate ovulation was examined in immature female rats which had been treated on day 22 of age with a non-ovulatory dose (12 i.u.) of pregnant mare serum gonadotrophin. An increased incidence of ovulation occurred in rats treated on the morning of day 24 with 20α-DHP. However, a dose of 20α-DHP three to four times that of progesterone was required to induce ovulation in all animals. In contrast, neither 20α-hydroxy-5α-pregnan-3-one (at doses ranging from 0·5 to 5·0 mg) nor 5α-pregnane-3α,20α-diol (tested at 3·0 or 4·0 mg) facilitated ovulation. Therefore both 20α-DHP and progesterone, the two major progestins of the rat oestrous cycle, have the ability to facilitate ovulation. It is concluded that the ability of 20α-DHP to facilitate ovulation does not appear to be by way of conversion to its 5α-reduced metabolites.

SUMMARY

In an attempt to locate the site(s) of action of the positive feedback of oestrogen for ovulation, a potent anti-oestrogen, I.C.I. 46474, was stereotaxically implanted into various parts of the brain or into the anterior pituitary. A dose of 5 μg of the anti-oestrogen when implanted into the cerebral cortex or injected subcutaneously on the morning of the day before pro-oestrus in 4-day cyclic rats was only marginally active in interfering with ovulation. By contrast, when the same amount was implanted into the median eminence region or the anterior pituitary, ovulation failed to occur in 80–100% of the rats (P < 0·05). Implantation of the cocoa butter vehicle alone into these regions interfered with ovulation in less than 35% of animals. Introduction of the anti-oestrogen into the anterior hypothalamic or mammillary region gave equivocal results. The data suggest that both the median eminence and the anterior pituitary contain receptors which can be blocked by the anti-oestrogen with resultant inhibition of ovulation. It is concluded that the positive feedback of oestrogen for ovulation is exerted both at the pituitary and the hypothalamic levels.

SUMMARY

Carp pituitary extracts obtained from the puntius carp, Puntius gonio-notus, the grass carp, Ctenopharyngodon idellus, and the big head carp, Aristichthys nobilis, were subjected to gel filtration on Sephadex G-100. The water-soluble proteins in the pituitary extracts resolved into three distinct components, referred to as fractions I, II and III. They were tested for their gonadotrophic activity in the female catfish, Heteropneustes fossilis, by evaluating ovarian maintenance and ovulation in hypophysectomized gravid specimens during the spawning season (July—August), and the induction of oogenesis and vitellogenesis in the regressed ovaries of hypophysectomized specimens during the preparatory period (February). Daily treatment with the puntius carp pituitary fraction II (100 or 300 μg) reinitiated and restored oogenesis and vitellogenesis. The grass carp pituitary fraction II (300 μg) induced vitellogenesis but its potency was much less than that of the puntius carp pituitary fraction II. The big head carp pituitary fraction II was ineffective. Daily treatment with the puntius carp pituitary fraction II (1 or 5 μg) maintained the yolky oocytes in the gravid ovaries and prevented atresia. Ovulation and spawning were induced in hypophysectomized gravid catfish by a single dose of the puntius carp pituitary fraction II (100 or 150 μg). The first and third fractions from the three species of carps were ineffective. Thus, all the reproductive processes in the hypophysectomized female catfish were initiated and/or maintained solely by the puntius carp pituitary fraction II.

Prostaglandin analogues were used to induce luteal regression simultaneously in a number of ewes, thereby synchronizing the final stages of follicular maturation in these animals. Some of the ewes were anaesthetized for 24 h immediately after the injection of prostaglandin (experiment 1), and others for 15 h, starting 24 h after the injection of prostaglandin (experiment 2). In both experiments administration of anaesthetic significantly delayed the onset of oestrus and the time of ovulation relative to prostaglandin-treated control animals. The results from assays of blood samples collected at regular intervals in experiment 1 indicated that the preovulatory peak in the concentration of LH and the periovulatory changes in the concentration of FSH were similarly delayed and that during anaesthesia the level of LH was significantly reduced. It is suggested that the reduced level of LH, which probably resulted from a reduction in the secretion of releasing factor due to anaesthesia, failed to support oestrogen production by the Graafian follicle(s), thereby delaying the occurrence of oestrus and ovulation.

ABSTRACT

In order to relate various prepubertal events in a group of 95 late prepubertal female rats, the following data were obtained during the last 10 days before the day of first ovulation: (1) amounts of ovarian inhibin-like activity (ILA) in some animals (n=47); (2) size and numbers of healthy (antral) follicles with a volume ≥100× 10⁵ μm³ (or diameter ≥260 μm) present per ovary in their litter-mates (n=48); (3) serum FSH concentrations in both groups.

Rats were unilaterally ovariectomized to obtain an ovary for either estimation of ILA content or for histological procedures and counting of follicles. At the time of unilateral ovariectomy they were bled to obtain serum for estimation of FSH concentrations. Rats were kept until the day after the day of first ovulation to determine the time-interval between the day of unilateral ovariectomy and first ovulation. They were studied between 10 and 1 days (days −10 to − 1, maturational age) before first ovulation. In addition, adult cyclic rats were bilaterally ovariectomized on different days of the oestrous cycle for estimation of ovarian ILA content.

The amount of ovarian ILA was estimated in steroid-free ovarian cytosols using an in-vitro bioassay system with dispersed anterior pituitary cells and subsequent measurement of FSH and LH in the spent medium.

The amount of ovarian ILA was about 83 units/ ovary from days −10 to −5, and subsequently increased (P < 0·005) to reach a maximum on day − 1, the day of pro-oestrus (213 units/ovary). Inhibin-like activity in adult rat ovaries at pro-oestrus amounted to 374 units/ovary. A significant relationship was found between ovarian ILA content and total volume of follicles of classes III–V (≥350 × 10⁵ μm³) (r= 0·9683, P<0·005) except for the period between days −7 and −5 when this volume increased earlier than did the ILA content.

The total volume of all follicles ≥ 100 × 10⁵ μm³ was steady from days −10 to −7. On day −6 this volume increased, mainly as a result of an increase of total volume of class II follicles. Thereafter, the total volume of follicles in classes III–V started to increase and was maximal on day −1, while the total volume of follicles in classes I plus II decreased and reached a minimum on day −1.

The serum FSH concentration declined between days −10 and −1 from 400 to 100 μg/l (P<0·001); the presence of follicles of classes III–V was always associated with FSH concentrations ≤200 μg/l (P<0·005). The presence of class I and II follicles was not related to FSH concentrations. This suggested that mainly follicles of classes III–V contribute to ovarian ILA.

The present data show that in immature rats ovarian ILA content increases towards the day of first pro-oestrus, as it does later during pro-oestrus in adult cyclic rats. Inhibin-like activity seems to be produced mainly by follicles of classes III–V which are present in the ovaries during the last 5 days preceding first ovulation. In this same period FSH concentrations are kept within narrow limits (<200 μg/l) as is the case during the adult cycle. Thus, ILA probably plays a role in the fine regulation of FSH secretion.

J. Endocr. (1986) 111, 159–166

SUMMARY

The stages of the maturation divisions, ovulation, fertilization and the first cleavage in the eggs of two strains of adult mice have been timed following superovulation treatment. Superovulation was induced by a priming injection of pregnant mares' serum followed after a 40 hr interval by human chorionic gonadotrophin (HCG).

The oocytes were in the dictyate (germinal vesicle) stage until 2 hr after the injection of HCG. They then completed the prophase of the first maturation division, and the first metaphase plates were found 30 min later. From approx. 4½ to 8 hr all oocytes were in metaphase. The first maturation division and extrusion of the first polar body were then rapidly completed just before ovulation.

Ovulation began, as judged by the presence of eggs with adherent cumulus in the uterine tubes, 11 hr after the injection of HCG and was virtually complete by 14 hr. One strain of mice ovulated slightly later than another strain. The time at which ovulation occurred was very similar in mated and unmated females.

Fertilization and the first cleavage were studied in females which mated before ovulation began, and the mean intervals between various stages were as follows. Spermatozoa penetrated through the cumulus and zona pellucida in approx. 1 hr, and remained in the perivitelline space for 50 min. Pronuclei were formed 4¼ hr later, and the first cleavage division occurred at approx. 25 hr after penetration of spermatozoa into the eggs. These estimates are similar to those reported by other workers.

The formation of oocytes in the adult mammalian ovary, the timing of early stages in the prophase of the first maturation division, and the relation between natural and induced ovulation are discussed.