Further investigation of the antiovulatory effects of the antiprogestational steroid RMI 12,936 in the rat.

Inhibition of ovulation by RMI 12,936 was associated with suppression of the pro-oestrous peak of hypothalamic dopamine. The antiovulatory effect was not reversed by administration of oestrogen, was partly reversed by progesterone and was fully reversed by oestrogen and progesterone. Hypophysial sensitivity to LH-RH, known to be reduced by RMI 12,936, remained low when ovulation was restored by steroid treatment. Administration of oestrogen did not restore the pro-oestrous peak of hypothalamic dopamine and ovulation was not induced following administration of L-DOPA in RMI 12,936-treated animals. It was concluded that RMI 12,936 is antioestrogenic as well as antiprogestational, that oestrogen is necessary for induction of full hypothalamic-hypophysial responsiveness to progesterone and that a hypothalamic dopaminergic pathway may have a non-essential role in the control of ovulation possibly associated with increasing hypophysial sensitivity to LH-RH.     

Androgenic effects of the antiprogestagen RMI 12,936

RMI 12,936 (7alpha-methyl-17beta-hydroxy-androst-5-en-one) was tested for androgenic activity in mouse kidney and for antiprogestational activity in guinea-pig uterus. RMI 12,936 stimulated an increase in kidney weight and in the activity of the androgen-responsive renal enzymes, beta-glucuronidase, alcohol dehydrogenase and arginase. RMI 12,936 was bound by the renal androgen receptor with a relative affinity approximately one-third that of testosterone. Although RMI 12,936 did not stimulate glycogen accumulation in guinea-pig endometrium in vivo, it was active in endometrial organ culture. When RMI 12,936 was combined with progesterone, glycogen accumulation in vitro was partly inhibited. RMI 12,936 was bound by the guinea-pig uterine progesterone receptor with a relative affinity of less than 1%. It is concluded that RMI 12,936 is an androgenic steroid with antifertility actions and in-vitro antiglycogenic activity.     

Norepinephrine stimulates LH-RH secretion into the hypophysial portal blood of the rat

The present study was designed to investigate the effect of intracerebroventricular infusion of norepinephrine (NE) on the secretion of luteinizing hormone-releasing hormone (LH-RH) into the hypophysial portal blood of steroid-primed ovariectomized rats. Saline infusion into the third ventricle caused no significant change in LH-RH levels. NE infusion (20 micrograms) resulted in a significant release of LH-RH (p less than 0.05) into the portal blood 10-30 min later. This endogenous LH-RH was similar to synthetic LH-RH when characterized by thin-layer chromatography. LH secretion in similarly treated rats but with intact portal vessels, also was significantly elevated (p less than 0.05) at 20 and 40 min after the start of NE infusion. These results show that NE stimulated the secretion of LH-RH into the hypophysial portal blood and this correlated with an enhanced release of LH.     

Some biological properties of RMI 12,936, a new synthetic antiprogestational steroid.

A new synthetic steroid, RMI 12,369, was examined for its contraceptive potential in rats and comparisons with ethinyloestradiol were also made. Marked differences in the biological effects of the compounds were found, RMI 12,936 having high antiprogestational but negligible oestrogenic activity. Administration of RMI 12,936 on Day 1 of pregnancy caused acceleration of egg transport, the initial changes being apparent within 12 hr of dosing. Termination of pregnancy was associated with a significant reduction in ovarian weight. On Day 8 of pregnancy, RMI 12,936 resulted in significant ovarian hypertrophy, apparent within 48 hr, possibly due to a luteotrophic stimulus of placental origin. Pregnancy could not be maintained by progesterone implants, indicating that utilization was inhibited. Egg transfer experiments indicated that the primary effects were probably on the maternal reproductive tract. Pregnancy was terminated after administration of RMI 12,936 ON Day 19. The compound also had antifertility activity following intravaginal administration.     

Effect of RMI 12,936, an antiprogestational steroid on decidual cell reaction and embryos in rat.

Subcutaneous administration of RMI 12,936 at a dose level of 2 mg/rat on day 5 of unilaterally pregnant rat having trauma-induced decidual cell reaction (DCR) in the contralateral uterine horn, suppresses DCR, induces resorption of implanted embryos and leads to decrease in the plasma level of progesterone. Progesterone replacement (D 5-8) in this situation reverses DCR suppressive effect of RMI 12,936 but fails to prevent resorption of implanted embryos. It is concluded that possibly the drug simultaneously exerts embryotoxic as well as luteolytic effects, but these effects are independent.     

Serum luteinizing hormone and ovulatory response to luteinizing hormone-releasing hormone in the estrous and anestrous domestic cat.

A heterologous double antibody radioimmunoassay was developed to measure changes in serum luteinizing hormone (LH) concentrations in estrous and anestrous queens (female domestic cats), following a single injection of varying doses (0--25 microgram) of luteinizing hormone-releasing hormone (LH-RH). No increase in serum LH was detected in any of the estrous or anestrous queens following a single saline injection. Treatment with LH-RH resulted in a sharp increase in serum LH concentration in both estrous and anestrous queens. Ovulations as observed by the presence of corpora lutea at laparoscopy occurred in none of four, one of four, two of four and four of four estrous queens receiving 0, 5, 10 or 25 microgram of LH-RH, respectively. Mean serum LH concentration of the ovulating queens was maintained at a higher level and did not return to basal level at the same time as that of nonovulating queens. The data show that: LH-RH can cause release of LH in both estrous and anestrous queens and induce ovulation in the estrous cat; the magnitude of LH response is influenced by the stage of the reproductive cycle; and the duration during which LH is maintained above basal level may play a significant role in ovulation induction in this coitus-induced ovulatory species.     

Comparison of mammalian luteinizing hormone releasing hormone (LH-RH), and of an analog (ICI 118630), on luteinizing hormone and ovarian steroid (progesterone, oestradiol) secretions in laying hens. (Gallus domesticus)

This experiment was conducted to compare the luteinizing hormone (LH), progesterone (P4) and oestradiol (E2) release in response to injections of various doses of synthetic mammalian luteinizing hormone-releasing hormone (LH-RH) and of an LH-RH agonist, ICI 118630, administered to laying hens 4 to 9 hours after a mid-sequence ovulation. Plasma LH increased significantly within 10 minutes of injection of either compound whereas any increases in plasma steroid concentrations were discerned later, at approximately minutes post-injection. No dose-response relationship was found for either compound with respect to LH release, but ICI 118630 appeared more potent than LH-RH. This analog also produced a greater mean incremental rise in plasma progesterone, but not oestradiol, than LH-RH, and this was found in animals injected at a time when the largest ovarian follicle was not mature. These result suggest that ICI 118630 is a more potent releasing hormone in the hen at the level of the pituitary, and that it may have a stimulating effect on ovarian progesterone secretion.     

Effects of third ventricular injection of beta-endorphin on luteinizing hormone surges in female rat: sites and mechanisms of opioid actions in the brain

The effects of third ventricular injection of beta-endorphin (beta-EP) on spontaneous, brain stimulation-induced and estrogen-induced LH surges were studied in the adult female rat. It was found that beta-EP blocked the preovulatory surge of LH release and ovulation, while it did not affect LH release in response to LH-RH injection. The site of the beta-EP blockade of ovulation was proved to be in the brain. Beta-EP completely blocked ovulatory LH release induced by the electrochemical stimulation of the medial amygdaloid nucleus and medial septum-diagonal band of Broca, but failed to block ovulation due to the stimulation of the medial preoptic area (MPO) or median eminence, though serum LH levels after the MPO stimulation were inhibited by beta-EP. In the spayed rats treated with estradiol benzoate (EB) on Day 1 and 4 of experiment, beta-EP given on Day 5 blocked the LH surge that normally occurred on that day and led to a compensatory surge of LH on the following day. Moreover, the LH surge on Day 5 was inhibited by beta-EP given either on Day 1 or Day 4. Present data suggest that beta-EP may act in inhibiting the preovulatory LH surges not only by suppressing the preoptic-tuberal LH-RH activities but also by affecting the initiation and development of stimulatory feedback of estrogen in the central nervous system.     

Genetic and environmental variation in the LH response of ovariectomized sheep to LH-RH.

The influence of breed and season on the sensitivity of the pituitary gland of sheep to LH-RH was assessed. Ovariectomized ewes of 3 breeds (Finnish Landrace, Scottish Blackface and Tasmanian Merino) with differing normal breeding seasons and with differing ovulation rates were injected (i.v.) with 3 doses of LH-RH (1.56, 6.25 or 25.0 micrograms) at 3 different times of the year covering the anoestrous and the breeding seasons of intact ewes; 9 ewes of each breed (3 per sub-class) were examined on the first and third occasions, 6 (2 per sub-class) on the second. The response was measured in terms of the concentration of LH in peripheral plasma 20, 40, 60 and 80 min after injection. Time of year, but not the breed of sheep, affected the magnitude of the response; the data indicated that the duration of LH secretion was greater during the breeding season than during anoestrus. It was concluded that changes in the spontaneous activity of the hypothalamus/hypophysis could contribute to seasonal changes in LH secretion independently of the modifying effects of gonadal steroids. Such variation in unmodulated activity apparently does not contribute to the differences in ovulation rate among the 3 breeds.     

Gonadotrophin-releasing hormone treatment for induction of follicular maturation and ovulation in amenorrhoeic women with anorexia nervosa.

Follicular maturation and ovulation can be induced in amenorrhoeic women with anorexia nervosa by long-term treatment with 500 mug of luteinizing hormone releasing hormone (LH-RH) every eight hours. In some women, however, treatment with LH-RH alone results in ovulatory menstrual cycles with indications of luteal phase insufficiency. Human chorionic gonadotrophin (HCG) was therefore given with LH-RH during three treatment cycles. This resulted in ovulation and normal corpus-luteum function, as shown by the occurrence of a single pregnancy in the only involuntarily sterile patient. During the prolonged LH-RH treatment the LH response to LH-RH increased in parallel with the increased oestrogen secretion while the follicle-stimulating hormone response to LH-RH decreased. These changes in the pituitary responsiveness to LH-RH may result from modulating effects on the pituitary by the sex steroids.     

Inhibition of ovarian function in subordinate female marmoset monkeys (Callithrix jacchus jacchus)

Plasma concentrations of progesterone, cortisol, LH and prolactin were measured in dominant and subordinate female marmosets in 10 well-established peer groups. Subordinate females never ovulated, had a reduced LH response to LH-RH and showed no positive feedback LH surge after oestrogen administration. There was no evidence of elevated plasma cortisol levels or hyperprolactinaemia in subordinates and all showed a similar prolactin response to TRH in comparison with dominants. However, subordinates showed a reduced prolactin response to metoclopramide. These results clearly indicate that high circulating levels of cortisol or prolactin are not responsible for the inhibition of ovulation in female marmosets.     

Augmentation, by naloxone, of the frequency and amplitude of LH-RH pulses in hypothalamo-hypophyseal portal blood in the castrated ram

The effect of naloxone administration on the LH-RH secretion in hypophyseal portal blood and LH secretion in peripheral blood was studied in four short term castrated rams (between 2 to 4 days after castration). For two animals (A and B) given a single naloxone injection, an increase of LH-RH pulse amplitude was observed (A, 22.3 to 80.5 pg/ml and B, 22.5 to 34.5 pg/ml) with only a small (nonsignificant) increase in LH-RH pulse frequency. For animals C and D given four injections of naloxone, both LH-RH pulse amplitudes and LH-RH pulse frequency were increased. Means of LH-RH pulse amplitude increase from 29.3 to 65.1 pg/ml and from 34.6 to 50.8 pg/ml for animals C and D respectively and the number of LH-RH pulses detected during the 3 hrs. before and after the first injection of naloxone were respectively 3 vs. 5 and 3 vs. 7. Whereas all LH pulses were preceded with a LH-RH pulse in animals A and B, after the multiple naloxone injections in animals C and D, a rapid LH-RH pulse frequency was associated with a sustained increment of LH secretion in peripheral blood in such a way that individual LH pulses were not clearly defined. The present report is the first documentation on naloxone increasing the release of LH-RH secretion in hypophyseal portal blood of conscious, unrestrained, short-term castrated rams. The results indicate: (1) that the opiate antagonist naloxone is able to increase both the amplitude and the frequency of LH-RH discharge by the hypothalamus and (2), when the LH-RH pulse frequency exceeds one pulse every 30 min., discrete LH secretory episodes are not observed in peripheral blood.     

Effect of prolonged administration of small doses of LH-RH and LH-RH analogue [D-Ser (But)6] Des Gly-NH210 ethylamide on the release of LH and induction of ovulation in mid-anoestrous ewes

A natural process of LH release and induction of ovulation in anoestrous ewes was simulated by prolonged administration of small doses of LH-RH and its analogue [D-Ser(But)⁶] Des Gly-NH₂¹⁰ ethylamide. In the first series of experiments on 40 Merino ewes infusions of LH-RH were made into the maxillaris interna artery for 6 consecutive days for 6 h each day. Total doses of 24.0, 26.0, 28.0 and 32.0 μg per animal of varying and progressively increasing daily quantities of the hormone were administered in groups I, II, III and IV, respectively. In group V the animals were infused with a total dose of 28.0 μg LH-RH and injected additionally i.m. with 3.0 μg 17β-oestradiol on days 4 and 5 of the infusion of LH-RH. Ovulation did not occur earlier than days 4, 5 and 6 after the beginning of infusions. The highest number of positive reactions occurred in group IV ($\text{8}\text{10}$) and in group V ($\text{7}\text{8}$ animals). The pattern of LH peaks in general was correlated with the time of ovulations. The LH concentrations of the preovulatory peaks in experimental ewes were mostly lower than those in naturally ovulating animals. The corpora lutea were functional during the first 7 days after ovulation.In the second series of experiments on 26 Merino ewes the LH-RH analogue [D-Ser -(But)⁶] Des Gly-NH₂¹⁰ ethylamide was injected i.m. or i.a. for 6 consecutive days. Total doses of 15.5, 9.5 and 7.5 μg of the analogue per animal, administered at varying and progressively increasing daily doses in respective groups, induced several surges of LH in the same individuals for 2 or even 3 consecutive days. Corpora lutea and degenerating follicles in the form of cysts were found in the ovaries of animals of these groups. Very small daily doses ranging from 0.1 μg administered during the first 3 days, to 1.5 μg on day 5 of the treatment, released one surge of LH on day 5 of the treatment in all individuals with peaks ranging from 30.0 to 58.0 ng/ml and induction of ovulation with almost normal luteal function. On the basis of these experiments it is suggested that the evaluation of the effect of active substance (LH-RH or its analogue), its suitability and application of rightly chosen doses to induce the full physiological process of ovulation should be based not only on the release of LH and luteal function but also on tests of the ability of the released ovum to undergo fertilization and its further development.     

Suppression of gonadotropin secretion and prevention of ovulation in the rat by antiserum to synthetic gonadotropin-releasing hormone

Administration of an antiserum (0.10–0.25 ml/rat) to the synthetic decapeptide “luteinizing hormone releasing hormone” (LH-RH) suppressed the cyclic surge of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in proestrous rats and prevented ovulation; exogenous LH reversed the block of ovulation. Serum prolactin levels remained unaffected. In ovariectomized rats, the antiserum suppressed the elevated serum levels of both gonadotropins. These findings are compatible with the view that the synthetic decapeptide is identical with the natural hypothalamic hormone that regulates the secretion of both LH and FSH.     

Inhibitory control of the pituitary LH secretion by LH-RH in male rats.

Luteinizing hormone-releasing hormone (LH-RH) was administered to prepubertal male rats (intact, castrate or castrate-adrenalectomized, 60 g body weight) for 28 days (1 microgram LH-RH/day, s.c.), at a 10-fold physiological dose, as compared to the minimal FSH-releasing dose of 100 ng/rat s.c. In intact rats, serum LH and weight of androgen-dependent organs (vented prostate, seminal vesicles) were reduced after 14 days of treatment. In castrate rats, the postcastration rise in serum LH was abolished by treatment. Pituitary LH content, FSH secretion and prolactin secretion were not suppressed. Hypothalamic LH-RH was increased at 14 and 21 days. In castrate adrenalectomized male rats, LH secretion was also suppressed by 1 microgram LH-RH s.c. x 28 days. The hypothalamic LH-RH content did not increase. The pituitary LH-RH receptor level was not down-regulated after 14 days treatment either in intact or castrate male rats. Pituitary inhibition (LH release) in rats by a supraphysiological dose of LH-RH given for 28 days indicates that the optimal regime for chronic treatment has to be determined by monitoring LH release at regular intervals. Direct pituitary inhibition by LH-RH may explain some of the unexpected antifertility effects observed with high doses of LH-RH.     

Effect of bromocriptine on the hypothalamo-pituitary function in adult male rats.

Daily oral administration of bromocriptine (50 μg/kg) to adult male rats, suppressed serum prolactin levels. The pituitary prolactin levels remained unaltered. Serum FSH levels as well as pituitary FSH levels showed no significant change as compared to the controls. Serum LH levels were significantly decreased in spite of the high pituitary LH levels, in bromocriptine treated rats. In the drug treated rats, $\text{in vitro}$ sensitivity of the pituitary to the exogenous LH-RH was not altered; whereas hypothalamic LH-RH content was considerably lowered. These observations suggest the possible effect of bromocriptine on the synthesis of LH-RH in the hypothalamus which leads to the accumulation of LH in the pituitary and decline of serum LH.     

Action of luteinizing hormone-releasing hormone and synthesis of prostaglandin in the pituitary gland.

The possibility that prostaglandin E2 (PGE2) may play a role in luteinizing hormone (LH) release was examined using an in vitro model. Addition of luteinizing hormone-releasing hormone (LH-RH) to the culture medium stimulated cyclic AMP accumulation and LH-release by incubated hemipituitaries, but did not affect the level of PGE2 or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE2 or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE2 content in the pituitary, but did not impair the stimulatory action of LH-RH on either cyclic AMP accumulation or LH-release. Flufenamic acid on its own caused LH-release, but the drug abolished the effect of LH-RH on cyclic AMP accumulation. The mechanism of this action of flufenamic acid is not understood. It is concluded that the stimulatory action of LH-RH on pituitary cyclic AMP production and LH release is not mediated by prostaglandins.     

Site of negative feedback action of estrogen on gonadotropin secretion in normal women

We have reported that iv administration of conjugated estrogens results in no significant change in the plasma LH-RH level during the negative feedback phase of LH, suggesting that estrogen does not suppress LH by decreasing hypothalamic LH-RH. To determine the site of estrogen action during the negative feedback phase, we studied the pituitary response to a small amount of LH-RH after estrogen administration in normal cyclic women in the mid-follicular phase. The pituitary responses to an iv bolus of 2.5 micrograms of synthetic LH-RH were evaluated by measuring serum LH and FSH 2 h before and 8 h after administration of 20 mg of conjugated estrogens (Premarin). The mean levels of serum LH and FSH were significantly (p less than 0.05) decreased 8 h after the injection. The peak responses of LH and FSH to LH-RH were also significantly (p less than 0.05) reduced after Premarin administration. These findings suggest that the negative feedback effect of estrogen on gonadotropin secretion is caused by its direct suppression on the pituitary response to LH-RH.     

Cytogenetic analysis of mouse oocytes after experimental induction of follicular overripening

Graafian follicle overripening was induced in (1) adult mice by inhibiting the ovulatory discharge of gonadotrophins with antibodies to LH-RH and (2) immature mice by injection of PMSG to promote follicular maturation before the neuroendocrine system was competent to produce an ovulatory stimulus. The numbers of follicles capable of meiotic maturation after exogenous LH were sharply reduced during the period of overripening and there was a corresponding increase in the proportion of cystically enlarged follicles, many of which were undergoing atresia. Freshly ovulated ova were collected after delaying ovulation for 2 days and prepared for cytogenetic study of metaphase chromosomes. The incidence of non-disjunction and other errors was indistinguishable from that of ova collected after spontaneous ovulation during 4- or 5-day cycles.     

Investigation into the hypogonadism of the obese mouse (genotype ob/ob)

Features of the reproductive axis in the genetically hypogonadal, obese mouse (genotype, ob/ob) were examined at 5-8 months of age and compared with those of wild-type litter mates. Hypothalamic concentrations of dopamine and 5-hydroxytryptamine were normal. Those of 5-hydroxyindoleacetic acid, noradrenaline and LH-RH were raised. LH-RH was biologically active. Pituitary concentration of LH was normal, but that of FSH was raised. Serum concentrations of LH and FSH, compared with those of wild-type animals, were normal and low, respectively. Gonad and accessory sex organs weights were reduced. These findings suggest that the release of FSH but not LH is defective in the ob/ob mouse. Preliminary in-vitro experiments indicated that the pituitary gland responded normally or even supernormally towards LH-RH in its release of LH. The defect in the reproductive axis of the obese mouse may be due to inadequate release of LH-RH although an insensitivity of the pituitary gland towards LH-RH in its release of FSH cannot be excluded.