Effect of an inhibitor of aromatization, 1,4,6 androstatriene-3,17-dione (ATD) on LH release and steroid binding in hypothalamus of adult female rats.

Prevention of testosterone aromatization in the female rat pups by perinatal treatment with 1,4,6 androstatriene-3,17-dione (ATD) induces an important defeminization as shown by a reduction of fluctuations of LH release after castration and estradiol implantation. The fact that, under our in vitro experimental conditions, ATD is able to displace testosterone binding in the hypothalamus whereas estradiol does not, confirms the hypothesis that ATD acts on aromatase. The most attractive explanation for the defeminization effect of ATD is then an estrogen-like action of ATD.

The effect of ovine pineal compounds prepared under red or green light on the activity of male rat anterior pituitaries in vitro.

A high molecular weight fraction XM100R (MW A 100,000) was prepared by ultrafiltration from ovine pineals using two different extraction methods under red light conditions (lambda greater than 600 nm). This fraction stimulates the release of radioimmunologically active luteinizing hormone (LH) of anterior pituitaries in vitro. The ultrafiltration fraction PM30R (MW greater than 30,000 and less than 100,000) was found to be radioimmunologically active only when the "Bensinger" extraction procedure was applied. However, when comparable fractions were prepared under green light and incubated with half-pituitaries, all the incubation media of the ultrafiltrated fractions, XM100R, PM30R, PM10R (MW greater than 10,000 and less than 30,000) UM2R (MW greater than 1000 and less than 10,000), UM05R (MW greater than 500 and less than 1000) and UM05F (MW greater than 500), reacted with anti-LH. This may mean that under green light conditions the high molecular weight ovine pineal compounds in XM100R are disintegrated and/or split up into small molecules which can stimulate the release of LH, or crossreact with the anti-LH serum.

Biphasic pattern of follicle stimulating and luteinizing hormone responses to gonadotropin-releasing hormone in vitro.

Increasing concentrations of LHRH or its active analog des-gly10 (D-ala6) LHRH induced a bimodal pattern of FSH and LH release from incubated male rat pituitaries. A low amplitude response (40% increase of LH over baseline levels) was observed for concentrations of LHRH and the agonist in the range of 10(-12) and 10(-13) M respectively. After a plateau of gonadotropin stimulation, a further high amplitude response (180-240% increase over baseline levels) occurred between 3.10(-10) and 10(-8) for LHRH and 3.10(-11) and 3.10(-9) M for des-gly10 (D-ala6) LHRH. Corresponding half maximal effective concentrations (ED50) were 2 and 0.3 nM respectively. Stimulation of FSH release closely paralleled that of LH. The low amplitude, high apparent affinity response was only obtained when the peptides were diluted in low concentrations of acidic tissue extracts. A preliminary study indicated that this method of dilution minimized peptide loss by adsorption. In addition, the low amplitude, high affinity response was never observed on pituitaries sampled from castrates. These experiments suggest the presence of two distinct populations of LHRH recognition sites on pituitary gonadotrophs. Under our experimental conditions, the appearance of the higher affinity response was dependent upon prior exposure to sex steroids. This could be due to a direct action of the steroid on the expression of a high affinity LHRH receptor.

Importance of perinatal testosterone in sexual differentiation in the male rat.

Testosterone secretion in the male rat was high during the late fetal and immediate postnatal periods. It then showed a rapid decrease 3h after birth and remained low until puberty. Male rats from mothers given daily injections of an antibody to testosterone during the week before delivery displayed an LH peak when they were adult, orchidectomized and implanted with oestradiol. However, the amplitude of the peak was far smaller than in female rats from the same mothers treated in the same manner. Thus, the critical period during which testosterone triggers hypothalamic sexual differentiation is very close to birth, possibly starting at the end of the fetal period.

Effect of neonatal administration of steroids or gonadectomy upon oestradiol-induced luteinizing hormone release in rats of both sexes.

Implantation of oestradiol into adult rats of both sexes induced different patterns of LH secretion depending on the time at which gonadectomy or testosterone injection were performed. Castration 2 h after birth allowed an LH peak to occur daily at 18.00 h, but its amplitude was lower than that of adult gonadectomized female rats treated with oestradiol. Castration 24 h after birth elicited two kinds of response; a circadian discharge of LH lower than that of male rats gonadectomized 2 h after birth or a steady low level of LH. The LH rhythmicity induced by implantation of oestradiol was not seen after castration at 8 weeks of age. Neonatal administration of testosterone to female rats prevented the LH peak induced by oestradiol that was seen in adult ovariectomized rats. Neonatal or adult ovariectomy did not interfere with the rhythmical response of LH after implantation of oestradiol. Thus, it is concluded that sexual differentiation of the hypothalamus is primarily of masculine origin.

Interaction of the anti-estrogen CI-628 and estradiol on plasma LH and hypothalamic LH-RH in the female rat.

The effects of the anti-estrogen Ci-628 have been tested on both plasma LH and hypothalamic LH-RH content in ovariectomized or ovariectomized, estradiol (E2)-implanted rats, in order to correlate those parameters with the [3H]E2 retention in the hypothalamus and the pituitary. Incresing doses of CI-628 induced a dose-dependent inhibition of [3H]E2 retention in both cytosolic and nuclear fractions of the pituitary. In contrast, hypothalamic retention of [3H]E2 is only inhibited significantly with higher doses of CI-628 (2.4 and 24 mg/kg). In ovariectomized rats, only a high dose of CI-628 (24 mg/kg) is able to decrease elevated LH levels observed following castration. In the presence of E2, CI-628 has both estrogenic and anti-estrogenic properties on LH secretion. CI-628 acts at the pituitary level to decrease the tissue sensitivity to LH-RH, but has no effect on mediobasal hypothalamic (MBH) LH-RH content.

Circadian rhythm of luteinizing hormone secretion in the ovariectomized rat implanted with oestradiol.

Implantation of a solid source of oestradiol into ovariectomized rats produced constant plasma concentrations of the hormone over a long period of time. Under these conditions, LH is released in a circadian pattern with a very marked peak in the afternoon. This circadian rhythm is synchronized to the light--darkness cycle, since it follows exactly a shift in the nycthemeral cycle. The first peak appeared on day 3 after placement of the oestrogen implant; its amplitude was constant from days 3 to 9 after implantation, and decreased gradually during prolonged implantation. The afternoon peak was not correlated with changes in the pituitary sensitivity to exogenous LH releasing hormone (LH-RH), since the LH response to increasing doses of the peptide could be superimposed in the morning and in the afternoon. However, the decreased amplitude of the rhythm observed after more than 9 days of implantation seemed to depend upon a progressive desensitization of the pituitary gland to LH-RH. Pituitary LH content also decreased as a function of implantation time. It is concluded that, under conditions of constant plasma oestradiol concentrations and of constant pituitary sensitivity to LH-RH, a daily activation of the neural trigger releasing pituitary gonadotrophins occurs.

Brain serotonin and estradiol retention in the hypothalamus and pituitary of the rat.

In order to investigate whether the capacity of hypothalamic and anterior pituitary tissue to concentrate and retain estradiol is affected by serotonin (5-HT), 3H-estradiol (3HE2) retention in these structures was measured after 5-HT synthesis inhibition by either parachlorophenylalanine (PCPA) or 6-fluoro-tryptophane (6 FTrp), or after destruction of midbrain raphe nuclei containing 5-HT cell bodies, as well as after administration of the 5-HT precursor 5-hydroxytryptophane (5-HTP). No modification in 3HE2 retention was observed after tryptophane hydroxylase inhibitors of raphe lesions; administration of the precursor only increased the steroid retention at very high, nonphysiological dose levels. It is concluded that the interaction of 5-HT with gonadotropic release cannot be accounted for by a direct effect on specific estrogenic receptors, but occurs at a different level of gonadotropic release regulating structures or directly on LH-RH neurons.

Effects of two estradiol antagonists upon the estradiol uptake in the rat brain and peripheral tissues.

Two estradiol antagonists, Parke-Davis CI 628 and CI 680, were studied for their inhibitory potency against labeled estradiol uptake within the hypothalamus, cerebral cortex, pituitary and uterus of the ovariectomized rat. Both drugs, tested from 15 min to 24 h prior to estradiol, induced a fast and long-lasting decrease of the hormone uptake. Three degrees of inhibition were observed depending on the tissue studied: (a) the most important inhibition concerned uterus and pituitary, where the estradiol uptake was prevented by up to 90% by the largest doses of antagonists used (6.3 and 6.1 mg, respectively, for CI 268 and CI 680); (b) in the hypothalamus, the estradiol uptake was counteracted to a lower degree; the inhibition being 39% for anterior hypothalamus and 22% for medial posterior hypothalamus; (c) cerebral cortex uptake was completely unaffected by pretreatments with the antagonists. After subcellular fractionation it was observed that the nuclear uptake of estradiol was completely abolished in both hypophyseal and hypothalamic tissues if the hormonal injection was preceded by the administration of 6.3 mg of CI 628.