LH Secretion In Male Rats Research Articles

Dissection of Kisspeptin's Role in the Neuroendocrine Regulation of Gonadotropin Using Antagonists.Robert P. Millar, Ph.D.

Kisspeptin and its cognate receptor (GPR54) are central regulators of reproduction and are key mediators of nutritional, photoperiod, stress and steroid hormone regulation of the GnRH neuron. Kisspeptin also regulates trophoblast invasion and stimulates vasoconstriction. Thus, specific antagonists at GPR54 would be useful as tools to interrogate physiological and patho-physiological roles of kisspeptin. Antagonists would also have potential as therapeutic agents for treating a wide range of clinical conditions. We have systematically substituted single and combinations of amino acids in kisspeptin-10 in a detailed structure-activity-relationship study using receptor binding and inhibition of kisspeptin-10 stimulation of inositol phosphate (IP) mobilisation as outputs. These studies revealed an essential binding pharmacophore and identified residues required for GPR54 activation allowing for the subsequent derivation of a series of antagonists and partial agonists. The most potent antagonists had IC</SUB50>s in the nM range as measured by the inhibition of 10nM kisspeptin stimulation of IP. A selected antagonist potently inhibited (1nM) kisspeptin-10 stimulation of GnRH neuronal firing in mouse brain slices and GnRH secretion in thesus monkeys. It also inhibited kisspeptin-induced LH secretion in male rats and mice. The antagonist inhibited the post-castration LH rise in mice and rats when administered intracerebroventricularly, confirming that inhibition of kisspeptin signalling mediates gonadal steroid negative feedback. The antagonist had no effect on basal LH secretion in male rats and mice possibly indicating a lower tone of kisspeptin input. LH pulse frequency and amplitude were inhibited by intracerebroventricular antagonist administration to ovariectomized ewes. Antagonist also delayed puberty in female rats and inhibited the ovulatory LH surge confirming a role in these processes. In summary, we have developed GPR54 antagonists that provide useful tools to investigate the neuroendocrine regulation of reproduction and for potential therapeutic applications.

Alpha-Melanocyte-Stimulating Hormone through Melanocortin-4 Receptor Inhibits Nitric Oxide Synthase and Cyclooxygenase Expression in the Hypothalamus of Male Rats

There is evidence that α-melanocyte-stimulating hormone (α-MSH) has immunomodulatory and anti-inflammatory actions within the brain. In this study, we tested whether these actions are due to inhibition of the synthesis of nitric oxide (NO) and prostaglandins induced by lipopolysaccharide (LPS). Since melanocortin subtype MC4 receptor has been detected in the hypothalamus, we investigated the effect of central administration of α-MSH and HS024 (a selective MC4 receptor antagonist) on the gene expression of inducible, neuronal and endothelial NO synthase (iNOS, nNOS and eNOS) and on cyclooxygenase (COX-1 and COX-2) expression in the mediobasal hypothalamus (MBH) of LPS-treated male Wistar rats. Peripheral administration of LPS (250 µg/rat, 3 h) induced iNOS and COX-2 gene expression in the MBH. This stimulatory effect was reduced by α-MSH (3 nmol/rat) injected 30 min before LPS. α-MSH and HS024 (1 nmol/rat) alone had no effect on iNOS and COX-2 expression. The action of α-MSH on LPS-induced iNOS and COX-2 mRNA levels was not observed in the presence of HS024, suggesting that MC4-R may be involved in the modulatory effect of α-MSH. None of these treatments produced any modifications in nNOS, eNOS and COX-1 expression in MBH. The increase in serum corticosterone levels induced by LPS was attenuated by α-MSH. Both LPS and α-MSH decreased serum LH and prolactin levels. HS024 failed to modify the inhibitory effects of LPS and α-MSH on prolactin release but reverted the effect of LPS on LH secretion, indicating that MC4-R activation may be involved in the effects of α-MSH on LH secretion in male rats. When we examined the in vitro effect of LPS (10 µg/ml) and LPS plus interferon-γ (IFN-γ, 100 ng/ml) on iNOS expression in MBH, an increase in iNOS mRNA levels was observed only in the presence of LPS + IFN-γ. This stimulatory effect was attenuated in the presence of α-MSH (5 µM), which by itself had no effect. No changes were found in nNOS, eNOS, COX-1 or COX-2 expression. These results indicate that α-MSH reduces the induction of iNOS and COX-2 gene expression at the hypothalamic level during endotoxemia and suggest that endogenous α-MSH may exert an inhibitory tone on iNOS and COX-2 transcription via MC4 receptors acting as a local anti-inflammatory agent within the hypothalamus.

Testosterone-dependent effects of galanin on pituitary luteinizing hormone secretion in male rats.

Galanin is a 29-amino-acid peptide that colocalizes with GnRH in hypothalamic neurons. High concentrations of galanin are present in portal vessel blood of both male and female rats, and galanin receptors are present on gonadotropes in both sexes. Results from studies of female rats indicate that galanin acts at the level of the pituitary to directly stimulate LH secretion and also to enhance GnRH-stimulated LH secretion. The effects of galanin on pituitary LH secretion in male rats are relatively uncharacterized; thus, the present in vivo study was conducted 1). to examine the ability of galanin to affect basal or GnRH-stimulated LH secretion in male rats and 2). to determine whether the effects of galanin on LH secretion in male rats are testosterone-dependent. All three doses of galanin used (1, 5, and 10 micro g/pulse) significantly enhanced GnRH-stimulated LH secretion in intact male rats. Only the highest dose of galanin directly stimulated LH secretion (without GnRH coadministration) in intact males. Galanin did not directly stimulate LH secretion or enhance GnRH-stimulated LH secretion in castrated male rats. In fact, the highest dose of galanin inhibited GnRH-stimulated LH secretion in castrated males. Upon testosterone replacement, the ability of galanin to directly stimulate LH secretion and to enhance GnRH-stimulated LH secretion was restored in castrated males. These results suggest a role for galanin in the regulation of LH release in male rats and demonstrate that testosterone upregulates the ability of the pituitary to respond to the stimulatory effects of galanin.

Effect of corticotropin releasing factor (CRF) injected into the median eminence on LH secretion in male rats.

We determined the dose-response relationship and examined the time-related effect of CRF (corticotropin releasing factor) injected directly into the Median Eminence (ME) on LH secretion in conscious intact and castrated male rats. Doses of 0.25, 0.75, 1, and 1.5 nmol CRF dissolved in 1 microl of saline (or saline only in the controls) were injected into the ME and blood samples collected 30, 60, 90, and 120 min postinjection to determine by RIA serum LH. CRF at doses of 0.75, 1 and 1.5 nmol significantly decreased serum LH in castrated and intact animals. The lower dose of CRF did not decrease LH in the two groups studied. The results suggest that in males as in females, CRF inhibits by itself LH secretion, at least in part, by a central action in the ME; the inhibitory effect of CRF on LH is similar in castrated and intact males; the dose of 0.25 nmoles of CRF was ineffective in decreasing LH and finally that CRF at ME levels may participate in a variety of stress-related responses, including reproduction inhibition, through LH suppression.

Immunocytochemical studies on effects of LHRH on pituitary LH secretion in male rats

It is known that LHRH is able to increase LH storage and release (l), but how about the morphologic indices? It is generally considered that the LH mainly relates to the formation of secretory granules and granular release from the cell (2-6), however, in fact during the LH secretory process besides the changes of granules, the vacuolar structure also undergoes serial cyclic changes. According to our previous observation on granular and vacuolar structural changes during the estrous cyhcle in female rats, it was indicated that the storage and release of LH not only related to the granular changes but also to the vacuolar changes (7,8). In this work the LH release peak induced by synthetic LHRH in the male rat has been specifically studied by attention to the following points: a) to determine the morphological changes after LHRH effects on the pituitary LH secretion in male rats. b) to further confirm the relationship between the changes of vacuolar structure and function after LH secretion.

Influence of acute intracerebroventricular (i.c.v.) administration of adrenocorticotrophin (ACTH) on LH secretion in male rats: effect of pretreatment (i.c.v.) with ACTH antiserum on the serum LH response to an acute ether stress.

Adult male rats with chronic indwelling intracerebroventricular (i.c.v.) and jugular catheters were given an i.c.v. injection (over 1 min) of 1, 10, 100 ng or 1 microgram ACTH(1-24), or 300 ng ACTH(4-10) or saline, and blood samples were taken 0, 5, 15, 30 and 60 min later. Increasing dosages of ACTH(1-24) caused a dose-related rise in serum LH levels. Peak levels of serum LH (ranging from 157 to 473% of pretreatment levels) were reached 5-15 min after treatment, and then serum LH values returned to pretreatment levels by 60 min. The serum LH response to 1 microgram ACTH(1-24) did not differ from the response to 100 ng ACTH(1-24). Administration (i.c.v.) of 300 ng ACTH(4-10) was also effective in increasing serum LH values. Repeated withdrawal of blood during the experiment increased serum corticosterone values in all groups (including saline-treated), but i.c.v. administration of ACTH(1-24) or ACTH(4-10) did not further increase serum corticosterone levels. Two additional groups of rats were injected i.p. with either saline or pentobarbital (30 mg/kg body weight) 1 h before i.c.v. administration of 10 ng ACTH(1-24) and blood samples were taken as in the other groups. The animals in these groups did not show a rise in serum LH concentrations in response to ACTH(1-24). In a third experiment, rats were pretreated (i.c.v.) with either ACTH antiserum (ACTH-Ab) or normal rabbit serum 15 min before a 2-min ether stress. The ether stress evoked a significant rise in serum LH concentrations within 15 min.(ABSTRACT TRUNCATED AT 250 WORDS)

INHIBITION OF STEROID 5α-REDUCTASE IN VIVO: EFFECT ON SUPPRESSION OF LUTEINIZING HORMONE AND STIMULATION OF ACCESSORY SEX ORGANS BY TESTOSTERONE IN THE ORCHIDECTOMIZED RAT

An inhibitor of 5 alpha-reductase, the 17 beta-carboxylic acid derivative of testosterone (testosterone-17 beta CA), has been used to evaluate the importance of the 5 alpha-reduction of testosterone in its action on the suppression of LH secretion in male rats. The potential of testosterone-17 beta CA to inhibit the formation of 5 alpha-dihydrotestosterone (DHT) was first demonstrated in vitro. When homogenates of hypothalami or anterior pituitary glands were incubated with [3H]Testosterone in the presence of a 50-fold excess of testosterone-17 beta CA, the formation of labelled DHT was inhibited by more than 80%. Adult male rats that had been castrated for 1-2 months were fitted with chronic intravenous catheters and implanted with silicone elastomer sheets: one group received one sheet, 0.5-2.0 cm2 in size containing 1.6% testosterone, a second group received one 50 cm2 sheet containing 1.6% testosterone-17 beta CA and a third group received two sheets, one sheet 50 cm2 in size containing 1.6% testosterone-17 beta CA and the second ranging in size from 0.5 to 2.0 cm2 and containing 1.6% testosterone. Blood was withdrawn daily from each rat over a 4-5 day period after implantation of the steroids and the level of LH in the plasma was measured by radioimmunoassay. The seminal vesicles and the ventral prostate gland were removed at autopsy on day 4 or 5; the weights of these organs were shown to have increased progressively as the size of the implant of testosterone increased. In contrast, the level of LH in the plasma was suppressed to a comparable extent by implants of testosterone between 0.6 and 2 cm2, whereas a 0.5 cm2 implant of testosterone had no effect. Implants of testosterone-17 beta CA alone did not influence the weight of the accessory organs or the level of LH. When testosterone-17 beta CA and testosterone were implanted together, the growth-promoting effect of the latter on the accessory sex organs was significantly reduced. The effectiveness of testosterone in suppressing the level of LH in the plasma of these animals was not influenced by the presence of testosterone-17 beta CA and in certain instances the level was raised.