Pulsatile nature of luteinizing hormone release is maintained during luteinizing hormone-releasing hormone stimulation in normal male rats

The plasma LH concentration is believed to be reasonably steady in normal male rats. We found that LH is released in a regular pulsatile fashion. The overall mean concentration of plasma LH in normal male rats was 46.6 +/- 4.4 (mean +/- SEM) ng/ml. The normal male rats showed periodic LH pulses: the mean pulse amplitude was 144.4 +/- 25.5 ng/ml and the inter-peak interval was 22.5 +/- 2.0 min. Each pulse lasted 9.7 +/- 0.8 min. When LH-RH (1 microgram/kg) was injected as a bolus, the peak concentration was attained in 10-30 min reaching a peak concentration of 279.4 +/- 39.6 ng/ml. Distinct pulsatile bursts of plasma LH were discernible during the period of elevated plasma LH concentration. When a higher dose of LH-RH (5 micrograms/kg) was administered, the LH concentration slowly increased to a peak concentration of 400.2 +/- 38.7 ng/ml in 20-40 min. The pulsatile nature of the LH concentration was recognizable with distinct bursts. We have observed that: (a) normal male rats release LH in a pulsatile fashion with an approximate 20-min inter-peak interval; (b) mean LH pulses last less than 10 min, and (c) the LH pulses are visible even with elevated LH and LH-RH concentrations in the general circulation.     

Modulation of luteinizing hormone(LH) release by clonidine and opioid peptides in castrated male rats

The effect of clonidine, a central alpha-adrenergic agonist, on the suppression of LH release induced by beta-endorphin or FK33-824, an endogenous opioid peptide or its synthetic analog, was investigated in castrated male rats, with or without pretreatment with reserpine. Pulsatile LH secretion was inhibited by intravenous injection of FK33-824 (400 micrograms/kg), or intraventricular injection of beta-endorphin (5 micrograms). Without pretreatment with reserpine, intraperitoneal administration of clonidine (1 mg/kg) failed to reverse the inhibition of LH release induced by these peptides. However, with pretreatment with reserpine (10 mg/kg), clonidine abolished the inhibitory effect on LH secretion induced by these peptides in castrated male rats. These data indicate that, unlike the results in ovariectomized, steroid-primed rats, pretreatment with reserpine allows the alpha-adrenergic system to act more peripherally than the opioid neuronal system in a neuronal network-regulating LH release in castrated male rats.     

Synergistic effect of testosterone and of a luteinizing hormone-releasing hormone agonist on androgen receptor content in the ventral prostate of castrated rats

The aim of the present experiment was that of studying the effect of an LHRH agonist analog on the prostatic content of cytosol and nuclear salt-extractable and salt-resistant androgen receptors (AR). Castrated rats were treated for six days with the LHRH agonist WY 40972 (A), with testosterone enanthate (T) or with A plus T. Intact adult male rats and castrated rats treated with the vehicle served as controls. The animals were sacrificed 18 h after the last subcutaneous injection. The ventral prostates were quickly removed and submitted to subcellular fractionation for the determination of cytosol and nuclear AR content. In addition, the weights of the prostates and of the seminal vesicles were recorded, and serum levels of LH and FSH were evaluated by radioimmunoassay. The dissociation constants (Kd) of cytosol and nuclear AR, on the order of 1 x 10(-9) M, were not affected by the various treatments. Conversely, the combined treatment with T and A induced a significant increase of nuclear AR in the prostatic tissue, when compared to the levels found in castrated rats treated with T alone and in intact rats. The treatment with T was able to restore the reproductive organs to their normal weights. The treatment with A inhibited the hypersecretion of gonadotropins induced by castration. The results show that, under the conditions of the present experiment, A exhibits a synergistic effect with T on nuclear AR content in the rat ventral prostate. The results also suggest that A acts directly on this androgen-dependent structure.     

Facilitation of mounting behavior in male rats by intracranial injections of luteinizing hormone-releasing hormone

Luteinizing hormone-releasing hormone (LH-RH) administration has been reported to facilitate male sex behavior. This laboratory has previously reported development of the ‘mounting test’, a paradigm which reflects sexual arousal mechanisms. We have used this test to study the interaction of LH-RH with the central components of male copulatory behavior in the rat.Sixty 90-day-old Long-Evans male rats were screened for sex behavior and divided into 5 treatment groups. For all mounting tests, a local anesthetic was applied to the penis and mounts were scored during a 15-min exposure to a stimulus female. The animals were given 3 successive weekly tests. By the final test, a significant decrement in mounting behavior was noted, and those males given 50 ng LH-RH i.c.v. displayed more mounting in this test than animals given either no treatment or saline ($\text{P < 0.01}$). A slight but significant ($\text{P < 0.05}$) enhancement of performance was also noted in peptide-treated rats in test I. There was no significant difference in any of the tests between animals given lateral cerebroventricular (i.c.v.) injections of 2 μl acidified saline and those given no treatment. When blood samples were taken from similarly treated animals and assayed by radioimmunoassay for luteinizing hormone and testosterone, plasma levels of these hormones were not different at either 30 min or 2 h after injection of saline or LH-RH.Thus, in animals with diminished genital sensory input, LH-RH administration increases mounting behavior without inducing measurable reproductive endocrine changes. It is proposed that this effect results from an interaction of this peptide with the neural substrates of the arousal mechanism.     

Flutamide blocks the self-priming effect of luteinizing hormone-releasing hormone in pubertal male rats

Castration of pubertal or young adult male rats eliminates the self-priming effect of luteinizing hormone-releasing hormone on luteinizing hormone secretion. Testosterone, dihydrotestosterone, or estradiol will maintain this effect in castrated animals. In order to explore the mechanism by which both dihydrotestosterone and estradiol are capable of maintaining the effect, intact rats as well as castrated animals implanted with testosterone capsules were treated with the antiandrogen Flutamide. In both intact animals and castrated rats bearing testosterone-filled Silastic capsules, Flutamide blocked the self-priming effect. These data suggest that the androgen receptor is of primary importance in the maintenance of the self-priming effect.     

Evidence that a decrease in testosterone negative feedback mediates the pubertal increase in luteinizing hormone pulse frequency in male ferrets

Neuroendocrine mechanisms regulating luteinizing hormone (LH) secretion during puberty were investigated in intact male ferrets and ferrets castrated at 8 wk of age that received s.c. implants of either empty or testosterone-filled Silastic capsules. To synchronize puberty onset among individuals, ferrets were exposed to short days between 8 and 16 wk of age, and then transferred to long days. Testis growth began in intact ferrets soon after photoperiod transition. Blood samples were obtained at 11, 15, 19, and 23 wk of age. LH pulse frequency was low in intact ferrets at 11 and 15 wk of age (less than or equal to 0.27 pulses/h), but rose to 0.94 pulses/h by 23 wk of age. No age-related increase in LH pulse frequency was observed in untreated castrated ferrets. LH pulses were rare in testosterone-treated castrated ferrets at 11 and 15 wk of age; but by 23 wk of age, frequency rose to 0.33 pulses/h. Thus, testis maturation in ferrets is accompanied by a dramatic increase in LH pulse frequency. No steroid-independent developmental increase in LH pulse frequency occurs in castrated ferrets. Furthermore, doses of testosterone that prevent LH secretion in young castrated ferrets do not as effectively suppress LH pulses in older ferrets. These data suggest that a decrease in the efficacy of testosterone negative feedback mediates the pubertal rise in LH pulse frequency in male ferrets.     

Cellular observations and hormonal correlates of feedback control of luteinizing hormone secretion by testosterone in long-term castrated male rhesus monkeys

Testosterone at physiological levels cannot exert negative feedback action on LH secretion in long-term castrated male monkeys. The cellular basis of this refractoriness is unknown. To study it, we compared two groups of male rhesus macaques: one group (group 1, n = 4) was castrated and immediately treated with testosterone for 30 days; the second group (group 2, n = 4) was castrated and treated with testosterone for 9 days beginning 21 days after castration. Feedback control of LH by testosterone in group 1 was normal, whereas insensitivity to its action was found in group 2. Using the endpoints of concentrations of aromatase activity (P450(AROM) messenger RNA [mRNA]) and androgen receptor mRNA in the medial preoptic anterior hypothalamus and in the medial basal hypothalamus, we found that aromatase activity in both of these tissues was significantly lower, P: < 0.01, in group 2 compared with group 1 males. P450(AROM) mRNA and androgen receptor mRNA did not differ, however. Our data suggest that the cellular basis of testosterone insensitivity after long-term castration may reside in the reduced capacity of specific brain areas to aromatize testosterone. Because P450(AROM) mRNA did not change in group 2 males, we hypothesize that an estrogen-dependent neural deficit, not involving the regulation of the P450(AROM) mRNA, occurs in long-term castrated monkeys.     

Influence of testosterone on autotomy in castrated male rats

Sex-related differences exist in nociception and gonadal steroids influence the analgesic response in animals and humans. As we have shown previously, estrogen could modify autotomy in female rats using the sciatic nerve transection model. To further characterize the relationship between gonadal steroid and nociception, the role of testosterone on autotomy in sciatic nerve sectioned rats was investigated. Twenty male rats were subjected to orchiectomy (ORX). Then ten rats received subcutaneous sesame oil and the other ten were treated with testosterone propionate in sesame oil (TP; 500 microg/day/rat). All the rats underwent sciatic nerve resection in left hind limb. Degree of self-mutilation was measured daily for 8 weeks. TP reinstatement resulted in significantly lower autotomy scores in orchiectomized rats. The results demonstrated that testosterone could modify the autotomy behavior, an indicator of neuropathic pain, in rats after nerve injury.     

Effects of gonadal steroids on follicle-stimulating hormone and luteinizing hormone secretion by pituitary cells from castrated and intact male rats

The effectiveness of androgens in suppressing gonadotropin secretion declines with time following orchidectomy; however, the mechanism for this acquired resistance to androgen action is unknown. The role of the pituitary was studied by use of perifused rat pituitary cells and cells in monolayer culture. Pituitary cells from 7-wk-old intact male rats and rats that had been castrated 2 wk previously were treated with 10 nM testosterone (T) for 24 h; cells were then packed into perifusion chambers and stimulated with 2.5 nM GnRH for 2 min every hour for 8 h during which time T treatment was continued. T suppressed GnRH-stimulated LH secretion and LH pulse amplitude equally in both groups to approximately 60% of control values. Interpulse LH secretion was unchanged by T in either group. GnRH-stimulated FSH release was suppressed more (p less than 0.05) by T with cells from castrated rats than with cells from intact rats (76 +/- 4% vs. 90 +/- 2% of control; mean +/- SEM). By contrast, the action of T to increase interpulse basal FSH secretion was less (p less than 0.05) with cells from castrated rats (115 +/- 10% of control) than with cells from intact rats (146 +/- 6% of control). T treatment for 72 h also increased basal FSH secretion by pituitary cells in monolayer culture to a lesser extent with cells from castrated rats than with cells from intact rats (151 +/- 14% vs. 191 +/- 16% of control, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Amplitude and frequency modulation of pulsatile luteinizing hormone-releasing hormone release

Summary 1. A variety of neuroendocrine approaches has been used to characterize cellular mechanisms governing luteinizing hormone-releasing hormone (LHRH) pulse generation. We review recentin vivo microdialysis,in vitro superfusion, andin situ hybridization experiments in which we tested the hypothesis that the amplitude and frequency of LHRH pulses are subject to independent regulation via distinct and identifiable cellular pathways.2. Augmentation of LHRH pulse amplitude is proposed as a central feature of preovulatory LHRH surges. Three mechanisms are described which may contribute to this increase in LHRH pulse amplitude: (a) increased LHRH gene expression, (b) augmentation of facilitatory neurotransmission, and (c) increased responsiveness of LHRH neurons to afferent synaptic signals. Neuropeptide Y (NPY) is examined as a prototypical afferent transmitter regulating the generation of LHRH surges through the latter two mechanisms.3. Retardation of LHRH pulse generator frequency is postulated to mediate negative feedback actions of gonadal hormones. Evidence supporting this hypothesis is reviewed, including results ofin vivo monitoring experiments in which LHRH pulse frequency, but not amplitude, is shown to be increased following castration. A role for noradrenergic neurons as intervening targets of gonadal hormone negative feedback actions is discussed.4. Future directions for study of the LHRH pulse generator are suggested.     

Puberty in the male chimpanzee: time-related variations in luteinizing hormone, follicle-stimulating hormone, and testosterone

The onset of pubertal testicular growth (Po) occurred in 12 out of 20 male chimpanzees surveyed monthly for at least 3.7 yr. When animals were synchronized according to Po, the mean weight gain was found to be higher before than after Po, and testicular volume started to rise immediately after Po. The earlier significant hormonal events were a rapid rise in LH and a slight testosterone increase occurring 6 mo before Po. Thereafter, the levels of LH remained elevated while testosterone continued to rise in parallel with the testicular volume. FSH levels increased suddenly at Po, 6 mo after the LH increase. FSH remained elevated for only 9 mo, then dropped to prepubertal levels. The dissociation between onsets of pubertal increases in LH and FSH secretions suggests that the complete reawakening of the hypothalamic-pituitary unit lasts several months. The secondary drop of FSH, occurring at the time of spermarche, may be induced by factor(s) secreted by the testis.     

Serotonin involvement in the inhibition of luteinizing hormone (LH) release during immobilization in castrated male rats

The involvement of serotonin in mediating the inhibitory effect of immobilization stress on LH secretion in castrated male rats was examined by employing p-chlorophenylalanine (PCPA, 320 mg/kg, ip), an inhibitor of serotonin synthesis, and 5,6-dihydroxytryptamine (5,6-DHT, 50 micrograms, icv), a drug toxic to the indoleaminergic system. Immobilization stress suppressed pulsatile LH release and decreased mean plasma LH levels. Pretreatment with PCPA or 5,6-DHT apparently eliminated the inhibitory effect of immobilization stress on LH release. These results suggest the possible involvement of a serotoninergic mechanism in mediating the suppression of LH release induced by immobilization stress in castrated male rats.     

Pituitary responsiveness to luteinizing hormone-releasing hormone in prepubertal and postpubertal male ferrets

The pituitary response to three different doses of exogenously administered LHRH was examined in prepubertal (9-wk-old) and postpubertal (32-wk-old) male ferrets. The doses of 5, 10, and 15 ng LHRH/kg body weight tested in this study produced dose-related increases in circulating LH concentrations in both pre- and postpubertal groups. In addition, a significant effect of age on LH response was observed, with the prepubertal animals demonstrating significantly greater serum LH values in response to the two higher doses than the postpubertal males. Prepubertal ferrets also exhibited a significant increase in endogenous LH pulse amplitude in sampling periods following exogenous administration of LHRH compared to baseline pulse amplitudes in periods prior to the LHRH infusions. These results suggest that the low frequency of endogenous LH pulses previously observed in prepubertal ferrets is not due to unresponsiveness of the pituitary gland to LHRH. Thus, suppression of the hypothalamo-hypophyseal axis observed in the prepubertal ferret is probably mediated at the level of the hypothalamus.     

24-hour changes in circulating prolactin, follicle-stimulating hormone, luteinizing hormone, and testosterone in young male rats subjected to calorie restriction

This work analyzes the effect of calorie restriction on the 24 h variation of pituitary-testicular function in young male Wistar rats by measuring the circulating levels of prolactin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Control animals were provided an equilibrium calorie diet and the experimental animals a calorie-restriction diet equivalent to 66% of food restriction for four weeks starting on day 35 of life. Different groups of control and experimental rats were killed at 6 h intervals around the clock, beginning 1 h after light on (HALO). Compared to the control animals, the mean secretion of prolactin was augmented and that of LH and testosterone decreased in calorie-restricted rats, whereas FSH release remained unchanged. Significant changes in the 24 h secretory pattern of circulating prolactin, LH, and testosterone occurred in the calorie-restricted rats. These include the appearance of a second maximum of plasma prolactin at 21 HALO, blunting of the LH peak seen at 13 HALO, and phase-shift of the testosterone peak from 13 HALO in controls to 17 HALO in calorie-restricted rats. The significant positive correlation between individual LH and testosterone levels found in controls was no longer observed in calorie-restricted rats. Availability of nutrients presumably affects the mechanisms that modulate the circadian variation of the pituitary-gonadal axis in growing male rats.