Age-related decreases in serum gonadotropin levels and gonadotropin-releasing hormone gene expression in the medial preoptic area of the male rat are dependent upon testicular feedback

In the male rat, age-associated reproductive decline is thought to be due in part to diminished GnRH secretion. We tested the hypothesis that the age-related decrease in GnRH secretion is due to decreased GnRH gene expression by comparing GnRH mRNA and peptide content in the anterior forebrain of intact young and old male rats. Since sex steroids modulate GnRH secretion, we also determined hypothalamic-pituitary responsiveness to removal of testicular feedback by comparing GnRH mRNA and gonadotropin levels in intact and orchidectomized young and old rats. In an initial study, 10 20-micron coronal sections from the medial preoptic area (MPOA) were anatomically matched and compared in intact young (3-month-old) and old (24-month-old) male F344 rats (n = 5/group). In another group of young and old male rats (n = 8-12/group), animals were randomly assigned to be either orchidectomized or sham operated. Rats were killed 21 days after surgery, and comparisons were made in 12 anatomically matched sections of MPOA and 4 matched sections of diagonal band of Broca. In both studies, GnRH mRNA was quantitated by in situ hybridization, using a 35S-labeled oligodeoxynucleotide probe complementary to rat prepro-GnRH mRNA and a computerized image analysis system. In a third study, GnRH content was measured by RIA in microdissected regions of the arcuate nucleus and median eminence in intact 3- and 24-month-old male rats (n = 10 and 8, respectively). Serum LH, FSH, and testosterone (T) levels were measured by RIA in trunk blood of all animals. The number of neurons expressing the GnRH gene in the MPOA was significantly lower in sham-operated old rats (mean +/- SEM, 10.5 +/- 0.5 cells/section) than in young rats (13.7 +/- 0.7 cells/section; P less than 0.01), while cellular GnRH mRNA content was unchanged with age (103 +/- 1 vs. 103 +/- 2 grains/cell). Similar results were obtained in intact old and young rats. GnRH peptide content was significantly decreased in the arcuate nucleus of intact old (0.5 +/- 0.08 ng/mg protein) compared to young animals (2.3 +/- 0.7 ng/mg protein; P less than 0.05), with a trend toward a decrease in the median eminence of old (53 +/- 2 ng/mg protein) vs. young rats (69 +/- 7 ng/mg protein; P = 0.06).(ABSTRACT TRUNCATED AT 400 WORDS)     

Short-term starvation decreases POMC mRNA but does not alter GnRH mRNA in the brain of adult male rats.

Dietary restriction reduces circulating gonadotropin and testosterone levels in male rats, an effect thought to be mediated through reduced gonadotropin-releasing hormone (GnRH) secretion; however, the cellular mechanisms subserving this response are still unknown. We reasoned that if dietary restriction reduces GnRH secretion, this would be reflected by a decrease in GnRH synthesis and likewise cellular GnRH mRNA levels. We tested this hypothesis by comparing cellular levels of GnRH mRNA between ad libitum fed (n = 4) and starved (n = 4) adult male rats. Five days of starvation resulted in a 21% decrease in body weight and an 85% decline in serum testosterone levels (fed: 13.9 +/- 2.00 vs. starved: 2.1 +/- 0.70 nmol/l; p < 0.01). In situ hybridization and image analysis demonstrated that short-term starvation influenced neither GnRH cell number (fed: 148 +/- 16 vs. starved: 157 +/- 13 cells) nor cellular GnRH mRNA signal level (fed: 177 +/- 5 vs. starved: 160 +/- 7 grains/cell) in any region of the basal forebrain. Endogenous opioid peptides are known to exert an inhibitory effect on GnRH secretion and have been implicated in having a role in the starvation-induced effects on the reproductive system. We therefore also tested the hypothesis that alterations in proopiomelanocortin (POMC) gene expression are involved in the neuroendocrine response to starvation, by comparing cellular POMC mRNA levels in individual neurons (approximately 160 neurons/animal) of the arcuate and periarcuate nuclei between fed control (n = 4) and starved (n = 4) adult male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testosterone regulation of proopiomelanocortin messenger ribonucleic acid in the arcuate nucleus of the male rat

GnRH regulates the secretion of LH and FSH, which stimulate the secretion of testicular hormones. Acting in a reciprocal fashion, these hormones, including testosterone and inhibin, exert a negative feedback effect on GnRH and gonadotropin secretion. Endogenous opioid peptides (EOPs) have been implicated to play a role in steroid-mediated regulation of gonadotropin secretion. In this context, certain steroid hormones (e.g. testosterone) increase EOP activity and ultimately inhibit GnRH secretion; however, the cellular mechanism by which this occurs is unknown. beta-Endorphin is one of these EOPs, and it is derived from a larger precursor molecule, POMC. We tested the hypothesis that testicular hormones and testosterone, in particular, stimulate POMC gene expression in the arcuate nucleus of the male rat brain. First, we compared POMC mRNA levels between intact and castrated male rats. Adult male rats were killed 4 days (n = 4) and 21 days (n = 5) after castration. Intact animals (sham-operated; n = 6) were used as controls. Using in situ hybridization and a computerized image analysis system, we measured the POMC mRNA content in individual cells of the arcuate nucleus. POMC mRNA signal was significantly lower (P less than 0.0003) in both 4-day (126 +/- 2 grains/cell) and 21-day (117 +/- 5 grains/cell) castrates than in controls (142 +/- 2 grains/cell). In a second experiment we tested whether testosterone would reverse the castration-induced loss of POMC message. Again, we castrated animals and immediately implanted them with either empty (sham; n = 6) or testosterone-containing Silastic implants (n = 5) of a size that would deliver physiological levels of testosterone (3.6 +/- 1.5 ng/ml). We observed that testosterone-treated animals had significantly higher levels of POMC mRNA signal (121.8 +/- 3.8 grains/cell) than sham-treated castrates (111.4 +/- 3.6 grains/cell; P less than 0.03) and that the testosterone-treated castrates had POMC mRNA signal levels indistinguishable from those of intact controls (122.0 +/- 1.1 grains/cell). These observations lend credence to the theory that one mechanism by which testosterone may regulate GnRH secretion is by increasing the synthesis of POMC in the arcuate nucleus.     

Pubertal changes in gonadotropin-releasing hormone and proopiomelanocortin gene expression in the brain of the male rat

Pubertal development in mammals is in part attributable to a brain-dependent process, whereby increased pulsatile GnRH secretion leads to the awakening of the entire reproductive system. However, the brain mechanisms controlling this event are unknown. The apparent increase in GnRH secretion at puberty could reflect an autonomous change in the activity of GnRH neurons themselves or in the afferent networks leading to GnRH neurons. If there were a significant increase in the secretion of GnRH with puberty onset, we hypothesized that there would be a commensurate increase in the biosynthetic capacity of GnRH neurons to meet the increasing demand. We tested this hypothesis by comparing the level of cellular prepro-GnRH mRNA (GnRH mRNA) observed between prepubertal (25-day-old; n = 5) and adult (75-day-old; n = 4) male rats by in situ hybridization. We detected no significant change with puberty in GnRH mRNA signal levels in any of the anatomical areas examined, which included the vertical limb of the diagonal band of Broca, medial septum, lateral preoptic area, and medial preoptic area. Given the variance of our analytical technique, we determined that there was a greater than 90% probability that we would have detected a 20% increase in GnRH mRNA had there been one. Endogenous opioid peptides have been implicated in timing the onset of puberty in the rat, with the argument being that a loss in opioid tone could effect a disinhibition of GnRH secretion. One opioid peptide, beta-endorphin, is among several peptides cleaved from the precursor POMC. We hypothesized that with puberty, POMC neurons in the arcuate nucleus would have an attenuated capacity to produce beta-endorphin. We tested this hypothesis by comparing cellular pre-POMC mRNA (POMC mRNA) levels in the arcuate nuclei of prepubertal (n = 6) and adult (n = 7) male rats with in situ hybridization. We observed an increase in POMC mRNA levels with puberty; prepubertal rats had relative POMC mRNA signal levels of 119 +/- 10 grains/cell, while adult rats contained 167 +/- 12 grains/cell (P less than 0.02). This increase in cellular POMC mRNA was confined to the rostral portion of the arcuate nucleus. We conclude that the GnRH gene is fully expressed well before the time of normal puberty onset and that the increase in POMC mRNA that occurs with the onset of puberty may be important for the development of pulsatile GnRH secretion.     

The suppression of pulsatile luteinizing hormone secretion during lactation in the rat

The inhibition of LH secretion during lactation may be the consequence of a pituitary insensitivity to GnRH stimulation and/or an inhibition of GnRH release from the hypothalamus. To assess the contribution that these mechanisms may make to the suppression of LH secretion during lactation, we described the pattern of LH secretion in lactating rats and the magnitude of LH secretion in response to a GnRH stimulus. We assessed the effect of the strength of the suckling stimulus (two and eight pups), the length of lactation (5 and 10 days), and the presence of the ovaries on the pattern of LH secretion. We also examined the pattern of LH secretion after removal of a large suckling stimulus. In the intact rat, the pattern of LH secretion during lactation was uniformly nonpulsatile, despite significant differences between animals suckling two and eight pups in pituitary responsiveness to GnRH. In intact rats suckling two pups during day 10 of lactation, significant LH secretion was stimulated by 0.4-ng pulses of GnRH every 50 min, while animals with eight pups secreted little LH in response to the same stimulus. It was concluded that a two-pup suckling stimulus was sufficient to completely suppress pulsatile GnRH release without affecting pituitary function, whereas an eight-pup suckling stimulus also depressed pituitary sensitivity to GnRH. In ovariectomized (ovx) rats suckling two pups, seven of nine animals showed no postcastration rise in LH secretion or evidence of pulsatile LH secretion during day 5 of lactation. In the remaining two animals, a castrate pattern of pulsatile LH secretion was observed, with a LH interpulse interval of 31 +/- 6 min. By day 10 of lactation, all animals suckling two pups had castration patterns of LH secretion, with a LH interpulse interval of 35 +/- 2 min, which was significantly different from the LH interpulse interval of 26 +/- 1 min observed in ovx animals without pups. Therefore, a two-pup suckling stimulus is capable of retarding the increase in LH pulse frequency characteristically seen in the rat after castration. In ovx rats suckling eight pups, the postcastration rise in LH secretion was completely inhibited in all animals examined on days 5 and 10 of lactation, and the pattern of LH secretion was uniformly nonpulsatile. A consistent pattern of pulsatile LH secretion was not reinitiated until 72 h after removal of the suckling stimulus (LH interpulse interval, 31 +/- 2 min).(ABSTRACT TRUNCATED AT 400 WORDS)     

Regional specificity of testosterone regulation of proopiomelanocortin gene expression in the arcuate nucleus of the male rat brain

Endogenous opioid peptides have been implicated as mediators in the negative feedback action of gonadal steroids on GnRH secretion. We have previously demonstrated that testosterone stimulates POMC gene expression in neurons of the arcuate nucleus. However, the wide distribution and variety of actions attributed to the numerous arcuate POMC neurons suggest that these cells may be heterogeneous in their responsiveness to steroid hormones. We tested the hypothesis that testosterone modulates a select population of POMC neurons within the arcuate nucleus of the adult male rat by comparing POMC mRNA signal levels throughout the arcuate nucleus of intact, castrated, and castrated testosterone-replaced adult males. Adult male rats were castrated and implanted (sc) with a Silastic capsule (30 mm) that was either empty (n = 6) or filled with crystalline testosterone (n = 5). Control sham-operated animals (n = 6) were left intact. In each animal the arcuate nucleus was divided into four equal rostral-caudal areas within which we measured POMC mRNA content in individual cells. We report that the effects of castration and testosterone replacement are observed in POMC neurons located in the most rostral region of the arcuate nucleus. After castration, POMC mRNA content was reduced in cells of the most rostral arcuate area (intact, 152 +/- 5 grains/cell; castrate, 119 +/- 2 grains/cell; P less than 0.0005), and replacement with physiological levels of testosterone prevented the decline in POMC mRNA levels so that they remained equivalent to that of the intact animal (castrated testosterone-replaced, 153 +/- 6 grains/cell). There was no significant difference in POMC mRNA signal between intact and castrated testosterone-replaced animals in the most rostral area. POMC neurons in the more caudal aspect of the arcuate (75% of the nucleus) were unaffected by the treatments; alternatively, it is possible that a real change in POMC message content in a subpopulation of cells was obscured by larger numbers of nonresponding cells within the same tissue sections. Based on these observations we conclude that there is a heterogeneous population of POMC neurons in the arcuate nucleus and that testosterone regulates POMC gene expression in a select group of these cells located in the rostral portion of the arcuate nucleus.     

Regulation of hypothalamic gonadotropin-releasing hormone secretion in experimental uremia: in vitro studies.

Defective regulation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion is the primary defect leading to the inhibition of pituitary gonadotropin secretion and its consequences such as androgen deficiency and infertility in experimental uremia. Previous studies using indirect methods to study presumptive GnRH release and the function of GnRH-secreting neurons have suggested functional disturbances of GnRH neurosecretion; however, the precise biochemical mechanisms involved were not defined. Therefore, in order to clarify the mechanisms of aberrant regulation of hypothalamic GnRH secretion in experimental uremia, we examined basal secretion of GnRH from mediobasal hypothalamus (MBH) in vitro and the GnRH-secretory responses to naloxone, an opiate receptor antagonist in experimental uremia. Using a static incubation system, adult male rats, either intact or castrate, with subtotal nephrectomy demonstrated a significant reduction of GnRH secretion by 25% in intact and by 40% in castrate uremic male rats compared with their nonuremic controls. In contrast, hypothalamic GnRH content of uremic animals was increased significantly (6% in intact and 14% in castrate uremic rats). Despite the fall in basal GnRH release from MBH, the MBH GnRH release response to in vitro stimulation by an opioid blocker (naloxone) and a membrane-depolarizing agent (veratrine) were not diminished in uremic male rats. These findings suggest that the inhibition of pituitary gonadotropin secretion in experimental uremia is likely to be due to a functional defect in suprahypothalamic regulation of GnRH secretion rather than an intrinsic defect in the GnRH-secreting neurons. Further studies are required to clarify the nature of the neuromodulator interactions involved.     

Mechanisms of age-related changes in gonadotropin-releasing hormone receptor messenger ribonucleic acid content in the anterior pituitary of male rats.

To determine the mechanism(s) of age-related changes in gonadotropin release from pituitary gonadotrophs in male rats, we measured the number of GnRH (gonadotropin-releasing hormone) receptor containing cells and expression of GnRH receptor mRNA per cell in the anterior pituitary. An in situ hybridization procedure was performed using young (six months) and old (24-25 months) Wistar rats. An image analysis system was employed for the autoradiographic analysis. The number of pituitary cells increased during aging (approximately 45%, p < 0.01). On the other hand, the number of GnRH receptor mRNA-containing cells decreased (approximately 25%, p < 0.05). The percentage of these cells in old rats decreased to less than a half of that in young animals (p < 0.01). GnRH receptor mRNA per cell in old rats was only 7% lower than in young (p < 0.01). These results suggest that loss of pituitary gonadotroph GnRH receptors and response is primarily due to the loss of gonadotrophs, and that the death mechanism(s) are responsible for decreased stimulation of Gn release during aging.     

Effects of orchidectomy on gonadotropin and inhibin subunit messenger ribonucleic acids in the pituitary of the rhesus monkey (Macaca mulatta).

Our research programs required the preparation of hypophysectomized and orchidectomized rhesus monkeys. This afforded us the possibility to characterize and compare levels of the gonadotropin and inhibin subunit mRNAs in pituitaries from intact and castrate monkeys. Eighteen adult male monkeys, four of which had been bilaterally orchidectomized 5-9 months previously, were used in this study. Plasma concentrations of LH and FSH were, respectively, 188.5 +/- 5.3 and 246.8 +/- 25.2 ng/ml in the castrate monkeys and 25.8 +/- 4.5 and 4.1 +/- 1.1 ng/ml (mean +/- SEM) in the intact animals. Total pituitary RNA was hybridized to cDNA probes for cynomolgus monkey gonadotropin subunits (FSH beta, LH beta, and the common alpha-subunit) and for human inhibin subunits (alpha, beta B, and beta A) by Northern blot analysis, and mRNA levels were normalized by subsequent hybridization to cyclophilin. Each of the gonadotropin subunit probes hybridized to a single RNA species with the approximate sizes of 1.6 kilobases (kb; FSH beta), 0.7 kb (LH beta), and 0.8 kb (alpha). Levels of LH beta and alpha-subunit mRNAs in pituitaries from castrate monkeys were about 5- and 2-fold higher, respectively, than those in pituitaries from intact monkeys. FSH beta mRNA, on the other hand, was elevated about 27-fold in castrate monkeys [mean +/- SEM, 3176 +/- 408 cpm bound (n = 4 castrate) and 116 +/- 30 cpm bound (n = 8 intact]). Inhibin beta B-subunit mRNA was present in the monkey pituitary as a doublet of about 5 kb, and it was approximately twice as abundant in intact pituitaries as in castrate pituitaries. Hybridizations involving inhibin beta A cDNA revealed a faint band in the region expected for monkey beta A mRNA (6.5 kb) in three of six RNA samples from intact monkeys and a 0.3- to 0.4-kb mRNA species. mRNA encoding the inhibin alpha-subunit was undetectable by Northern blot hybridization. These results indicate that the postpubertal testis imposes an inhibition on the expression of the genes encoding FSH beta, LH beta, and glycoprotein hormone alpha-subunit and that this suppression of the FSH beta gene in the monkey is much greater than that in the rat. In addition, the monkey pituitary may be a source of activin, which may act locally to modulate FSH gene expression and secretion.     

Release of gonadotropin-releasing hormone from the Japanese quail hypothalamus in vitro

The regulation of GnRH (gonadotropin-releasing hormone) secretion in Japanese quail was studied by maintaining excised hypothalamic slices containing the median eminence region in a continuous flow superfusion system. GnRH released into the superfusate was measured by radioimmunoassay using an antibody generated against synthetic GnRH. GnRH secretion increased in a dose-response manner when hypothalamic slices from female quail were exposed to superfusion medium containing elevated potassium ion concentrations. The potassium-induced GnRH release was found to be calcium dependent. GnRH was also increased when either 1 × 10^?6M norepinephrine, epinephrine, or 1 × 10^?5M of the β-adrenergic agonist, isoproterenol was added to the superfusion medium. GnRH secretion from hypothalami of castrate and intact male quail was measured on Days 1, 10, and 21 of photostimulation and was elevated on Day 21. No differences were observed between the castrate and intact groups. It is concluded that GnRH is released from the quail hypothalamus in a manner analogous to mammals and that its release is subject to adrenergic stimulation.     

A surge of gonadotropin-releasing hormone accompanies the estradiol-induced gonadotropin surge in the rhesus monkey.

In several species, the ovulatory LH surge is preceded by a surge of GnRH. Although a role for estradiol in the initiation of the LH surge is well established in the primate, several observations in the rhesus monkey have questioned whether such an estradiol-induced neurosecretory event takes place. We report on GnRH measurements in cerebrospinal fluid (CSF) samples obtained from the third ventricle of intact and ovariectomized (OVX) conscious rhesus monkeys during control periods and throughout the estradiol-induced positive feedback phase. In the first experiment, we measured control GnRH concentrations in CSF collected at 15-min intervals uninterruptedly for a period of 1-5 days in tethered OVX monkeys (n = 4) in their cages without steroid priming. As had been demonstrated previously with the same method in restrained animals, CSF from the third ventricle contained detectable amounts of GnRH. Spontaneous GnRH secretion was pulsatile; overall mean pulse interval was 67.4 (+/- 2.2 SE) min for a total of 177 GnRH pulses. During 2 periods (8 and 6 h) when simultaneous blood and CSF samples were obtained, 14 out of 15 GnRH pulses were accompanied by an LH pulse. To evaluate the effects of an estrogen challenge on GnRH secretion, estradiol benzoate (E2B; 330 micrograms) was given to 4 intact (5 experiments) and to 2 OVX monkeys. CSF collection was initiated 8-24 h before E2B injection and continued for 72-84 h thereafter. E2B administration resulted in a surge of LH and of GnRH in all but one experiment. The mean time of onset of the GnRH surge was 22.0 (+/- 4.0) h after E2B, whereas that of the LH surge was 24.7 (+/- 3.4) h. In contrast to LH, which declined after a peak at 35.2 +/- 3.9 h, the increase in GnRH secretion persisted throughout most of the observation period. The magnitude of the GnRH response differed in the 2 groups; in the intact animals, mean peak GnRH concentration increased 8.9-fold but only 3.8-fold in the OVX monkeys. A similar GnRH surge was observed in 1 OVX monkey, receiving an iv infusion of E2, which produced more physiological concentrations of E2. In this animal, an initial suppression of GnRH concentration in the 24-48 h period after E2 (GnRH control, 14.6 +/- 1.9; post-E2, 4.0 +/- 0.5 pg/ml) preceded the initiation of the GnRH surge which occurred at 54 h after E2.(ABSTRACT TRUNCATED AT 400 WORDS)     

A gamma-aminobutyric acidB agonist reverses the negative feedback effect of testosterone on gonadotropin-releasing hormone and luteinizing hormone secretion in the male sheep

Infusion of baclofen, a GABA(B) agonist, into the medial basal hypothalamus (MBH) of castrated rams rapidly increases LH pulse amplitude without altering pulse frequency. The objectives of this study were to determine whether baclofen infusion increased LH in testosterone (T)-treated and intact rams, the increased LH was due to increased GnRH release, and FSH secretion also was increased. In the first experiment we tested the main effects and interaction of baclofen and T on FSH and LH pulse patterns in castrated rams (n = 7). In the second experiment we determined whether baclofen affected GnRH and LH pulses in intact males. Microdialysis guide cannulae were implanted bilaterally into the MBH. After recovery of the animal from surgery, the MBH was perfused using concentric microdialysis probes (2-mm tip) with artificial cerebrospinal fluid (aCSF) for a 3-h control period followed by either aCSF or 1 mM baclofen for 4 h. Blood samples were taken at 10-min intervals. T suppressed mean LH concentrations (10.4 +/- 1.3 vs. 3.3 +/- 1.3 ng/ml) such that LH pulses were undetectable in some T-treated animals during the control period. The change (control period vs. drug infusion period) in mean LH was greater in response to baclofen than in response to aCSF and was not altered by T. The baclofen x T interaction was nonsignificant. Mean FSH was decreased by T, but was not altered by baclofen. In the second experiment hypophyseal portal blood was collected coincident with microdialysis. Infusion of baclofen into the MBH of intact males (n = 7) resulted within 1 h in the onset of frequent and robust GnRH pulses (0.10/h before baclofen vs. 1.57/h after baclofen) that were followed either immediately or gradually by coincident LH pulses. One interpretation is that baclofen acts downstream of the site of action of T. GABA(B) receptors may regulate pulse amplitude in both the presence and absence of T and regulate pulse frequency by modulating the inhibitory effect of T.     

Hyperprolactinemia inhibits gonadotropin-releasing hormone (GnRH) stimulation of the number of pituitary GnRH receptors

Gonadotropin secretion is diminished in the presence of hyperprolactinemia, and previous studies have shown that PRL can reduce GnRH secretion and impair LH responses to GnRH. To investigate the mechanisms of the inhibitory effects of PRL on the pituitary, we administered intraarterial pulse injections of GnRH (25 ng/pulse every 30 min) to castrate testosterone-implanted male rats placed in restraint cages. Serum PRL, GnRH receptor (GnRH-R), and LH responses to GnRH were measured at intervals over 72 h. In control animals which received saline pulses, serum PRL was transiently elevated to the range of 100-150 ng/ml during the first 24 h, GnRH-R remained stable (approximately 300 fmol/mg protein) and serum LH was low (less than 10 ng/ml) throughout the 72 h. GnRH pulses in castrate testosterone-implanted animals increased GnRH-R to values (approximately 600 fmol/mg) similar to those in castrate controls (no testosterone implant, saline pulses) through 48 h, but GnRH-R declined to baseline values by 72 h in both groups. Serum LH responses to GnRH pulses were only present at 24 h. Administration of bromocriptine throughout the 72 h to immobilized castrate rats or to castrate testosterone-replaced animals treated with GnRH pulses suppressed serum PRL, and GnRH-R concentrations remained elevated through 72 h. Serum LH responses to GnRH pulses were 5- to 20-fold higher in bromocriptine-treated rats, and responses were present throughout the 72 h of the experiment. Delaying the start of bromocriptine treatment until 36 h (after the spontaneous PRL peak) resulted in reduced GnRH-R and LH responses at 72 h. Similarly, administration of ovine PRL (during the first 48 h) to bromocriptine-treated rats produced low GnRH-R concentrations at 72 h. Thus, the transient elevation of PRL seen in immobilized rats can inhibit the GnRH-stimulated increase in GnRH-R and is associated with reduced LH responses to GnRH. These results indicate that PRL has a direct inhibitory effect on the gonadotrope and suggest that impaired GnRH-R responses to GnRH are one of the mechanisms involved in the diminished gonadotropin secretion seen in hyperprolactinemia.     

Pro-opiomelanocortin messenger RNA in hypothalamic neurons is increased by testosterone through aromatization to estradiol

We have previously demonstrated that neurons in the rostral arcuate nucleus expressing the messenger RNA (mRNA) for pro-opiomelanocortin (POMC) are responsive to modulation by physiological levels of testosterone. It is uncertain, however, whether testosterone's action is mediated through direct activation of androgen receptors or through aromatization to estradiol and subsequent binding to estrogen receptors. We examined this question by evaluating the effectiveness of estradiol and dihydrotestosterone (DHT), a nonaromatizable androgen, in reversing the castration-induced diminution of POMC mRNA in the arcuate nucleus. Using in situ hybridization, we measured POMC mRNA content within arcuate neurons of intact, castrated, castrated testosterone-replaced, castrated estradiol-replaced, and castrated DHT-replaced male rats. Adult male rats were castrated and implanted (s.c.) with a Silastic capsule filled to one of the following specifications: crystalline testosterone (30 mm; n = 4); 17 beta-estradiol (E2) diluted 1:1 with cholesterol (5 mm; n = 4); DHT (40 mm; n = 4); or empty (30 mm; n = 4). Control, sham-operated animals (n = 4) were left intact. Analysis of the results showed that following castration, POMC mRNA content was significantly reduced in cells of the arcuate nucleus (intact: 152 +/- 3 grains/cell vs. castrate: 110 +/- 3 grains/cell). Replacement with physiological levels of testosterone prevented the decline of POMC mRNA levels (castrated testosterone-replaced: 143 +/- 6 grains/cell), as did replacement with physiological levels of estrogen (castrated estrogen-replaced: 149 +/- 8 grains/cell). Treatment with DHT failed to prevent the postcastration decline in POMC mRNA content (castrated DHT-treated: 118 +/- 4 grains/cell).(ABSTRACT TRUNCATED AT 250 WORDS)     

Testosterone regulates progonadotropin-releasing hormone levels in the preoptic area and basal hypothalamus of the male rat

GnRH is synthesized as part of a larger 92-amino acid prohormone (pro-GnRH). In the present study, we examined the effect of gonadectomy and hormone replacement on the processing of pro-GnRH to GnRH in adult male rats. Immunoreactive levels of pro-GnRH and GnRH in acid extracts of preoptic area (POA) and basal hypothalamus (BH) from intact, castrate, and testosterone (T)-treated castrate males were quantified by RIA. In addition, we used immunocytochemistry to detect pro-GnRH-and GnRH-positive neurons and determine the effects of hormone treatment. Three weeks after castration, the pro-GnRH content of the POA was 2-fold greater than the amount found in intact males. Treatment with T for 1 week lowered the prohormone content to intact levels. Although the pro-GnRH content in the BH was about 50% lower than that in the POA, the BH responded to castration and steroid replacement in a manner identical to the POA. The GnRH content of the BH, but not that of the POA, was significantly reduced after castration and increased by T treatment. On the other hand, the total number of pro-GnRH and GnRH cell bodies visualized by immunocytochemistry was not significantly altered by either treatment. These results show that changes in pro-GnRH content vary in inverse relation to changes in GnRH content and suggest that gonadectomy inhibits the enzymatic processing of precursor, while T treatment promotes it.     

Sexual dimorphism in the synaptic input to gonadotropin releasing hormone neurons

GnRH neurons form the final common pathway regulating the secretion of gonadotropins from the anterior pituitary. Since the patterns of gonadotropin release display profound sexual dimorphism among mammals including the rodent, we undertook an ultrastructural analysis to determine whether these neurosecretory cells were differentially innervated between the sexes. As a further exploration of the organization of the neurocircuitry integrating GnRH neurons with the central nervous system, we also determined the degree to which GnRH cells and their processes were innervated by terminals containing either the endogenous opiate, beta-endorphin (BE) or GnRH itself. Sections from the diagonal band of Broca and the preoptic area of adult male and diestrus II female rats were immunocytochemically processed for dual localization of GnRH and BE. GnRH neurons cut through the plane of the nucleus were identified in 1 micron sections. Serial ultrathin sections were made and analyzed for 1) total synaptic input to both cell bodies and dendrites; 2) BE input; and 3) input arising from GnRH itself. We report that GnRH neuronal cell bodies in females received approximately twice the number of synapses as did those of males. The input to the GnRH dendrites, when measured as percent of plasma membrane in synaptic contact, also showed a profound sexual dimorphism with the female having a larger proportion of the dendrite in synaptic apposition. BE innervation contributed to this dimorphism at the level of both the cell body and dendrite. In contrast, the distribution and number of GnRH terminals did not differ between the sexes. In both they were confined to the dendritic arbor. We hypothesize that the capacity of the female rodent GnRH system to show neurogenic derived alterations in GnRH output not seen in the male may be due in part to these anatomical differences.     

Impotence associated with low biological to immunological ratio of luteinizing hormone in a man with a pituitary stone

Quantitative reduction in LH secretion resulting from hypothalamic-pituitary dysfunction is a known cause of impotence. Qualitative abnormalities of secreted LH, however, have not been described under these circumstances. During evaluation of a 39-yr-old man with impotence and a calcified pituitary mass (pituitary stone), we detected a qualitative abnormality of LH characterized by a low ratio of bio- to immunoactivity (B:I). Initial work-up revealed basal morning serum testosterone levels of 2.14, 3.18, 3.97, and 3.11 ng/ml on 4 separate days, low to low normal urinary LH (300, 200, and 478 mIU/h), and normal GH, TSH, PRL, and ACTH secretion after provocative testing. The response of impotence to testosterone but not placebo in a double blind trial confirmed the clinical significance of the borderline low androgen levels. These findings prompted a systematic analysis of 24-h LH pulses as well as clomiphene and GnRH responsiveness. By RIA, mean serum LH levels [9.1 +/- 0.3 (+/- SE) mIU/ml] and all other response parameters were normal. In striking contrast, mean serum LH by bioassay was low (9.9 +/- 0.4 mIU/ml vs. 41.4 +/- 5.7 in normal subjects), as were B:I ratios (1.0 +/- 0.03 vs. control values of 3.1 +/- 0.5 to 5.3 +/- 0.3). Only during maneuvers designed to increase GnRH were B:I ratios increased to 3.3 +/- 0.22 (exogenous GnRH) and 1.8 +/- 0.12 (clomiphene). Mean testosterone levels before and after exogenous GnRH treatment were 3.28 +/- 0.24 and 4.76 +/- 0.16, respectively (P less than 0.001). The results suggest an association between the low LH B:I ratio and the anatomical disruption of the hypothalamic-pituitary portal system by the pituitary stone. The increased B:I ratio during GnRH or clomiphene administration indicates a functional link between pituitary GnRH exposure and the greater potency of the LH secreted.     

The frequency of gonadotropin-releasing hormone stimulation determines the number of pituitary gonadotropin-releasing hormone receptors

Gonadotropin-releasing hormone (GnRH) induces both synthesis and release of pituitary gonadotropins, but rapid or slow frequencies of stimulation result in reduced LH and FSH secretion. We determined the effects of frequency of GnRH stimulation on pituitary GnRH receptors (GnRH-R). Castrate male rats received testosterone implants (cast + T) to inhibit endogenous GnRH secretion. GnRH pulses were injected by a pump into a carotid cannula and animals received GnRH (25 ng/pulse) at various frequencies for 48 h. In control animals (saline pulses) GnRH-R was 307 +/- 21 fmol/mg protein (+/- SE) in cast + T and 598 +/- 28 in castrates. Maximum GnRH-R was produced by 30-min pulses and was similar to that seen in castrate controls. Faster or slower frequencies resulted in a smaller GnRH-R response and GnRH given every 240 min did not increase GnRH-R over saline controls. Equalization of the total GnRH dose/48 h (6.6 ng/pulse every 7.5 min or 200 ng/pulse every 240 min) did not increase receptors to the maximum concentrations seen after 30-min (25 ng) pulses. Serum LH responses after 48 h of injections were only present after 30-min pulses, and peak FSH values were also seen after this frequency. Serum LH was undetectable in most rats after other GnRH frequencies, even though GnRH-R was increased. These data show that GnRH pulse frequency is an important factor in the regulation of GnRH-R. A reduction of GnRH-R is part of the mechanism of down-regulation of LH secretion by fast or slow GnRH frequencies, but altered frequency also exerts effects on secretory mechanisms at a site distal to the GnRH receptor.     

Isolated gonadotropin-releasing hormone neurons harvested from adult male rats secrete biologically active neuropeptide in a regular repetitive manner

Immunochemical treatments for the recovery of viable GnRH neurons from adult male rats have previously been described by this laboratory. In the present report, efforts were made to limit cellular adhesion, as well as the proteolytic and mechanical damage which occurred during isolation of the neurons, in order to determine if such damage may account for failure of the isolated cells to exhibit spontaneous neuropeptide release. These modifications prevented the loss of assayable GnRH during the isolation process, and neurons recovered from individual rats in this study contained 10.7 +/- 2.5 ng GnRH. Further, all isolated neuronal preparations exhibited spontaneous peptide release which continued in a regular repetitive manner. When maintained in closed chambers, these preparations released 105 +/- 42 pg/ml biologically active GnRH at 18.9 +/- 0.4-min intervals. In contrast, GnRH release from heterologous preparations was characterized by erratic low level pulses. The results from this work suggest that independent neuroendocrine properties of GnRH neurons may be responsible for tonic gonadotropin secretion in castrated adult male rats and that the erratic patterns of gonadotropin release in gonadally intact males may be related, in part, to coupling between GnRH neurons and unidentified neuronal factors.