Nitric oxide control of steroidogenesis: endocrine effects of NG-nitro-L-arginine and comparisons to alcohol.

Recent studies suggest that nitric oxide (NO) may regulate hormone biosynthesis and secretion. This was tested by treating male rats with NG-nitro-L-arginine methyl ester (NAME), a NO synthase inhibitor, and measuring serum and testicular interstitial fluid testosterone and serum corticosterone, luteinizing hormone (LH), and prolactin (PRL). The effect of NG-nitro-L-arginine (NA), a less-soluble form of the same NO synthase inhibitor, on the reproductive suppressant actions of alcohol was also examined. NAME increased testosterone and corticosterone secretion dose-dependently without affecting LH and PRL secretion. The alcohol-induced suppression of testosterone or LH secretion was not altered by treatment with NA. Although effects of NAME and NA on other systems may be involved, these results indicate that testicular and adrenal steroidogenesis are negatively regulated by endogenous NO and that NO does not regulate LH and PRL secretion or inhibit the testicular steroidogenic pathway in the same way as alcohol.     

Rôle du peptide vasoactif intestinal (VIP) dans la stéroïdogenèse et le vieillissement testiculaire

VIP (vasoactive intestinal peptide) neuropeptide has long been considered to be putative regulator of testicular functions.In vitro evidence suggests that VIP could play an important role in testosterone biosynthesis. However, the endogenous role of VIP on testicular functions remained to be demonstrated. In C57BL/6 mice exhibiting complete disruption of the VIP gene, the authors observed that male fertility remained intact but serum testosterone levels were lower than those of WT littermates. At the age of 4 months, this phenotype was accompanied by reduced steroidogenesis due to inhibition of the expression of StAR (steroidogenic acute regulatory protein) and 3ßHSD (3ß-hydroxysteroid dehydrogenase) in the testis. In addition, serum levels of FSH (Follicle-stimulating hormone) but not LH (Luteinizing hormone) were reduced in young KO males. Testicular anatomy also revealed a subtle but significantly higher percentage of degenerated seminiferous tubules in 4-month-old VIP-/-animals compared to WT. In aging animals (15 months old), control males showed typical testicular aging including severe degeneration of seminiferous tubules, a dramatic decrease in serum testosterone levels and a reduction in StAR and 3ß-HSD gene expression. In age-matched VIP-/-males, serum levels of testosterone and steroidogenic enzymes were still very low. Interestingly, in contrast with young mice, testicular degeneration at 15 months was significantly less severe marked in VIP-/-mice than in WT mice. Altogether, these results suggest that: 1) VIP is an important factor for regulating testosterone biosynthesis and FSH secretion and 2) VIP regulates testicular aging.     

Reduced ability of naloxone to stimulate LH and testosterone release in aging male rats; possible relation to increase in hypothalamic met5-enkephalin

Blood luteinizing hormone (LH) and testosterone levels are lower in old than in young male rats. The specific opiate antagonist, naloxone, previously shown to increase serum LH in mature male rats, exhibited relatively little ability to raise serum LH and testosterone levels in old (18–20 mo) as compared to young (4–5 mo) male rats. The brain opiate, met⁵-enkephalin, which depresses LH, was found to be significantly higher in the hypothalamus of old than of young male rats. These observations suggest that hypothalamic opiates may be partially responsible for the lower serum LH and testosterone levels in old male rats, and for reduced release of these hormones in response to naloxone administration.     

Pituitary and gonadal function during physical exercise in the male rat

The effects of training and acute exercise on serum testosterone, luteinizing hormone (LH) and corticosterone levels and on testicular endocrine function in male rats were studied. In the first part of the study, the rats were trained progressively on a treadmill, over 8 weeks. Training did not change the basal levels of serum testosterone, LH and corticosterone, or the testicular concentrations of testosterone and its precursors progesterone and androstenedione. The levels of testicular LH (30.3 +/- 2.6 ng/g wet wt, mean +/- SEM) and lactogen (150 +/- 14 pg/g) receptors were unchanged after training. However, the capacity of testicular interstitial cell suspensions to produce cAMP and testosterone increased by 20-30% during in vitro gonadotropin stimulation. In the second part, the trained and untrained control animals underwent acute exhaustive exercise. Serum testosterone levels decreased by 74 and 42% in trained and untrained rats, respectively (P less than 0.02), and corticosterone rose by 182% in trained and 146% in untrained rats (P less than 0.01), whereas the LH level was unchanged. Testicular levels of testosterone and its precursors decreased, with the exception of unchanged androstenedione, in trained rats; the cAMP concentration was unchanged. In both trained and untrained rats, acute exercise decreased the capacity of interstitial cell suspensions to produce cAMP, whereas there were no consistent effects on testosterone production. Acute exercise had no effect on LH or lactogen receptors in testis tissue. In conclusion, training had no effect on serum or testicular androgen concentrations, but increased Leydig cell capacity to produce testosterone and cAMP. Acute exercise decreased serum and testicular testosterone concentrations without affecting serum LH. A direct inhibitory effect of the increased serum corticosterone level on the hypothalamic-pituitary level and/or testis may be the explanation for this finding.     

Changes in LH and prolactin levels in diabetic male rats and the role of the opiate system in the control of their secretion

Diabetic male rat has low serum levels of luteinizing hormone (LH) and testosterone (T), which are accompanied by atrophy of the testes and accessory glands. The present study investigated changes in the serum levels of LH, prolactin (PRL) and glucose, following diabetes induction by streptozotocin. In addition, involvement of the opiate system in the control of LH and PRL secretion was evaluated. There was no difference in PRL levels between diabetic and control animals, except at 8 hours after streptozotocin injection. In contrast, the diabetic animals had consistently lower levels of LH, starting on the second day of diabetes. Blockade of the opiate system by naltrexone caused a sharp increase of LH levels in normoglycemic rats, while only a gradual decrease was observed in hyperglycemic animals. PRL secretion was inhibited by naltrexone, both in diabetic and control groups. It is concluded that, unlike normoglycemic rats, inhibition of LH secretion in diabetes is not under the control of the opiate system, probably as a result of T deficiency. In contrast, PRL secretion in diabetic rats, as in the control group, is under the influence of endogenous opiates.     

The adrenal gland and progesterone stimulates testicular steroidogenesis in the rat in vivo

Administration of pharmacological doses of glucocorticoid to male rats in vivo suppresses adrenal steroidogenesis and inhibits testicular steroidogenesis by inhibiting the anterior pituitary secretion of LH. In contrast, administration of ACTH to these pharmacologically-suppressed rats stimulates the adrenal secretion of progesterone and testicular steroidogenesis. The mechanism by which ACTH increases testicular steroidogenesis is dependent on the presence of the adrenal gland and is reproduced by the administration of progesterone. The conclusion from these data is that the adrenal gland has an important role in generating external signals that modulate the hypothalamic-pituitary-gonadal axis in male rats. The adrenal secretion of glucocorticoid acts as a negative signal to testicular steroidogenesis whereas progesterone acts as a positive signal. The adrenal secretion of progesterone and its conversion to testosterone by steroidogenic enzymes in the cytoplasm of the Leydig cell may provide an alternative pathway for testosterone biosynthesis and may account for the increased plasma testosterone levels during the acute phase of stress and mating.     

Direct effects of propylthiouracil on testosterone production in monkeys

Propylthiouracil (PTU) is an anti-thyroid drug. However, the direct effects of PTU on the endocrine functions of non-thyroid glands are unclear. In the present study, we examined the acute effects of PTU on testosterone secretion in monkeys. Male monkeys were infused intravenously with PTU for 30 min. Blood samples were collected at several time intervals. Monkey testicular interstitial cells were cultured with PTU, human chorionic gonadotropin (hCG), or forskolin, at 34 degrees C for 1 h. In another study, steroidogenesis in monkey testicular interstitial cells were examined. PTU decreased plasma testosterone but not plasma thyroxine (T4) and luteinizing hormone (LH) levels in monkeys. Administration of PTU resulted in a dose-dependent inhibition of basal and hCG-, as well as forskolin-stimulated testosterone release by monkey testicular interstitial cells. PTU also diminished the stimulatory effects induced by androstenedione. These results suggest that PTU inhibits testosterone secretion via a mechanism independent of the secretion of T4 and LH in primates. The inhibitory mechanism of PTU on testosterone production involves a decreased activity of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and post-cAMP pathways.     

Endocrine actions of opioids

The widespread occurrence of opioid peptides and their receptors in brain and periphery correlates with a variety of actions elicited by opioid agonists and antagonists on hormone secretion. Opioid actions on pituitary and pancreatic peptides are summarized in Table 1. In rats opioids stimulate ACTH and corticosterone secretion while an inhibition of ACTH and cortisol levels was observed in man. In both species, naloxone, an opiate antagonist, stimulates the release of ACTH suggesting a tonic suppression by endogenous opioids. In rats, a different stimulatory pathway must be assumed through which opiates can stimulate secretion of ACTH. Both types of action are probably mediated within the hypothalamus. LH is decreased by opioid agonists in many adult species while opiate antagonists elicit stimulatory effects, both apparently by modulating LHRH release. A tonic, and in females, a cyclic opioid control appears to participate in the regulation of gonadotropin secretion. Exogenous opiates potently stimulate PRL and GH secretion in many species. Opiate antagonists did not affect PRL or GH levels indicating absence of opioid control under basal conditions, while a decrease of both hormones by antagonists was seen after stimulation in particular situations. In rats, opiate antagonists decreased basal and stress-induced secretion of PRL. Data regarding TSH are quite contradictory. Both inhibitory and stimulatory effects have been described. Oxytocin and vasopressin release were inhibited by opioids at the posterior pituitary level. There is good evidence for an opioid inhibition of suckling-induced oxytocin release. Opioids also seem to play a role in the regulation of vasopressin under some conditions of water balance. The pancreatic hormones insulin and glucagon are elevated by opioids apparently by an action at the islet cells. Somatostatin, on the contrary, was inhibited. An effect of naloxone on pancreatic hormone release was observed after meals which contain opiate active substance. Whether opioids play a physiologic role in glucose homeostasis remains to be elucidated.     

Central, stereoselective receptors mediate the acute effects of opiate antagonists on luteinizing hormone secretion

Although a central site of acute opiate action in regulating luteinizing hormone (LH) secretion has been suggested by the ability of centrally implanted opiate antagonists to increase LH levels, opiate antagonists are lipophilic and could influence the pituitary in situ. Also, the physiological significance of opiate receptor blockade with antagonists rests on the assumed, but untested, stereoselectivity of these receptors. Therefore, a lipophobic quaternized derivative of naltrexone (MRZ 2663-Naltrexone methobromide) and dextro- (+) and levo- (-) stereoisomers of naloxone were used to study the site- and stereoselectivity of gonadotropin responses to opiate antagonists in vivo. Male rats were injected intracerebroventricularly (icv) or intravenously (iv) with the quaternary or tertiary congeners of naltrexone and subcutaneously (sc) with (-) or (+)-naloxone. Rats injected icv with 20 ug of quaternary naltrexone displayed significant increases in serum luteinizing hormone (LH). The onset of the response was rapid with serum LH levels being significantly elevated 15 minutes after the injection and returning to basal levels 30 minutes later. Rats injected iv with 10 mg/kg of quaternary naltrexone failed to show significant LH responses. Rats injected either centrally or periphally with equivalent doses of tertiary naltrexone showed LH responses that were similar to those found in animals injected icv with quaternary naltrexone. As little as 0.5 mg/kg of (-)-naloxone resulted in significant elevations in serum LH that were higher than those elicited by up to 10 mg/kg of (+)-naloxone, indicating that this effect of naloxone is stereoselective. These data support the argument that opioids can acutely modulate LH secretion through actions at stereoselective opioid receptors in the central nervous system.     

Luteinizing hormone secretion following intracerebroventricular administration of morphine in the prepuberal gilt

Antagonism of endogenous opioids with naloxone stimulates luteinizing hormone (LH) release in mature but not prepuberal gilts. The present report demonstrates that the opiate agonist morphine (500 micrograms), administered intracerebroventricularly (ICV), reduced LH secretion in both ovariectomized mature and prepuberal gilts. We suggest that opioid receptors are functionally coupled to the GnRH secretory system in prepuberal gilts even though endogenous opioid peptide modulation of LH secretion was not demonstrable in our previous studies.     

Absence of direct effects of GnRH on testicular steroid secretion in the ram

Effects of GnRH, administered via the testicular artery, on testicular steroidogenesis were studied in rams during the non-breeding season. Concentrations of testosterone and 17-hydroxyprogesterone in testicular venous blood showed similar profiles which were identical for GnRH-treated (0.5 ng infused over 60 min or 25 ng injected) and control testes. Increases of testicular venous concentration of both hormones were only marginally reflected in peripheral venous concentrations. Peripheral administration of hCG (200 i.u., i.v.) stimulated testosterone secretion to a larger extent than 17-hydroxyprogesterone secretion in 10/11 rams, GnRH-treated and control testes showing identical responses. High testicular venous concentrations of both hormones after administration of GnRH were paralleled by increased concentrations of endogenous LH. These LH peaks were evoked by 25 ng GnRH in 7/8 rams. The observed effects of GnRH treatment on testicular steroid secretion thus cannot be considered to be the result of direct stimulation of steroidogenesis by GnRH.     

Ethanol inhibits the naloxone-induced release of luteinizing hormone-releasing hormone from the hypothalamus of the male rat

It has been inferred that ethanol suppresses the secretion of luteinizing hormone (LH) in the male by depressing the release of LH-releasing hormone (LH-RH) from the hypothalamus. Direct support for this inference has been difficult to obtain, however, because of significant technical difficulties in measuring LH-RH release under in vivo conditions. To circumvent these problems, we made use of the opiate antagonist naloxone, as a neuroendocrine probe, to elicit the release of LH-RH under in vivo conditions. We found that ethanol was a potent suppressor of the increase in serum LH levels evoked by naloxone at extremely low blood ethanol concentrations ( less than 60 mg/dl). Furthermore, we observed that the antagonism between ethanol and naloxone appeared to be competitive in nature since a fixed dose of ethanol (1 g/kg, blood ethanol concentration 60 mg/dl) shifted the naloxone dose-response curve significantly to the right and high doses of the antagonist overcame ethanol's effects. Finally, we found that the interaction between ethanol and naloxone took place at the level of the hypothalamus. Our results, therefore, seem to provide the first in vivo evidence supporting the widely-held hypothesis that ethanol reduces serum LH levels by depressing the hypothalamically-medicated release of LH-RH. The mechanisms underlying ethanol's depression of naloxone-induced increases in the release of LH-RH are not fully understood at this time, but one prominent possibility is that ethanol enhances the synthesis or release of endogenous opioids which in turn override naloxone's effects.     

Cross-talk between G protein-coupled and epidermal growth factor receptors regulates gonadotropin-mediated steroidogenesis in Leydig cells

Gonadal steroid production is stimulated by gonadotropin binding to G protein-coupled receptors (GPCRs). Although GPCR-mediated increases in intracellular cAMP are known regulators of steroidogenesis, the roles of other signaling pathways in mediating steroid production are not well characterized. Recent studies suggest that luteinizing hormone (LH) receptor activation leads to trans-activation of epidermal growth factor (EGF) receptors in the testes and ovary. This pathway is critical for LH-induced steroid production in ovarian follicles, probably through matrix metalloproteinase (MMP)-mediated release of EGF receptor (EGFR) binding ectodomains. Here we examined LH and EGF receptor cross-talk in testicular steroidogenesis using mouse MLTC-1 Leydig cells. We demonstrated that, similar to the ovary, trans-activation of the EGF receptor was critical for gonadotropin-induced steroid production in Leydig cells. LH-induced increases in cAMP and cAMP-dependent protein kinase (PKA) activity mediated trans-activation of the EGF receptor and subsequent mitogen-activated protein kinase (MAPK) activation, ultimately leading to StAR phosphorylation and mitochondrial translocation. Steroidogenesis in Leydig cells was unaffected by MMP inhibitors, suggesting that cAMP and PKA trans-activated EGF receptors in an intracellular fashion. Interestingly, although cAMP was always needed for steroidogenesis, the EGFR/MAPK pathway was activated and necessary only for early (30-60 min), but not late (120 min or more), LH-induced steroidogenesis in vitro. In contrast, 36-h EGF receptor inhibition in vivo significantly reduced serum testosterone levels in male mice, demonstrating the physiologic importance of this cross-talk. These results suggest that GPCR-EGF receptor cross-talk is a conserved regulator of gonadotropin-induced steroidogenesis in the gonads, although the mechanisms of EGF receptor trans-activation may vary.     

Chronic administration of corticosterone impairs LH signal transduction and steroidogenesis in rat Leydig cells

The mechanism involved in the inhibitory actions of chronic corticosterone treatment on Leydig cell steroidogenesis was studied in adult Wistar rats. Rats were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days and another set of rats was treated with corticosterone plus ovine luteinizing hormone (oLH) (100 microg/kg body weight, s.c., daily) for 15 days. Chronic treatment with corticosterone increased serum corticosterone but decreased serum LH, testosterone, estradiol and testicular interstitial fluid (TIF) testosterone and estradiol concentrations. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone and estradiol. Leydig cell LH receptor number, basal and LH-stimulated cAMP production were diminished by corticosterone treatment which remained at control level in the corticosterone plus LH treated rats. Activities of steroidogenic enzymes, 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta-HSD and 17beta-HSD) were significantly decreased in corticosterone treated rats. LH plus corticosterone treatment did not affect 3beta-HSD activity but decreased 17beta-HSD activity, indicating a direct inhibitory effect of excess corticosterone on Leydig cell testosterone synthesis. The indirect effect of corticosterone, thus, assume to be mediated through lower LH which regulates the activity of 3beta-HSD. Basal, LH and cAMP-stimulated testosterone production by Leydig cells of corticosterone and corticosterone plus LH treated rats were decreased compared to control suggesting the deleterious effect of excess corticosterone on LH signal transduction and thus steroidogenesis.     

Short-term fasting leads to inhibition of responsiveness to LH-stimulated testosterone secretion in the adult male bonnet monkey

A variety of stressors including fasting profoundly inhibit reproductive function in mammals. Although the effect of short-term fasting on gonadotropic axis is well established, the direct effects of fasting on gonads have not been reported. The objectives of the present experiments were to examine the effect of short-term fasting on circulating luteinizing hormone (LH) and testosterone (T) secretion, and to determine the responsiveness of testis to exogenous recombinant human (rh) LH treatment in male bonnet monkeys. In addition, an experiment was carried out to examine whether brief inhibition of endogenous LH secretion causes alteration in testicular responsiveness. Adult male monkeys were fasted for 1 day for examining the circulating endocrine hormone concentrations and challenged with rhLH injection 1 day after fasting. Food withdrawal for 1 day resulted in significant (P<0.05) decrease in LH, T and increase in cortisol concentrations. Surprisingly, T secretion in response to direct stimulation of Leydig cells by LH was not observed in fasted monkeys. In fed monkeys, treatment with Antide (a specific gonadotropin releasing hormone receptor antagonist to inhibit pituitary LH secretion) for 1 day did not compromise T secretion stimulated by rhLH, suggesting that loss of responsiveness of testis to exogenous LH treatment in fasted monkeys was not because of interruption in pituitary LH stimulation of the testis. The results indicate that short-term fasting in adult male monkeys cause inhibition of LH and T secretion, and inhibition of responsiveness of testis to LH stimulation.     

Physiological role of putative testicular gonadotrophin releasing hormone (GnRH)

Evidence suggests that exogenous GnRH and agonist analogues have short-term stimulatory effects on rat Leydig cell function - when administered intratesticularly. Since rat Leydig cells possess GnRH receptors and their endogenous ligand has not yet been identified the physiological importance of the observations for testis function is unknown. To address this issue we have determined the consequences of blockade of testis GnRH receptors on Leydig cell function under both normogonadotrophic and hypogonadotrophic stimulation of the testis in vivo. A GnRH antagonist (ANT) was used to achieve receptor blockade but during continuous systemic infusion ANT occupied pituitary GnRH receptors and markedly reduced serum LH, FSH, testosterone, and intratesticular testosterone in adult and 30 d old immature male rats. These results were similar to those obtained by administration of a GnRH antiserum which did not bind to testis GnRH receptors. Thus, blockade of testis GnRH receptors during hypogonadotrophism did not produce additional inhibition of steroidogenesis by Leydig cells. However, direct continuous infusion of ANT into one testis produced greater than 90% occupancy of GnRH receptors while reducing GnRH receptors by only 50% in the contralateral testis. Unilateral intratesticular infusion did not reduce serum LH, FSH, Prolactin or testosterone levels despite 75% occupancy of pituitary GnRH receptors. Thus, both ANT infused and saline infused testes were exposed to the same gonadotrophic stimulants but in the former GnRH-R were essentially non-existent. Compared to the control testis, the ANT infused testis showed a 20-30% reduction in LH, FSH, lactogen receptors and 30-40% fall in testosterone content. Identical results were obtained in adult and 30 d-old male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of endogenous opiates on anterior pituitary function

In general, the endogenous opioid peptides (EOP), morphine (MOR), and related drugs exert similar effects on acute release of pituitary hormones. Thus administration of opiates produces a rapid increase in release of prolactin (PRL), growth hormone (GH), adrenocorticotropin (ACTH), and antidiuretic hormone (ADH), and a decrease in release of gonadotropins and thyrotropin (TSH). Although not yet fully established, there is growing evidence that the EOP participate in the physiological regulation of pituitary hormone secretion. Thus naloxone (NAL), a specific opiate antagonist, has been shown to reduce basal serum levels of PRL and GH, and to elevate serum levels of LH and follicle stimulating hormone in male rats. Other reports have shown that NAL can inhibit the stress-induced rise in serum PRL, raise the castration-induced increase in serum LH to greater than normal castrate values, and counteract the inhibitory effects of estrogen and testosterone on LH secretion. Opiates appear to have no direct action on the pituitary, but there is evidence that they can alter activity of hypothalamic dopamine and serotonin in modulating secretion of pituitary hormones.     

Effects of intracerebroventricular administration of irisin on the hypothalamus–pituitary–gonadal axis in male rats

Irisin is a product of fibronectin type III domain-containing protein 5 (FNDC5) and plays an important role in energy homeostasis. In this study, we aimed to determine effects of intracerebroventricular administration of irisin on the hypothalamus–pituitary–gonadal axis by molecular, biochemical, and morphological findings. Fourty male Wistar-Albino rats were used and divided into four groups including control, sham (vehicle), 10, and 100 nM irisin infused groups (n = 10). Hypothalamic gonadotropin releasing hormone (GnRH) level and serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels were determined. Testicular tissue histology and spermiogram analysis were also performed. Both irisin concentrations significantly reduced hypothalamic GnRH messenger RNA (mRNA) and protein levels (p < 0.05). It was found that serum LH, FSH, and testosterone levels and Sertoli and Leydig cell numbers were decreased by irisin administration (p < 0.05). In addition, irisin administration reduced sperm density and mobility (p < 0.05). However, it did not cause any change in testicular and epididymis weights and tubular diameter. Our results reveal that irisin can play a role in the central regulation of reproductive behavior and also reduces testosterone levels by suppressing LH and FSH secretion. These results suggest that the discovery of irisin receptor antagonists may be beneficial in the treatment of infertility.     

Opioid regulation of luteinizing hormone secretion in the male rat

Current evidence suggests that endogenous opioid peptides (EOPs) tonically inhibit secretion of luteinizing hormone (LH) by modulating the release of gonadotropin-releasing hormone (GnRH). Because of their apparent inhibitory actions, EOPs have been assumed to alter both pulse frequency and amplitude of LH in the rat; and it has been hypothesized that EOP pathways mediate the negative feedback actions of steroids on secretion of GnRH. In order to better delineate the role of EOPs in regulating secretion of LH in the male rat, we assessed the effects of a sustained blockade of opiate receptors by naloxone on pulsatile LH release in four groups: intact male rats, acutely castrated male rats implanted for 20 h with a 30-mm capsule made from Silastic and filled with testosterone, acutely castrated male rats implanted for 20 h with an osmotic minipump dispensing 10 mg morphine/24 h, and male rats castrated approximately 20 h before treatment with naloxone. We hypothesized that if EOPs tonically inhibited pulsatile LH secretion, a sustained blockade of opiate receptors should result in a sustained increase in LH release. We found that treatment with naloxone resulted in an immediate but transient increase in LH levels in intact males compared to controls treated with saline. Even though mean levels of LH increased from 0.15 +/- 0.04 to a high of 0.57 +/- 0.14 ng/ml, no significant difference was observed between the groups in either frequency or amplitude of LH pulses across the 4-h treatment period. The transient increase in LH did result in a 3- to 4-fold elevation in levels of plasma testosterone over baseline. This increase in testosterone appeared to correspond with the waning of the LH response to naloxone. The LH response to naloxone was eliminated in acutely castrated rats implanted with testosterone. Likewise, acutely castrated rats treated with morphine also failed to respond to naloxone with an increase in LH. These observations suggest that chronic morphine and chronic testosterone may act through the same mechanism to modulate secretion of LH, or once shut down, the GnRH pulse-generating system becomes refractory to stimulation by naloxone. In acutely castrated male rats, levels of LH were significantly increased above baseline throughout the period of naloxone treatment; this finding supports the hypothesis that the acute elevation in testosterone acting through mechanism independent of opioid is responsible for the transient response of LH to naloxone in the intact rat.