Action Of Luteinizing Hormone-releasing Hormone Research Articles

Mechanism of action of luteinizing hormone-releasing hormone in rat ovarian cells.

The initial step in the signal transduction of luteinizing hormone-releasing hormone (LHRH) in rat ovarian cells is the hydrolysis of membrane polyphosphoinositides into inositol phosphates and 1,2-diacylglycerol. The former compounds, especially inositol 1,4,5-triphosphate, are known to cause the release of calcium from intracellular stores, while diacylglycerol is a potent activator of protein kinase C. LHRH causes a rapid and transient increase in intracellular concentrations of free calcium ions, by approximately 4.5-fold, in the majority of granulosa cells as assessed by fura-2 microspectrofluorimetry. Like LHRH, a calcium ionophore (A23187) and activators of protein kinase C attenuate the steroidogenic response of the cells to follicle-stimulating hormone, but enhance the formation of gonadotropin-induced prostaglandin formation. These results support the concept that stimulation of polyphosphoinositide hydrolysis is intimately involved in the direct action of LHRH at the level of the ovary.

Synergistic Stimulation Of Prostaglandin E2 Production by Calcium lonophore and Protein Kinase C Activator in Rat Granulosa Cells1

The effects of luteinizing hormone-releasing hormone (LHRH) and its putative intracellular mediators on progesterone (P) and prostaglandin E2 (PGE2) formation were studied in rat granulosa cells. A calcium ionophore (A23187), 12-0-tetradecanoylphorbol-13-acetate (TPA), and melittin (a phospholipase A2-stimulator) were used to later intracellular calcium, protein kinase C, and arachidonic acid levels, respectively. During a 5-h incubation, LHRH increased basal P levels but failed to affect the formation of P induced by cholera toxin (CT). On the other hand, both basal and CT-stimulated PGE2 formation were increased by LHRH. Treatment of the cells with A23187 or TPA attenuated the formation of P induced by CT or FSH. By contrast, A23187 or TPA significantly augmented CT- or FSH-stimulated PGE2 formation. Interestingly, the effects of A23187 and TPA on PGE2 were synergistic, whether or not FSH or CT was present during the incubation. This synergy was not observed with regard to P formation. Melittin also increased basal P and PGE2 levels, and enhanced the stimulation of PGE2 by A23187 or TPA. However, in the combined presence of A23187 and TPA, melittin failed to further enhance the high levels of PGE2 accumulated. These findings further support a role for the intracellular calcium, protein kinase C, and arachidonic acid metabolic pathways in the multiple actions of LHRH in the ovary.

Differential role of protein kinase C in the action of luteinizing hormone-releasing hormone on hormone production in rat ovarian cells

This study was undertaken to determine the involvement of arachidonic acid and protein kinase C in the actions of luteinizing hormone-releasing hormone on steroid and prostaglandin formation in the ovary. In primary culture of rat granulosa cells, treatment with 3 x 10(-7) mol/L melittin stimulates progesterone and prostaglandin E2 accumulation after a 5-hour culture period. Concomitant treatment of the cells with melittin and luteinizing hormone-releasing hormone or 12-0-tetradecanoylphorbol 13-acetate further enhances the stimulatory action of either luteinizing hormone-releasing hormone or 12-0-tetradecanoylphorbol 13-acetate by itself on prostaglandin E2 production. In contrast, no synergistic effects are observed on progesterone production by the same treatments. Treatment with luteinizing hormone-releasing hormone for 24 hours significantly decreases follicle-stimulating hormone-induced progesterone production by approximately 50%. Treatment of the cells with either follicle-stimulating hormone or luteinizing hormone-releasing hormone stimulates prostaglandin E2 production at least tenfold in the same cultures. When follicle-stimulating hormone and luteinizing hormone-releasing hormone are present concomitantly, they synergistically enhance prostaglandin E2 formation (p less than 0.01). Similar effects are observed with the phorbol ester, 12-0-tetradecanoylphorbol 13-acetate, which causes a dose-dependent inhibition of progesterone production by follicle-stimulating hormone whereas follicle-stimulating hormone-stimulated prostaglandin E2 formation is enhanced. Thus luteinizing hormone-releasing hormone-induced activation of protein kinase C may play multiple roles (stimulatory or inhibitory) in hormone production in the ovary.

Effect of 5α-dihydrotestosterone and flutamide on the facilitation of lordosis by LHRH and naloxone in estrogen-primed female rats

Although 5α-dihydrotestosterone (DHT) is a potent inhibitor of lordosis behavior in ovariectomized estrogen-primed female rats, the mechanism(s) by which this steroid has this action is unknown. The present experiments sought to determine whether DHT inhibits lordosis by preventing the known facilitatory actions of luteinizing hormone-releasing hormone (LHRH), naloxone, and Substance P on lordosis. Lordosis behavior was examined in ovariectomized, estrogen-primed rats prior to or 30, 60, 90, and 180 min following intracerebroventricular (ICV) infusion of LHRH (500ng), naloxone (1 μg), Substance P (1 μg), or saline and 0.01 N acetic saline vehicles, and the effects of DHT (2.5 mg/rat) following similar treatment were examined. In Experiment 1, LHRH and naloxone increased lordosis within 30 min after infusion, while Substance P and the saline or acetic saline vehicles had no effect. Treatment with DHT in combination with estrogen prevented the facilitation of lordosis by LHRH and naloxone. In Experiment 2, ovariectomized, estrogen-primed females shown to be responsive to LHRH during a first screening test were tested for lordosis after receiving either DHT or DHT in combination with the androgen receptor antagonist, Flutamide (7.5 mg/injection × 3). Again, DHT prevented the facilitatory action of LHRH; however, Flutamide did not counteract that effect. In Experiment 3, Flutamide did not counteract the inhibitory effect of DHT on estrogen and progesterone-induced lordosis. These results demonstrate that the inhibitory effect of DHT cannot be overridden by neuroactive peptides which themselves stimulate receptivity. It seems unlikely that DHT inhibits lordosis either by interfering with the behavioral action of these peptides or by activation of the androgen receptor.

Inhibitory effect of central LHRH on LH secretion in the ovariectomized ewe.

The role of central luteinizing hormone releasing hormone (LHRH) in the control of pulsatile LHRH and luteinizing hormone (LH) secretion was investigated in ovariectomized adult ewes. Injection of LHRH (2.1-21 pmol) into the third cerebral ventricle caused a delayed but sustained inhibition of LH secretion. Pulse frequency, pulse amplitude and mean LH levels were reduced significantly when compared with the responses to the control injection of saline (50 microliters). The inhibitory effect of centrally administered LHRH was not accompanied by a reduction in the pituitary responsiveness to intravenous LHRH. In contrast to the effect on LH, plasma levels of follicle-stimulating hormone (FSH) and prolactin were unaffected by central LHRH. The inhibitory action of LHRH was antagonized by prior injection of an LHRH antagonist ([N-Ac-D-Nal(2)1, D-p-Cl-Phe2, D-Trp3, D-hArg (Et2)6, D-Ala10] LHRH, 69 pmol) into the third ventricle. Central injection of the LHRH antagonist alone (at the same concentration) did not influence any characteristic of pulsatile LH secretion. In conclusion, these data indicate that exogenous administration of LHRH into the brain exerts a dose-related and receptor-mediated inhibition of LHRH pulse generator activity. However, the physiological significance of endogenous LHRH in the regulation of the LHRH pulse generator remains unresolved.

Inhibition of follicle-stimulating hormone- and adenosine-3′,5′-cyclic monophosphate-induced progesterone production by calcium and protein kinase C in the rat ovary

In this study we examined the effects of A23187 (a calcium ionophore) and 12-O-tetradecanoyl phorbol-13-acetate, a known activator of protein kinase C, on progesterone production. Granulosa cells obtained from pregnant mare serum gonadotropin-primed rats were maintained in primary culture. Treatment with follicle-stimulating hormone (0.5 μg/ml), 8-bromo-adenosine-3′,5′-cyclic monophosphate (2 mmol/L), or cholera toxin (0.1 μg/ml) for 5 hours or 24 hours markedly stimulated progesterone production. The concomitant presence of A23187 attenuated the elevated levels of progesterone induced by follicle-stimulating hormone, 8-bromo-adenosine-3′,5′-cyclic monophosphate, or cholera toxin, with or without the presence of a phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (0.2 mmol/L). Likewise, treatment of the cells with 12-O-tetradecanoyl phorbol-13-acetate suppressed follicle-stimulating hormone-induced progesterone production, whether or not 1-methyl-3-isobutylxanthine was present in the cultures. The effect of 12-O-tetradecanoyl phorbol-13-acetate was not mimicked by phorbol-13-monoacetate or 4α-phorbol-12, 13-didecanoate. These results indicate that both A23187 and 12-O-tetradecanoyl phorbol-13-acetate inhibit follicle-stimulating hormone-induced progesterone production, in part at a step or steps beyond adenosine-3′,5′-cyclic monophosphate generation and degradation. They further support a role of calcium and protein kinase C in the intraovarian action of luteinizing hormone-releasing hormone.

Modulatory actions of luteinizing hormone-releasing hormone on electrical activity of preoptic neurons in brain slices

Single unit activity was recorded from 378 neurons, in two preoptic nuclei rich in luteinizing hormone-releasing hormone neurons, using in vitro brain tissue slices which were prepared from either ovariectomized or ovariectomized plus estradiol-treated rats. To test possible transmitter-like actions, agents were injected into the perfusion medium. Luteinizing hormone-releasing hormone excited 46%, inhibited 7% and evoked biphasic responses in 2% of the 250 units tested. By comparison, two other peptides, thyrotropin-releasing hormone and cholecystokinin-octapeptide sulfated were exclusively excitatory, acting on 55 and 67% of the neurons, respectively. The response to thyrotropin-releasing hormone, cholecystokinin-octapeptide sulfated and neurotransmitters were prompt, large and consistent from trial to trial. In contrast, responses to luteinizing hormone-releasing hormone were usually delayed, small and variable. Responses to the agents tested were not affected by in vivo estradiol treatment. Possible modulatory actions of luteinizing hormone-releasing hormone were tested by comparing the responses of single neurons to norepinephrine and serotonin before and after an application of luteinizing hormone-releasing hormone. In 39 and 20% of the 119 neurons tested, the norepinephrine responses were potentiated and attenuated, respectively, by luteinizing hormone-releasing hormone. In 46 serotoninresponsive neurons, 28% were potentiated and 22% attenuated. These neuromodulatory actions of luteinizing hormone-releasing hormone were specific in affecting only certain responses of certain neurons, and they were not duplicated on the same neurons by thyrotropin-releasing hormone. It appears that luteinizing hormone-releasing hormone may be a neuromodulator in the preoptic area.

Hypothalamic LH-RH release after acute and chronic treatment with morphine studied in a combined in vivo/in vitro model

Rats were treated with the opiate agonist, morphine, and the release of luteinizing hormone-releasing hormone (LH-RH) from their hypothalami was studied in vitro. Within 16–24 h after morphine treatment, basal LH-RH release rates were observed to be higher compared to those from hypothalami derived from opiate-naive rats, suggesting that dependence had occurred in the neural mechanisms underlying LH-RH release. Maintenance of tissues exposed to morphine in vivo in medium containing morphine in vitro did not alter the increased basal release of LH-RH, but because this was significantly greater than control rates, tolerance is not believed to have occurred. Addition of the opioid antagonist naloxone in vitro resulted in a 220% increase in the release of LH-RH by hypothalami exposed to morphine for 48 h in vivo, whereas it caused a 50% reduction in LH-RH release from tissues exposed to morphine for 96 h in vivo. This latter result shows parallels with our previous finding that naloxone paradoxically decreases serum LH levels of chronically morphine-treated rats. In view of recent evidence for presynaptic feedback inhibitory effects operating on opioid neurons, it is suggested that, following chronic exposure to morphine, the opioid neurons which normally inhibit LH-RH neurons are inhibited; upon treatment with naloxone, they are disinhibited and release more opioid peptides which then act to switch off LH-RH neuronal activity.

Testosterone stimulates LH-RH-like mRNA level in the rat hypothalamus

To elucidate whether testosterone (T) replacement to castrated male rats may promote luteinizing hormone-releasing hormone (LH-RH) biosynthesis in the hypothalamus, an RNA-blot hybridization assay was employed. Poly(A) mRNA fractions from hypothalami (40–50) of intact, castrated plus vehicle, and castrated plus T groups were isolated, blotted on nitrocellulose paper and hybridized with 32P-end labelled LH-RH oligonucleotides (29 mer) which is complementary to the rat LH-RH mRNA. LH-RH-like mRNA level markedly attenuated 2 weeks following castration and T replacement significantly increased LH-RH-like mRNA level, comparable to about 80% of that observed in intact male rats. In addition, LH-RH contents and release from hypothalami were studied, to examine the correlation with LH-RH gene expression and secretory activity of LH-RH. Castration significantly reduced LH-RH contents and LH-RH release in vitro. T replacement restored LH-RH contents and release comparable to those shown in the intact group. This study demonstrates for the first time that T replacement increases LH-RH-like mRNA level in the hypothalami of castrated rats suggesting that T may act at the pretranslational level. This change in LH-RH gene expression is parallel with alteration of LH-RH content and release.

モルモット心筋収縮能に対するｌｕｔｅｉｎｉｚｉｎｇ ｈｏｒｍｏｎｅ‐ｒｅｌｅａｓｉｎｇ ｈｏｒｍｏｎｅ（ＬＨ‐ＲＨ）の作用

モルモット心筋収縮能に対するｌｕｔｅｉｎｉｚｉｎｇ ｈｏｒｍｏｎｅ‐ｒｅｌｅａｓｉｎｇ ｈｏｒｍｏｎｅ（ＬＨ‐ＲＨ）の作用

Role of protein kinase C in luteinizing hormone-releasing hormone (LHRH)-stimulated progesterone production in rat granulosa cells

Treatment of rat granulosa cells with LHRH or the phorbol ester TPA stimulated progesterone (P) production during a 5-h incubation. The concomitant presence of a potent inhibitor of protein kinase C attenuated the LHRH effect by ca. 60% and completely blocked the stimulatory effect of TPA. Addition of TPA (10 −9, 10 −8 or 10 −7M) or dioctanylglycerol (100 μg/ml) increased P production; these stimulatory effects were not potentiated by the concomitant presence of a calcium ionophore (A23187, 10 −7 or 10 −6M). These data support the hypothesis that protein kinase C activation have a role in the steroidogenic action of LHRH in the ovary.

Absence of specific luteinizing hormone-releasing hormone (LHRH) receptors in the ovaries of Djungarian hamsters (Phodopus sungorus).

High affinity binding sites for luteinizing hormone-releasing hormone (LHRH) were characterized in Djungarian hamsters. Scatchard analysis was used to demonstrate specific LHRH-binding in hamster and, serving as controls, rat pituitaries (dissociation constant, KD = 0.6 nM, binding capacity, BM = 2.5 +/- 0.7 fmol/mg tissue; KD = 0.6 nM, BM = 6.9 +/- 1.9 fmol/mg tissue, respectively). In contrast to results obtained with rat ovaries (KD = 0.9 nM, BM = 3.0 +/- 0.9 fmol/mg tissue), no specific LHRH-binding was detected in hamster ovaries. Thus, it seems that direct gonadal action of LHRH in the Djungarian hamster is not involved in ovarian regulation.

Use of the Cutaneous Anaphylactoid Test to Detect Differences in Mast Cell Mediator-Releasing Activity among LHRH Peptides

The cutaneous anaphylactoid test was evaluated as a screen for assessing mast cell mediator-releasing (MCMR) activity of luteinizing hormone-releasing hormone (LHRH) peptides. As expected, LHRH and D-Trp6-LHRH, an agonist of LHRH, were nonreactive at concentrations of antagonist which induced pronounced skin lesions. Differences between LHRH and antagonists were obvious, but it was difficult to detect differences among antagonists using arithmetic means. However, when the geometric means of the highest positive dilution were calculated, the MCMR activities of the antagonists differed and these differences appeared to be related to the hydrophobicities of their N-termini. Thus, it appears that the cutaneous anaphylactoid test is an appropriate screen to assess the MCMR activity of LHRH peptides and should assist in the development of clinically useful inhibitory analogs of LHRH.

Electrophysiological test of an amphiphilic β-structure in LHRH action

Micropipettes were used to record electrical activity from single neurons in hypothalamic tissue slices, in the hypothalamic arcuate nucleus (ARC), and in the periventricular and suprachiasmatic preoptic nuclei (POA). Responses were measured following in vitro application of luteinizing hormone releasing hormone (LHRH), and two analogues: LHRH models 1 and 3. Model 1 (pyroGlu-His-Trp-Ser-Phe-Thr-Ile-Lys-Ile-ThrNH 2) had amino acid substitutions in residues 5–10 designed to form an amphiphilic β-strand structure. Model 3 (pyroGlu-His-Trp-Ser-Phe-Gly-Ile-Lys-Pro-SerNH 2) was also designed to possess amphiphilic characteristics, but also more closely to resemble the native peptide. Electrical recording results showed that LHRH was able both to excite or inhibit different hypothalamic neurons, and that it was more effective in the preoptic area than in the arcuate nucleus. Responses to LHRH model 3 were strongly correlated with responses to LHRH, in their occurrence and their direction, for both of the brain regions studied. Moreover, LHRH models 1 and 3 also retained the neuromodulatory effects of LHRH on cellular responses to norepinephrine and serotonin. Thus, LHRH analogues, designed to possess an amphiphilic β-structure, preserved some of the properties of LHRH when tested electrophysiologically in the central nervous system.

Regulation of lordosis behaviour in the female rat by corticotropin-releasing factor, β-endorphin/corticotropin and luteinizing hormone-releasing hormone neuronal systems in the medial preoptic area

The role of corticotropin-releasing factor (CRF) and opiocortin neuronal systems and a possible functional relationship between the two in the control of luteinizing hormone-releasing hormone (LH-RH) activity in the medial preoptic area (MPOA) for the regulation of lordosis behaviour were assessed in ovariectomised oestrogen-progesterone-treated female rats. Lordosis behaviour (assessed as the lordosis quotient) triggered by male mounting was significantly inhibited by either CRF or β-endorphin infused into the MPOA in animals treated with normal doses of oestradiol benzoate (OEP) (5 μg) and progesterone (500 μg). Saline-treated animals exhibited high levels of lordosis. The inhibition of lordosis produced by either CRF or β-endorphin could be reversed by LH-RH microinfusions into the MPOA. While naloxone pretreatment of the MPOA site prevented the inhibitory effects of β-endorphin, neither the opiate antagonist nor anti-β-endorphin-γ-globulin (even in high concentrations) infused into the MPOA was effective in completely preventing the inhibition of lordosis produced by CRF. These findings suggest that the inhibition of LH-RH neuronal activity and lordosis behaviour by CRF may be due to a direct action and may not be the result of activation of β-endorphin release. The possibility that the two peptidergic systems may act in a synergistic fashion is supported by the data showing that combined CRF-β-endorphin treatment in the MPOA completely abolished lordosis. This is further supported by the finding that CRF totally abolished lordosis in animals pretreated with anti-corticotropin (ACTH-γ-globulin although this result could suggest that CRF could preferentially stimulate the release of ACTH in the MPOA. Conversely, naloxone, anti-β-endorphin-γ-globulin, anti-CRF-γ-globulin and ACTH infused into the MPOA produced high levels of lordosis in female rats normally showing low levels of lordosis by treatment with low doses of OEB (2 μg) and normal doses of progesterone (500 μg). In each case the facilitation could be blocked by pretreatment of the MPOA site with a potent antagonist analogue of LH-RH. The results indicate the key role of LH-RH in the action of endogenous CRF, β-endorphin and ACTH in the regulation of lordosis behaviour. Each of these substances may act directly on the LH-RH neurone. The postulated presynaptic relationship between CRF and β-endorphin neuronal systems that seem to exist in the mediobasal hypothalamus and the central gray may be less predominant in the MPOA. The results, however, provide clear further evidence for the potent effects of CRF and opiocortin peptides in the regulation of LH-RH and reproduction.

Degradation of luteinizing hormone-releasing hormone (LHRH) and an LHRH agonist by the rat testis

Degradation of LHRH and [ d-Ser(tBu) 6,des-Gly-NH 10 2]LHRH ethylamide (LHRH-A), during incubation with high-speed supernatants of rat testes, as assessed by reversed-phase (RP)-HPLC fractionation of the iodinated peptides and by radioimmunoassays for LHRH or LHRH-A, was principally due to a neutral 43000 Da peptidase with apparent K m values at 25°C of 0.15 μM for LHRH and 1.19 μM for LHRH-A. The peptidase was inhibited by sulphydryl reagents, TLCK, 1,10-phenanthroline, EDTA, bacitracin, other LHRH analogues, oxytocin, [Lys 8]vasopressin and somatostatin. It was predomantly located in seminiferous tubule supernatants (98% of recovered activity), with much lower levels in interstitial fluid (2%), interstitial tissue or testicular paniculate fractions (< 0.8%). Extracts of cultured immature Sertoli cells produced LHRH- and LHRH-A-degradation profiles, as assessed by RP-HPLC, that were identical to those produced by testicular supernatants. Similar levels of peptidase activity/mg protein were observed in immature and adult rat testes. These studies indicate that the principal LHRH-peptidase in the rat testis is produced by cells of the seminiferous epithelium, chiefly the Sertoli cell, and may play an important role in regulating the activity of LHRH and other peptide hormones in the testis.

CAMP augmentation of secretagogue-induced luteinizing hormone secretion

Whether adenosine 3',5'-cyclic monophosphate (cAMP) acts as a mediator for luteinizing hormone-releasing hormone (LHRH) in either its immediate LH release action or in its self-priming action was investigated. Pituitary pieces from either proestrous or estrous rats were superfused in vitro in the presence of dibutyryl cAMP [(Bu)2cAMP], 8-bromo-cAMP (8BrcAMP), forskolin, or control for 2-3 h. For proestrous but not estrous pituitary pieces, a slight increase in base-line LH secretion rate occurred at approximately 70 min of exposure to elevated cAMP; in the same system LHRH caused an increase in LH secretory rate within 2 min in either proestrous or estrous tissue. In contrast to its ineffectiveness as a secretagogue, cAMP elevation resulted in a severalfold augmentation of both LHRH- and elevated K+-induced LH secretion from proestrous but not estrous pituitary pieces; for these experiments, superfusion with a cAMP analogue or forskolin for varying times preceded a 10-min pulse of either 8 nM LHRH or 47 mM K+. Augmentation was evident after 30 min of cAMP elevation; longer exposures were coincident with greater potentiation. Cycloheximide prevented (Bu)2cAMP augmentation of LHRH-induced secretion. These data show that cAMP does not mediate the immediate LH release action of LHRH, but cAMP does augment LHRH- or K+-induced LH secretion with characteristics in common with the self-priming action of LHRH.

Antagonistic Analogs of Luteinizing Hormone-Releasing Hormone Are Mast Cell Secretagogues

The histamine-releasing activity of luteinizing hormone-releasing hormone (LHRH) antagonistic analogs has been documented. Antagonists of LHRH elicited in vitro histamine release from mast cells obtained from previously unexposed rats. Intradermal injection of the antagonists caused increased local skin permeability. Anaphylactoid reactions followed subcutaneous injection of the antagonists and in some cases these edematous reactions were accompanied by increased serum histamine levels. These studies show that these small peptides can cause mast cell degranulation and suggest that the neuropeptide, LHRH, may have modulating effects on the immune system.

Sexual behavior in the female rat following removal of the vomeronasal organ

We used female Wistar rats from which the vomeronasal organ (VNO) had been removed. The first question addressed was whether such females would show deficits in sexual behavior. The data left no doubt that removal of the VNO severely depressed lordosis as well as such proceptive behaviors as “darting” and “hopping.” We then injected estrogen and progesterone and, in still another experiment, estrogen and luteinizing hormone-releasing hormone (LHRH). Estrogen and progesterone significantly enhanced lordosis, but only estrogen and LHRH raised the lordosis quotient of VNO-ablated females to that of the control females. We discuss the results in terms of a theoretical model involving the complementary action of estrogen and LHRH in inducing lordosis in the intact female.

The isolation and measurement of luteinizing hormone-releasing hormone (LHRH) from the rat testis

Although LHRH has been implicated in the direct control of rat Leydig cell function, LHRH has not been previously detected in the rat testis. An optimized fractionation procedure, which involved acid extraction, bulk fractionation on ODS-silica, extraction in chloroform/ethanol, ether extraction, gel filtration on Sephadex G-15 and RP-HPLC, was employed to isolate LHRH from lyophilized adult rat testes. LHRH activity was assessed by an in vitro LHRH bioassay system employing rat anterior pituitary cells in monolayer culture and several radioimmunoassays for LHRH. LHRH recoveries were monitored by the addition of exogenous LHRH to some samples of lyophilized testes prior to extraction. LHRH bioactivity was non-detectable in the LHRH region of the gel filtration profile of testis extracts in the absence of exogenous LHRH; however, using the most specific LHRH RIA procedure, LHRH immunoactivity which co-chromatographed with LHRH following RP-HPLC was found in all extracts. Based on an average LHRH recovery of 31.0%, as determined from the extracts containing exogenous LHRH, the level of endogenous LHRH immunoactivity in the extracts was determined to be the equivalent to 5.6 pg LHRH/g dry weight or 1.0 pg LHRH/testis. These results indicate that the levels of LHRH in the adult rat testis are considerably less than those of the hypothalamus. Based on these findings a simplified fractionation/assay procedure with sufficient sensitivity can be devised for the quantitation of testicular LHRH as a means of clarifying its physiological role in gonadal function.