Stimulation of luteinizing hormone release from chicken pituitary cells by analogues of luteinizing hormone-releasing hormone.

The luteinizing hormone (LH) releasing activities of luteinizing hormone-releasing hormone (LH-RH) and four related analogues were compared using isolated chicken anterior pituitary cells. The analogues, des-Gly¹⁰-LH-RH and Phe⁵-LH-RH, exhibited a greater potency than LH-RH (150 and 237%, respectively), whereas LH-RH(OH) was much less active (1.1%). The potency of Phe⁵-LH-RH was reduced to 0.9% by the insertion of a tyrosine molecule at position 11, indicating that chain length is a significant feature of the biological activity of the molecule. des-Gly¹⁰-LH-RH and Phe⁵-LH-RH were more active in the present system, than is indicated by available information for the rat.     

Gonadotropin-releasing hormone-mediated desensitization of cultured rat anterior pituitary cells can be uncoupled from luteinizing hormone release

GnRH stimulates LH release from pituitary gonadotropes. Prolonged exposure of these cells to GnRH results in decreased sensitivity to further stimulation by the releasing hormone both in vivo and in vitro. Chelation of extracellular Ca++ with EGTA blocks GnRH-stimulated LH release but does not prevent subsequent desensitization. Desensitization occurs when cells are preincubated in EGTA containing 10(-7) M GnRH for a variety of times (20 min to 12 h) or when cells are preincubated for 3 h in EGTA with 10(-10), 10(-9), or 10(-8) M GnRH. A GnRH antagonist does not cause desensitization to GnRH and blocks desensitization in response to GnRH in the Ca++-free medium. Preincubation in EGTA containing 10(-7) M GnRH for 3 h did not alter sensitivity of cells to sn 1,2 dioctanoylglycerol (a protein kinase C activator), Ca++ ionophore A23187, or veratridine (an activator of endogenous ion channels). These results suggest that desensitization results from occupancy of the GnRH receptor by an agonist and may be uncoupled from LH release.     

Specific release of a novel pituitary polypeptide, 7B2, from rat anterior pituitary cells in vitro by luteinizing hormone-releasing hormone

We have recently purified a novel pituitary polypeptide, designated 7B2. Subsequently, we developed a sensitive and specific radioimmunoassay (RIA) for this novel polypeptide. Our aim in the present study was to investigate the release of 7B2 from rat pituitary induced by various hypothalamic factors [luteinizing hormone-releasing factor (LH-RH), corticotropin-releasing factor (CRF), and growth hormone-releasing factor (GRF)]. The anterior pituitaries were removed from rats and immediately dispersed enzymatically (a mixture of collagenase/dispase/deoxyribonuclease/chicken serum) and plated on collagen-coated multiwell plates in culture medium containing 10% fetal bovine serum. After 2 days of attachment period, the medium was replaced with fresh medium every 24 h. The primary cell culture was incubated with various concentrations of LH-RH, CRF or GRF. Subsequently, the concentrations of IR-7B2, IR-LH, IR-FSH, and IR-ACTH released into the medium were quantified by specific RIA. LH-RH, at a concentration as low as 7.5 ng/ml (6 X 10(9) M: dose range 7.5-60 ng/ml) stimulated the release of IR-7B2, IR-LH, and IR-FSH, by 2- to 3-fold, 17- to 18-fold, and 3-fold, respectively, over basal levels. No significant increase of IR-7B2 was observed when stimulated by CRF or GRF at doses as high as 100 ng/ml. In addition, K+ (50 mM) stimulated the release of all the peptides measured. In conclusion, our studies suggest that the novel peptide 7B2 is under LH-RH control and indirectly confirm the immunohistochemical results of its cellular co-localization in FSH and LH cells.     

Gonadal steroid modulation of signal transduction and luteinizing hormone release in cultured chicken pituitary cells

The mechanism whereby gonadal steroids modulate GnRH-stimulated LH secretion by primary cultures of chicken pituitary cells was investigated. Estradiol (10(-8) M), testosterone (10(-7) M), and progesterone (10(-7) M) inhibited LH release stimulated by GnRH (10(-7) M) by 56%, 61%, and 53%, respectively, and the inhibitory effects required prolonged preincubation (24-48 h) with the steroids. The steroids inhibited the spike (0-3 min) and plateau (9-30 min) phases of LH release to a similar degree. The ED50 values of estradiol, testosterone, and progesterone for inhibition of GnRH-stimulated LH release were 7 x 10(-11), 2 x 10(-9), and 1 x 10(-9) M, respectively. Estradiol, testosterone, and progesterone inhibited the maximal LH response to GnRH, but the ED50 of GnRH (4 x 10(-9) M) was not altered by steroid pretreatment. Steroid pretreatment did not cause a change in cellular LH content, suggesting that the steroids do not inhibit LH synthesis. Combinations of two or three of the steroids were not additive, suggesting that all three steroids affect GnRH-stimulated LH release via the same mechanism. In experiments investigating their mechanism of action, the steroids inhibited LH release stimulated by GnRH and Ca2+ ionophore A23187, but generally had no effect on the responses to phorbol ester (12-O-tetradecanoylphorbol-13-acetate), forskolin, K+, Bay K8644, or veratridine. Estradiol inhibited GnRH-stimulated 45Ca2+ efflux, but its inhibitory effect on GnRH-induced inositol phosphate production was not significant. Estradiol had no effect on binding of 125I-[His5,D-Tyr6]GnRH to a pituitary cell preparation. These findings suggest that the site of steroid modulation of GnRH action is distal to binding of GnRH to its receptor, and that the inhibitory effects are exerted at two intracellular sites: 1) the coupling events linking receptor activation to mobilization of Ca2+, and 2) a site distal to Ca2+ mobilization.     

Similar luteinizing hormone-releasing hormone binding sites in rat anterior pituitary and ovary.

To study the luteinizing hormone-releasing hormone (LH-RH; luliberin) receptors in the rat anterior pituitary gland and ovary, 125I-labeled [D-Ser(TBU)6des-Gly-NH2(10)]LH-RH ethylamide was used as a labeled ligand. The binding characteristics were assessed by Scatchard analysis of labeled ligand binding and by potency displacement with unlabeled peptides. Similar Kd values, ranging from 0.1 to 0.3 nM, were found for the labeled and unlabeled peptides in both tissues. A similar order of potency was observed between the finding affinity of 15 peptides in anterior pituitary and ovarian homogenates and their biological activity on luteinizing hormone release in rat anterior pituitary cells in culture. These data demonstrate that the LH-RH receptors present in the rat ovary have a specificity similar to that of the anterior pituitary LH-RH receptor controlling secretion of luteinizing hormone and follicle-stimulating hormone. Moreover, the binding affinities of the LH-RH agonists and antagonists can account, at least up to a large extent, for their relative biological potencies. Although there definitely are specific LH-RH receptors in the ovary which may play a role in the antifertility effects observed after administration of LH-RH agonists, the possible physiological significance of these ovarian receptors is still unknown and of great biological interest.     

Dissociation of the porcine anterior pituitary: the kinetics of luteinizing hormone release in response to luteinizing hormone-releasing hormone.

A method has been developed for disaggregating porcine anterior pituitary tissue and for providing dissociated preparations which have good, stable viability in culture. The secretory capacity for these preparations in terms of their ability to release luteinizing hormone in response to luteinizing hormone-releasing hormone has been documented in detail. In following the short-term kinetics of LH secretion a biphasic pattern of release has been demonstrated in which a sharp initial peak, maximal at 2 min, is followed by a second, more prolonged phase of release reaching a maximum between 10 and 20 min.     

Effects of suramin on hormone release by cultured rat anterior pituitary cells

Suramin is a polyanionic compound which has been used in the treatment of trypanosomiasis and acquired immunodeficiency syndrome (AIDS), while preliminary success has been reported in the treatment of cancer. However, suramin also causes adrenal insufficiency. We have previously reported that suramin selectively inhibited corticotropin (ACTH)-stimulated corticosterone release by dispersed adrenal cells in a dose-dependent manner via a direct interaction with the ACTH molecule. The present study was undertaken in order to investigate the effect of suramin on hormone release by dispersed rat anterior pituitary cells. Suramin at a concentration of 100 microM inhibited both basal and secretagogue-stimulated ACTH release by cells cultured in minimal essential medium (MEM) only, while it had no effect on ACTH release by cells cultured in MEM + 10% fetal calf serum (FCS) or MEM + 0.1% bovine serum albumin (BSA). In addition, suramin also caused a parallel decrease of prolactin (PRL) and growth hormone (GH) release by cells cultured in MEM only, suggesting a toxic, rather than a selective effect of suramin on anterior pituitary cells cultured in MEM only. In addition, suramin potentiated the effect of thyrotropin-releasing hormone (TRH) on PRL release by cells cultured in MEM + 10% FCS and suppressed the inhibitory effect of dopamine (DA) on PRL release by cells cultured in MEM + 10% FCS and in MEM + 0.1% BSA. Comparable suppressive effects of suramin on growth hormone-releasing hormone (GHRH)-stimulated and somatostatin (SRIH)-inhibited GH release were found in cells cultured in MEM + 0.1% BSA but not in cells cultured in MEM + 10% FCS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leukotriene C4 as a mediator of luteinizing hormone release from rat anterior pituitary cells.

This study demonstrates that leukotriene C4, at concentrations in the picomolar range, released luteinizing hormone (LH) but not growth hormone (GH) from dispersed rat anterior pituitary cells. Leukotriene B4, another lipoxygenase pathway product of arachidonic acid, had no effect on LH or GH release. The stimulatory effect of leukotriene C4 could be seen after 0.5 but not after 3 hr of incubation. This was in contrast to the dose-dependent LH-releasing hormone (LHRH)-induced LH release that was not measurable after 0.5 hr but was fully established after incubation for 3 hr. Furthermore, the LH-releasing ability of leukotriene C4 was blocked in the presence of high doses of LHRH. The immunohistochemical analysis revealed leukotriene C4-immunoreactive fibers at all levels of the median eminence, mainly in the lateral parts. These fibers exhibited a marked overlap distribution with LHRH-immunoreactive fibers and elution-restaining experiments revealed identity of at least a large proportion of the leukotriene C4- and LHRH-immunoreactive fibers. Furthermore, cell bodies in the preoptic area contained both leukotriene C4- and LHRH-like immunoreactivities, suggesting localization of these two compounds in the same neurons.     

Desensitization of luteinizing hormone release in cultured pituitary cells by gonadotropin-releasing hormone

Preincubation of cultured pituitary cells with GnRH caused a marked decrease in subsequent LH release. The rate of desensitization increased when the preincubating concentration of GnRH and the preincubation time were increased. Pituitary cells obtained from male rats were not as sensitive to GnRH as cells obtained from female rats and the extent of desensitization was also smaller in cells from male rats. Densensitization was found to be a long-lasting effects, without any change in the viability of the cells. A superactive analogue of GnRH (D-Phe6-GnRH) caused almost complete desensitization of LH secretion, while a competitive inhibitory analogue of GnRH caused a much smaller decrease in LH response which could be overcome by increasing the concentration of GnRH used for reincubation. These data suggest that the desensitization is closely related to the biological activity of GnRH and does not correlate with receptor binding. High concentrations of potassium also induced desensitization, although to a lower extent than GnRH. Since K+ induces LH release by a different mechanism than GnRH, our data suggest that the desensitization phenomenon cannot be explained only at the receptor level. The time curve of desensitization supports the idea that GnRH action has two-phases: an acute effect which cannot be desensitized, and a secondary phase which can be densensitized.     

Requirement of extracellular calcium in fish pituitary gonadotropin release by gonadotropin hormone-releasing hormone

Influence of extracellular calcium on gonadotropin hormone-releasing hormone (GnRH)-stimulated gonadotropin hormone (GtH) release from a teleostean fish (Channa punctatus) pituitary was examined in vitro by preparing enzymatically dispersed pituitary cell incubation. Effect of Ca2+ on GnRH-augmented GtH release was evaluated with partially purified C. punctatus GnRH (cGnRH) and synthetic mammalian GnRH (mGnRH). Cells were dispersed by 0.3% collagenase plus 0.05% trypsin in culture medium and a high yield of viable cells were obtained. Addition of cGnRH (10 micrograms/ml) to pituitary cells in Ca2+-free medium resulted in a significant increase in GtH release, but the addition of Ca2+ (2 mM) enhanced it to about four- and threefold over cGnRH and mGnRH, respectively. Increasing concentrations of Ca2+ (0.1-2.0 mM/well) with fixed concentrations of GnRH (10 micrograms/ml) or increasing doses of GnRH (2.5 to 20 micrograms/ml) with fixed amount of Ca2+ (2 mM/well) resulted in a dose dependent increase in GtH release. EDTA or EGTA (2 mM/well) completely suppressed the Ca2+-augmenting effect of GnRH-stimulated GtH release. Addition of lanthanum (La3+, 4 mM/well), a competitive inhibitor of Ca2+, reduced 60% of the Ca2+ (2 mM/well) stimulation. Verapamil, a specific Ca2+ channel blocker, when added in increasing concentrations (1-100 microM/well) to pituitary cell incubations containing GnRH-stimulated GtH release in Ca2+-free medium could be waived by EGTA (2 mM/well), indicating availability of extracellular calcium from tissue sources. The uptake of radioactive Ca2+ by pituitary cells was greatly enhanced by GnRH while the addition of verapamil (10 microM/well) not only inhibited the GnRH-stimulated uptake, but also reduced it below the control level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and release of luteinizing hormone by rat anterior pituitary cells: effects of cytochalasins B and D

We determined the role of microfilaments in regulating LH synthesis (translation or glycosylation) and release from cultured rat anterior pituitary cells under basal and GnRH-stimulated conditions. Cells were pretreated for 2 h with microfilament-disrupting drugs, cytochalasin B (CB; 2 and 20 microM) or cytochalasin D (CD; 1 and 10 microM). LH synthesis and release were measured after 4 h of incubation with or without 1 nM GnRH and drugs. LH translation and glycosylation were monitored by measuring the incorporation of [14C]alanine and [3H]glucosamine, respectively, into total (cell and medium) immunoprecipitable LH. Immunoreactive LH (IRLH) in medium and cells was measured by RIA. GnRH at 1 nM significantly (P less than 0.01) increased the release of IRLH and total [3H]LH (glycosylation), but had no effect on total [14C]LH (translation), uptake, or incorporation of precursors into total protein. Neither CB (2 and 20 microM) nor CD (10 microM) altered basal or GnRH-stimulated IRHL release. Neither drug altered basal medium concentrations of [3H]LH or [14C]LH. In contrast, both CB and CD reduced (P less than 0.01) GnRH-stimulated [3H]LH in the medium and total system (LH glycosylation). CB reduced (P less than 0.01) [3H]glucosamine uptake, total [3H]protein synthesis, and basal level of total [3H]LH, while CD had no effects on these parameters. Thus, CD exerted a more specific inhibitory effect on GnRH-stimulated LH glycosylation than CB. CB (2 and 20 microM) increased (P less than 0.01), while CD (10 microM) decreased (P less than 0.01) [14C]alanine uptake, total [14C]LH, and [14C]protein under both basal and GnRH-stimulated conditions. These results demonstrated that while the cytochalasins did not inhibit either basal or GnRH-stimulated IRLH release, they did inhibit GnRH-stimulated LH glycosylation, although the effect of CB was due partially to reduced [3H]glucosamine uptake. Integrity of microfilaments appears to be important for GnRH-enhanced LH glycosylation, but not for GnRH-enhanced LH release.     

Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells

The purpose of this study was to investigate the effects of lowering the extracellular calcium concentration on GnRH-stimulated LH glycosylation and LH translation, as measured by the incorporation of [3H]glucosamine (3H-Gln) and [35S]methionine (35S-Met) into immunoprecipitable LH. Cultured anterior pituitary cells, previously exposed to estradiol (5 X 10(-10) M) to maximize precursor incorporation were incubated for 4 h in normal calcium (2.5 mM) or low calcium medium (less than 15 microM) containing radiolabeled precursors with or without 1 nM GnRH. In the presence of normal calcium, GnRH significantly increased 3H-Gln-labeled LH in the medium (278%) and cells (290%), as well as total (cells plus medium) 3H- Gln LH (280%) compared to the control value (no GnRH). GnRH also significantly increased the 35S-Met LH released into the medium (164%) and total 35S-Met LH (186%) over control values. Depletion of extracellular calcium completely inhibited GnRH-stimulated 3H-Gln LH and 35S-Met LH production. Total immunoreactive LH (iLH), as measured by RIA, was also increased significantly by GnRH treatment in the presence of calcium, but this response was prevented by removal of calcium from the medium. Lowering extracellular calcium had no effect on cellular uptake or incorporation of 3H-Gln or 35S-Met into total trichloroacetic acid-precipitable protein. Approximately 80% of newly synthesized LH was released into the medium in all treatment groups independent of whether calcium or GnRH was present. The specific activity (disintegrations per min/microgram iLH) of radiolabeled LH released into the medium was significantly reduced by treatment with GnRH due to the large amount of unlabeled iLH released into the medium. However, when the cells were incubated in low calcium, the SA of 3H-Gln LH and 35S-Met LH in the medium was unaltered by GnRH, whereas GnRH-stimulated iLH release was inhibited. We conclude that GnRH stimulation of LH glycosylation and LH apoprotein synthesis involves extracellular calcium-dependent events, and the release of newly synthesized LH is closely coupled to LH biosynthesis and is less dependent on extracellular calcium, whereas the GnRH-stimulated release of previously synthesized, stored LH is dependent on extracellular calcium.     

Stimulation by phorbol ester and diacylglycerol of luteinizing hormone glycosylation and release by rat anterior pituitary cells

We studied the effects of protein kinase C (PKC) activators on LH glycosylation and release and the effect of 17 beta-estradiol on PKC activator-induced LH release. Rat anterior pituitary cells were incubated for 4 h with diluent, GnRH, and the PKC activators, phorbol 12-myristate 13-acetate (PMA), L-alpha-1,2-dioctanoyl glycerol (C8), and 1-oleoyl-2-acetyl-glycerol. LH translation and glycosylation were monitored by measuring incorporation of [14C]alanine ([14C]A) and [3H]glucosamine ([3H]GA), respectively, into total (medium + cell) immunoprecipitable LH. Immunoreactive LH (IRLH) was measured by RIA. PMA (10(-9) M) and 1-oleoyl-2-acetyl-glycerol (50-200 microM) had no significant effects. PMA at 10(-7) M elevated (P less than 0.01) medium IRLH, medium and total [3H]GA-LH, and medium but not total [14C]A-LH. PMA at 10(-7) M increased (P less than 0.01) uptake and incorporation of [3H]GA, but not [14C]A, into total pituitary protein. C8 increased both medium IRLH and total [3H]GA-LH (P less than 0.01) without altering total [14C]A-LH. Two hundred micromolar C8 increased medium concentrations of [3H]GA-LH (P less than 0.01) and [14C]A-LH (P less than 0.05). C8 (50-200 microM) had no detectable effects on uptake and incorporation of precursors into protein. GnRH (1 nM) enhanced (P less than 0.01) both medium IRLH and total [3H]GA-LH, but had no effect on total [14C]A-LH. Pretreatment of pituitary cells with 17 beta-estradiol (6 X 10(-10) M) greatly enhanced LH release induced by C8. In conclusion, PMA and C8, like GnRH, stimulated both LH glycosylation and release. These results suggest that PKC may regulate both LH release and glycosylation and may be important in estrogen modulation of LH release.     

Prolactin suppresses luteinizing hormone secretion and pituitary responsiveness to luteinizing hormone-releasing hormone by a direct action at the anterior pituitary

In pathological or experimental hyperprolactinemia, the elevated circulating levels of PRL are the usual cause of the impairment in gonadotropic function. The present study was undertaken to determine whether PRL could suppress basal LH secretion and LHRH-stimulated LH release by a direct action at the anterior pituitary. Anterior pituitaries from ovariectomized rats were incubated in medium 199 alone or in medium 199 containing ovine PRL, and basal and the LHRH-stimulated LH release were followed for 2 or 3 h in vitro. Ovine PRL at 40 and 80 micrograms/ml suppressed basal LH release by 41% and 72%, respectively, at 2 h of incubation. This suppressive effect of both concentrations of PRL continued to the third hour of incubation. LHRH at 5 ng/ml increased the release of LH from pituitaries incubated in medium alone by 57%, 61%, and 107% at 1, 2, and 3 h of incubation, respectively. However, in the pituitaries treated with 40 micrograms/ml ovine PRL, the stimulatory effects of LHRH were diminished at all time points measured. Pretreatment of anterior pituitaries with ovine PRL for 6 h significantly inhibited by 81% the LHRH (5 ng/ml) stimulation of LH release at 2 h of incubation. On the other hand, inhibition of endogenous PRL release by 10(-6) M bromocriptine enhanced the stimulatory effects of 5 ng/ml LHRH by 2.5-fold at 2 h of incubation. The inhibitory effects of PRL on basal and stimulated LH secretion appeared unique, since neither BSA nor vasopressin could elicit similar suppressive effects on LH. These results suggest that in anterior pituitaries exposed to elevated levels of PRL, LH secretion and pituitary responsiveness to LHRH could be impaired. This phenomenon may contribute in part to the antigonadotropic effects of PRL.     

Activation of luteinizing hormone release from pituitary cells by polycations

In the present work we examined the effect of cationic polymers on the pituitary gonadotrope. Such polymers are widely used to anchor gonadotropes and other cell types to culture dishes and other substrata to which they are not normally adherent. Homopolymers of Lys (eight size classes from 4,000-700,000 daltons) stimulate Ca+2-dependent LH release from pituitary cell cultures. In contrast, release does not occur in response to the epsilon-CBZ or succinyl derivatives (which have no internal charge) or in response to polymers of L-Glu, D-Glu, or Gly. The observation that polymers of D-Lys, L-Lys, and L-Arg all stimulate LH release with similar efficacy and potency indicates that simple charge interactions, rather than interaction with specific polymer-binding sites, are the cause of LH release. Since monomeric Lys neither stimulates LH release nor competitively inhibits release in response to Lys polymers, it appears that multiple charge coordination by Lys polymers is responsible for activation of the release mechanism. Putrescine, spermine, and spermidine (which have more closely spaced charges) do not stimulate LH release, suggesting that a certain minimal distance of charge separation must occur to obtain efficacy. The reduced potency of heteropolymers of Lys (spaced with Ala or Tyr) suggests that a maximal effective distance also exists. Consecutive and concomitant incubation studies indicate that LH released in response to poly-L-Lys or GnRH comes from the same pool as that released by GnRH. The time courses of release are similar for the two compounds.     

Growth hormone-releasing hormone stimulates cAMP release in superfused rat pituitary cells.

The release of growth hormone (GH) and cAMP was studied in superfused rat pituitary cells by infusing growth hormone-releasing hormone (GHRH) at different doses or a combination of GHRH and somatostatin 14 (SS-14). Three-minute pulses of GHRH caused a dose-dependent GH and cAMP release (effective concentration of 50% of the maximal biological effect is 0.21 nM and 52.5 nM, respectively). The lowest effective doses of GHRH in the superfusion system were 0.03 nM for GH release and 0.3 nM for cAMP discharge when 3-min pulses were applied. The amount of cAMP liberated from the cells was not proportional to GH release: cAMP responses to low doses of GHRH were disproportionally small, and the gradual increase in the release of cAMP after high doses of GHRH was not followed by a parallel rise in GH release. The desensitization induced by repeated pulses or prolonged infusion of GHRH resulted in a greater reduction in GH release than in cAMP liberation. A simultaneous infusion of SS-14 completely blocked GH release stimulated by GHRH but did not inhibit the immediate release of cAMP caused by GHRH. An abrupt decrease in GHRH-stimulated GH release induced by SS-14 was followed by only a minimal reduction in cAMP liberation 9 min later. Our findings indicate that a discharge of cAMP is stimulated after a GHRH pulse, but this effect alone cannot maintain the release of GH. Other steps of the signal transduction mechanisms that are independent of the cAMP route may participate in the process of GH release. The nature of the mechanisms involved in the mediation of GH release may vary with the doses of GHRH used.     

Inhibition of luteinizing hormone release by morphine and endogenous opiates in cultured pituitary cells

Morphine sulfate was found to have a direct inhibitory effect on both basal and GnRH-stimulated LH release by cultured rat pituitary cells. The inhibitory effect of morphine on LH release was prevented by the opiate antagonist naltrexone, and treatment of cells with naltrexone or beta-endorphin antiserum significantly increased basal LH release. Also, incubation of pituitary cells with CRF caused a significant decrease in basal LH release, an effect that was reversed by naltrexone. Saturable opiate-binding sites were demonstrated in enriched gonadotrophs by [3H]etorphine binding studies. The ability of morphine to inhibit gonadotropin secretion through a direct action on pituitary opiate receptors suggests that long term exposure to exogenous opiates may suppress reproductive function at the hypophyseal level. In addition, the converse effects of CRF and naltrexone or beta-endorphin antiserum on LH release indicate that intrapituitary opioid peptides could exert a paracrine inhibitory action on the gonadotroph.