Ultrastructural observations of granule extrusion from rat anterior pituitary cells after injection of LH-releasing hormone

Summary In order to observe the ultrastructural aspects of granule extrusion from gonadotrophs, the authors injected pure, natural porcine LH-releasing hormone (LH-RH) into persistent-estrous (PE) rats. Clear-cut extrusions of secretory granules appeared from LH-gonadotrophs 2.5, 5 and 15 minutes after the injection. Massive extrusion was observed at 15 minutes. Serum LH and FSH were also estimated by radioimmunoassay on blood samples taken at the same times after the injections. LH was increased in the blood sera after the injection of LH-RH, but serum FSH was not significantly different among the various treated rats. The rise in serum LH after LH-RH injection was well correlated with the ultrastructural phenomenon of granule extrusion from LH-gonadotrophs.The study was supported by USPHS Grants AM 12583, AM 09094 and AM 07467.The authors acknowledge the able assistance of Mrs. Martha Castilleja in San Antonio and of Meredith M. Nickel in New Orleans.     

Age-related changes in the secretory pattern of FSH and LH in response to LH-RH in prepubertal female rats

Developmental changes in the pituitary responsiveness and the secretory pattern of FSH and LH in response to a single injection of LH-RH (100 ng/rat, s.c.) as estimated by increases in plasma concentrations of FSH and LH 10, 30 and 60 min after the injection were studied in female rats at 5, 10, 15, 20, 25 and 30 days of age. The pituitary responsiveness to LH-RH for both FSH and LH release increased from 5 to 15 days of age, reached a maximum on 15 days of age and declined thereafter, whereas a marked increase in the amount of these hormones in the pituitary occurred between 15 and 20 days of age. An apparent change in the secretory pattern of both FSH and LH was observed from 20 days of age onward. In groups up to 15 days of age, plasma concentrations of FSH and LH remained elevated 60 min after the injection of LH-RH, though the plasma concentration of these hormones returned to preinjection concentrations in groups at 20 days of age or later. These results indicate that the age-related changes in the secretory pattern of LH and FSH in response to LH-RH as well as changes in the pituitary responsiveness were apparent during the prepubertal period.     

Subcellular localization of gonadotrophic hormones LH and FSH in frog adenohypophysis using double-staining immunocytochemistry

We applied double post-embedding immunocytochemical methods using specific antibodies against bullfrog (Rana catesbeiana) luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with immunogold staining (5- and 20-nm particles) to determine the subcellular localization of both gonadotropins and to observe their immunostaining patterns in anterior pituitary of the frog Rana pipiens. Results showed that individual gonadotrophs may store either one or both gonadotropins in a given secretory granule and in large globules (lysosomes?). Most gonadotrophs (50-88%) contain both hormones; 12-50% contain only FSH, and only a few (0-7%) contain LH alone. Individual secretory granules, even in cells that contain both hormones, may contain only one or both gonadotropin molecules. Evaluation of the percentage of monohormonal and multihormonal secretory granules revealed that multihormonal secretory granules were the most numerous and that LH monohormonal secretory granules were the least numerous. These results indicate that cellular storage of gonadotropin in amphibian pituitary is similar to that described for mammals, where a single cell type containing both gonadotropins predominates. Variability in hormone content both of cells and of granules in all individuals is consistent with the hypothesis that frog pituitary possesses a single multipotential gonadotroph.     

Differential sorting of lutropin and the free alpha-subunit in cultured bovine pituitary cells.

The glycoprotein hormones lutropin (LH) and follitropin (FSH) are both synthesized by gonadotrophs in the anterior pituitary but are stored in separate secretory granules prior to secretion. Despite having highly homologous beta-subunits and alpha-subunits with the identical amino acid sequence, the Asn-linked oligosaccharides on LH terminate with SO4-GalNAc while those on FSH terminate with sialic acid-Gal. In addition to LH and FSH, gonadotrophs secrete uncombined (free) alpha-subunit which bears the same sulfated oligosaccharides as LH. We have examined the synthesis and secretion of LH and free alpha-subunit in primary cultures of bovine pituitary cells in order to determine if the sulfated oligosaccharides have any impact on sorting. Our results show that newly synthesized free alpha-subunit is secreted exclusively via the constitutive pathway with a t1/2 of 1.8 h and is never found in dense-core secretory granules. In contrast, LH dimer is secreted by both the constitutive and the regulated pathways. Constitutive secretion and arrival in a dense secretory granule both occur with t1/2 values of 1-1.5 h for newly synthesized LH. Sulfation occurs immediately prior to arrival of LH in the secretory granule and is followed by a period of 1-1.5 h before the LH-containing granules become sensitive to release by gonadotropin releasing hormone. As a result the t1/2 for LH secretion in the presence of gonadotropin releasing hormone is 3.5 h. Sulfation of the free alpha-subunit oligosaccharides is not, therefore, sufficient to direct the alpha-subunit to secretory granules, and the information required for directing LH to granules must reside either in the beta-subunit or the alpha beta-complex.     

Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs

Summary Chromogranins (Cg)/secretogranins (Sg) are representative acidic glycoproteins in secretory granules of many endocrine cells where they are co-stored and co-released with resident amines or peptides. The exact distribution of these proteins in the rat anterior pituitary is unknown. Therefore, pituitaries from untreated male rats were investigated by light- and electron-microscopical immunocytochemistry for the cellular and subcellular localization of CgA, CgB, and SgII. Endocrine cells, identified light-microscopically as gonadotrophs in adjacent semithin sections immunostained for follicle-stimulating hormone (FSH) and luteinizing hormone (LH), concomitantly were immunoreactive for CgA, CgB, and SgII. Ultrastructurally, gonadotrophs exhibited two types of secretory granules which varied in their immunoreactivities for gonadotropins and Cg/Sg. Large-sized (500 nm), moderately electron-dense granules showed antigenicities for FSH, LH, and CgA. Smaller-sized (200 nm), electron-dense granules were immunoreactive exclusively for LH and SgII. The distinct localization of CgA and SgII to morphologically and hormonally different secretory granules indicates the existence of two regulated secretory pathways in rat pituitary gonadotrophs. Hence, these proteins are considered as valuable tools to analyze the intracellular trafficking during granule biogenesis and the possible different regulation of FSH and LH secretion.     

Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs.

Chromogranins (Cg)/secretogranins (Sg) are representative acidic glycoproteins in secretory granules of many endocrine cells where they are co-stored and co-released with resident amines or peptides. The exact distribution of these proteins in the rat anterior pituitary is unknown. Therefore, pituitaries from untreated male rats were investigated by light- and electron-microscopical immunocytochemistry for the cellular and subcellular localization of CgA, CgB, and SgII. Endocrine cells, identified light-microscopically as gonadotrophs in adjacent semithin sections immunostained for follicle-stimulating hormone (FSH) and luteinizing hormone (LH), concomitantly were immunoreactive for CgA, CgB, and SgII. Ultrastructurally, gonadotrophs exhibited two types of secretory granules which varied in their immunoreactivities for gonadotropins and Cg/Sg. Large-sized (500 nm), moderately electron-dense granules showed antigenicities for FSH, LH, and CgA. Smaller-sized (200 nm), electron-dense granules were immunoreactive exclusively for LH and SgII. The distinct localization of CgA and SgII to morphologically and hormonally different secretory granules indicates the existence of two regulated secretory pathways in rat pituitary gonadotrophs. Hence, these proteins are considered as valuable tools to analyze the intracellular trafficking during granule biogenesis and the possible different regulation of FSH and LH secretion.     

Progesterone and LH variations after LH-RH administration in the luteal phase of the menstrual cycle

We have investigated the pituitary and luteal responses to LH-RH and their related changes. 11 normal women were studied during the luteal phase (day +4/+11). Blood samples were collected every 15 min for a basal period of 180 and 120 min after the intravenous administration of 25 micrograms of LH-RH. Progesterone (P) and LH were assayed by radioimmunoassay. Data were analyzed as maximum peak and its percent increase (delta max), integrated secretory area (ISA) and percent increase of ISA (delta A) in respect to basal values for both P and LH. LH-RH elicited a secretory response of both hormones in all cases. ISA of LH was significantly greater after LH-RH administration in respect to basal values (p less than 0.001) and delta max accounted to 475 +/- (SE) 36% of the basal concentration. Luteal responsiveness varied from about 115-130% to more marked increments. ISA of P differed from basal to stimulated conditions (p less than 0.05) and delta max was 166 +/- (SE) 14%. The analysis of temporal relationship between P and LH secretion showed that LH promptly rose after LH-RH, while the enhancement of P plasma levels occurred within 31 +/- 19 min after LH rise. Then P levels reached a plateau, values of which were statistically different from those observed before LH-RH administration. In two cases where luteal function was blunted or absent, in spite of marked increments of LH, P secretion did not occur. These data are consistent with the presence of close relationships between hypothalamic, pituitary and luteal functions and strengthen the contention about the usefulness of LH-RH during luteal phase for the lifespan and maintenance of corpus luteum.     

Crystalline inclusions in the pituitary gonadotrophs of estrogen-primed, spayed rhesus monkeys (Macaca mulatta)

17-β-Estradiol or cholesterol was subcutaneously implanted in bilaterally oophorectomized rhesus monkeys for 37 days, after which the pituitary glands were analyzed ultrastructurally for evidence of granule disposal. Plasma estrogen returned to a physiologic level of 97.6 ± 2.8 pg/ml in the estrogen-treated animals but not in the cholesterol-treated controls. The hypertrophied gonadotrophs in the controls contained numerous large vesicles and a few secretory granules. The gonadotrophs in the experimental animals showed a reduction in cell size, a diminution of the dilated endoplasmic reticulum, and an accumulation of secretory granules. Moreover, crystalline inclusions composed of parallel and orthogonally arranged fibers were present in some gonadotrophs, most frequently in animals with lower levels of circulating estrogen. These inclusions are thought to be involved in the process of intracellular secretion in monkeys.     

Ultrastructural immunocytochemical localization of LH and FSH in the pituitary of the untreated male rat

Summary Rapid freeze-substitution fixation was employed in immunocytochemical studies on the localization of LH and FSH in the typical gonadotrophs of the anterior pituitary in the untreated male rat; a modification of a recently described ferritin antibody method (Inoue et al. 1982) was used in these studies. It was shown that rapid freeze-substitution fixation provides good preservation not only of the ultrastructure but also of the antigenicity. Both LH and FSH were clearly demonstrated in the same gonadotrophic cells, but the subcellular localization of these gonadotrophins differed: (i) LH was mainly located in small secretory granules, 250–300 nm in diameter; (ii) FSH was mainly present in large secretory granules, up to 500 nm in diameter. In the pituitary gland of the adult male rat, all gonadotrophs that react to antibodies against gonadotrophins are characterized by small and large secretory granules. Other types of cells of the anterior pituitary containing either small secretory granules or resembling corticotrophs with secretory granules assembled at cell periphery did not react to either anti-LH beta or anti-FSH beta serum.For light microscopy, the peroxidase antibody method was used. All of the gonadotrophin-positive cells contain both LH and FSH. None of the pituitary cells reacted to antibody against only one gonadotrophin. However, some cells are LH-rich while other cells are FSH-rich.     

Morphological changes of pituitary gonadotrophs and thyrotrophs following treatment with LH-RH or TRH in vitro

Summary To investigate the relationship between LH-RH and TRH and the formation of castration cells and thyroidectomy cells, pituitary glands of 14-day old female rats were cultured with LH-RH or TRH for 10 days. Observed in these glands were external and internal zones, the former containing active, healthy appearing cells, and the latter consisting of many degenerative and necrotic cells. Gonadotrophs and thyrotrophs were readily demonstrated in the external zone of the organs by immunocytochemistry. Hypertrophie gonadotrophs (castration cells) and thyrotrophs (thyroidectomy cells) were observed only in the external zone by electron microscopy. Neither the typical signet-ring gonadotrophs nor intracisternal granules of thyroidectomy cells were seen in the present study. However, the hypertrophic gonadotrophs or thyrotrophs were similar to castration cells or thyroidectomy cells observed in the pituitary gland following castration or thyroidectomy. The results indicate that LH-RH and TRH directly stimulate the secretion of hormones and alter the morphological features of pituitary target cells.Supported by USPHS Grant HD 11826. The author wishes to express his thanks to NIAMDD for providing pituitary hormones     

Binding and internalization of native gonadoliberin (GnRH) by anterior pituitary gonadotrophs of the rat

Summary To identify anterior pituitary cell types containing GnRH binding sites and to study the internalization process of this peptide by target cells under physiological conditions, autoradiography was performed on rat anterior pituitaries removed at specific time intervals (2–60 min) after intravenous injection of mono-radioiodinated ¹²⁵I-GnRH into intact males. At electron-microscopic level, gonadotrophs and lactotrophs appeared to contain silver grains. Concomitant administration of an excess of unlabeled GnRH with the radioiodinated hormone prevented this localization indicating the specificity of the reaction. The time-course study in gonadotrophs showed that 2 min after injection silver grains could be found over the plasma membrane, secretory granules and nuclear membrane. Similar results were observed 5 and 15 min after injection. Extensive label was observed over the nucleus and nuclear membrane 15 to 60 min after injection. The injection of a radioiodinated GnRH agonist [D-Trp⁶, Pro⁹ (Net), DesGly¹⁰]-GnRH produced comparable results. In contrast, the injection of ¹²⁵I-[D-pGlu¹, D-Phe², Trp^3,6]-GnRH, an antagonist of GnRH, produced positive labeling only at the plasma membrane without internalization. These results indicate that, after binding with receptors on the plasma membrane, GnRH is rapidly internalized, accumulating in secretory granules, and localizing over the nuclear membrane and later, in the nucleus. Association of radioactivity with secretory granules could be related to a specific action of GnRH at this level or to receptor recycling, and presence of label in the nucleus may be related to stimulation of neosynthesis of LH and GnRH receptors.     

Mechanism of LH release in cultured rat pituitary cells

To investigate the mechanisms of the synthesis and the release of gonadotropin, rat anterior pituitary cells were stimulated in vitro with luteinizing hormone releasing hormone (LH-RH), [D-Ser(tBu)]6 des-Gly-NH2(10) ethylamide (Buserelin) and 12-0-tetradecanoyl phorbol-13-acetate (TPA), and then the LH and LH-beta subunit released into the medium were determined by radioimmunoassay. Buserelin showed its biological activity at a much lower concentration than LH-RH, but both of them caused the release of LH and LH-beta subunit in a dose-dependent manner. Furthermore, intracellular LH synthesis from LH-beta subunit by stimulation with LH-RH or Buserelin was also found. After inducing various degrees of desensitization by stimulation with LH-RH or Buserelin in a dose-dependent manner (the first stimulation), pituitary cells were stimulated with a fixed dose of TPA (the second stimulation) and the released LH was assayed. LH was released almost constantly by the second stimulation, regardless of the dose used for the first stimulation. These results suggest that the C-kinase pathway was unaffected by the desensitization induced with LH-RH or Buserelin.     

Immunocytochemical localization of cathepsin B in rat anterior pituitary endocrine cells, with special reference to its co-localization with renin and prorenin in gonadotrophs

We examined by immunocytochemistry the localization of cathepsin B in endocrine cells of rat anterior pituitary lobe, using a monospecific antibody to cathepsin B. By light microscopy, granular immunodeposits for cathepsin B were detected in most endocrine cells of anterior pituitary lobe. Cells immunoreactive for luteinizing hormone (LH) were diffusely immunostained by anti-cathepsin B. By electron microscopy, immunogold particles for cathepsin B were localized in lysosomes of thyrotrophs, somatotrophs, and mammotrophs. In mammotrophs, immunogold particles for cathepsin B were also detected in crinophagic bodies. Double immunostaining co-localized immunogold particles for LH and cathepsin B in secretory granules of gonadotrophs. Immunocytochemistry was also applied to demonstrate localization of renin and prorenin in LH-producing gonadotrophs; immunogold particles for renin were co-localized with those for LH, cathepsin B, or prorenin in their secretory granules. Immunogold particles for prorenin were also co-localized with those for LH or cathepsin B in secretory granules, but prorenin-positive granules appeared less frequently than renin-positive granules. These results suggest that cathepsin B not only plays a role in the protein degradation in lysosomes of anterior pituitary endocrine cells but also participates in the activation of renin in gonadotrophs, as has been demonstrated in secretory granules of juxtaglomerular cells.     

Pituitary responsiveness to synthetic LH-RH and pituitary LH content at various reproductive stages in the sheep.

In sheep the basal concentration of LH in jugular vein plasma was significantly higher during the first 50 days of gestation in late pregnancy or at parturition. The pituitary response to a single i.v. injection of 200 microng synthetic LH-RH was determined at different stages of gestation and compared with that of anoestrous and cyclic sheep. Pituitary response to LH-RH decreased progressively with advancing gestation: by 56 days after mating the response had declined to 35% and by parturition to 14% of the value in anoestrous sheep. The pituitary response to LH-RH increased after parturition and the pattern of recovery differed in non-lactating and lactating sheep. By 63 days postpartum the response to LH-RH in non-lactating and lactating animals had returned to values similar to those in sheep during anoestrus and sheep during the luteal phase of the oestrous cycle. A decrease in pituitary responsiveness during pregnancy was associated with a decrease in pituitary content of LH. The quantity of LH released in response to a standard injection of LH-RH was linearly related to pituitary LH content.     

Site of the in vivo stimulatory effect of prostaglandins on LH release

A possible direct effect of prostaglandins E₁ and E₂ (PGE₁ and PGE₂) on luteinizing hormone (LH) release at the pituitary level was studied using anterior pituitary cells in primary culture, a system approximately 10-fold more sensitive to stimulation of LH release than previously used hemipituitaries. No effect of PGE₁ or PGE₂ could be detected on the time course of basal or LH-RH-stimulated LH release or on the LH responsiveness to LH-RH. This absence of a direct effect of PGEs at the pituitary level on LH release was confirmed by experiments using female rats under Surital anesthesia in the afternoon of proestrus. After intravenous injection, under these conditions, 15(S)-15-methyl PGE₂ was 3–5 times more potent than PGE₂ to increase plasma LH levels while PGE₁ had about 50% the potency of PGE₂. Injection of sheep anti-LH-RH serum one hour before PGE₁ or PGE₂ injection not only lowered basal plasma LH levels but prevented the rise induced by PGEs. These data indicate clearly that the increased plasma LH levels observed after PGE injection are secondary to a stimulation of LH-RH release while PGEs do not appear to have a significant effect on LH release at the pituitary level.     

Atrial natriuretic factor-induced cGMP accumulation in rat anterior pituitary cells in culture is not coupled to hormonal secretion

While atrial natriuretic factor (ANF) does not influence ACTH secretion, it was reported to have a marked stimulatory effect on the intracellular accumulation of cGMP in rat anterior pituitary cells in culture. Since many biological actions of ANF appear coupled to its excitatory action on target cell guanylate cyclase, the current study was designed to characterize the ANF-induced cGMP response in anterior pituitary with a view to determining whether the nucleotide plays a regulatory role in the secretory function of this gland. A 3 min exposure of cells in primary culture to 300 nM ANF (99-126) or 100 microM sodium nitroprusside (SNP), a stimulator of guanylate cyclase, causes maximal 10- and 3-fold elevations of cGMP levels, respectively. Following a progressive decrease, 6- and 2-fold increases over basal cGMP levels are still observed after 180 min of incubation with ANF (99-126) and SNP, respectively. The half-maximal stimulation of cGMP accumulation induced by a 10 min exposure to ANF (99-126), or rat atriopeptin II (ANF 103-125) is observed at 9 +/- 2 and 125 +/- 22 nM, respectively. ANF fragments (99-109) and (111-126), as well as human cardiodilatin (hANF 1-16), do not alter cGMP levels. Basal and ANF-induced cGMP levels are at least 10-fold higher in cell populations enriched in gonadotrophs compared to gonadotroph-impoverished preparations. A 3 h incubation of cells with ANF (0.1-1000 nM), however, fails to modify spontaneous or LHRH-induced LH secretion. Similarly, ANF does not alter spontaneous release of GH, TSH or PRL. The data suggest indirectly that gonadotrophs represent a principal site at which ANF acts to stimulate cGMP synthesis, but that the nucleotide is not a specific regulator of the LH secretory process; nor is it generally involved as a second messenger in the secretory function of any cell type of the anterior pituitary gland.     

Ultrastructural and immunocytochemical changes of prolactin cells of grafted pituitary after the injection of dopamine in the albino rat

Anterior pituitary glands were homografted into the anterior chamber of the eye in female rats. The pituitary grafts survived and were well vascularized three weeks after the transplantation. The prolactin cells were morphologically active as shown by their well-developed Golgi complexes and granular endoplasmic reticulum and the exocytosis of secretory granules. The injection of dopamine into the common carotid artery of the graft-bearing rat rapidly suppressed the granule extrusion and then gradually induced a remarkable morphological atrophy in the prolactin cells.     

Phasic changes in immunocytochemical stainability of pituitary luteinizing hormone cells associated with their ultrastructural changes during estrous cycle in the rat

Changes in the immunoreactivity of pituitary luteinizing hormone (LH) cells and in their fine structure were studied in 4-day-cyclic female rats along with the radioimmunoassay of pituitary and serum LH. Pituitary LH increased during diestrus (DE) and in early proestrus (PE) to a maximal level at noon of PE, followed by a marked decrease by 2100 h PE. Serum LH stayed at low levels in estrus (E) and in DE, while they displayed a significant increase at PE. Light microscopic immunocytochemistry distinguished intensely and weakly stained cells using rat LH beta antiserum. The populations of intensely stained cells were 80% at PE, 30% at E and 75% at DE. This suggests that all of the LH cells do not secrete LH synchronously on the afternoon of PE. Immunoreactivity of LH cells was related to the amount of secretory granules stored in the cells as determined by the superimposition technique. Analysis of the LH storage site by the protein A-gold method confirmed that the small secretory granules, which accumulated in LH cells at DE or PE, certainly contain LH. At least two LH cell types were distinguished: one is the oval or polygonal cell with flattened rER numerous mitochondria, abundant small secretory granules (about 200 nm), a well developed Golgi complex, and a round nucleus. The other has similar structural characteristics along with large secretory granules which are more than 300 nm in diameter. At noon of PE almost all of the LH cells were the first type while the second ones were mainly found at DE or E. The relationship of these LH cell types of the male gonadotrophs is discussed.     

Gonadotropin release and redistribution of calcium-activated, phospholipid-dependent protein kinase in phorbol-stimulated rat pituitary cells

The effect of phorbol esters on calcium-activated, phospholipid-dependent kinase (protein kinase C) and luteinizing hormone (LH) secretion was examined in cultured rat anterior pituitary cells. The potent tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) stimulated LH secretion and activated pituitary protein kinase C in the presence of calcium and phosphatidylserine. The enzyme activity present in cytosol and particulate fractions was eluted at about 0.05 M NaCl during DE52-cellulose chromatography. Preincubation of pituitary cells with TPA markedly decreased cytosolic protein kinase C activity and increased enzyme activity in the particulate fraction. The maximal TPA-induced change in enzyme activity, with a 76% decrease in cytosol and a 4.3-fold increase in the particulate fraction, occurred within 10 min. The dose-dependent changes in protein kinase C redistribution in TPA-treated cells were correlated with the stimulation of LH release by the phorbol ester. These results suggest that activation of protein kinase C by TPA is associated with intracellular redistribution of the enzyme and is related to the process of secretory granule release from gonadotrophs.     

Effects of in vivo pretreatment with D-Trp-6-LH-RH on prolactin and LH secretion by pituitary glands in vitro

In order to study a possible direct action of LH-RH analogs on the pituitary lactotrophs, we investigated the effect of long-term in vivo pretreatment with D-Trp-6-LH-RH on in vitro secretion of PRL and luteinizing hormone (LH) by the pituitary glands from male and female rats. In vivo pretreatment with D-Trp-6-LH-RH (50 micrograms/day, SC) for 15 days greatly reduced basal in vitro PRL release (p less than 0.01) in female, but not in male pituitary glands. TRH-stimulated PRL secretion was not affected by pretreatment with D-Trp-6-LH-RH in female rats, but was impaired in male pituitaries. Acute in vitro exposure to D-Trp-6-LH-RH did not modify PRL secretion by female pituitary glands pretreated in vivo with the analog. However, this same in vivo pretreatment greatly decreased PRL release from male pituitaries (p less than 0.01). Basal in vitro LH release by male pituitary glands was partially lowered by in vivo pretreatment with D-Trp-6-LH-RH, as compared to controls (p less than 0.01), while basal LH release in female pituitaries remained at control levels. Finally, D-Trp-6-LH-RH-induced stimulation of in vitro LH release was severely impaired in female pituitaries (p less than 0.01) but only slightly reduced in the males.