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.     

Origin and destination of globules and irregular masses in the gonadotropin cells from the pituitary of the African catfish,Clarias gariepinus: a morphological study

The possible function of globules and irregular membrane-bound masses in the gonadotropin cells of the pituitary of Clarias gariepinus was studied. Strong secretory stimulation led to the disappearance of the secretory granules from gonadotropin cells but globules and irregular masses remained present. Acid phosphatase was detected enzyme-cytochemically in both globules and irregular masses. Radiolabelling with tritiated amino acids followed by autoradiography demonstrated that globules received radioactive material after secretory granules. The latter received radioactive material within 75 min of administration of radioactive amino acids but globules and irregular masses did not. Although some globules became radioactively labelled within 24 h of the administration of radioactive amino acids, irregular masses remained unlabelled during this period. Secretory granules reacted positively with antisera against and gonadotropin subunits, whereas globules and irregular masses only reacted with the antiserum against the subunit. A moderate anti-7B2 immunoreactivity was demonstrated in secretory granules and globules, whereas irregular masses labelled strongly. The combined cytological results indicate that globules and irregular masses are degradative, possibly crinophagic structures which develop by fusional events from secretory granules to globules and then to irregular masses.     

Role of endogenous opiates in the modulation of gonadotropin secretion in infant monkeys (Macaca mulatta): effects of naltrexone

A potential inhibitory role of endogenous opioids in the gonadotropin decline from infancy to the prepubertal period in primates was assessed by examining the effect of the specific opioid antagonist naltrexone on gonadotropin levels in infant rhesus monkeys. Paradoxically, both chronic administration of naltrexone to neonatally castrate males as well as acute administration of graded doses to intact infant females resulted in gonadotropin suppression compared to appropriate vehicle-treated controls. Thus, naltrexone behaves as a gonadotropin secretory antagonist in infant monkeys and cannot be used to unmask a putative inhibitory mechanism involving endogenous opiates.     

Regulation of gonadotropin secretion in the anterior pituitary

Studies on the regulation of gonadotropin secretion in dissociated pituitary cell cultures are described. Initial studies employing a ferritin-labelled analogue of gonadotropin hormone releasing hormone (GnRH) to localize its receptor sites on the gonadotropin cell surface that while these receptor sites initially have a random monodisperse distribution, binding of the ligand causes coarse aggregation and internalization of the GnRH receptor. These events are not due to the multivalency of the ligand and probably reflect redistributive events in vivo. By using an octapeptide analogue GnRH that binds to the GnRH receptor but lacks gonadotropin releasing activity in conjunction with sequence-specific antisera it is shown that antibodies that bind the octapeptide can induce the octapeptide to release gonadotropin. These data suggest that receptor aggregation is important in GnRH stimulation. Finally immunocytochemical studies are described in which golg-protein-A-antibody complexes are used to identify gonadotropins on ultrathin frozen sections of porcine pituitary cells. These studies indicate that in porcine gonadotropin cells the majority of the secretory granules contain both luteinizing hormone and follicle-stimulating hormone.     

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.     

Effects of brain lesions on gonadotrop ultrastructure and serum gonadotropin levels in goldfish

Summary Brain lesions that destroyed the anterior preoptic region or the pituitary stalk in sexually mature (= completed ovarian recrudescence) goldfish caused a significant increase in serum gonadotropin levels for at least 11 days postoperatively. These results confirmed previous findings indicating the presence of a gonadotropin release-inhibitory factor. Electron-microscopic investigation revealed that the gonadotrops were depleted of the small secretory granules, had marked dilations of the cisternae of the endoplasmic reticulum and extensive development of the Golgi apparatus. This indicated both secretion and synthesis, and correlated with the prolonged increase in serum gonadotropin resulting from the lesions.Supported by grants from Natural Sciences and Engineering Research Council of Canada to W.S. Hoar and R.E. Peter     

Loss of pulsatile luteinising hormone secretion in men with chronic renal failure.

In an attempt to determine the nature of hypothalamic and pituitary dysfunction in renal failure the secretory patterns of luteinising hormone were measured in men with end stage renal disease and compared with those in healthy controls and renal transplant recipients of similar age distribution. Mean luteinising hormone and oestradiol concentrations were significantly higher and the number of luteinising hormone secretory pulses was significantly lower in uraemic men compared with controls. Plasma testosterone and oestradiol concentrations were significantly lower in renal transplant recipients than normal men, but there were no significant differences in mean gonadotropin concentrations or the number of pulses of luteinising hormone between the two groups. As pulses of luteinising hormone are thought to reflect episodic gonadotropin releasing hormone from the hypothalamus these data suggest that uraemia interferes with central mechanisms controlling synchronised release of gonadotropin releasing hormone. This defect appears to be reversible after successful transplantation.     

In vivo pretreatment with human chorionic gonadotropin fails to reverse the dysfunction of isolated Leydig cells from chronically uremic rats

In order to further investigate the previously reported hypogonadal state of chronically uremic rats, we examined the effects of in vivo pretreatment with human chorionic gonadotropin (hCG) on in vivo and in vitro Leydig cell function, comparing paired intact rats with rats made chronically uremic by 5/6 nephrectomy. The in vitro testosterone (T) secretory responses to varying concentrations of hCG or dibutyryl cAMP and the number of gonadotropin receptors were determined following hemicastration. The rats were then treated with hCG for 3 days and the remaining testes were removed and studied as before. Compared with intact rats, the uremic rats had higher serum concentrations of urea nitrogen (P less than 0.001); serum T concentrations were lower in uremic rats before (P less than 0.001), but not after (P greater than 0.6) treatment. Treatment produced increases in serum T only in uremic rats (P less than 0.001). Serum LH was lower in uremic rats before treatment (P less than 0.001) and was reduced (P less than 0.001) to similar levels (P greater than 0.8) in both groups after treatment. Baseline in vitro T secretion was lower (P less than 0.001) from Leydig cells of uremic than intact rats both before and after treatment. Analysis of variance of dose-response curves showed pre- and post-treatment T secretory responses to hCG or dibutyryl cAMP in vitro to be less from Leydig cells of uremic rats (P less than 0.01). Before treatment, Leydig cell gonadotropin receptor number was lower in uremic than intact rats (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

The developing Xenopus oocyte specifies the type of gonadotropin-stimulated steroidogenesis performed by its associated follicle cells

As a response to gonadotropin, amphibian ovarian follicles primarily synthesize and secrete estradiol-17 β (E2) during vitellogenesis and progesterone (P) when fully grown. Stage IV (vitellogenic) and stage VI (full-grown) ovarian follicles from Xenopus laevis, as well as intermediate sizes, were used to explore this change in steroidogenesis. Optimum steroidogenesis occurred in both stage IV and stage VI follicles exposed for 6 h to 20 IU human chorionic gonadotropin/mL. Although the total amounts of steroid found were about the same, the E2/P ratios ranged from 26 to 35 for intact stage IV follicles, but only 0.02–0.03 for intact stage VI follicles. Steroid-producing follicle cells were isolated from stage IV and stage VI follicles by non-enzymatic procedures, were washed and were tested for steroidogenic activity in the absence of oocytes. In both cases, P was the predominant steroid produced (E2/P = 0.004–0.04), so the presence of stage IV, but not stage VI, oocytes appears to be necessary for E2 production as a response to gonadotropin. Octanol had no significant effect on the E2/P ratio of intact stage IV follicles. Dissected oocyte/follicle cell preparations from stage IV follicles were also periodically challenged with gonadotropin over 72 h, during which time most follicle cells detached from the oocyte and formed a monolayer over the bottom of the culture dish. The relatively high E2/P ratios for such preparations showed no significant change when stimulated with gonadotropin at various times over the 72 h, as long as the medium was not replaced. We conclude that the estrogenic effect of stage IV oocytes is most likely mediated by a secretory product rather than by gap junctions or by cell contact. Because the X. laevis oocyte has been shown to be a self-differentiating cell, the steroidogenic shift that occurs in developing ovarian follicles appears to be fundamentally regulated by the growing oocyte as it undergoes a physiological change rather than by different gonadotropins.     

TREATMENT OF THE INADEQUATE ENDOMETRIUM IN INFERTILITY

In many cases of sterility in women, the usual routine investigation does not reveal abnormality sufficient to be considered the cause of the condition. In such cases thorough study of the endometrium is indicated, for in some it will be noted that there is not a consistently good secretory development and conditions are not conducive to indation.Intensive hormonal therapy with chorionic gonadotropin, estrogens and corpus luteum preparations may prove helpful where endometrial defects are found, both in sterility and, preconceptionally, in habitual abortion.     

Demonstration of specific secretory granules for human chorionic gonadotropin in placenta

Existence of secretory granules and exocytosis during secretion of human chorionic gonadotropin (hCG) in human placenta has been a point of controversy. Using two methods, the highly sensitive avidin-biotin complex (ABC) method and the protein A-gold technique, for immunochemical identification of beta-hCG on electron microscopic sections, we have examined placentas at 8-10 weeks gestation and at term for the presence of secretory granules. First-trimester placentas demonstrated plentiful syncytiotrophoblast cytoplasmic granules, some undergoing exocytosis, when stained using specific beta-hCG antiserum in the ABC and protein A-gold methods. Term placentas did not show positive reaction product. The data demonstrate that the classic secretory granule-exocytosis pathway mediates placental hCG secretion. However, clear morphological differences exist between placenta granules and hormone secretory granules observed in pituitary, consistent with known functional differences between these organs. This methodology will be useful for further studies of the secretory pathways for placental peptides.     

Porcine pregnancy-associated glycoprotein family (pPAGs) - as in vitro-produced chorionic ligands for luteal and uterine gonadotropin receptors

This paper compares proteomic interaction-types and binding-effectiveness of secretory chorionic ligands (including pPAGs) with other proteins, i.e. gonadotropin membrane receptors (Rc) isolated from luteal-phase corpora lutea, uterine myometrium and endometrium of cyclic (cCLRc, cMYORc and cENDRc) or pregnant (pCLRc, pMYORc and pENDRc) pigs. Binding-effectiveness of miscellaneous in vitro-produced chorionic ligands (+pPAGs) was compared by radioreceptor assay (RRA) with endometrial (END) proteins of cyclic, pseudopregnant and pregnant gilts - as negative control ligands and porcine LH and hCG - as positive control ligands. The binding-comparison suggests that the pPAGs may play an important role as potential antiluteolytic or luteoprotective chorionic-origin signals during pregnancy, according to the binding-effectiveness of secretory chorionic ligands (+pPAGs) that was relatively comparable to LH/hCG - as classical ligands competing for luteal and uterine gonadotropin receptors of cyclic and pregnant pigs.     

Higher ghrelin and lower leptin secretion are associated with lower LH secretion in young amenorrheic athletes compared with eumenorrheic athletes and controls

Amenorrhea is common in young athletes and is associated with low fat mass. However, hormonal factors that link decreased fat mass with altered gonadotropin pulsatility and amenorrhea are unclear. Low levels of leptin (an adipokine) and increased ghrelin (an orexigenic hormone that increases as fat mass decreases) impact gonadotropin pulsatility. Studies have not examined luteinizing hormone (LH) secretory dynamics in relation to leptin or ghrelin secretory dynamics in adolescent and young adult athletes. We hypothesized that 1) young amenorrheic athletes (AA) would have lower LH and leptin and higher ghrelin secretion than eumenorrheic athletes (EA) and nonathletes and 2) higher ghrelin and lower leptin would be associated with lower LH secretion. This was a cross-sectional study. We examined ghrelin and leptin secretory patterns (over 8 h, from 11 PM to 7 AM) in relation to LH secretory patterns in AA, EA, and nonathletes aged 14-21 yr. Ghrelin and leptin were assessed every 20 min and LH every 10 min. Groups did not differ for age, bone age, or BMI. However, fat mass was lower in AA than in EA and nonathletes. AA had lower LH and higher ghrelin pulsatile secretion and AUC than nonathletes and lower leptin pulsatile secretion and AUC than EA and nonathletes. Percent body fat was associated positively with LH and leptin secretion and inversely with ghrelin. In a regression model, ghrelin and leptin secretory parameters were associated independently with LH secretory parameters. We conclude that higher ghrelin and lower leptin secretion in AA related to lower fat mass may contribute to altered LH pulsatility and amenorrhea.     

In vitro stimulation and inhibition of steroid hormone release from postovulatory follicles of the tilapia,Oreochromis mossambicus

Summary Postovulatory follicles of the tilapia, Oreochromis mossambicus, were incubated with graded doses of salmon gonadotropin to identify the steroid hormones released by this tissue. In addition, the effects of either cytochalasin B or colchicine on steroid hormone release were studied. After the incubation, the tissue was examined by electron microscopy. Postovulatory follicles released testosterone and estradiol-17B in a dose-dependent manner with gonadotropin. There was no detectable release of progesterone or 17a-OH-progesterone. When stimulated with high doses of gonadotropin, the steroidogenic cells showed an increase in smooth endoplasmic reticulum, Golgi complexes, and lipid droplets. Also, microfilaments became arranged in orderly bundles and were found close to the numerous secretory vesicles and lipid droplets. Upon incubation with gonadotropin and either colchicine or cytochalasin B, the cells still appeared steroidogenic, but the filaments were not organized nor associated with vesicles or lipid droplets. Release of steroid hormone decreased significantly. Also in these tissues, vesicles were no longer numerous in the apical region of the granulosa cells, but were located primarily near smooth endoplasmic reticulum and Golgi complexes. This suggests that disruption of the cytoskeleton results in reduced steroid hormone synthesis or release.     

Gonadotropin production and release in female goldfish (Carassius auratus) after administration of pimozide and an LHRH analogue as studied by electron microscopy and radioimmunoassay

Summary The effect of pimozide and an LHRH-analogue (LHRH-A) on gonadotropic cells of the goldfish pituitary gland were described qualitatively and quantitatively. A scale of four categories was devised to reflect various ultrastructural appearances of the cells. Experimental animals were divided into a control group, a group injected with LHRH-A alone, pimozide alone, and groups receiving these two substances in combination. Fish injected with the single substance were killed 12 h after injection while the groups receiving the combined treatments were killed at 4, 12 and 48 h. Serum levels of gonadotropin measured by radioimmunoassay were used to indicate whether an increase in hormone release had occurred. An immunocytochemical technique, the protein A-gold procedure, assured that the cells studied were gonadotropes. The control group showed variation in the profiles of gonadotropic cells. The single treatment groups showed some increase in secretory inclusions. At 4 h after injection the combined treatment caused a significant increase in hormone granules; at 12 and 48 h there was a gradual decrease in content of secretory products, and an increase in vacuolization. The results indicate that the combined pimozide and LHRH-A treatment stimulated gonadotropin production as well as release.     

An immunogold,cryoultrastructural study of sites of synthesis and storage of chorionic gonadotropin and placental lactogen in human syncytiotrophoblast

Summary The sites of intracellular synthesis and storage of human placental lactogen (hPL) and human chorionic gonadotropin (hCG) are controversial. We have used one of the most sensitive methods, cryoultramicrotomy and immunogold labelling, to localise these hormones at the electron-microscopic level. In both 12-week and term placentas hCG and hPL are present throughout the rough endoplasmic reticulum cisternae, in the Golgi bodies, and in the infrequent small dense granules of the syncytiotrophoblast. Previous assays have shown that hCG is at a higher concentration in early pregnancy and hPL peaks in late pregnancy, and our results corroborate these findings. No significant localisation of either hormone was seen in the cytotrophoblast or villous stroma. The results suggest that both hCG and hPL are synthesised and packaged by the classical secretory pathway, although the level of hormone stored in granules at any one time is small.     

Cell-surface expression of a membrane-anchored form of the human chorionic gonadotropin alpha subunit

We carried out experiments designed to generate a novel cell-surface protein from a small glycosylated secretory protein. DNA encoding the entire precursor of human chorionic gonadotropin (hCG, alpha subunit) was fused precisely to DNA encoding the transmembrane and cytoplasmic domains of the vesicular stomatitis virus glycoprotein. When expressed in animal cells this DNA encoded the 92-amino acid hCG-alpha subunit anchored in cellular membranes by an extension composed of the 49 carboxyl-terminal amino acids of vesicular stomatitis virus glycoprotein. This hybrid protein was transported efficiently to the plasma membrane of animal cells. The two asparagine-linked glycans on the anchored form of hCG-alpha were large and heterogeneous when compared to those on the secretory form. Experiments employing in vitro mutagenesis and the glycosylation inhibitor tunicamycin established that the presence of at least one of the two asparagine-linked glycans was required for expression of the anchored molecule on the cell surface. However, as reported previously, secretion of hCG-alpha occurred in the absence of glycosylation. Also, mutations eliminating the second glycosylation site (at amino acid 78) in both the anchored or secreted forms apparently led to partial denaturation or a conformational change interfering with transport of the protein.     

Relationship between characteristics of immunoreactive LH/FSH cells and the levels of gonadotropin in the female rat

Morphological and functional changes of pituitary LH/FSH cells in the female rat were investigated using the parameters on the radioimmunoassay, immunohistochemistry and ultrastructure. Changes in immunostainability, populations of intensely immunostained LH and FSH cells and total volume of secretory granules were correlated with the changes in pituitary LH and FSH contents during the estrous cycle. The immunohistochemical feature of gonadotropin release is the transformation of intensely immunostained gonadotrophs into the weakly stained ones. Secretory granules of small diameter (less than 150 nm) were numerous just before LH and FSH surges then sharply declined along with LH and FSH surges. The number of secretory granules of large diameter (larger than 150 nm) also decreased when LH and FSH surges took place. Then the number increased progressively until 17.00 h on the day of diestrus, corresponding to the increase in pituitary LH and FSH contents. It is suggested that small secretory granules are a release pool while large ones are a reserve pool.     

Regulation of gonadotropin-releasing hormone (GnRH) gene expression in hypothalamic neuronal cells

Summary 1. Gonadotropin-releasing hormone (GnRH) is the hypothalamic releasing factor that controls pituitary gonadotropin subunit gene expression and indirectly gametogenesis and steroidogenesis from the gonad, which results in reproductive competence.2. GnRH is synthesized in only about 1000 neurons in the hypothalamus and released in an episodic fashion down the median eminence to regulate gonadotropin biosynthesis.3. Although much is known about the secretory dynamics of GnRH release, little is known about the pretranslational control of GnRH biosynthesis due to lack of appropriate model systems. The recent availability of immortalized neuronal cell lines that produce GnRH allows investigators for the first time to begin to dissect the factors that directly regulate GnRH gene expression.4. This article reviews the current state of knowledge concerning the mechanisms that direct tissue-specific and peptide hormone control of GnRH biosynthesis.     

Stereological study of gonadotropes in the frog,Rana pipiens,after GnRH stimulation in vitro

Summary Previous physiological results have indicated the existence of two releasable pools of gonadotropins in amphibian pituitaries: an acute releasable pool that appears independent of protein synthesis, and a storage pool involved in chronic release that depends on protein synthesis. To elucidate the ultrastructural localization of these pools and the morphological changes induced in gonadotrope cells after treatment with gonadotropin-releasing hormone, we carried out a morphometric study of immuno-identified gonadotrope cells using an in vitro superfusion system. Treatment with gonadotropin-releasing hormone induced a degranulation of small (110–255 nm) and medium (236–360 nm) secretory granules as well as hypertrophy of the endoplasmic reticulum and Golgi complex. Simultaneous incubation with gonadotropin-releasing hormone and cycloheximide inhibited the release of secretory granules although the endoplasmic reticulum and Golgi complex were hypertrophied. These morphological results strongly suggest: (1) that gonadotropin-releasing hormone induces degranulation and hypertrophy of the biosynthetic machinery in gonadotrope cells; and (2) that the activation of the endoplasmic reticulum and Golgi complex by stimulation with gonadotropin-releasing hormone is independent of protein synthesis, while the release of secretory granules is protein synthesis-dependent. In addition, the second or storage pool of gonadotropin is associated mainly with the small and medium secretory granules.