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.     

Evidence for an association between calmodulin and membrane patches containing gonadotropin-releasing hormone — receptor complexes in cultured gonadotropes

Summary Participation of calmodulin, clathrin, and actin in receptor mediated endocytosis of gonadotropin-releasing hormone (GnRH) was studied in an in vitro system of dispersed pituitary cells with a triple staining procedure. Cells were incubated in D-Lys⁶-Pro⁹-Des¹⁰-GnRH-biotin and stained with avidin-peroxidase-diaminobenzidine. Calmodulin, clathrin, and actin as well as luteinizing hormone were identified by indirect immunofluorescence with FITC- and rhodamine-labeled second antibody. The results indicate a close spatial association of calmodulin, but not of clathrin and actin, with GnRH-containing plasma membrane patches.Supported by PHS grants NIH NS1761401, HS 09914, and HD 19899     

Cytochemical characterization of pituitary target cells for biotinylated gonadotropin releasing hormone

These studies describe the application of new cytochemical stains that co-localize a biotin-labeled gonadotropin releasing hormone (GnRH) analog and FSH or LH in the same field or cell. Pituitary monolayer cells were stimulated with the [D-Lys6] GnRH analog or the same analog labeled with biotin. Biotinylated [D-Lys6] GnRH exhibited a higher affinity and was 7-10 X more potent than unlabeled [D-Lys6] GnRH. The avidin-biotin peroxidase complex technique (ABC) was applied to localize the biotinylated GnRH on the cells with the use of a dense black peroxidase substrate. Specificity tests showed that the stain could be eliminated by competition with unlabeled [D-Lys6] GnRH. The GnRH stain was followed by immunocytochemical stains for LH beta, FSH beta or 25-39ACTH with a different peroxidase substrate (amber or orange-red). Stain for GnRH was found on the surfaces of 16% of the cells and 60-90% of the GnRH stained cells also stained for one of the gonadotropins. Most (90-100%) of the gonadotropes showed stain for GnRH. Our studies demonstrate that a potent biotinylated GnRH analog binds cells that can be identified specifically as gonadotropes.     

Effects of gonadotropin-releasing hormone (GnRH) on the cytodifferentiation of gonadotropes in rat adenohypophysial primordia in organ culture

The effects of gonadotropin-releasing hormone (GnRH) on the development of gonadotropes were investigated by the use of organ culture and by means of immunocytochemistry and radioimmunoassay. Pituitary primordia from rat fetuses were cultured in a medium with or without 10^-9 M GnRH during the first 24 h of culture. The ratio of the number of immunoreactive LH cells to the total number of cells in the explants derived from 13.5-day fetuses was increased by the GnRH treatment after 6 or 8 days of culture, while the total number of cells was not altered. LH released into the medium and LH content of explants were not affected by the GnRH treatment. Subsequent treatment with 10^-9 M GnRH for 4 h after 7 days of culture resulted in a marked release of LH, accompanying a significant decline in LH content, in both explants exposed or unexposed to the first GnRH treatment. However, the former explants contained a lower amount of LH than the latter explants. The present results indicate that pituitary primordia at 13.5 days of gestation are capable to respond to GnRH, and that GnRH is effective in stimulating the responsiveness of gonadotropes to GnRH during early pituitary cytodifferentiation.     

Estrogen target cells in the pituitary of platyfish,Xiphophorus maculatus

Summary The distribution of estradiol-concentrating cells in the pituitary of the platyfish, Xiphophorus maculatus, is studied after the injection of ³H estradiol-17 by thaw-mount autoradiography. Autoradiograms prepared 2–8 h after the injection show nuclear concentration of radioactivity in certain cells of the proximal pars distalis, while no nuclear labeling is found in cells of the rostral pars distalis, pars intermedia and pars nervosa. Radioactively labeled cells are identified as gonadotropes by a combined technique of thaw-mount autoradiography and immunoperoxidase staining with antiserum to ovine LH. Approximately 80% of the immunoreactive LH cells show a concentration of radioactivity in their nuclei. These observations indicate that in teleosts, as in mammals, estradiol has a direct effect on pituitary gonadotropes.Supported by PHS grant NS09914. The authors are grateful to Dr. D.G. Humm for providing the platyfish     

Transient receptor potential (TRP) channel function in the reproductive axis

Transient receptor potential (TRP) channels play important functional roles in the signal transduction machinery of hormone-secreting cells and have recently been implicated in reproductive physiology. While expression studies have demonstrated TRP channel expression at all levels of the hypothalamic–pituitary–gonadal (hpg) axis, functional details about TRP channel action at the level of the individual cells controlling reproduction are just beginning to emerge. Canonical TRP (TRPC) channels are prominently expressed in the reproductive center of the neuroendocrine brain, i.e. in kisspeptin and gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin neurons are depolarized by leptin via activation of TRPC channels and kisspeptin depolarizes GnRH neurons through TRPC4 activation. Recent studies have functionally identified TRPC channels also in gonadotrope cells in the anterior pituitary gland, which secrete gonadotropins in response to GnRH and thus regulate gonadal function. TRP channel expression in these cells exhibits remarkable plasticity and depends on the hormonal status of the animal. Subsequent functional analyses have demonstrated that TRPC5 in gonadotropes contributes to depolarization of the plasma membrane upon GnRH stimulation and increases the intracellular Ca²⁺ concentration via its own Ca²⁺ permeability and via the activation of voltage-gated Ca²⁺ channels. However, conditional gene targeting experiments will be needed to unambiguously dissect the physiological role of TRPC channels in the different cell types of the reproductive axis in vivo.     

Mitotic activity of gonadotropes in the anterior pituitary of the castrated male rat

Summary The proliferation of gonadotropes in the anterior pituitary of the castrated male rat was examined immunohistochemically after colchicine treatment. The results show a more than 10-fold increase in mitotic frequency in gonadotropes 1 or 2 weeks after castration, as compared with controls. This result explains the increase in the population of immunoreactive LH cells in castrated male rats. The gonadotropes decreased significantly 1 month after castration. The mitotic activity of gonadotropes was almost completely suppressed in castrates implanted with a silastic tube filled with testosterone.     

Ultrastructural and biochemical study of extracellular matrix vesicles in normal alveolar bone of rats

Summary The immunocharacteristics of androgen target cells in the anterior pituitary of male rats are assessed by a combined thaw-mount autoradiography and immunoperoxidase staining method permitting the simultaneous identification of steroid-hormone target cells and protein or polypeptide hormone-producing cells in the same preparation. After injection of ³H-dihydrotestosterone, nuclear concentration of radioactivity is observed in cells of the anterior lobe, in ectopic cells in the intermediate and posterior lobes as well as in certain pituicytes. The radioactively labeled cells in the anterior lobe are identified immuno-histochemically as gonadotropes and thyrotropes with the use of antiserum to ovine LH or its subunit -LH and bovine -TSH. The results suggest genomic effects of androgen on gonadotropes and thyrotropes as well as pituicytes.Supported by U.S. PHS Grant NS09914     

Morphometric analysis of the folliculostellate cells and luteinizing hormone gonadotropic cells of the anterior pituitary of the men during the aging process

The aim of this research was to quantify the changes in the morphology and density of the anterior pituitary folliculostellate (FS) and luteinizing hormone (LH) cells. Material was tissue of the pituitary gland of the 14 male cadavers. Tissue slices were immunohistochemically stained with monoclonal anti-LH antibody and polyclonal anti-S100 antibody for the detection of LH and FS cells, respectively. Digital images of the stained slices were afterwards morphometrically analyzed by ImageJ. Results of the morphometric analysis showed significant increase of the FS cells volume density in cases older than 70 years. Volume density of the LH cells did not significantly change, whereas their area significantly increased with age. Nucleocytoplasmic ratio of the LH cells gradually decreased and became significant after the age of 70. Finally, volume density of the FS cell significantly correlated with LH cells area and nucleocytoplasmic ratio. From all above cited, we concluded that in men, density and size of the FS cells increase with age. Long-term hypertrophy of the LH cells results in their functional decline after the age of 70. Strong correlation between FS cells and LH cells morphometric parameters might point to age-related interaction between these two cell groups.