Marian Jutisz

Binding of gonadotropin-releasing hormone (LH-RH) to the pituitary plasma membranes and the problem of adenylate cyclase stimulation

Specific binding studies of tritium-labeled LH-RH to sheep anterior pituitary membranes at 37 degrees C showed a maximum of binding capacity of 110 +/- 20 mol/mg protein with an association rate constant of 2 +/- 1 X 10(5) M-1 S-1 and a dissociation rate constant of 0.7 +/- 0.4 X 10(-2) S-1. The Scatchard plot data showed a single type of binding site with Kass = 1.1 +/- 0.4 X 10(8) M-1 in good agreement with the kinetic studies. Various doses of LH-RH in the presence or absence of Ca2+, were unable to stimulate adenylate cyclase either in the rat anterior pituitary homogenates, or in the purified sheep anterior pituitary plasma membranes. To explain these results, it may be argued that the proportion of gonadotrophs in the pituitary gland is too small to show a significant increase in the LH-RH-induced adenylate cyclase activity. Another possibility is the disconnection of the hormonal receptor from the site of activation of adenylate cyclase during the preparation of plasma membranes. Finally, cAMP may not be involved in LH release by LH-RH.

Effect of gonadectomy on pituitary levels of mRNA encoding gonadotropin subunits and secretion of luteinizing hormone.

Using cell-free translation of pituitary mRNAs we have investigated how, following gonadectomy in rats, the translational capacity of the specific messages encoding precursors to gonadotropin subunits alpha, LH-beta and FSH-beta increases with time. In parallel, serum LH was assayed in order to compare release and synthesis patterns. We observed a rapid rise in the rate of synthesis of all three precursors, with a significant increase already detectable 4 days after gonadectomy, and a plateau reached after 21 days. The kinetics were similar in both males and females, but maximal translational values for alpha-subunit were slightly higher in males. During the same time period, serum LH rapidly increased in the males, while in the females the rise of circulating LH was somewhat delayed. Although no direct correlation seems to exist between synthesis and release processes of gonadotropins, it is evident from our previous findings and the present data that both phenomena are dependent on gonadal steroids. In this respect, estradiol has been shown to regulate negatively, via different routes, the synthesis as well as the secretion of pituitary gonadotropins.

Biosynthesis of gonadotropins by rat pituitary cells in culture and in pituitary homogenates: Effect of gonadotropin-releasing hormone

The incorporation of labeled amino acids and glucosamine into LH and FSH by cultured rat anterior pituitary cells and anterior pituitary homogenates is reported. There was a significant augmentation in this incorporation by cells after 6 days of culture in the presence of GnRH. Tritiated LH and FSH were found in the cell extracts as well as in the media by the method of immunoprecipitation. An increase of approximately 7--13-fold in the release of LH and FSH into the cell incubation medium was observed in the presence of GnRH (3 ng/ml). The rate of incorporation of [3H]proline was higher than that of [3H]glucosamine into LH and FSH. At the same time a higher incorporation of labeled amino acids was observed in the case of FSH than with LH. Cycloheximide inhibited completely the incorporation of labeled proline but the inhibition was partial for the incorporation of labeled glucosamine. Freshly dispersed cells, short-time cultures maintained for 20 h and pituitary homogenates also incorporated labeled amino acids into LH and FSH, but GnRH had no effect on this incorporation. Pituitary homogenates also incorporated [3H]glucosamine into LH and FSH with an optimal incorporation after 30 min of incubation. Three different concentrations of GnRH had no effect on the incorporation of [3H]proline by homogenates. Cycloheximide and puromycin inhibited this incorporation completely.

Studies of enzymatic degradation of luteinizing hormone-releasing hormone by different tissues

The isolation and structure identification of luteinizing hormone-releasing hormone (LH-RH) [ 1,2] together with its synthesis had led to the use of this hormone for many biochemical and physiological studies [3]. In the course of our research on the in vitro biosynthesis of LH-RH in the rat hypothalamus, we observed that this hormone undergoes a rapid inactivation as determined by radioimmunoassay. Subsequently, the examination of the incubates by thin layer chromatography allowed us to observe the presence of several degradation products. There are some reports on inactivation of LH-RH by plasma [4], brain tissue and hypothalamus [5-71. Lipmann has also recently reported the degradation of another hypothalamic hormone TRH by hypothalamic tissue [8]. The aim of this work was to measure the inactivation of LH-RH by homogenates of median eminence, of hypothalamic tissue without median eminence, of pituitary gland and of brain cortex and to find inhibitor(s) preventing its inactivation.

Estradiol regulates mRNAs encoding precursors to rat lutropin (LH) and follitropin (FSH) subunits

Anterior pituitary mRNA was prepared using a microscaled method and translated in a wheat-germ cell-free system in the presence of [35S] labeled cysteine and methionine. Translation products, immunologically related to LH beta, FSH beta and the common subunit alpha, were isolated as precursors with antisera to denatured subunits and characterized by SDS-polyacrylamide gel electrophoresis and fluorography. The radioactive bands were excised from the gel and counted for quantitative evaluation. Our data show that translation of pituitary mRNAs from ovariectomized (ovx) rats results in precursor levels increased by 10 fold for alpha and 14 fold for LH beta as compared to the levels in normal rats. The increase in FSH beta precursor was impossible to evaluate as a specific immunoprecipitation product was undetectable in the case of normal rats. Estradiol, but not progesterone, administered in vivo to ovariectomized rats, reversed the stimulatory effect of ovariectomy on the expression of mRNAs coding for gonadotropin subunit precursors 48 h after injection. These results suggest that estradiol, but not progesterone, negatively regulates the synthesis of the pituitary gonadotropins in the female rats via changes in specific mRNA levels. This hormonal control probably occurs at the transcriptional level.

Kinetics of release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) by primary cultures of dispersed rat anterior pituitary cells. Chronic effect of synthetic LH and FSH releasing hormone

Abstract The kinetics of release of LH and FSH have been followed in primary cultures of dispersed rat anterior pituitary cells maintained up to two months, either in control media or in media containing 2ng/ml of LH-RH. In control cultures the LH and FSH contents in the media dropped during the first seven days, then decreased more slowly, and finally reached a low or undetectable level with some irregular variations. Starting on the 6th or 7 th day, the FSH/LH ratio increased and in some series FSH alone remained at a detectable level. The LH-RH induced an increase of both LH and FSH release. This increase appeared only after the first 5 days of culture. The stimulatory effect of LH release reached a maximum on day 11 to 13 and thereafter progressively decreased to the level of the control. The stimulatory effect of FSH release reached a maximum later than did that of LH, and generally remained significant for a longer time. The maximum of the FSH response was close or inferior to that of the LH response.

Gonadotropes in a novel rat pituitary tumor cell line, RC-4B/C. Establishment and partial characterization of the cell line.

SummaryAn epithelial cell line (RC-4B/C) was established from a pituitary adenoma obtained from a 3-yr-old (ACI/fMai × F344/fMai)F1 male rat. Before Year 5 in vitro, RC-4B/C cells could not be viably recovered from cryogenic storage. Recovery of viable cells from cryogenic storage in Year 5 was associated with a more transformed phenotype, including the appearance of endogenous C-type rat retroviral particles. The ultrastructural appearance of the cells was similar to that of differentiated anterior pituitary cell; the cultured cells contained numerous, electron dense, secretory granules, Golgi complexes, and extended arrays of rough endoplasmic reticulum. Immunocytochemical study showed that all cell types present in the rat anterior pituitary gland were present in the cell line. The percentage of luteinizing hormone beta (LHβ) cells in the cell line was higher (19.9%) and that of growth hormone cells was lower (12.2%) than in normal male rat pituitary, whereas the cell line contained a comparable percentage of follicle stimulating hormone beta (FSHβ), prolactin (PRL), ACTH, and thyrotropin beta cells. Radioimmunoassay data demonstrated the PRL content of the cells was comparable to that of normal male rat pituitary gland, whereas the content of LH and FSH was 70- and 800-fold lower, respectively. Assay of specific receptor sites for gonadotropin releasing hormone (GnRH) using Scatchard plots of the data established the RC-4B/C cells contained GnRH receptor sites of the same affinity as in the pituitary gland, but of twofold lower capacity. These data suggest the RC-4B/C cell line warrants further study as a model for the induction and maintenance of the gonadotropic function of the pituitary gland.

Cloning and sequence analysis of the cDNA for the pituitary glycoprotein hormone α-subunit of the European eel

A cDNA library constructed using mRNAs isolated from pituitary glands of estradiol-treated eels was screened with a cDNA fragment for the rat glycoprotein hormone alpha-subunit. Three out of 10,000 cDNA clones were revealed and subcloned in pUC13 for characterization and sequencing. All three had the same nucleotide sequence except for a single, silent change in the coding sequence for one of them, and for the location of the poly(A) tail. Analysis of the deduced amino acid sequence strongly suggests that these cDNA clones encode the precursor for the eel common glycoprotein hormone alpha-subunit. This precursor would therefore consist of a 93 amino acid apoprotein preceded by a 24 amino acid long signal peptide. Alignment with glycoprotein hormone alpha-subunits from fish and mammals reveals high homology, ranging from 60 to 90%. Particularly, the ten cysteines and the two putative N-linked glycosylation sites were at the same position. Comparison between fish and mammals shows also that two regions are highly conserved, comprising about half of the protein length. This high conservation rate through evolution argues for the importance of these regions in the conservation of biological properties of the alpha-subunits. In contrast, other regions are highly variable and could be responsible for the immunological specificity. Northern blot analysis of pituitary RNA from control and estradiol-treated eels showed that estradiol treatment strongly increases the pituitary content of mRNA encoding the glycoprotein hormone alpha-subunit.

Characterization of the precursors of α and >β subunits of follitropin following cell-free translation of rat and ovine pituitary mRNAs

Abstract Poly(A + )RNA was prepared from anterior pituitary glands of ovariectomized (ovx) ewes and rats and the mRNAs were translated in a wheat-germ cell-free system in the presence of [ 35 S]-labeled cysteine and methionine. Specific antisera raised against denaturated (RCXM) ovine FSHβ and α-subunits were used to demonstrate the in vitro synthesis of FSH subunits. Anti-RCXM FSHβ precipitated a single polypeptide, exhibiting a M r ⋍ 19,000 by SDS-polyacrylamide gel electrophoresis whether its synthesis was directed by ewe or rat mRNA. A M r of 17,000–17,500 was found for the α-polypeptide. FSHβ-polypeptides represented about 0.015–0.019% of the total radioactivity incorporated in response to mRNA from ovx ewes and 0.046–0.050% in the case of mRNA from ovx rats. LHβ-polypeptides represented, under the same conditions, respectively, about 0.81% and 0.44% and α-polypeptides, 1.19% and 1.33%. Further, our results indicate that FSHβ is synthesized as a precursor with a size larger than the authentic apopeptide and that the β-subunits of either LH or FSH, as well as their common subunit α are encoded by distinct mRNAs.

Cell-free translation of the rat pituitary messenger RNA coding for the precursors of α and β subunits of lutropin

Lutropin is a glycoprotein belonging to a group of pituitary glycoprotein hormones which includes also follitropin and thyrotropin. It is composed of two dissimilar, noncovalently linked subunits, (Y and /3. The amino acid sequences of (Y and fl subunits of glycoprotein hormones from many species have been established and are well known. Within a species, the sequence is identical or nearly identical for (Y subunits of all pituitary hormones. In the human being, it is also identical to that of a placental glycoprotein hormone, chorionic gonadotropin. The amino acid sequence of the hormone-specific fl subunits differs, although considerable homology exists in some portions of their primary structure (reviewed in [ 1 I). Each subunit of LH and hCG contains two N-asparaginelinked oligosaccharides. Astructure has been proposed for asparagine-linked oligosaccharides of human LH and CC; these might be identical [2.3]. Messenger RNA has been extracted from placental tissue and has been translated in vitro by systems derived from wheat-germ embryo, ascites tumors or reticulocyte lysates [4-71. The first evidence for the cell-free synthesis of LHa and LHp from bovine origin by separate mRNAs appeared in [8,9].