Maïthé Corbani

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.

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.

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.

Estrogen-like effects of 7,12-dimethylbenz(a)anthracene on the female rat hypothalamo-pituitary axis

We have recently demonstrated that 7,12-dimethylbenz(a)anthracene (DMBA), a potent inducer of mammary tumors in rodents, can in vitro decrease the number of membrane dopamine D2 receptors and stimulate prolactin (PRL) release, by direct estrogen-like actions on anterior pituitary. In the present study, we tested the ability of DMBA to mimic the in vivo estradiol (17 beta E2) effects on pituitary D2 receptors and on PRL as well as LH release. We have found that DMBA, like 17 beta E2, when injected to ovariectomized rats, induced a decrease in the number of anterior pituitary D2 receptors, a release of PRL and exerted a biphasic (acute negative and longer term positive) action on LH secretion. We thus examined the ability of DMBA to interact with 17 beta E2 receptors in the hypothalamo-pituitary axis: DMBA binds to the pituitary cytosolic estrogen receptors with an affinity 0.001% that of 17 beta E2. Finally [3H]DMBA binds to hypothalamus-containing brain sections. This binding was displaced partially by RU 2858 a pure estrogen agonist and totally by tamoxifen, a purported estrogen antagonist. No competition for [3H]DMBA binding was observed with an androgen (RU 1881) or a glucocorticoid (RU 26988) agonist. From these data, it may be concluded that DMBA can act as a partial estrogen in pituitary and hypothalamic tissues.

Biosynthesis of gonadotropin releasing hormone (GnRH): Present status

Although the mechanism of GnRH synthesis has not yet been elucidated, many experimental data argue in favour of a ribosomal pathway resulting in the biosynthesis of a precursor with a size larger than the decapeptide. The existence of high mol. wt (HMW) immunoreactive forms of GnRH in hypothalamic extracts has been reported but without any evidence that they are precursors of the hypothalamic hormone. Some results obtained with immunocytological methods also suggest that at least a part of immunoreactive GnRH in perikarya might be extended on one or both its terminal ends. In our laboratory, we first investigated the process of GnRH biosynthesis in vivo by infusing tritiated amino acids into the third ventricle of rats. The results, along with the demonstration of the existence of HMW GnRH, support a ribosomal mechanism of GnRH synthesis. Also, preliminary data from our laboratory demonstrate that poly (A+) enriched RNA preparations from rat hypothalami contain compounds capable of hybridizing to a synthetic oligodeoxynucleotide probe which is complementary to a part of the GnRH sequence.

Bioactivity studies on atypical natural opioid hexapeptides processed from proenkephalin (PENK) precursor polypeptides

Endogenous opioids are derived from four related polypeptide precursors: proenkephalin (PENK), prodynorphin (PDYN), pronociceptin (PNOC) and proopiomelanocortin (POMC). In mammals PENK encodes for four copy of Met-enkephalin, one octapeptide Met-enkephalin-Arg-Gly-Leu, one heptapeptide Met-enkephalin-Arg-Phe and a single copy of Leu-enkephalin. Our detailed bioinformatic search on the existing PENK sequences revealed several atypical hexapeptide Met-enkephalins in different vertebrate animals. They are located either in the second enkephalin unit or in the seventh enkephalin core position at the C-terminus. Altogether four different hexapeptide sequences were obtained representing eleven animal species: Met-enkephalin-Arg(6) (YGGFMR) in the bird zebra finch, Met-enkephalin-Asp(6) (YGGFMD), Met-enkephalin-Ile(6) (YGGFMI) in zebrafish; and Met-enkephalin-Ser(6) (YGGFMS) in two pufferfish species. All novel peptides were chemically synthesized and studied in receptor binding and G-protein activation assays performed on rat brain membranes. The four novel enkephalins were equipotent in stimulating G-proteins. Affinities of the peptides determined by equilibrium competition assays in receptor binding experiments were statistically different. At the MOP receptors the highest affinity (Ki 4nM) was obtained with the zebra finch peptide Met-enkephalin-Arg(6). The pufferfish Met-enkephalin-Ser(6) exhibited the highest affinity (Ki 6.7nM) at the DOP receptor. Phylogenetic neuropeptide libraries, defined here as a collection of mutationally different species variants of orthologous and paralogous peptide sequences, represent the natural molecular diversity of the neuropeptides. Such libraries can provide a wide range of structural information establishing comparative functional analyses. Since DNA sequencing data are rapidly increasing, more development in the natural peptide library concept is expected.

Hormonal Regulation of Pituitary Gonadotropin Gene Expression

Lutropin (LH) and follitropin (FSH) are two members of a family of structurally related polypeptide hormones, that also includes pituitary thyrotropin (TSH) and placental human chorionic gonadotropin (hCG). Each hormone of this family contains two non identical, non covalently linked subunits, α and β. The primary structure of the a subunits is identical among hormones within a species, whereas the primary structures of the β subunits differ greatly and confer to the hormones their specific biological activities (Pierce and Parsons, 1981). Although they are not identical, the β subunits show enough homology in their primary structures to suggest that they arose by duplications and mutations of a single ancestral gene (Fontaine and Burzawa-Gerard, 1977).

Cell-Free Synthesis of Rat Lutropin Subunits

Lutropin (LH) is a member of a family of glycoprotein hormones that includes follitropin (FSH), thyrotropin (TSH), and a placental hormone, human chorionic gonadotropin (hCG). Each one of these hormones is built up of two dissimilar subunits α and β, attached noncovalently in the native hormone. Within a given species, the α subunit is common to all glycoprotein hormones; only the β subunits, which confer to a hormone its biological and immunological specificity, differ (Giudice and Pierce, 1978).

Mise au point sur la biogenese des gonadotropines hypophysaires

We have studied the regulation of the biosynthesis of pituitary gonadotropins in the rat by gonadal steroids and a hypothalamic hormone, GnRH. The methodology used for studying the action of steroids, was either cell-free translation of pituitary messenger RNAs, or hybridization (Northern blot) with synthetic oligodeoxynucleotides (ODN), and for studying the effect of GnRH, primary anterior pituitary cell culture. Our results show that gonadectomy increases and injection of gonadal steroids into gonadectomized rats diminishes the rate of synthesis of the gonadotropin subunit precursors. Progesterone acts only after induction of its pituitary receptors in ovariectomized rats with estradiol benzoate. Thyroxine modulates the action of steroids. Hybridization experiments suggest that gonadal steroids act on the expression of genes encoding the precursors of gonadotropin subunits. GnRH significantly increases incorporation of the labeled amino acids into polypeptide chains of both alpha and LH beta subunits. Intracellular mediators of hormone action, such as cyclic AMP and diacylglycerols, mimic the stimulatory action of GnRH on the synthesis of LH subunits. However, we have no evidence that these products intervene in the effect of GnRH on the LH subunit synthesis. In conclusion, the synthesis of LH and FSH subunits is regulated, with opposite effects, by gonadal steroids which exert their negative control at the genomic level and by GnRH which proceeds via different, yet unknown mechanisms.

In Vivo Regulation by Estradiol of the Messenger RNAs Encoding LH and FSH Subunits and the Secretion of Gonadotropins

Gonadotropins are glycoproteins composed of two dissimilar, non-covalently linked α- and β-subunits. Within a given species, the amino acid sequences of the α-subunits of LH and FSH are identical, while those of the hormone specific β-subunits differ (Pierce and Parsons, 1981).