Light and electron microscope localization of binding sites of antibodies against ovine luteinizing hormone and its two subunits in rat adenohypophysis using peroxidase-labeled antibody technique.

The binding sites of antisera generated in the guinea pig against ovine luteinizing hormone (oLH) and its two subunits (oLHalpha and oLHbeta) have been localized in rat anterior pituitaries taken from normal or castrated males and from ovariectomized females with the peroxidase-labeled antibody method, using light and electron microscopy. With the light microscope, the cells positive with antiserum to ovine luteinizing hormone (A-oLH) were violet after the Alcian blue-periodic acid-Schiff (AB-PAS) staining; they were also positive for A-oLHalpha and for A-oLHbeta and, from castrated males, they displayed an increased affinity for A-oLHbeta. Another cell type which was blue after the AB-PAS method reacted with the A-oLHalpha only; these cells, presumably thyrotropic cells, were retracted after castration and, besides their affinity for A-oLHalpha, acquired an affinity for A-oLHbeta. As seen through the electron microscope, two cell types were positive for A-oLH, A-oLHbeta, and A-oLHalpha and may be identified as luteinizing hormone-secreting cells. Type A cells were characterized by two classes of rounded, secretory granules. Type B cells were smaller and contained only small secretory granules. 1 mo after the rats were castrated the type A cells were hypertrophied and vacuolized. In both cases the secretory granules were the main sites of the antigenicity with the three antisera. A positive reaction was also found in the cytoplasm, particularly in hypertrophied cells from ovariectomized females and with A-oLHbeta. The cisternae of the rough endoplasmic reticulum were usually negative, except in highly degranulated cells from ovariectomized females and with A-oLHbeta.

Characterization and partial purification of a substance in the pineal gland which inhibits cell multiplication in vitro.

A substance which inhibits the in vitro multiplication of 3 cell strains, 37 RC, KB and NCTC clone 929, was characterized in the sheep pineal gland and partially purified using three successive chromatography techniques, respectively on Sephadex G-25, CM-cellulose and Biogel P 60 columns. The sheep cerebral cortex and liver also contain, but at much lower concentration than in the pineal, substance(s) that behave in different tests like the factor isolated from the pineal. The nature of the antimitotic substance from the pineal is as yet unknown. It is not destroyed by treatment with proteolytic enzymes, nor by boiling with 6 M HC1. It was established that it is different from the known antiblastic drugs such as Daunomycin and Methotrexate and from some active substances known to be present in the pineal, such as melatonin, secotonin and norepinephrine, which, in the same conditions, did not show any antimitotic activity. It was shown that when the concentration of the pineal factor in the culture medium was high enough (10 mug/ml), the inhibition of the KB cells multiplication was complete and irreversible. Microscopic examination of the treated cells showed that the morphological alteration was rapid (3--6 h) and deep, with shrinkage of both cytoplasm and nucleus, while with antiblastic drugs, morphological alteration proceeded slower (1--3 days) and appeared less pronounced.

Regulation of pituitary gonadotropin gene expression outline of intracellular signaling pathways.

Genes encoding pituitary gonadotropins, luteinizing hormone, and follicle-stimulating hormone, as well as their transcripts, have been isolated from different species and characterized. The position of introns in the genes is highly conserved in different species, and it appears that sequences encoding gonadotropin subunits evolved from an ancestral gene. Expression of gonadotropin genes is regulated mainly by gonadotropin-releasing hormone (GnRH) and by gonadal steroids (estradiol and testosterone). Other factors, notably thyroid hormones and gonadal peptides (follistatin, inhibin, and activin), also play a role in this regulation. Although the intracellular mechanisms of GnRH signal transduction are still unclear, now a body of evidence suggests the involvement of protein kinases C and A in intermediary steps of gonadotropin synthesis and release.

Cyclic adenosine monophosphate and phorbol ester, like gonadotropin-releasing hormone, stimulate the biosynthesis of luteinizing hormone polypeptide chains in a nonadditive manner.

We have previously reported that GnRH stimulates the synthesis of both the alpha- and beta-polypeptide chains of LH. In the present study, in order to investigate the mechanisms involved in the GnRH regulation of LH subunit synthesis, we have explored the effects of cAMP and a phorbol ester [12-O-tetradecanoyl phorbol 13-acetate (TPA)] using anterior pituitary cells in primary culture incubated in the presence of [35S]methionine. The radioactivity incorporated into alpha and LH beta immunologically related polypeptides was measured after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled material immunoextracted from cells and media with specific antisera. The cAMP analog 8-Br-cAMP (at concentrations 0.25-2 mM), the cholera toxin (6-60 nM), and forskolin (10-100 microM) induced, like GnRH, an increase in the [35S]methionine incorporation into alpha- and LH beta-subunits. On the other hand, TPA (50-100 nM) also enhanced the synthesis of LH subunits. After a 5-h incubation in the presence of GnRH, 8-Br-cAMP, and TPA in different combinations, no cumulative effect was observed. These results demonstrate that intracellular cAMP and TPA are potent activators of both alpha- and LH beta-polypeptide chain synthesis, suggesting that cAMP as well as diacylglycerols may act as intracellular mediators of the GnRH effect on LH subunit synthesis.

Gonadotropin-releasing hormone stimulates the synthesis of the polypeptide chains of luteinizing hormone.

The effect of GnRH on the synthesis of the polypeptide chains of LH was reevaluated using the incorporation of labeled methionine by pituitary cells in culture, followed by specific immunoprecipitation of LH-related subunits and sodium dodecyl sulfate-polyacrylamide gel analysis of immuno-precipitated peptides. Fluorography and counting of labeled subunits separated on the gels demonstrated that the presence of GnRH in the medium significantly enhanced the radioactivity incorporated into both alpha- and LH beta-subunits after a 5-h incubation period. Cycloheximide completely inhibited [35S]Met incorporation in the absence or presence of GnRH, whereas actinomycin D only prevented the stimulatory effect of GnRH on this incorporation. The increase of potassium ion concentration in the medium to 59 mM was without any effect on the synthesis of LH subunits. These data demonstrate that GnRH specifically stimulates synthesis of the polypeptide chains of LH and suggest that GnRH action is mediated by RNA synthesis. Whether the expression of specific messenger RNAs encoding the LH subunits is affected by GnRH is now under investigation.

Estradiol has inverse effects on pituitary glycoprotein hormone alpha-subunit messenger ribonucleic acid in the immature European eel and the gonadectomized rat.

In teleosts, the pituitary contains a single glycoprotein gonadotropic hormone (GTH) composed of two dissimilar alpha- and beta-subunits. The European eel, Anguilla anguilla L, is sexually immature at the silver stage due to a deficiency in GTH synthesis and secretion. In previous studies we (S.D., YA.F.) have demonstrated a strong stimulatory action of estradiol (E2) on eel pituitary GTH content. In contrast, we (R.C., M.J.) have shown that in the rat E2 negatively regulates gonadotropin subunit synthesis via changes in specific mRNA levels. The purpose of our present work was to check for such effects of E2 on the synthesis of GTH alpha- and beta-subunits in the European eel. Eel pituitary mRNA was translated in a cell-free system in the presence of [35S]Met + Cys. We demonstrate that one of the translated polypeptides, characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, cross-reacts with an antiserum to denatured bovine alpha-subunit. Its apparent mol wt (18.5K), which is slightly higher than that of the corresponding rat alpha-precursor, suggests that it represents the precursor of the alpha-subunit of eel glycoprotein hormones. The specificity of immunoprecipitation was confirmed by competition with ovine alpha (but not with ovine LH beta or bovine TSH beta). Quantitative evaluation of the putative eel alpha-subunit precursor showed that it represents 0.2% of the total protein translated by RNA from the normal silver eel. Chronic treatment of eels for 3 weeks with 17 beta-E2 increased by 8.0- to 8.5-fold the proportion of the putative alpha-subunit precursor among translation products. Due to the lack of cross-reactivity with the presumed GTH beta precursor, no radioactive material could be specifically detected in translation medium of eel pituitary mRNA using antisera to either denatured bovine LH beta or ovine FSH beta. Our data suggest that E2, depending on vertebrate group and probably on sexual status, may exert either positive or negative control on gonadotropin synthesis by opposite effects on the levels of specific mRNA.

Molecular cloning and sequence analysis of the cDNA for the putative beta subunit of the type-II gonadotrophin from the European eel.

Oestradiol treatment enhances type-II gonadotrophin (GTH-II) synthesis in the European eel (Anguilla anguilla) at the silver stage. As a first step in studying the molecular mechanisms involved in this stimulation, we cloned and characterized the cDNA encoding the beta subunit of eel GTH-II. A cDNA library was constituted in lambda gt10 from oestradiol-treated eels. It was screened using an oligodeoxyribonucleotide mixed probe designed to be complementary to a highly conserved region of cDNAs from several LH-related beta subunits. Several clones were obtained and four were subcloned in pUC13 and sequenced. The longest clones comprised a 420 bp coding sequence, plus 5' and 3' untranslated regions of 36 and 172 bp respectively. Comparison with GTH-II from other teleost fish permitted the localization of the putative cleavage site of a 24 amino acid signal peptide. The resulting 116 amino acid apopeptide had well-conserved cysteine positions and a putative N-linked glycosylation site; homology was 70-80% with GTH-II from other fish, 45% with LH from mammals and birds, 38% with mammalian FSH and only 35% with fish GTH-I. Preliminary results indicated a strong positive effect of oestradiol treatment on the level of the putative GTH-II beta-subunit mRNA. This supports our proposal that the European eel provides a suitable model for studying the positive regulation of gonadotrophin synthesis by gonadal steroids.

Synergistic effects of progesterone and oestradiol on rat LH subunit mRNA.

The effects of oestradiol and progesterone on LH-subunit mRNA levels were investigated in ovariectomized rats. Four weeks after ovariectomy, rats were implanted with silicone elastomer capsules containing oestradiol and/or injected daily with progesterone in oil (5 mg/rat) for 8 days. The levels of pituitary mRNA encoding alpha and LH-beta were determined using direct hybridization with specific [32P]cDNA probes. After oestradiol implantation in ovariectomized rats, both alpha and LH-beta mRNA decreased with time, with maximum inhibition after 6-8 days of treatment. Progesterone injected alone did not show any effect on alpha and LH-beta mRNA. Cytosolic progesterone receptors, determined using [3H]methyl-17 alpha-progesterone as ligand, were undetectable in control ovariectomized rats. In contrast, 2 days after oestradiol implantation, the number of receptors increased to 287.5 +/- 35.4 (S.E.M.) fmol/pituitary and reached a plateau of 400 +/- 21.8 fmol/pituitary after 4 days. The effects of progesterone were therefore examined by first implanting ovariectomized rats with oestradiol to induce progesterone receptors and then injecting progesterone daily for a further period of 6 days. As a result of this treatment, progesterone induced a decrease in the pituitary gland contents of both alpha and LH-beta mRNAs, and LH release was significantly greater than that observed in the group receiving oestradiol alone. Moreover, the mRNA levels in the animals treated with oestradiol plus progesterone were lower after 8 days of treatment than those observed in ovariectomized rats treated with a tenfold higher dose of oestradiol alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of gonadal steroids and cycloheximide on the release of gonadotrophins by rat pituitary cells in culture.

The effects of oestradiol-17beta, testosterone and progesterone alone and together with cycloheximide on the basal and gonadotrophin releasing hormone (Gn-RH)-induced release of gonadotrophins were studied in cultured dispersed rat pituitary cells. In the control group (no steroid treatment), GnRH significantly stimulated the release of LH and FSH; cycloheximide partially inhibited this response, although it had no effect on the basal secretion of gonadotrophins. A dose of 5 ng oestradiol/ml had no significant effect on the response to GnRH; at a dose of 100 ng/ml the GnRH-induced release of LH was significantly augmented whereas the release of FSH was inhibited. Cycloheximide blocked the augmenting effect of oestradiol. The basal release of LH was slightly but significantly inhibited in response to 10 ng testosterone/ml and increased in response to progesterone (200 ng/ml). Testosterone at both dose levels and progesterone significantly inhibited the GnRH-induced release of LH and FSH and in testosterone and progesterone-treated groups, the response to GnRH was inhibited by cycloheximide, but not beyond the levels observed in the control group. It is concluded that steroids can act directly on the pituitary cells, that oestradiol stimulates the GnRH-induced release of LH and that cycloheximide blocks this stimulatory effect. Testosterone and progesterone, on the other hand, partially inhibit the response to GnRH.

Effect of thyroid hormones on gonadotrophin release and biosynthesis using rat pituitary cell cultures.

The effect of thyroid hormones, tri-iodothyronine (T3) and thyroxine (T4) on the gonadotrophin releasing hormone (GnRH)-induced release of LH and FSH has been studied using dispersed rat pituitary cell cultures. Both T3 and T4 significantly inhibited the release of gonadotrophins in the presence of GnRH. Actinomycin D and cycloheximide also inhibited synergistically the release of LH and FSH. The incorporation of labelled amino acids and glucosamine into LH and FSH which was stimulated by GnRH was also inhibited by T3 and T4. Gonadotrophin releasing hormone had no effect on the incorporation of labelled amino acids and glucosamine into total poteins but this incorporation was also significantly inhibited by T3 and T4, the former being the more potent.