Reduction of testicular luteinizing hormone/human chorionic gonadotropin receptors by [D-Trp6]-luteinizing hormone releasing hormone in hypophysectomized rats.

Adult and immature male rats were hypophysectomized and injected daily with saline or 0.2 or 2 μg of superactive Luteinizing Hormone Releasing Hormone (LHRH) agonist, [D-Trp⁶]-LHRH subcutaneously for seven days - with, or without, concomitant treatment of 1 IU Human Chorionic Gonadotropin (hCG) or 50 IU Pregnant Mare Serum. The administration of [D-Trp⁶]-LHRH reduced Luteinizing Hormone/Human Chorionic Gonadotropin receptors in all cases. The magnitude of this reduction was dose-related. As small a dose as 0.2 μg of the peptide resulted in approximately a 72% reduction of the receptors. The results suggest a direct action of [D-Trp⁶]-LHRH on the testis. It also indicated that reduction of testicular Luteinizing Hormone/Human Chorionic Gonadotropin receptors by the peptide is not necessarily due to the over-stimulation of Luteinizing Hormone (LH) release from the pituitary through a “down regulation” mechanism.     

Messenger RNA synthesis in the testis of immature rats: effect of gonadotropins and cyclic AMP.

Two hours after the intratesticular injection of FSH, hCG or cyclic AMP, the incorporation of labeled uridine into poly(A)-rich RNA was increased. Pretreatment with actinomycin D inhibited the incorporation of uridine into mRNA. After the seminiferous tubules and interstitial cells were separated by treatment with collagenase, FSH treatment increased mRNA synthesis only in the tubules whereas hCG stimulated mRNA synthesis only in the interstitial cells. Cyclic AMP increased the synthesis of mRNA in both interstitial cells and seminiferous tubules. These results suggest a differential action of the two gonadotropic hormones in the cells of the testis; both effects appear to be mediated by cyclic AMP.     

Hypophyseal regulation of hepatic histidase during postnatal development and adulthood: II. Pituitary-estrogen interrelationship

Estrogen, which augments rat liver histidase activity in pre- and postpubertal rats, is ineffective in hypophysectomized animals, in which enzyme activities are already elevated. This failure of estrogen action has been demonstrated in hypophysectomized weanling, as well as adult females, the former of which manifest enzyme activities considerably below maximum possible values. The following agents, which are known to suppress histadase activity under certain conditions, were ineffective in permitting estrogen stimulation of histadase in hypophysectomized rats: glucocorticoid, testosterone, adrenocorticotrophin acting extra-adrenally and via the adrenals, growth, and preparations of whole pituitaries of bovine or female rat origin. The following materials, which do not themselves alter enzymic activity, were likewise incapable of permitting estrogenic enhancement of histadase activity in these animals: follicle-stimulating hormone and luteinizing hormone acting extra-gonadally, thyrotrophic hormone, and bovine posterior pituitary preparations.     

Immunocytological localization of LH,FSH, TSH and their subunits in the pituitary of normal and anencephalic human fetuses

Summary Immunostaining with antisera to oLH, hCG, hLH, pLH, hFSH, hFSH, hTSH and bTSH was used to delineate the gonadotropic and thyrotropic cells of the human fetal anterior pituitary. Hypophyses from 29 normal fetuses, 3 newborn infants, and 5 totally ancencephalic fetuses were used. Several controls to check for the specificity of the immunocytological reaction were made.In normal fetuses, observations showed that: 1) the subunit was detected from the eighth week and throughout gestation without sex differences; 2) intact LH was detected during the third month, however, age and sex differences were observed during the fourth and fifth months; 3) intact FSH was detected in female fetuses from the beginning of the fourth month, a sex difference was observed; 4) LH and FSH were detected in the same cells; 5) the thyrotropic cells were detectable from 15 weeks of gestation and their number increased during gestation without sex difference; 6) at birth the gonadotropic cells were scarce and were located in the ventromedian zone of the anterior pituitary, while the thyrotropic cells remained numerous and were located in the dorsomedian zone.In anencephalic fetuses: 1) the subunit existed at each stage studied; 2) the reaction induced by anti-pLH and anti-hFSH sera was always very weak regardless of sex or age; 3) the thyrotropic cells were more numerous in comparison to the gonadotropic cells. These data are discussed in terms of the relationship of the hypophysiotropic hypothalamic factors to the appearance and evolution of the glycoprotein hormones and their subunits.     

A model system for the biochemical study of luteinizing hormone/chorionic gonadotropin receptor synthesis

A model system for the biochemical study of LH/CG receptor synthesis has been developed. Culture conditions for porcine granulosa cells were adapted that maximized the selective induction of LH/CG receptors by cAMP-inducing stimuli with an elimination of background LH/CG receptor appearance. It was found that the addition of FSH (1.5 μg/ml) or cholera toxin (10 ng/ml) 1 day after plating resulted in optimal induction of the LH/CG receptor (20–60 pg [¹²⁵I]CG bound/μg DNA 72 h after addition) with virtually no LH/CG receptor appearance in the absence of added stimuli. Later additions of FSH or cholera toxin required insulin (1.0 μg/ml) which alone caused background LH/CG receptor appearance in the absence of any additional stimuli. Furthermore, insulin increased the general rate of cellular protein synthesis, whereas FSH or cholera toxin each decreased it. Thus, the use of FSH or cholera toxin, without insulin, may enable one to detect the synthesis of the LH/CG receptor by metabolic labeling techniques where background protein synthesis has been lowered.     

In vitro translation of distinct mRNAs coding for the precursors of porcine LH subunits

Conditions are described to characterize and estimate the precursors of porcine LH alpha and beta subunits and indirectly their specific mRNAs. Poly(A) RNAs extracted from castrated male pig anterior pituitaries were translated in a wheat-germ system in the presence of [35S] cysteine and [35S] methionine. The translation products were precipitated by antisera directed against reduced and carboxymethylated LH alpha and beta subunits and analyzed by high resolution electrophoresis. It is shown that the precursors of pLH alpha and beta subunits are located in two distinct congruent to 15 K proteins and represent--on the basis of the incorporation of the [35S] labeled aminoacids into proteins--congruent to 0.12% and 0.05% respectively of the total translation products. It is suggested that in the pig, as in other species, the LH alpha and beta subunits are encoded by two distinct mRNAs, and at variance with other species the leader sequence of LH alpha mRNA is longer than that of LH beta mRNA.     

Homeodomain transcription factor Hesx1/Rpx occupies Prop-1 activation sites in porcine follicle stimulating hormone (FSH) beta subunit promoter

Homeodomain repressor factor Hesx1/Rpx plays a crucial role in the formation of Rathke's pouch at the start of pituitary organogenesis and represses the Prop-1-dependent expression of Pit-1 gene, which promotes the differentiation of Pit-1-dependent hormone producing cells. Recently, we discovered a novel function of Prop-1 by which it activates the porcine follicle stimulating hormone beta subunit (FSHbeta) gene through Fd2 region (-852/-746). The present study aimed to determine whether Hesx1 exerts its role in the Prop-1-dependent activation of FSHbeta gene. Transient transfection assay for the porcine FSHbeta promoter -985/+10, electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis for Fd2 region were carried out. Transfection assay in GH3 cells demonstrated that expression of Hesx1 alone does not change the promoter activity but the coexpression with Prop-1 represses the Prop-1-dependent activation of FSHbeta promoter. Similar results were obtained for the mutant reporter vector deleting the region -745/-104 indicating that Fd2 region is a target site of Hesx1 as well as Prop-1. EMSA and DNase I footprinting analysis using recombinant Hesx1 and Prop-1 protein demonstrated that Hesx1 and Prop-1 certainly bind to the AT-rich regions in a different manner. These results suggest that Hesx1 blocks the advanced expression of FSHbeta gene in the early stage of pituitary development, and Prop-1 thereafter appears and activates this gene.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I₅₀ = 2 μg/ml) when compared to other naturally occurring glycosaminoglycans. This inhinibition was also appparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E ₂. Heparin was also found to inhibit glucagon-sensitive rat hepatice adenylate cyclase, and the prostaglandin E₁-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfade polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.     

Sialic acid moiety is responsible for the charge heterogeneity and the biological potency of rat lutropin

To elucidate a possible role of sialic acid moiety in the electrical heterogeneity of rat pituitary lutropin, seven components separated were individually treated with neuraminidase. Some intermediates with isoelectric points corresponding to the native components were concomitantly seen at the serial stages of the enzyme treatment. All the treated components showed an isoelectric point of about 10.0 which was the same to the isoelectric point of one of the seven components. Desialylation of the components with less biological activity caused enhancement of the in vitro cyclic AMP producing- and testosterone producing-activities as well as the binding activity to the receptor. It is concluded that the number of sialic acid moiety in lutropin is responsible for the charge heterogeneity and the biological potency of the hormone.     

Actions of releasing factors on isolated secretory granules mediated by calcium.

Synthetic TRH, crude hypothalamic extract and partially purified prolactin releasing factor stimulated prolactin and growth hormone release from isolated secretory granules. Somatostatin and partially purified prolactin release-inhibiting factor inhibited release of both hormones. Calcium promoted hormone release from granules; its releasing action was potentiated by TRH and ionophore A23187 but reduced by somatostatin.     

Gonadal receptors I. Evidence for irreversibility in the binding of human chorionic gonadotropin and human luteinizing hormone

The binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors of rat testes has generally been assumed to follow an equilibrium model similar to that proposed for many enzyme systems. Our work shows that equilibrium dissociation constant (K_d) and number of hormone binding sites (B_max) are highly sensitive to changes in hormone and/ or receptor concentration and to treatment received by tissue or receptor preparation prior to the assay. The results of binding assays obtained using receptor preparation pretreated with hormone (labeled as well as unlabeled) indicated that the binding reaction between hormone and receptor was irreversible and that pretreatment of the tissue with hormone greatly alters the number of high affinity gonadotropin binding sites in the testicular homogenate. Data from studies involving increasing receptor concentrations revealed that increasing the mass of particulate receptors in the binding assays leads to higher K_d as well as B_max values. These findings are incompatible with a binding model based upon occupancy of receptor sites and the state of equilibrium implied. The incompatibilities are analyzed and an alternate model advanced (Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim. Biophys. Acta 584, 436–453).     

Isoelectric focusing of rat pituitary gonadotropins]

Isoelectric focusing of rat gonadotropins has been studied using a small scale column and various pH gradients. Hormones were detected by radioimmunoassay. FSH focuses as a single peak, the pI being 2.8. It is thus slightly more acidic than the pI of FSH from other species. LH is more heterogeneous, the main activity focusing in the pH 9.0 area, whereas a second activity appears, for some samples, in the acidic part of the gradient. TSH exhibits a broad zone of activity between pH 7.0 and 10.0. The fractionation of pituitary glycoproteins using a pH 3-10 gradient followed by removal of sucrose and ampholytes through Sephadex G 50 chromatography allows the recovery with good yields of a purified rat FSH fraction devoid of LH activity as estimated by radioimmunoassay.     

Interference of gonadotropin-receptor interaction by synthetic estrogens.

In in vitro studies, the synthetic estrogens diethylstilbestrol and diethylstilbestrol sodium phosphate inhibited the binding of ¹²⁵I ovine lutropin to the rat ovarian receptor and ¹²⁵I ovine follitropin to the bovine testicular receptor. As the lutropin binding to receptor is affected to a greater extent, a preferential inhibitory effect may be suggested. Removal of the estrogens from the incubation medium by washing does not restore gonadotropin binding ability, indicating a strong effect. The two compounds were effective in displacing the labeled gonadotropin from the preformed receptor-hormone complex. This effect increased with time of incubation. It appears unlikely that the interference of gonadotropin-receptor interaction may be because of increased hormone and/or receptor degradation by the two compounds.     

A novel heterozygous SOX2 mutation causing congenital bilateral anophthalmia,hypogonadotropic hypogonadism and growth hormone deficiency

Heterozygous de novo mutations in SOX2 have been reported in approximately 10–20% of patients with unilateral or bilateral anophthalmia or microphthalmia. An additional phenotype of hypopituitarism, with anterior pituitary hypoplasia and hypogonadotropic hypogonadism, has been reported in patients carrying SOX2 alterations. We report a novel heterozygous mutation in the SOX2 gene in a male affected with congenital bilateral anophthalmia, hypogonadotrophic hypogonadism and growth hormone deficiency. The mutation we describe is a cytosine deletion in position 905 (c905delC) which causes frameshift and an aberrant C-terminal domain. Our report highlights the fact that subjects affected with eye anomalies and harboring SOX2 mutations are at high risk for gonadotropin deficiency, which has important implications for their clinical management.     

Role of luteinizing hormone β-subunit gene variants among South Indian women with polycystic ovary syndrome

Abnormal luteinizing hormone (LH) secretion and action are known to affect ovarian steroidogenesis and thus playing a crucial role in manifestation of polycystic ovary syndrome (PCOS). This study is first of its kind to study association of LH β-subunit gene variants with PCOS among South-Indian women. 250 PCOS cases and 299 controls were recruited for the study. All the exons of LH β gene were screened. Allele and genotype frequencies of the SNPs were compared between the cases and controls. We identified seven SNPs in the LH β gene; one SNP in exon 3 (rs#1056917) exhibited significant difference in the allele frequency between the PCOS cases and controls (p = 0.015). Although, the LH β variants that are found to be more frequent among PCOS cases are silent in nature and not of any functional significance, they might influence other significant functional polymorphisms in the hypothalamic-pituitary-gonadal axis which needs to be explored.