Maria L. Dufau

Hormonal regulation of peptide receptors and target cell responses

Regulation of plasma membrane receptors for peptide hormones by the prevailing ligand concentration often causes altered target cell function. Receptor number is determined by hormone-induced changes in membrane conformation, irreversible ligand binding, and processing of ligand–receptor complexes during hormone action.

Regulation of peptide hormone receptors and gonadal steroidogenesis.

Publisher Summary This chapter discusses regulation of peptide hormone receptors and gonadal steroidogenesis. The control of gonadal function by gonadotropins and prolactin is expressed through activation of plasma-membrane effector systems following hormone binding to specific, high-affinity receptors on the target-cell surface. Hormonal stimulation of characteristic gonadal responses by gonadotropins is predominately mediated by the adenylate cyclase protein kinase pathway, whereas the nature and mechanism of prolactin actions are less defined. Peptide hormones comprise one of the major groups of chemical signals by which intercellular communications operate within the organism. The low concentrations at which hormones circulate are commensurate with the high affinities of their cell-surface receptor sites, and the marked specificities of such receptors permit recognition of the circulating hormone amid a vast excess of other protein molecules. These well-defined binding properties of peptide receptors are similar to those of antigen-antibody reactions. In B lymphocytes, the immunoglobulin molecule serves as a surface receptor comparable to the peptide hormone receptor. The ability of peptide hormones to regulate the concentrations of their specific receptor sites in endocrine target cells is demonstrated in numerous tissues.

Expression of functional leptin receptors in rodent Leydig cells.

Several studies indicate that the size of body fat stores and the circulating levels of the adipocyte-derived hormone leptin are able to influence the activity of the hypothalamic-pituitary-gonadal axis. The leptin-hypothalamic-pituitary-gonadal interactions have been mainly studied at the level of the central nervous system. In this study, we investigated the possibility that leptin may have direct effects on the rodent Leydig cell function. To probe this hypothesis, we first analyzed the expression of leptin receptors (OB-R) in rodent Leydig cells in culture. RT-PCR studies showed that rat Leydig cells express both the long (OB-Rb) and short isoform (OB-Ra) of leptin receptor, whereas MLTC-1 cells (a murine Leydig tumor cell line) express only the long isoform. Short-term (30 ‐90 min) incubation of rat Leydig cells with increasing concentrations of leptin (2‐500 ng/ml) led to a significant and dose-dependent inhibition of human (h)CGstimulated testosterone (T) production (;60% reduction, IC50 5 20 ng/ml) but no change in basal androgen release. Also, leptin (150 ng/ml) amplified hCG-induced intracellular cAMP formation (1- to 2-fold) without modifying basal cAMP levels. Subsequent experiments showed that leptin inhibited 8Br-cAMP-stimulated T production, indicating that leptin’s effect is exerted beyond cAMP. The inhibitory effect of leptin on hCG-induced T secretion was accompanied by a significant reduction of androstenedione and a concomitant rise of the precursor metabolites pregnenolone, progesterone, and 17-OHprogesterone, conceivable with a leptin-induced lesion of 17,20 lyase activity. Separate experiments performed with the MLTC-1 cells (not expressing cytochrome P450 ‐17a) showed that leptin, though amplifying hCG-stimulated cAMP production, did not modify hCG-stimulated pregnenolone and progesterone release. These results further indicate that leptin action on steroidogenesis occurs downstream of progesterone synthesis. Northern Blot experiments showed no acute effect of leptin on cytochrome P450 ‐17a messenger RNA accumulation in rat Leydig cells in basal and hCG-stimulated conditions, excluding that the rapid changes observed were caused by messenger RNA degradation. In conclusion, these findings, for the first time, show that leptin has direct, receptor-mediated actions on rodent Leydig cells in culture, at concentrations within the range of obese men. (Endocrinology 140: 4939 ‐ 4947, 1999)

Gonadotropin receptors and regulation of steroidogenesis in the testis and ovary.

Publisher Summary This chapter discusses the gonadotropin receptors and regulation of steroidogenesis in the testis and ovary. The regulation of testicular and ovarian function by gonadotropic hormones is mediated by specific, high-affinity receptors that are located in the plasma membrane of the respective target cells. A common feature of receptors for gonadotropins and other protein hormones is their extremely high affinity for the respective ligands, with association constants of about 1010 M−1. The initial demonstration of gonadotropin receptors in the rat testis and ovary by isotopic tracer methods was performed by in vivo localization of radioiodinated lutenizing hormone (LH) or human chorionic gonadotropin (hCG) upon the LH receptor sites. The morphological evidence for the existence of gonadotropin receptor sites in the gonads has been complemented by a considerable body of in vitro data demonstrating that specific hormone binding sites are present in slices, cell suspensions, and homogenates of the testis and ovary. The location of LH receptors in the plasma membrane of the Leydig cell of the testis and the luteinized cells of the corpus luteum is also elaborated in the chapter.

Radioimmunoassay of plasma testosterone

Abstract Radioimmunoassay of testosterone in plasma extracts purified by thin layer chromatography has been applied to the development of a highly sensitive and specific method for measurement of plasma testosterone. Antibodies to testosterone coupled to bovine gamma globulin via the 3 position were raised in sheep, and an antiserum of high association constant ( K = 4.5 × 10 9 M −1 ) was employed for assay. Isolation of plasma testosterone prior to assay was performed by thin layer chromatography, and extraction losses were corrected for recovery of tritiated testosterone indicator. For assay, antiserum (1:2500) and tritiated testosterone (40 000 dpm) were incubated at 4° for 16 h, followed by separation of bound and free tracer with 500 μg dextrancharcoal. The sensitivity of the assay was 10 pg, between-assay precision was ± 12% and testosterone recovery was 95–100%. Plasma testosterone levels in individual samples were similar to those obtained by competitive binding assay, and the levels observed in males (709 ± 143; 450–1100 ng/100 ml) and females 54 ± 22; 25–80 ng /100 ml ) were in agreement with those reported by other methods. The combination of radioimmunoassay and thin layer chromatography provides a sensitive and reliable method for the specific determination of testosterone in small plasma samples.

Interaction of glycoprotein hormones with agarose-concanavalin A

Abstract The ability of agarose-coupled concanavalin A to bind carbohydrate and glycoprotein molecules has been applied to chromatography of glycoprotein hormones. Human chorionic gonadotrophin and subunits were bound strongly by Sepharose-concanavalin A, and could be displaced by 0.2 M methyl α- d -glucopyranoside. Desialylated human chorionic gonadotrophin was eluted more slowly than the intact hormone, and asialo-agalacto-human chorionic gonadotrophin was not readily dissociable from combination with concanavalin A. Human luteinizing hormone and follicle stimulating hormone were also adsorbed by Sepharose-concanavalin A columns, and eluted with methyl α- d -glucopyranoside. Prior combination with specific antibody did not prevent 125I-labeled human chorionic gonadotrophin binding to concanavalin A, and subsequent elution with methyl glucopyranoside released the antigen-antibody complex in high yield. Chromatography of human chorionic gonadotrophin on Sepharose-concanavalin A was applied to preparation of 125I-labeled human chorionic gonadotrophin with improved binding affinity for antibody and gonadal receptor sites, and to purification and isolation of human chorionic gonadotrophin from crude urine extracts and human pregnancy plasma. Comparison of plasma and urinary human chorionic gonadotrophin revealed an elution profile consistent with partial desialylation of the urinary hormone. These experiments have shown that the group-specific interactions of glycoprotein hormones with agarose-concanavalin A are of potential value for the purification and isolation of such hormones and their derivatives, and for the preparation of radioiodinated gonadotrophins of improved binding characteristics.

Estrogen Dependence of a Gonadotropin-induced Steroidogenic Lesion in Rat Testicular Leydig Cells

Leydig cells isolated from the testes of rats treated with intravenous exogenous gonadotropin (hCG) or subcutaneous gonadotropin-releasing hormone (GnRH) show markedly decreased luteinizing hormone (LH) receptors and a partial block in testicular 17,20 desmolase activity. In contrast, Leydig cells from animals with equivalent degrees of LH receptor loss induced by subcutaneous hCG treatment show no change in 17,20 desmolase activity. These findings indicated that the acuteness of gonadotrophic stimulation, rather than the extent of LH receptor loss, was responsible for the steroidogenic lesion. A role of estradiol in the enzymatic block produced in vivo by acute elevation of circulating gonadotropin (intravenous hCG or GnRH-stimulated endogenous LH) was suggested by rapid elevations of testicular 17beta-estradiol within 30 min after intravenous hCG, whereas more gradual increases in estradiol occurred 4-8 h after subcutaneous hCG. The inhibitory effect of endogenous estrogen on testicular steroidogenesis was confirmed by the ability of an estrogen antagonist (Tamoxifen) to prevent the reduction of testosterone responses caused by intravenous hCG and subcutaneous GnRH. In addition, Tamoxifen significantly increased the number of LH receptors in Leydig cells from both control and gonadotropin-desensitized animals. These findings indicate that the acute elevations of intratesticular estrogen produced by treatment with hCG or GnRH are responsible for the steroidogenic lesion seen in gonadotropin-desensitized Leydig cells. These results also suggest that locally produced estrogens contribute to the regulation of testicular LH receptors and 17,20 desmolase activity.

Silencing of Transcription of the Human Luteinizing Hormone Receptor Gene by Histone Deacetylase-mSin3A Complex

Modification of chromatin structure by histone acetylases and deacetylases is an important mechanism in modulation of eukaryotic gene transcription. The present study investigated regulation of the human luteinizing hormone receptor (hLHR) gene by histone deacetylases. Inhibition of histone deacetylases (HDACs) by trichostatin A (TSA) increased hLHR promoter activity by 40-fold in JAR cells and markedly elevated endogenous hLHR mRNA levels. Acetylated histones H3 and H4 accumulated in TSA-treated cells and associated predominantly with the hLHR promoter. Furthermore, TSA significantly enhanced the recruitment of RNA polymerase II to the promoter. One of the two Sp1 sites essential for basal promoter activity was identified as critical for the TSA effect, but the binding of Sp1/Sp3 to this site remained unchanged in the absence or presence of TSA. A multiprotein complex was recruited to the hLHR promoter via interaction with Sp1 and Sp3, in which HDAC1 and HDAC2 were docked directly to Sp1-bound DNA and indirectly to Sp3-bound DNA through RbAp48, while mSin3A interacted with both HDACs. HDAC1 and HDAC2 were shown to potently repress the hLHR gene transcription, and mSin3A potentiated the inhibition mediated by HDAC1. Our studies have demonstrated that the HDAC-mSin3A complex has an important role in the regulation of hLHR gene transcription by interaction with Sp1/Sp3 and by region-specific changes in histone acetylation and polymerase II recruitment within the hLHR promoter.

Corticotropin-releasing factor: an antireproductive hormone of the testis.

Corticotropin‐releasing factor (CRF), the key neuropeptide in the stress cascade, has major inhibitory actions on testicular function in addition to its known antireproductive effects at the central level (inhibition of sexual behavior and LH secretion). CRF is secreted by the Leydig cells of the testis and acts through high‐affinity receptors at the Leydig cell membrane as a potent negative regulator of LH action, inhibiting gonadotropin‐induced cAMP generation and androgen production. CRF is also a primary stimulus of β‐endorphin secretion by the Leydig cells, which in turn exerts paracrine inhibition of FSH action in the tubular compartment of the testis through high‐affinity receptors in the Sertoli cells. CRF action in the Leydig cells involves a pertussis toxin‐insensitive guanyl nucleotide regulatory unit. In contrast to CRF receptors in the brain, pituitary, and other peripheral tissues, those in the Leydig cell are not coupled to Gs. The inhibitory action of CRF in the Leydig cell is exerted through protein kinase C, at the level of the catalytic subunit of adenylate cyclase. The secretion of CRF by the Leydig cell is stimulated by LH, acting via release of serotonin (5HT) and autocrine activation of 5HT2 receptors. Serotonin acts on 5HT2 receptors in the Leydig cell to stimulate CRF secretion via a pertussis toxin insensitive G‐protein and presumably through activation of phosphoinositide hydrolysis. The diversity of the biochemical responses to CRF and 5HT2 receptor activation (i.e., inhibition of adenylate cyclase at the cytoplasmic aspect of the cell membrane vs. stimulation of CRF release from secretion granules) may reflect the stimulation of different protein kinase C isoenzymes. The LH å 5HT å CRF inhibitory loop serves to continuously buffer the stimulation of androgen production by gonadotropin. 5HT, the immediate stimulus of testicular CRF secretion, is released during stress and is locally increased in the testis in pathological conditions associated with impaired testicular function (i.e., orchitis, varicocele). Also, propranolol, the β‐adrenergic antagonist frequently used in the control of blood pressure in patients with hypertension and often associated with impotence, acts via a serotonergic mechanism to stimulate CRF secretion and causes marked inhibition of LH‐induced cAMP production and steroidogenesis in cultured Leydig cells. These basic studies of 5HT and CRF are relevant to the pathogenesis of testicular dysfunction and for the development of antagonist therapies to block CRF production and its local antireproductive effects.— Dufau, M. L., Tinajero, J. C., Fabbri, A. Corticotropin releasing factor: an antireproductive hormone of the testis. FASEB J. 7: 299‐307; 1993.

Retention of in vitro biological activities by desialylated human luteinizing hormone and chorionic conadotropin

Summary The marked reduction of biological activity after desialylation of human luteinizing hormone and chorionic gonadotropin is not accompanied by a corresponding loss of activity in vitro. The desialylated gonadotropins show increased affinity for gonadal binding sites, and retain a considerable degree of steroidogenic activity when incubated with the rat testis in vitro. These findings indicate that the sialic acid residues of gonadotropic hormones are not essential for biological activity at the target cell level.