Hyponatremia, hypernatremia and impairment of functional, psychological and sexual domains.

OBJECTIVE: To determine the influence of serum sodium on physical, psychologic and sexual function. METHODS: This is a cross-sectional survey on 3340 community-dwelling men aged 40-79 years from a prospective cohort study in eight European countries, the European Male Ageing Study (EMAS). Participants filled-out the Short Form-36 (SF-36), the Physical Activity Scale for the Elderly (PASE), and the EMAS sexual function questionnaire. For all the analyses, serum sodium corrected for glycaemia ([Na+]G) was used. RESULTS: The relationship between [Na+]G and SF-36 physical function score (F = 3.99; p = 0.01), SF-36 mental health score (F = 7.69; p < 0.001), and PASE score (F = 14.95; p < 0.001) were best described by a quadratic equation, with worse scores for [Na+]G in either the lowest or the highest ends of the range. After dividing the sample into [Na+]G < 136 mmol/L (n = 81), 136-147 mmol/L (n = 3223) and > 147 mmol/L (n = 36), linear regression analyses with linear spline functions adjusted for confounders did not confirm these relationships. Similarly, erectile dysfunction and [Na+]G, were in a quadratic relationship (F = 9.00; p < 0.001). After adjusting for confounders, the linear regression with spline functions denoted a significantly worsened erectile function for increases in serum [Na+]G > 147 mmol/L (B = 0.15 [0.04;0.26], p < 0.01) but no relationship with [Na+]G < 136 mmol/L. Likewise, the relationship of [Na+]G with concerns about sexual dysfunction was confirmed only for men with serum [Na+]G > 147 mmol/L. CONCLUSIONS: This is the first study supporting an association between [Na+]G and sexual function. A worsening of erection and concerns about sexual function were observed for the highest values of [Na+]G, independently of other relevant factors.

Lower bone turnover and relative bone deficits in men with metabolic syndrome: a matter of insulin sensitivity? The European Male Ageing Study.

We examined cross-sectional associations of metabolic syndrome and its components with male bone turnover, density and structure. Greater bone mass in men with metabolic syndrome was related to their greater body mass, whereas hyperglycaemia, hypertriglyceridaemia or impaired insulin sensitivity were associated with lower bone turnover and relative bone mass deficits. INTRODUCTION: Metabolic syndrome (MetS) has been associated with lower bone turnover and relative bone mass or strength deficits (i.e. not proportionate to body mass index, BMI), but the relative contributions of MetS components related to insulin sensitivity or obesity to male bone health remain unclear. METHODS: We determined cross-sectional associations of MetS, its components and insulin sensitivity (by homeostatic model assessment-insulin sensitivity (HOMA-S)) using linear regression models adjusted for age, centre, smoking, alcohol, and BMI. Bone turnover markers and heel broadband ultrasound attenuation (BUA) were measured in 3129 men aged 40-79. Two centres measured total hip, femoral neck, and lumbar spine areal bone mineral density (aBMD, n = 527) and performed radius peripheral quantitative computed tomography (pQCT, n = 595). RESULTS: MetS was present in 975 men (31.2 %). Men with MetS had lower ß C-terminal cross-linked telopeptide (ß-CTX), N-terminal propeptide of type I procollagen (PINP) and osteocalcin (P < 0.0001) and higher total hip, femoral neck, and lumbar spine aBMD (P ≤ 0.03). Among MetS components, only hypertriglyceridaemia and hyperglycaemia were independently associated with PINP and ß-CTX. Hyperglycaemia was negatively associated with BUA, hypertriglyceridaemia with hip aBMD and radius cross-sectional area (CSA) and stress-strain index. HOMA-S was similarly associated with PINP and ß-CTX, BUA, and radius CSA in BMI-adjusted models. CONCLUSIONS: Men with MetS have higher aBMD in association with their greater body mass, while their lower bone turnover and relative deficits in heel BUA and radius CSA are mainly related to correlates of insulin sensitivity. Our findings support the hypothesis that underlying metabolic complications may be involved in the bone's failure to adapt to increasing bodily loads in men with MetS.

Men homozygous for an inactivating mutation of the follicle-stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility.

Gonadal function is controlled by the two pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). While LH mainly regulates gonadal steroidogenesis, FSH is considered essential for folliculogenesis in the female and spermatogenesis in the male. We recently discovered that an inactivating point mutation in the FSH receptor (R) gene causes a recessively inherited form of hypergonadotropic ovarian failure in homozygous females. This 566C-->T mutation, predicting an alanine to valine substitution, is located in exon 7 of the FSHR gene, in the region encoding the extracellular domain of the receptor molecule. Functional testing showed a clear-cut reduction in ligand binding and signal transduction by the mutated receptor. Hence, lack of FSH function is incompatible with ovarian follicular maturation and female fertility. In the male, FSH is generally considered essential for the pubertal initiation of spermatogenesis and maintenance of quantitatively normal sperm production in adults. We report here the first characterization of males homozygous for an inactivating FSHR mutation. They have variable degrees of spermatogenic failure, but, surprisingly, do not show azoospermia or absolute infertility. These results question the essential role of FSH for the initiation of spermatogenesis, and demonstrate that FSH is more important for female than for male fertility.

Thyroid hormones and male sexual function.

The role of thyroid hormones in the control of erectile functioning has been only superficially investigated. The aim of the present study was to investigate the association between thyroid and erectile function in two different cohorts of subjects. The first one derives from the European Male Ageing Study (EMAS study), a multicentre survey performed on a sample of 3369 community-dwelling men aged 40-79 years (mean 60 ± 11 years). The second cohort is a consecutive series of 3203 heterosexual male patients (mean age 51.8 ± 13.0 years) attending our Andrology and Sexual Medicine Outpatient Clinic for sexual dysfunction at the University of Florence (UNIFI study). In the EMAS study all subjects were tested for thyroid-stimulating hormone (TSH) and free thyroxine (FT4). Similarly, TSH levels were checked in all patients in the UNIFI study, while FT4 only when TSH resulted outside the reference range. Overt primary hyperthyroidism (reduced TSH and elevated FT4, according to the reference range) was found in 0.3 and 0.2% of EMAS and UNIFI study respectively. In both study cohorts, suppressed TSH levels were associated with erectile dysfunction (ED). Overt hyperthyroidism was associated with an increased risk of severe erectile dysfunction (ED, hazard ratio = 14 and 16 in the EMAS and UNIFI study, respectively; both p < 0.05), after adjusting for confounding factors. These associations were confirmed in nested case-control analyses, comparing subjects with overt hyperthyroidism to age, BMI, smoking status and testosterone-matched controls. Conversely, no association between primary hypothyroidism and ED was observed. In conclusion, erectile function should be evaluated in all individuals with hyperthyroidism. Conversely, assessment of thyroid function cannot be recommended as routine practice in all ED patients.

Influence of age and sex steroids on bone density and geometry in middle-aged and elderly European men.

SUMMARY: The influence of age and sex steroids on bone density and geometry of the radius was examined in two European Caucasian populations. Age-related change in bone density and geometry was observed. In older men, bioavailable oestradiol may play a role in the maintenance of cortical and trabecular bone mineral density (BMD). INTRODUCTION: To examine the effect of age and sex steroids on bone density and geometry of the radius in two European Caucasian populations. METHODS: European Caucasian men aged 40-79 years were recruited from population registers in two centres: Manchester (UK) and Leuven (Belgium), for participation in the European Male Ageing Study. Total testosterone (T) and oestradiol (E(2)) were measured by mass spectrometry and the free and bioavailable fractions calculated. Peripheral quantitative computed tomography was used to scan the radius at distal (4%) and midshaft (50%) sites. RESULTS: Three hundred thirty-nine men from Manchester and 389 from Leuven, mean ages 60.2 and 60.0 years, respectively, participated. At the 50% radius site, there was a significant decrease with age in cortical BMD, bone mineral content (BMC), cortical thickness, and muscle area, whilst medullary area increased. At the 4% radius site, trabecular and total volumetric BMD declined with age. Increasing bioavailable E(2) (bioE(2)) was associated with increased cortical BMD (50% radius site) and trabecular BMD (4% radius site) in Leuven, but not Manchester, men. This effect was predominantly in those aged 60 years and over. In older Leuven men, bioavailable testosterone (Bio T) was linked with increased cortical BMC, muscle area and SSI (50% radius site) and total area (4% radius site). CONCLUSIONS: There is age-related change in bone density and geometry at the midshaft radius in middle-aged and elderly European men. In older men bioE(2) may maintain cortical and trabecular BMD. BioT may influence bone health through associations with muscle mass and bone area.

Gonadal sex steroid status and bone health in middle-aged and elderly European men.

SUMMARY: The influence of sex steroids on calcaneal quantitative ultrasound (QUS) parameters was assessed in a population sample of middle-aged and elderly European men. Higher free and total E(2) though not testosterone, were independently associated with higher QUS parameters. INTRODUCTION: The aim of this study was to investigate the association between QUS parameters and sex steroids in middle-aged and elderly European men. METHODS: Three thousand one hundred forty-one men aged between 40 and 79 years were recruited from eight European centres for participation in a study of male ageing: the European Male Ageing Study. Subjects were invited by letter to attend for an interviewer-administered questionnaire, blood sample and QUS of the calcaneus (Hologic-SAHARA). Blood was assessed for sex steroids including oestradiol (E(2)), testosterone (T), free and bio-available E(2) and T and sex hormone binding globulin (SHBG). RESULTS: Serum total T was not associated with any of the QUS parameters. Free T and both free and total E(2) were positively related to all QUS readings, while SHBG concentrations were negatively associated. These relationships were observed in both older and younger (<60 years) men. In a multivariate model, after adjustment for age, centre, height, weight, physical activity levels and smoking, free E(2) and SHBG, though not free T, remained independently associated with the QUS parameters. After further adjustment for IGF-1, however, the association with SHBG became non-significant. CONCLUSION: Higher free and total E(2) are associated with bone health not only among the elderly but also middle-aged European men.

Investigating the determinants of international differences in the prevalence of chronic widespread pain: evidence from the European Male Ageing Study.

OBJECTIVES: To determine whether among middle-aged and elderly men there is evidence of international differences in the prevalence of chronic widespread pain (CWP) and whether any such differences could be explained by psychological, psychosocial factors or differences in physical health status. METHODS: The European Male Ageing Study (EMAS) sampled from population registers in cities (centres) of eight European countries. Each centre recruited an age-stratified sample of men aged 40-79 years. Information on pain was collected by questionnaire and subjects were classified according to whether they satisfied the American College of Rheumatology definition of CWP. Information was collected on social status, mental health, recent life events and co-morbidities. RESULTS: Across all centres 3963 subjects completed a study questionnaire, with participation rates ranging from 24% in Hungary to 72% in Estonia. There were significant differences in prevalence: between 5% and 7% in centres in Italy, England, Belgium and Sweden, 9-15% in centres in Spain, Poland and Hungary and 15% in Estonia. There were strong relationships between poor mental health, adverse recent life events, co-morbidities and CWP. Adjustment for these factors explained between half and all of the excess risk in the eastern European centres: the excess risk in Poland was explained (odds ratio (OR) 1.1, 95% CI 0.9 to 1.2) but there remained excess risk in Hungary (OR 1.6, 95% CI 1.4 to 1.8) and Estonia (OR 2.6, 95% CI 2.2 to 2.9). CONCLUSIONS: This study is the first directly to compare the occurrence of CWP internationally. There is an excess prevalence in countries of eastern Europe and this excess is associated with adverse psychosocial factors as well as poorer psychological and physical health.

Novel epididymal proteins as targets for the development of post-testicular male contraception.

Apart from condoms and vasectomy, modern contraceptive methods for men are still not available. Besides hormonal approaches to stop testicular sperm production, the post-meiotic blockage of epididymal sperm maturation carries lots of promise. Microarray and proteomics techniques and libraries of expressed sequence tags, in combination with digital differential display tools and publicly available gene expression databases, are being currently used to identify and characterize novel epididymal proteins as putative targets for male contraception. The data reported indicate that these technologies provide complementary information for the identification of novel highly expressed genes in the epididymis. Deleting the gene of interest by targeted ablation technology in mice or using immunization against the cognate protein are the two preferred methods to functionally validate the function of novel genes in vivo. In this review, we summarize the current knowledge of several epididymal proteins shown either in vivo or in vitro to be involved in the epididymal sperm maturation. These proteins include CRISP1, SPAG11e, DEFB126, carbonyl reductase P34H, CD52, and GPR64. In addition, we introduce novel proteinases and protease inhibitor gene families with potentially important roles in regulating the sperm maturation process. Furthermore, potential contraceptive strategies as well as delivery methods will be discussed. Despite the progress made in recent years, further studies are needed to reveal further details in the epididymal sperm maturation process and the factors involved, in order to facilitate the development of new epididymal contraceptives.

Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function.

The recent unraveling of structures of genes for the gonadotropin subunits and gonadotropin receptors has provided reproductive endocrinologists with new tools to study normal and pathological functions of the hypothalamic-pituitary-gonadal axis. Rare inactivating mutations that produce distinctive phenotypes of isolated LH or FSH deficiency have been discovered in gonadotropin subunit genes. In addition, there is a common polymorphism in the LHbeta subunit gene with possible clinical significance as a contributing factor to pathologies of LH-dependent gonadal functions. Both activating and inactivating mutations have been detected in the gonadotropin receptor genes, a larger number in the LH receptor gene, but so far only a few in the gene for the FSH receptor. These mutations corroborate and extend our knowledge of clinical consequences of gonadotropin resistance and inappropriate gonadotropin action. The information obtained from human mutations has been complemented by animal models with disrupted or inappropriately activated gonadotropin ligand or receptor genes. These clinical and experimental genetic disease models form a powerful tool for exploring the physiology and pathophysiology of gonadotropin function and provide an excellent example of the power of molecular biological approaches in the study of pathogenesis of diseases.

Elevated luteinizing hormone induces expression of its receptor and promotes steroidogenesis in the adrenal cortex.

Transgenic (TG) female mice expressing bLHbeta-CTP (a chimeric protein derived from the beta-subunit of bovine luteinizing hormone [LH] and a fragment of the beta-subunit of human chorionic gonadotropin [hCG]) exhibit elevated serum LH, infertility, polycystic ovaries, and ovarian tumors. In humans, increased LH secretion also occurs in infertility and polycystic ovarian syndrome, often concomitant with adrenocortical dysfunction. We therefore investigated adrenal function in LH overexpressing bLHbeta-CTP female mice. The size of their adrenals was increased by 80% with histological signs of cortical stimulation. Furthermore, adrenal steroid production was increased, with up to 14-fold elevated serum corticosterone. Primary adrenal cells from TG and control females responded similarly to ACTH stimulation, but, surprisingly, the TG adrenals responded to hCG with significantly increased cAMP, progesterone, and corticosterone production. LH receptor (LHR) expression and activity were also elevated in adrenals from female TG mice, but gonadectomized TG females showed no increase in corticosterone, suggesting that the dysfunctional ovaries of the intact TG females promote adrenocortical hyperfunction. We suggest that, in intact TG females, enhanced ovarian estrogen synthesis causes increased secretion of prolactin (PRL), which elevates LHR expression. Chronically elevated serum LH, augmented by enhanced PRL production, induces functional LHR expression in mouse adrenal cortex, leading to elevated, LH-dependent, corticosterone production. Thus, besides polycystic ovaries, the bLHbeta-CTP mice provide a useful model for studying human disorders related to elevated LH secretion and adrenocortical hyperfunction.

Ontogeny of pituitary-gonadal interactions: current advances and controversies.

The different compartments of the fetal hypothalamic-pituitary-gonadal axis, the hypothalamus, anterior pituitary, and gonads, probably start their embryonic development independently, and become fully interactive as the last link o f their maturation. The developing hypothalamic-pituitary-gonadal axis offers a good model for studies on the mechanisms of regulation of fetal hormonal systems. It is evident that fetal hormonal functions are not the same as those of the adult on a smaller scale, but that there are fundamental differences between the fetus and adult in basic features of the mechanisms o f reproductive hormone action.

Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin alpha-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines.

To establish in vivo gonadal tumor models and permanent lines of gonadal somatic cells we produced transgenic (TG) mice expressing the Simian virus (SV) 40 T-antigens (T-ag), driven by 6 or 2.1 kilobase fragments of the mouse inhibin alpha-subunit promoter. Hitherto, altogether 44 TG mice, one of which carried the shorter transgene, have produced gonadal tumors. Two founder females expressing the longer transgene, KK1 and KK3, and three established TG mouse lines were studied in detail. Penetrance of the phenotype in IT6-M and IT6-F mouse lines was 100% (tumors/TG: IT6-M 22/22, IT6-F 14/14). The T-ag mRNA was strongly expressed in the gonads, adrenal glands, pituitary, and brain. The KK-1 and KK-3 ovarian tumor cells immunostained with anti-SV40 large-T antibody. The KK-1 cells possessed high-affinity LH receptors [equilibrium association constant (Ka = 7.8 x 10(10) liters/mol] and responded to human CG by elevated cAMP and progesterone production. Also FSH slightly stimulated their cAMP and estradiol production (P < 0.01). These cells expressed cytochrome P450arom and inhibin alpha mRNA, but not cytochrome P450c17 alpha. In conclusion, the KK-1 cells are immortalized luteinizing granulosa cells expressing endogenous gonadotropin receptors, steroidogenic enzymes, and inhibin alpha. These cells will be useful in studies on the molecular aspects of granulosa cell function. The present study indicates that the 6-kilobase fragment of the inhibin alpha promoter described in this article contains the elements directing tissue-specific expression in vivo and is useful for targeted expression of other genes in the gonads.

Associations of obesity with socioeconomic and lifestyle factors in middle-aged and elderly men: European Male Aging Study (EMAS).

BACKGROUND: Social and lifestyle influences on age-related changes in body morphology are complex because lifestyle and physiological response to social stress can affect body fat differently. OBJECTIVE: In this study, we examined the associations of socioeconomic status (SES) and lifestyle factors with BMI and waist circumference (WC) in middle-aged and elderly European men. DESIGN AND SETTING: A cross-sectional study of 3319 men aged 40-79 years recruited from eight European centres. OUTCOMES: We estimated relative risk ratios (RRRs) of overweight/obesity associated with unfavourable SES and lifestyles. RESULTS: The prevalence of BMI ≥ 30 kg/m(2) or WC ≥ 102 cm rose linearly with age, except in the eighth decade when high BMI, but not high WC, declined. Among men aged 40-59 years, compared with non-smokers or most active men, centre and BMI-adjusted RRRs for having a WC between 94 and 101.9 cm increased by 1.6-fold in current smokers, 2.7-fold in least active men and maximal at 2.8-fold in least active men who smoked. Similar patterns but greater RRRs were observed for men with WC ≥ 102 cm, notably 8.4-fold greater in least active men who smoked. Compared with men in employment, those who were not in employment had increased risk of having a high WC by 1.4-fold in the 40-65 years group and by 1.3-fold in the 40-75 years group. These relationships were weaker among elderly men. CONCLUSION: Unfavourable SES and lifestyles associate with increased risk of obesity, especially in middle-aged men. The combination of inactivity and smoking was the strongest predictor of high WC, providing a focus for health promotion and prevention at an early age.

Induction and maintenance of gonadotropin and lactogen receptors in hypoprolactinemic rats.

The effect of 2-bromo-alpha-ergocriptine (BR)-induced hypoprolactinemia on the induction and maintenance of testicular gonadotropin and lactogen receptors was studied in 60-day-old rats after receptor regulation by a gonadotropin-releasing hormone agonist analog [[D-Ser-(tBu)6]des-Gly10-GnRH N-ethylamide (GnRH-A)] and in young animals during sexual maturation. In adult animals, BR treatment delayed the reappearance of LH binding in the testis after GnRH-A injection, but had no effect on the recovery of lactogen binding. BR treatment also inhibited the increase of LH binding that occurred in control animals during the experimental period, but did not affect lactogen binding. Furthermore, BR potentiated the effect of GnRH-A on the decrease of Leydig cell testosterone synthesis observed 2 days later in vitro. Eight days after GnRH-A injection, concomitant BR treatment significantly inhibited the recovery of Leydig cell cAMP production. In peripubertal (25- to 46-day-old) animals, BR diminished the normal rise in testicular LH receptors, but did not affect the increase in lactogen receptors. Serum testosterone levels and other features of pubertal development, such as balano-preputial separation and spermatogenesis, were unaffected by hypoprolactinemia. In neonatal female animals, significant lactogen binding was detected at 3 days of age, whereas hCG binding was not demonstrable until 9 days after birth. These findings indicate that the expression of lactogen receptors precedes that of LH receptors in the developing gonad, and that the increase of LH binding in the testis during pubertal development requires normal circulating PRL levels. In adult animals, hypoprolactinemia potentiates GnRH-A-induced desensitization of steroidogenesis and cAMP formation, as well as LH receptor down-regulation, delaying the recovery of these phenomena. Although decreased serum PRL levels were associated with a marked reduction in testicular LH receptors both during development and in adult life, the absence of changes in lactogen receptors indicates that the latter sites are largely independent of the circulating PRL concentration.

Effects of gonadotropin-releasing hormone receptor blockade on rat testicular gonadotropin and lactogen receptors, steroidogenesis, and responses to human chorionic gonadotropin stimulation.

Testicular endocrine regulation was studied in adult male rats after treatment with a potent GnRH antagonist analog [N-Ac-D-p-Cl-Phe1,2, D-Trp3, D-Lys6, D-Ala10-GnRH (Ant.)] given in doses of 1 mg/kg at 0, 12, and 24 h. One group of animals also received an injection of human CG (hCG) (600 IU/kg) at 12 h, and all animals were killed at 36 h for hormone and receptor (R) measurements. Treatment with Ant. blocked greater than 95% of the pituitary and testicular GnRH-R, and decreased serum LH concentration by greater than 90%. Testicular lactogen-R content was decreased by 60% (P less than 0.01), but there was no change in LH-R and FSH-R concentrations. Ant. decreased serum and testicular testosterone levels by 90%, and testicular capacity to produce testosterone in vitro by 50% (P less than 0.01). No decrease was observed in production rates of cAMP and progesterone. hCG alone abolished testicular LH-R, decreased lactogen-R by 55% (P less than 0.01), and GnRH-R by 65% (P less than 0.01). Desensitization of cAMP and testosterone production, and an increase in progesterone-testosterone ratio, were seen after hCG. hCG + Ant. treatment resulted in R, cAMP, and steroid responses that were indistinguishable from those seen after hCG alone. These findings indicate that: 1) Ant.-induced hypogonadotropism decreases testicular lactogen-R concentration and testosterone production; 2) testicular GnRH-R and lactogen-R are subject to heterologous down-regulation by hCG; and 3) inhibition of the putative GnRH-mediated regulation of testis by Ant. blockade of the GnRH-R does not change testicular response to hCG-treatment in vivo. Hence, the present observations still leave the physiological role of testicular GnRH-R open.

Gonadotropin-releasing hormone agonist analog-induced steroidogenic lesion in the neonatal rat testis: evidence for direct gonadal action.

In this study we sought to determine whether a GnRH agonist analog (GnRH-A) could influence steroidogenesis by a direct effect on the neonatal rat testis. Five-day-old male rats were given a single sc injection of hCG (600 IU/kg BW), GnRH-A [D-Ser(tBu)6]des-Gly10-GnRH N-ethylamide (4 micrograms/kg BW), or their combination. Testicular testosterone (T) was increased (3-fold) only 6 h post-GnRH-A treatment, whereas after hCG administration testicular T remained elevated (3 to 6-fold) for 48 h. Testicular progesterone (P) increased by 40% 6-72 h after hCG treatment, but was not raised after GnRH-A injection. In vitro T production by testes from control and GnRH-A-treated (injection 24 h earlier) animals was stimulated 5 to 8-fold by hCG (7.9 nM) or 8-bromo-cAMP (8-Br-cAMP; 1 mM). hCG and 8-Br-cAMP did not further stimulate T production from testes of animals treated with hCG in vivo 24 h earlier. While hCG and 8-Br-cAMP had only a small stimulatory effect (1.5 to 2-fold) on in vitro P production by testes from control or hCG-treated animals, their stimulation of P production from testes of GnRH-A-treated animals was dramatic (20 to 30-fold). In vitro P production from testes of animals receiving combined treatment with hCG and GnRH-A in vivo reached a high hCG-stimulated rate similar to that found after GnRH-A treatment alone; the unstimulated values were also considerably elevated (5-fold) compared to those of untreated animals. The ability of GnRH-A treatment to stimulate testicular P production in the presence of a high concentration of hCG strongly suggests a direct gonadal action of the peptide. The possibility of such action was corroborated by the finding of abundant GnRH receptors in the neonatal testis. These results indicate that the steroidogenic lesion seen in adult rat testis after gonadotropic stimulation (blockade of C21 steroid side-chain cleavage with compensatory accumulation of P) can be reproduced in neonatal rat testes by a direct action of GnRH-A, but not by hCG.

Gonadotropin binding and Leydig cell activation in the rat testis in vivo.

Testicular LH receptor occupancy and steroidogenic responses were measured in adult male rats after intracardiac injections of [125I]iodo-hCG (0.5--5 x 10(6) cpm) mixed with known amounts of nonradioactive hCG to yield doses ranging from 10 ng to 300 micrograms. Uptake of the hormone by the testis was measured in the whole tissue or the 20,000 x g homogenate, with correction for nonspecific binding in animals injected with a 100-fold excess of unlabeled hCG. The steroidogenic response to hCG was followed by measurements of serum and testicular testosterone. Maximum specific uptake of [125I]iodo-hCG by the testes was observed 4--6 h after hormone injection. Of the specific counts, 80% were recovered in the 20,000 x g pellet of the tissue homogenate. The testicular contents of hCG-binding sites were similar when measured by in vivo occupancy of the receptors and by the in vitro receptor assay, indicating the physiological validity of the receptor measurements in tissue homogenates. Serum and testicular testosterone levels reached a maximum at 1 h, independent of the hCG dose used. When receptor occupancy in vitro after injection of hCG was compared with stimulation of steroidogenesis, a significant (P less than 0.05) 3-fold elevation of serum testosterone was seen when only 0.05% of the receptors were occupied. The maximal testosterone response was reached with 0.8% receptor occupancy. It is concluded that the same number of testicular LH receptors can be occupied by the circulating hormone in vivo and in tissue homogenates in vitro. The spare receptor concept also applied to the in vivo situation, since stimulation of steroidogenesis in the intact animal requires occupancy of only a few receptors per Leydig cell. This may be a general feature of hormonal activation of endocrine target cells in vivo.

Functional assessment of the calcium messenger system in cultured mouse Leydig tumor cells: regulation of human chorionic gonadotropin-induced expression of the steroidogenic acute regulatory protein.

The steroidogenic acute regulatory (StAR) protein, a 30-kDa mitochondrial factor, is a key regulator of steroid hormone biosynthesis, facilitating the transfer of cholesterol from the outer to the inner mitochondrial membrane. StAR protein expression is restricted to steroidogenic tissues, and it responds to hormonal stimulation through different second messenger pathways. The present study was designed to explore the mechanisms of extracellular calcium (Ca2+) involved in the hCG-stimulated expression of StAR protein and steroidogenesis in a mouse Leydig tumor cell line (mLTC-1). Extracellular Ca2+ (1.5 mmol/liter) enhanced the hCG (50 microg/liter)-induced increases in StAR messenger RNA (mRNA) and protein levels (1.7 +/- 0.3-fold; 4 h), as monitored by quantitative RT-PCR and immunoblotting. The potentiating effect of Ca2+ on the hCG-stimulated StAR response correlated with the acute progesterone (P) response. In accordance, omission of Ca2+ from the extracellular medium by specific Ca2+ chelators, EDTA or EGTA (4 mmol/liter each), markedly diminished the hCG-stimulated P production. The Ca2+ effect on hCG-induced StAR mRNA expression was dramatically suppressed by 10 micromol/liter verapamil, a Ca2+ channel blocker. The Ca2+-mobilizing agonist, potassium (K+; 4 mmol/liter), greatly increased the hCG responses of StAR expression and P production, which conversely were attenuated by Ca2+ antagonists, further supporting the involvement of intracellular free Ca2+ ([Ca2+]i) in these responses. The interaction of Ca2+ or K+ with hCG accounted for a clear increase in the StAR protein level (1.4-1.8-fold; 4 h) compared with that after hCG stimulation. Inhibition of protein synthesis by cycloheximide (CHX) drastically diminished the hCG-induced StAR protein content, indicating the requirement for on-going protein synthesis for hCG action. The transmembrane uptake of 45Ca2+ was increased by 26% with hCG and was strongly inhibited by verapamil. [Ca2+]i moderately augmented the response to hCG in fura-2/AM-loaded mLTC-1 cells within 30-40 sec, reaching a plateau within 1-3 min. Interestingly, the calcium ionophore (A23187) clearly increased (P < 0.01) StAR mRNA expression, in additive fashion with hCG. Northern hybridization analysis revealed four StAR transcripts at 3.4, 2.7, 1.6, and 1.4 kb, with the 1.6-kb band corresponding to the functional StAR protein; all of them were up-regulated 3- to 5-fold upon hCG stimulation, with a further increase in the presence of Ca2+. The mechanism of the Ca2+ effect on hCG-stimulated StAR expression and P production was evaluated by assessing the involvement of the nuclear orphan receptor, steroidogenic factor 1 (SF-1). Stimulation of hCG significantly elevated (2.1 +/- 0.3-fold) the SF-1 mRNA level, which was further augmented in the presence of Ca2+, whereas EGTA and verapamil completely abolished the increase caused by Ca2+. Cells expressing SF-1 marginally increased StAR expression, but coordinately elevated StAR mRNA levels in response to hCG and hCG plus Ca2+ compared with those in mock-transfected cells. On the other hand, overexpression of the nuclear receptor DAX-1 remarkably diminished (P < 0.0001) the endogenous SF-1 mRNA level as well as hCG-induced StAR mRNA expression. In summary, our results provide evidence that extracellular Ca2+ rapidly increases [Ca2+]i after hCG stimulation, presumably through opening of the transmembrane Ca2+ channel. Neither extracellular Ca2+ nor K+ alone has a noticeable effect on StAR expression and steroidogenesis, whereas they clearly potentiate hCG induction. The Ca2+-mediated increase in hCG involved in StAR expression and P production is well correlated to the levels of SF-1 expression. The stimulatory effect of hCG that rapidly increases [Ca2+]i is responsible at least in part for the regulation of SF-1-mediated StAR expression that consequently regulates steroidogenesis in mouse Leydig tumor cells.

Biphasic action of prolactin in the regulation of murine Leydig tumor cell functions.

We investigated in this study the effects of ovine PRL on endocrine functions of cultured murine Leydig tumor cells (mLTC-1). The parameters studied were the activation of signal transduction systems involving cAMP and intracellular free Ca(2+), the expression of Janus kinase 2 (JAK2), expression and function of LH and PRL receptors (R), expression of the steroidogenic acute regulatory (StAR) protein, and stimulation of steroidogenesis. Very similar biphasic dose- and time-dependent responses of all the parameters studied were found upon PRL stimulation, comprising a fast inhibition within 24 h in response to high PRL doses (>/=30 microgram/liter), and a slow stimulation, between 48-72 h, in response to lower PRL doses (1-10 microgram/liter). In addition, extracellular Ca(2+) (1.5 mmol/liter) increased the effect of PRL on human CG (hCG)-stimulated StAR messenger RNA expression and progesterone (P) production. Importantly, the biphasic effects of PRL on LHR gene expression and hCG-mediated P production were abolished in the presence of anti-PRL antiserum, demonstrating specificity of PRL action. The PRL effects on StAR expression, and steroid and cAMP production, apparently reflect its effects on LHR function. The relevance of the PRL effects observed in mLTC-1 cells was supported by demonstration of similar PRL responses in hCG-stimulated testosterone production of isolated mouse Leydig cells. Collectively, these findings clearly demonstrate the biphasic regulatory actions of PRL, and clarify some facets of the controversial role of this hormone in Leydig cell function.