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.

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.

Serum sex hormone-binding globulin and testosterone in relation to cardiovascular disease risk factors in young men: a population-based study.

OBJECTIVE: Reduced sex hormone-binding globulin (SHBG) concentration predicts insulin resistance and type 2 diabetes, but its association with cardiovascular disease (CVD) risk is unclear. We examined the association between SHBG and cardiovascular risk factors, independently of total testosterone (TT), in young men. DESIGN: Observational, cross-sectional study. SETTING: General community. PARTICIPANTS: The study included 2716 men aged 31 years in the Northern Finland Birth Cohort in 1996 with clinical examination data and fasting blood samples. OUTCOME VARIABLES: Blood pressure (BP), lipids and C-reactive protein (CRP) as biological CVD risk markers. RESULTS: SHBG concentration was significantly and inversely related to systolic and diastolic BP, triglycerides and CRP, but positively to HDL cholesterol after adjusting for insulin, BMI, waist circumference, smoking, education and physical activity (all P<0.05). These linearly graded associations persisted with additional adjustment for TT. SHBG was significantly associated with total cholesterol only with adjustment for covariates and TT (P<0.05). The direction and magnitude of associations between TT and risk factors were variable, but further adjustment for insulin, adiposity and SHBG showed positive associations between TT and BP, total and LDL-cholesterol and triglycerides and an inverse association with CRP (all P<0.05), but its relation with HDL-cholesterol was no longer significant. CONCLUSIONS: In this cohort of young adult men, higher SHBG concentration was associated with a more favourable CVD risk profile, independently of TT. SHBG concentration modified the associations of TT with CVD risk factors.

Late-onset hypogonadism and mortality in aging men.

CONTEXT: Late-onset hypogonadism (LOH) has recently been defined as a syndrome in middle-aged and elderly men reporting sexual symptoms in the presence of low T. The natural history of LOH, especially its relationship to mortality, is currently unknown. OBJECTIVE: The aim of this study was to clarify the associations between LOH, low T, and sexual symptoms with mortality in men. DESIGN, SETTING, AND PARTICIPANTS: Prospective data from the European Male Aging Study (EMAS) on 2599 community-dwelling men aged 40-79 years in eight European countries was used for this study. MAIN OUTCOME MEASURE(S): All-cause, cardiovascular, and cancer-related mortality was measured. RESULTS: One hundred forty-seven men died during a median follow-up of 4.3 years. Fifty-five men (2.1%) were identified as having LOH (31 moderate and 24 severe). After adjusting for age, center, body mass index (BMI), current smoking, and poor general health, compared with men without LOH, those with severe LOH had a 5-fold [hazard ratio (HR) 5.5; 95% confidence interval (CI) 2.7, 11.4] higher risk of all-cause mortality. Compared with eugonadal men, the multivariable-adjusted risk of mortality was 2-fold higher in those with T less than 8 nmol/L (irrespective of symptoms; HR 2.3; 95% CI 1.2, 4.2) and 3-fold higher in those with three sexual symptoms (irrespective of serum T; compared with asymptomatic men; HR 3.2; 95% CI 1.8, 5.8). Similar risks were observed for cardiovascular mortality. CONCLUSIONS: Severe LOH is associated with substantially higher risks of all-cause and cardiovascular mortality, to which both the level of T and the presence of sexual symptoms contribute independently. Detecting low T in men presenting with sexual symptoms offers an opportunity to identify a small subgroup of aging men at particularly high risk of dying.

Age-associated changes in hypothalamic-pituitary-testicular function in middle-aged and older men are modified by weight change and lifestyle factors: longitudinal results from the European Male Ageing Study.

OBJECTIVE: Health and lifestyle factors are associated with variations in serum testosterone levels in ageing men. However, it remains unclear how age-related changes in testosterone may be attenuated by lifestyle modifications. The objective was to investigate the longitudinal relationships between changes in health and lifestyle factors with changes in hormones of the reproductive endocrine axis in ageing men. DESIGN: A longitudinal survey of 2736 community-dwelling men aged 40-79 years at baseline recruited from eight centres across Europe. Follow-up assessment occurred mean (±S.D.) 4.4±0.3 years later. RESULTS: Paired testosterone results were available for 2395 men. Mean (±S.D.) annualised hormone changes were as follows: testosterone -0.1±0.95  nmol/l; free testosterone (FT) -3.83±16.8  pmol/l; sex hormone-binding globulin (SHBG) 0.56±2.5  nmol/l and LH 0.08±0.57  U/l. Weight loss was associated with a proportional increase, and weight gain a proportional decrease, in testosterone and SHBG. FT showed a curvilinear relationship to weight change; only those who gained or lost ≥15% of weight showed a significant change (in the same direction as testosterone). Smoking cessation was associated with a greater decline in testosterone than being a non-smoker, which was unrelated to weight change. Changes in number of comorbid conditions or physical activity were not associated with significant alterations in hypothalamic-pituitary-testicular (HPT) axis function. CONCLUSIONS: Body weight and lifestyle factors influence HPT axis function in ageing. Weight loss was associated with a rise, and weight gain a fall, in testosterone, FT and SHBG. Weight management appears to be important in maintaining circulating testosterone in ageing men, and obesity-associated changes in HPT axis hormones are reversible following weight reduction.

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.

Chronic widespread pain is associated with slower cognitive processing speed in middle-aged and older European men.

Evidence from clinic-based studies suggests that the fibromyalgia syndrome (FMS) is associated with impairment in cognitive function though the mechanism is unclear. The aim of this analysis was to determine whether there is a similar association between chronic widespread pain (CWP), a cardinal feature of FMS, and impaired cognition in a community setting. Men (n=3369, 40-79 years) were recruited from population registers in eight centres for participation in the European Male Ageing Study (EMAS). The subjects completed a pain questionnaire and pain manikin, with the presence of CWP defined using the American College of Rheumatology criteria. The cognitive functions measured were visuospatial-constructional ability and visual memory (Rey-Osterrieth Complex Figure [ROCF]); visual recognition (Camden Topographical Recognition Memory test [CTRM]); and psychomotor processing speed (Digit-Symbol Substitution test [DSST]). We restricted our analysis to those subjects reporting pain that satisfied the criteria for CWP and those who were pain free. Of these 1539 men [mean (SD) age 60 (11) years], 266 had CWP. All cognitive test scores declined cross-sectionally with age (P<0.05). In age-adjusted linear regressions men with CWP had a lower DSST score (ß=-2.4, P<0.001) compared to pain-free subjects. After adjustment for lifestyle and health factors the association between pain status and the DSST score was attenuated but remained significant (ß=-1.02, P=0.04). There was no association between CWP and the ROCF-copy, ROCF-recall or CTRM scores. CWP is associated with slower psychomotor processing speed among community-dwelling European men. Prospective studies are required to confirm this observation and explore possible mechanisms for the association.

The effects of neurokinin B upon gonadotrophin release in male rodents.

Growing evidence suggests the tachykinin neurokinin B (NKB) may modulate gonadotrophin secretion and play a role in sex-steroid feedback within the reproductive axis. NKB signalling has recently been identified as being necessary for normal human reproductive function, although the precise mechanisms underpinning this role remain to be established. We have used rodents to explore further the role of NKB within the reproductive axis. In particular, we have studied its interactions with kisspeptin, a neuropeptide essential for reproductive function in rodent and human with close anatomical links to NKB within the hypothalamus. Intraperitoneal administration of NKB (50 nmol) to male mice had no effect on circulating luteinsing hormone (LH) levels and, although i.p. kisspeptin (15 nmol) increased LH five-fold, co-administration of NKB and kisspeptin was indistinguishable from kisspeptin alone. Intracerebroventricular administration of NKB (10 nmol) to male mice also had no effect on LH levels, with 1 nmol kisspeptin i.c.v. significantly increasing LH compared to control (0.37 +/- 0.18 versus 5.11 +/- 0.28 ng/ml, respectively). Interestingly, i.c.v. co-administration of NKB and kisspeptin caused a significant increase in LH concentrations compared to kisspeptin alone (8.96 +/- 1.82 versus 5.11 +/- 0.28 ng/ml respectively). We used hypothalamic explants from rats to assess the effect of NKB on gonadotrpohin-releasing hormone (GnRH) secretion ex vivo. Doses of NKB up to 1000 nm failed to stimulate GnRH secretion, whereas 100 nm kisspeptin robustly increased GnRH secretion. Of note, co-administration of NKB with kisspeptin abrogated the effect of kisspeptin, producing no GnRH release above basal state. Finally, we analysed the expression of Tac2/Tacr3 (genes encoding NKB and NK3R, respectively) within the arcuate nucleus in different nutritional states. After a 48-h fast, the expression of both Tac2 and Tacr3 showed a significant increase, in contrast to levels of Kiss1 and Kiss1r mRNA, which remained unchanged. In male rodent models, NKB and kisspeptin have different effects upon gonadotrophin release and appear to interact in a complex manner.

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.

Ovarian and adrenal steroid production: regulatory role of LH/HCG.

BACKGROUND: The contribution of the adrenal glands to the total circulating steroid pool in women is not well known. There is evidence that human adrenals express the LH receptor gene and that LH may affect adrenal androgen secretion. METHODS: HCG stimulation tests (a single dose of 5000 IU i.m.) were performed in women at reproductive age (group 1, n = 6, age 21--39 years) before and after treatment with a GnRH agonist for 3 weeks, and in oophorectomized post-menopausal women (group 2, n = 6, 47--59 years) during and after estrogen replacement therapy (ERT). RESULTS: HCG did not stimulate the secretion of cortisol, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) in group 2. In contrast, in group 1, the basal concentrations of serum 17-hydroxyprogesterone (17-OHP), androstenedione, testosterone and estradiol (E(2)) were stimulated significantly (17-OHP 105%, androstenedione 31%, testosterone 20%, E(2) 136%) by HCG, and the treatment with GnRH agonist decreased the responses. The basal serum concentrations of these steroids were significantly lower in oophorectomized women (17-OHP 57%, androstenedione 46%, testosterone 25%), and HCG did not increase these levels. It can be approximated that the ovarian contribution to the circulating levels of 17-OHP, androstenedione and testosterone is 25--30%, and that the adrenals are the primary source of cortisol, DHEA and DHEAS. CONCLUSION: LH/HCG does not have a major role in the regulation of adrenal steroid synthesis in endocrinologically healthy women.

Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis.

The steroidogenic acute regulatory (StAR) protein, a novel phosphoprotein, is a crucial factor involved in intramitochondrial cholesterol transportation, the rate-limiting step in steroidogenesis. The present investigations were undertaken to elucidate involvement of thyroid hormone and StAR protein in the regulation of steroidogenesis in mouse Leydig cells. Treatment of cells with triiodothyronine (T(3)) coordinately augmented the levels of StAR protein, StAR mRNA, and steroid production, and these responses were progressively dependent on expression of steroidogenic factor 1 (SF-1). With regard to steroidogenesis and StAR expression, the T(3) response requires both on-going mRNA and protein synthesis. In addition, the effects of T(3) were acutely modulated at the steroidogenic machinery and luteinizing hormone receptor (LHR) function, while these levels were suppressed following longer periods of exposure to T(3). Furthermore, the inhibition of SF-1 expression by DAX-1 markedly abolished T(3)-mediated StAR expression in a time frame, which was consistent with decreased steroid biosynthesis. Specific involvement of SF-1 was further confirmed by assessing the 5'-flanking region of the mouse StAR gene, which identified a region between -254 and -110 bp that was essential for T(3) function. Importantly, it was found that the SF-1 binding site at position -135 bp of the 5'-flanking region was greatly involved in T(3)-mediated reporter activity. Electrophoretic mobility shift assays (EMSA) also demonstrated involvement of SF-1 in T(3) function. The relevance of T(3)-mediated LHR function was investigated in mice rendered hypo-and hyperthyroid, which accounted for up-regulation in the former and down-regulation in the latter group, respectively. These findings demonstrate a key role of thyroid hormone in maintaining mouse Leydig cell function, where thyroid hormone and StAR protein coordinately regulate steroid hormone biosynthesis.

Gonadal tumors of mice double transgenic for inhibin-alpha promoter-driven simian virus 40 T-antigen and herpes simplex virus thymidine kinase are sensitive to ganciclovir treatment.

We have previously produced transgenic (TG) mice expressing the mouse inhibin alpha-subunit promoter/Simian virus 40 T-antigen (Inhalpha/Tag) fusion gene. The mice develop gonadal somatic cell tumors at the age of 5-7 months; the ovarian tumors originate from granulosa cells, and those of the testes from Leydig cells. In the present study another TG mouse line was produced, expressing under the same inh-alpha promoter the herpes simplex virus thymidine kinase gene (Inhalpha/TK). Crossbreeding of the two TG mouse lines resulted in double TG mice (Inhalpha/TK-Inhalpha/Tag), which also developed gonadal tumors. The single (Inhalpha/Tag) and double TG (Inhalpha/TK-Inhalpha/Tag) mice, both bearing gonadal tumors, were treated at the age of 5.5-6.5 months with ganciclovir (GCV, 150 mg/kg body weight twice daily i.p.) for 14 days, or with aciclovir (ACV, 300-400 mg/kg body weight per day perorally) for 2 months. During GCV treatment, the total gonadal volume including the tumor, decreased in double TG mice by an average of 40% (P<0.05), while in single TG mice, there was a concomitant increase of 60% in gonadal size (P<0.05). GCV was also found to increase apoptosis in gonads of the double TG mice. Peroral treatment with ACV was less effective, it did not reduce significantly the gonadal volume. We also analyzed the in vitro efficacy of ACV and GCV treatments in transiently HSV-TK-transfected KK-1 murine granulosa tumor cells, originating from a single-positive Inhalpha/Tag mouse. GCV proved to be more effective and more specific than ACV in action. These results prove the principle that targeted expression of the HSV-TK gene in gonadal somatic cell tumors is potentially useful for tumor ablation by antiherpes treatment. The findings provide a lead for further development of somatic gene therapy for gonadal tumors.

Luteinizing hormone, its beta-subunit variant, and epithelial ovarian cancer: the gonadotropin hypothesis revisited.

The gonadotropin hypothesis postulates that excessive gonadotropin stimulation results in increased proliferation and subsequent malignant transformation of ovarian epithelium. The authors evaluated this hypothesis by analyzing the association between serum levels of wild-type luteinizing hormone (LH) and ovarian cancer risk. They also examined the relation between a variant of LH containing two missense point mutations (Trp(8)Arg and Ile(15)Thr) in its beta-subunit and ovarian cancer risk. Fifty-eight cases of epithelial ovarian cancer and 116 controls matched on age, menopausal status, and date of blood donation were included in a case-control study nested within the New York University Women's Health Study, a prospective cohort enrolled between 1985 and 1991 in New York City. Wild-type serum levels and variant LH status were determined by immunofluorometric assays in which monoclonal antibodies specific for wild-type and variant LH were used. Compared with women in the lowest tertile of wild-type LH, women in the highest tertile had a lower risk of ovarian cancer, after adjustment for potential confounders (odds ratio = 0.42, 95% confidence interval: 0.09, 2.09). Women heterozygous for variant LH were not at increased risk (adjusted odds ratio = 0.95, 95% confidence interval: 0.27, 3.34). The results suggest that neither wild-type LH levels nor variant LH status is associated with increased risk of epithelial ovarian cancer.

Improved technique for detection of enhanced green fluorescent protein in transgenic mice.

One of the most exciting recent advances in cell biology is the possibility to use the green fluorescent protein and its various mutated forms as reporter proteins in studies carried out in vitro and in vivo. In the present study, several detection techniques for the enhanced green fluorescent protein (EGFP) were compared in transgenic mice, using fluorescence and confocal microscopy. In addition, different tissue preparation techniques (squash preparations, vibratome sections, frozen sections) were evaluated. As a model we used transgenic mice expressing EGFP under the control of a 5.0-kb fragment of the glutathione peroxidase isoenzyme 5 protein promoter (GPX5-EGFP) or under a 3.8-kb fragment of the cysteine rich protein-1 promoter (CRISP1-EGFP). In the GPX5-EGFP mice, expression of EGFP was observed in the distal part of the caput epididymis, while the CRISP1 promoter directed EGFP expression in the tubular compartment of the testis. Among the various tissue preparation procedures tested, the best morphological and histological preservation, and reproducibility in EGFP detection, were obtained using frozen sections after a slow tissue-freezing protocol developed in the present study. After slow tissue freezing, specimens of testis and epididymis could be stored at -70 degrees C for at least six weeks without any affect on EGFP fluorescence. Hence, the method developed offers the possibility to analyze EGFP fluorescence in tissues several weeks after specimen collection. The sensitivity achieved was equal to that found in immunohistochemistry, applying biotin-streptavidin-FITC detection. Confocal microscopy is known to have the advantage that fluorescence can be detected from cells in different layers. This was found to be important as regards detecting EGFP fluorescence because the fluorescence was destroyed at the cut surfaces of sections produced by either vibratome or cryomicrotome.

Evaluation of the 5'-flanking regions of murine glutathione peroxidase five and cysteine-rich secretory protein-1 genes for directing transgene expression in mouse epididymis.

Based on strong epididymal expression of the mouse glutathione peroxidase 5 (GPX5) and cysteine-rich secretory protein-1 (CRISP-1) genes, we evaluated whether the 5.0-kilobase (kb)-long GPX5 and 3.8-kb-long CRISP-1 gene 5'-flanking regions could be used to target expression of genes of interest into the epididymis in transgenic mice. Of the two candidate promoters investigated, the CRISP-1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene was highly expressed in the tubular compartment of the testis in all stages of the seminiferous epithelial cycle between pachytene spermatocytes at stage VII to elongated spermatids at step 16. In contrast to CRISP-1, the 5.0-kb 5' region of the mouse GPX5 gene directed EGFP expression to the epididymis. In the various GPX5-EGFP mouse lines, strongest expression of EGFP mRNA was found in the epididymis, but low levels of reporter gene mRNA were detected in several other tissues. Strong EGFP fluorescence was found in the principal cells of the distal caput region of epididymis, and few fluorescent cells were also detected in the cauda region. No EGFP fluorescence was detected in the corpus region or in the other tissues analyzed. Hence, it is evident that the 5.0-kb 5'-flanking region of GPX5 promoter is suitable for directing the expression of structural genes of interest into the caput epididymidis in transgenic mice.

Ovarian tumorigenesis in mice transgenic for murine inhibin alpha subunit promoter-driven Simian Virus 40 T-antigen: ontogeny, functional characteristics, and endocrine effects.

We previously reported formation of ovarian granulosa cell tumors with 100% penetration in a transgenic mouse model with murine inhibin alpha subunit promoter-driven (inhalpha)/Simian Virus 40 T-antigen (Tag). The tumor-bearing inhalpha/Tag mice showed highly elevated serum levels of immunoreactive inhibin. To investigate the onset of tumorigenesis and related endocrine consequences, 6-8 female mice of two inhalpha/Tag lines and their mating control littermates were killed monthly between 1 and 6 mo of age. We also investigated tumorigenesis-related fertility aspects of these two mouse lines. The ontogeny and progression of tumors could be monitored in both inhalpha/Tag lines by alterations of ovarian weights and serum hormone levels. Serum progesterone levels increased in both inhalpha/Tag lines in an age-dependent manner as ovarian tumorigenesis progressed, and a reciprocal decrease occurred in serum LH and FSH. Neither serum estradiol (E(2)) nor uterine weights were significantly altered during tumorigenesis, suggesting that the ovarian tumors represented late stages of granulosa cell differentiation. In conclusion, the present findings show in the inhalpha/Tag TG mice a relation between endocrine consequences of granulosa cell tumorigenesis, and a connection of onset of tumor formation with aberrant steroidogenesis and gonadotropin secretion. These findings indicate that tumors are endocrinologically active and able to exert enhanced negative feedback effects on pituitary function. The tumors provide a good model for endocrinologically active hormone-dependent tumors.

Disparate response of wild-type and variant forms of LH to GnRH stimulation in individuals heterozygous for the LHbeta variant allele.

There is a common genetic variant of LH due to two amino acid changes in the LHbeta subunit, Trp(8)Arg and Ile(15)Thr. In order to compare the relative activities of wild type (wt-LH) and variant LHbeta (v-LHbeta) genes in LH production and secretion, we performed gonadotrophin-releasing hormone (GnRH) stimulation tests for healthy females (n = 7) and males (n = 10) heterozygous for the v-LHbeta allele. Blood samples were drawn up to 180 min after injection of GnRH. The serum samples were subjected to two immunofluorometric assays, one detecting wt hormone, the other detecting equally both LH types. The wt/total ratio increased significantly (P < or = 0.016) after GnRH injection in males. This indicates that the proportion of wt-LH increases in the circulation in men but not in women, and that women consequently secrete relatively more v-LH. An in-vitro bioassay was performed on 0 and 60 min samples, and the bio/immunoreactivity (B/I) ratio decreased in both sexes (P = 0.010-0.012). This supports the previously reported lower B/I ratio of wt. than v-LH, since wt-LH is expected to accumulate in circulation because of its longer half-life. In conclusion, these findings demonstrate that wt.- and v-LH respond differently to GnRH stimulation in men and women heterozygous for v-LHbeta. These results are in agreement with previously documented differences of the two forms in circulation, as well as with different promoter activities of the two LHbeta alleles.

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.