[Show abstract][Hide abstract] ABSTRACT: Background:
G protein-coupled receptors (GPCRs) represent a physiologically and pharmacologically important family of receptors that upon coupling to GαS stimulate cAMP production catalyzed by adenylyl cyclase. Thus, developing assays to monitor cAMP production is crucial to screen for ligands in studies of GPCR signaling. Primary cell cultures represent a more robust model than cell lines to study GPCR signaling since they physiologically resemble the parent tissue. Current cAMP assays have two fundamental limitations: 1) absence of cAMP kinetics as competition-based assays require cell lysis and measure only a single time-point, and 2) high variation with separate samples needed to measure consecutive time points. The utility of real-time cAMP biosensors is also limited in primary cell cultures due to their poor transfection efficiency, variable expression levels and inability to select stable clones. We therefore, decided to develop an assay that can measure cAMP not only at a single time-point but the entire cAMP kinetics after GPCR activation in untransfected primary cells.
CANDLES (Cyclic AMP iNdirect Detection by Light Emission from Sensor cells) assay for monitoring cAMP kinetics in cell cultures, particularly in primary cultures was developed. The assay requires co-culturing of primary cells with sensor cells that stably express a luminescent cAMP sensor. Upon GPCR activation in primary cells, cAMP is transferred to sensor cells via gap junction channels, thereby evoking a luminescent read-out. GPCR activation using primary cultures of rat cortical neurons and mouse granulosa cells was measured. Kinetic responses of different agonists to adrenergic receptors were also compared using rat cortical neurons. The assay optimization was done by varying sensor-test cell ratio, using phosphodiesterase inhibitors and testing cell-cell contact requirement.
Here we present CANDLES assay based on co-culturing test cells with cAMP-detecting sensor cells. This co-culture setup allows kinetic measurements, eliminates primary cell transfections and reduces variability. A variety of cell types (rat cortical neurons, mouse granulosa cells and established cell lines) and receptors (adrenergic, follicle stimulating hormone and luteinizing hormone/chorionic gonadotropin receptors) were tested for use with CANDLES. The assay is best applied while comparing cAMP generation curves upon different drug treatments to untransfected primary cells.
Cell Communication and Signaling 11/2014; 12(1):70. · 4.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During epididymal sperm maturation, the lipid content of the sperm membrane is modified, which facilitates sperm motility and fertility. However, little is known about the mechanisms regulating the maturation process. By generating a conditional knockout (cKO) of Dicer1 in the proximal part of the mouse epididymis, we studied the role of RNA interference in epididymal functions. The Dicer1 cKO epididymis displayed an altered lipid homeostasis associated with a 0.6-fold reduction in the expression of the gene elongation of very long chain fatty acids-like 2, an enzyme needed for production of long-chain polyunsaturated fatty acids (PUFAs). Furthermore, the expression of several factors involved in cholesterol synthesis was up-regulated. Accordingly, the Dicer1 cKO sperm membrane showed a 0.7-fold decrease in long-chain PUFAs, whereas the amount of cholesterol in acrosome-reacted sperm displayed a 1.7-fold increase. The increased cholesterol:PUFA ratio of the sperm membrane caused breakage of the neck and acrosome region and immotility of sperm. Dicer1 cKO mice sperm also displayed reduced ability to bind to and fertilize the oocyte in vitro. This study thus shows that Dicer1 is critical for lipid synthesis in the epididymis, which directly affects sperm membrane integrity and male fertility.-Björkgren, I., Gylling, H., Turunen, H., Huhtaniemi, I., Strauss, L., Poutanen, M., Sipilä, P. Imbalanced lipid homeostasis in the conditional Dicer1 knockout mouse epididymis causes instability of the sperm membrane.
[Show abstract][Hide abstract] ABSTRACT: 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: We examined the associations of socioeconomic status (SES) and lifestyle factors with body mass index (BMI) and waist circumference (WC) in middle-aged and elderly European men. Design and setting: Cross-sectional study of 3,319 men aged 40-79 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 BMI30kg/m2 or WC102cm rose linearly with age, except in the 8th decade when high BMI, but not high WC, declined. Among men aged 40-59y, compared to non-smokers or most active men, centre and BMI adjusted RRRs for having a WC between 94-101.9cm increased by 1.6-fold in current smokers, and 2.7-fold in least active men, maximal at 2.8-fold in least active men who smoked. Similar patterns but greater RRRs were observed for men with WC≥102cm, notably 8.4-fold greater in least active men who smoked. Compared to men in employment, those who were not in employment had increased risk of having a high WC by 1.4-fold in the 40-65y group and by 1.3-fold in the 40-75y 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.
European Journal of Endocrinology 10/2014; · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The advent of technologies to genetically manipulate the mouse genome has revolutionised research approaches, providing a unique platform to study the causality of reproductive disorders in vivo. With the relative ease of generating genetically modified mouse models, the last two decades have yielded multiple loss-of-function and gain-of-function mutation mouse models to explore the role of gonadotrophins and their receptors in reproductive pathologies. This work has provided key insights into the molecular mechanisms underlying reproductive disorders with altered gonadotrophin action, revealing the fundamental roles of these pituitary hormones and their receptors in the hypothalamic-pituitary-gonadal axis. This review will describe genetically modified mouse models of gonadotrophins and their receptors with enhanced or diminished actions, specifically focussing on the male. We will discuss the mechanistic insights gained from these models into male reproductive disorders, and discuss the relationship and understanding provided into male human reproductive disorders originating from altered gonadotrophin action.
[Show abstract][Hide abstract] ABSTRACT: Background Androgens acting via the androgen receptor (AR) stimulate production of prostate specific antigen (PSA), which is a clinical marker of prostate cancer (PCa). Since genetic variants in the AR may have a significant impact on the risk of being diagnosed with PCa, the aim was to investigate if AR-variants were associated with the risk of having PSA above clinically used cut-off thresholds of 3 or 4 ng/mL in men without PCa. Methods Men without PCa history (n=1744) were selected from the European Male Ageing Study (EMAS) cohort of 40-80 year old men from 8 different European centers. Using linear and logistic regression models, with age and center as covariates, we investigated whether AR-variants (CAG repeat-length and/or SNP genotype) were associated with having serum PSA concentrations above 3 or 4 ng/mL, which often are set as cut-off concentrations for further investigation of PCa. Results Carriers of the SNP rs1204038 A-allele (16% of the men) were more likely to have PSA>3 and 4 ng/mL (OR; 95%CI 1.65; 1.13-2.40 and 1.87; 1.18-2.96, respectively) than G-allele carriers. They also had shorter CAG-repeats (median 20 vs. 23, p<0.0005), but CAG repeat length per se did not affect the PSA concentrations. Conclusion The A-allele of the SNP rs1204038 gives a 65% higher risk of having PSA above 3 ng/mL than the G-allele in men without PCa, and thereby an increased risk of being referred for further examination on suspicion of PCa. Impact Serum PSA as a clinical marker could be improved by adjustment for AR-genotype.
[Show abstract][Hide abstract] ABSTRACT: Background: Both low total testosterone (T) and sex hormone binding globulin (SHBG) have been associated with an increased risk for metabolic syndrome (MetS) in men. However, serum concentrations of T are strongly linked to SHBG levels, and both are decreased in MetS. Whether the risk for MetS associated with low T is independent of SHBG, remains unclear. Furthermore, T is aromatised to estradiol (E2). The potential impact of variations in E2 on the risk for MetS has not been investigated.
Objective: To study the longitudinal association of T and E2 and the risk for MetS in a large European cohort of healthy middle aged and elderly men and to assess if this risk is independent of SHBG.
Methods: The European Male Ageing Study (EMAS) included 2736 community-dwelling men from 8 European countries, aged 40-79 years at baseline, followed-up for 4.3 years (range 2.95-5.7 years). MetS was defined using the ATP III criteria. Serum total T and E2 levels were measured by respectively liquid and gas chromatography/mass spectrometry. The association between baseline SHBG and sex steroids and the development of incident MetS at follow-up was assessed using logistic regression with adjustments made for age, study centre, smoking status, physical activity and general health. Results were expressed as standardised odds ratios (OR) with 95% confidence intervals (CI).
Results: In the EMAS cohort, 282 men developed MetS between baseline and follow up, while 1387 men did not satisfy criteria for MetS at either time point. Men with a higher level of SHBG or total T had a significantly lower risk of developing MetS (OR 0.59 (CI 0.49-0.70, p˂0.001) and 0.58 (CI 0.50-0.68, p˂0.001), respectively). Calculated free T showed similar findings to total T. E2 was not associated with incident MetS (OR 0.99, CI 0.86-1.13, p=0.867). However, the T/E2 ratio was strongly inversely associated with incident MetS (OR 0.55, CI 0.46-0.66, p˂0.001). The inverse association between MetS and SHBG became non-significant if T was added to the model (OR 0.80, CI 0.64-1.01, p=0.064), while the association with T persisted after addition of SHBG (OR 0.67, CI 0.55-0.83). The association between E2 and incident MetS remained non-significant after adjustment for SHBG (OR 1.14, CI 0.99-1.32, p=0.074), but became significant after adjustment for T (OR 1.40, CI 1.20-1.64, p˂0.001). The T/E2 ratio remained strongly inversely associated with incident MetS even after SHBG was added to the model (OR 0.62, CI 0.51-0.75, p˂0.001).
Conclusions: In men, low T, independently of SHBG and E2, predicts the development of MetS. A higher T/E2 ratio, reflecting lower aromatisation of T into E2, is linked with a reduced risk for MetS.
[Show abstract][Hide abstract] ABSTRACT: Transgenic female mice overexpressing the α- and β- subunits of human chorionic gonadotropin (hCGαβ+) exhibited precocious puberty, as evidenced by early vaginal opening. Chronically elevated hCG in 21-day-old hCGαβ+ females stimulated gonadal androgen production, which exerted negative feedback over the endogenous gonadotropin synthesis, and activated the hypothalamic GnRH pulsatility and gene expression. Transgenic females also exhibited elevated hypothalamic aromatization in the preoptic area (POA), which is the sexually-differentiated area that controls the LH surge in adulthood. Ovariectomy at 14 days of age was unable to rescue this phenotype. However, the blockade of androgen action by flutamide from postnatal day 6 onwards reduced the aromatase levels in the POA of hCGαβ+ females. Our results suggest that early exposure of females to androgen action during a critical period between postnatal days 6 to 14 induces sex-specific organizational changes of the brain, which affect the aromatase expression in the POA at the onset of precocious puberty.
Molecular and Cellular Endocrinology 06/2014; · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Andropause or late-onset hypogondism (LOH) is a situation where a middle-aged or older man has low serum testosterone (T) in conjunction with diffuse symptoms reminiscent of those of genuine male hypogonadism (e.g. reduced sexual function, loss of vigor, muscle weakness, depression). Opinions about the diagnostic criteria, prevalence and treatment options of andropause vary considerably amongst experts. We review here some salient findings on the prevalence, diagnostic criteria and impact on health of andropause, obtained from the European Male Ageing Study (EMAS), a multicenter study of ageing among community-dwelling middle-aged and older men.
Annales d Endocrinologie 04/2014; · 0.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Observational, cross-sectional study SETTING: General community PARTICIPANTS: 2,716 31-year old men in the 1966 Northern Finland Birth Cohort with clinical examination data and fasting blood samples.
Blood pressure (BP), lipids and C-reactive protein (CRP) as biological CVD risk markers.
SHBG concentration was significantly and inversely related to systolic and diastolic BP, triglycerides and CRP but positively to high-density lipoprotein (HDL) cholesterol after adjusting for insulin, body mass index, 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 blood pressure, total and low-density lipoprotein cholesterol and triglycerides and an inverse association with CRP (all P<0.05), but its relation with HDL-cholesterol was no longer significant.
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.
European Journal of Endocrinology 03/2014; · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Testosterone (T), alone or in combination with progestin, provides a promising approach to hormonal male contraception. Its principle relies on enhanced negative feedback of exogenous T to suppress gonadotropins, thereby blocking the testicular T production needed for spermatogenesis, while simultaneously maintaining the extragonadal androgen actions, such as potency and libido, to avoid hypogonadism. A serious drawback of the treatment is that a significant proportion of men do not reach azoospermia or severe oligozoospermia, commensurate with contraceptive efficacy. We tested here, using hypogonadal luteinizing hormone/choriongonadotropin receptor (LHCGR) knockout (LHR(-/-)) mice, the basic principle of the T-based male contraceptive method, that a specific T dose could maintain extragonadal androgen actions without simultaneously activating spermatogenesis. LHR(-/-) mice were treated with increasing T doses, and the responses of their spermatogenesis and extragonadal androgen actions (including gonadotropin suppression and sexual behavior) were assessed. Conspicuously, all dose responses to T were practically superimposable, and no dose of T could be defined that would maintain sexual function and suppress gonadotropins without simultaneously activating spermatogenesis. This finding, never addressed in clinical contraceptive trials, is not unexpected in light of the same androgen receptor mediating androgen actions in all organs. When extrapolated to humans, our findings may jeopardize the current approach to hormonal male contraception and call for more effective means of inhibiting intratesticular T production or action, to achieve consistent spermatogenic suppression.-Oduwole, O. O., Vydra, N., Wood, N. E. M., Samanta, L., Owen, L., Keevil, B., Donaldson, M., Naresh, K., Huhtaniemi, I. T. Overlapping dose responses of spermatogenic and extragonadal testosterone actions jeopardize the principle of hormonal male contraception.
[Show abstract][Hide abstract] ABSTRACT: The development of genetically modified animals has been useful to understand the mechanisms involved in the regulation of the gonadotropin function. It is well known that alterations in the secretion of a single hormone is capable of producing profound reproductive abnormalities. Human chorionic gonadotropin (hCG) is a glycoprotein hormone normally secreted by the human placenta, and structurally and functionally it is related to pituitary LH. LH and hCG bind to the same LH/hCG receptor, and hCG is often used as an analog of LH to boost gonadotropin action. There are many physiological and pathological conditions where LH/hCG levels and actions are elevated. In order to understand how elevated LH/hCG levels may impact on the hypothalamic-pituitary-gonadal axis we have developed a transgenic mouse model with chronic hCG hypersecretion. Female mice develop many gonadal and extragonadal phenotypes including obesity, infertility, hyperprolactinemia, and pituitary and mammary gland tumors. This article summarizes recent findings on the mechanisms involved in pituitary gland tumorigenesis and hyperprolactinemia in the female mice hypersecreting hCG, in particular the relationship of progesterone with the hyperprolactinemic condition of the model. In addition, we describe the role of hyperprolactinemia as the main cause of infertility and the phenotypic abnormalities in these mice, and the use of dopamine agonists bromocriptine and cabergoline to normalize these conditions.
[Show abstract][Hide abstract] ABSTRACT: G protein-coupled receptors (GPCRs) transduce signals from a wide variety of extracellular stimuli such as ions, photons, odors, tastants, hormones, and neurotransmitters and hence are central players in communication between the cells of multicellular organisms and their environment. Characterization of these receptors at the molecular level has shown that GPCRs form dimers or oligomers (for simplicity, herein called dimers), yet their function is just beginning to emerge. The activation of GPCRs as dimers presents the opportunity for trans-activation of receptors, where a ligand-bound GPCR can change the activity of a neighboring GPCR. Trans-activation of GPCRs can take place either via transmembrane domains (TMDs) of two or more receptors or through a ligand-bound extracellular domain (ECD) of one receptor to the TMD of another (referred to as intermolecular cooperation), for GPCRs with a distinct ligand-binding ECD such as the glycoprotein hormone receptors. Before explaining the phenomenon of intermolecular cooperation between domains of GPCR dimers, this chapter first discusses the molecular interactions between GPCR dimers, which leads to trans-activation, and then explains various experimental strategies employed to study the significance of intermolecular cooperation either in rescuing the signaling of mutant GPCRs by complementary receptors or in changing the allosteric properties of GPCR dimers. The physiological significance of intermolecular cooperation is also discussed in detail, as well as its clinical significance that lies in the use of agents that selectively activate or inhibit particular signaling pathways or affect GPCR dimer interactions.
01/2014: chapter 13: pages 239-255; Humana Press., ISBN: 9781627037785
[Show abstract][Hide abstract] ABSTRACT: Constitutively active mutants (CAMs) of gonadotropin receptors are, in general, rare conditions. Luteinizing hormone-choriogonadotropin receptor (LHCGR) CAMs provoke the dramatic phenotype of familial gonadotropin-independent isosexual male-limited precocious puberty, whereas in females, there is not yet any identified phenotype. Only one isolated follicle-stimulating hormone receptor (FSHR) CAM (Asp567Gly) has so far been detected in a single male patient, besides other FSHR weak CAMs linked to pregnancy-associated ovarian hyperstimulation syndrome or to impaired desensitization and internalization. Several animal models have been developed for studying enhanced gonadotropin action; in addition to unraveling valuable new information about the possible phenotypes of isolated FSHR and LHCGR CAMs in women, the information obtained from these mouse models has served multiple translational goals, including the development of new diagnostic and therapeutic targets as well as the prediction of phenotypes for mutations not yet identified in humans. Mutagenesis and computational studies have shed important information on the physiopathogenic mechanisms leading to constitutive activity of gonadotropin receptors; a common feature in these receptor CAMs is the release of stabilizing interhelical interactions between transmembrane domains (TMDs) 3 and 6 leading to an increase, with respect to the wild-type receptor, in the solvent accessibility at the cytosolic extension of TMDs 3, 5, and 6, which involves the highly conserved Glu/Asp-Arg-Tyr/Trp sequence. In this chapter, we summarize the structural features, functional consequences, and mechanisms that lead to constitutive activation of gonadotropin receptor CAMs and provide information on pharmacological approaches that might potentially modulate gonadotropin receptor CAM function.
Advances in pharmacology (San Diego, Calif.) 01/2014; 70:37-80.
[Show abstract][Hide abstract] ABSTRACT: We previously reported that in male patients consulting for sexual dysfunction, low prolactin (PRL) levels were associated with metabolic syndrome (MetS), arteriogenic erectile dysfunction, and incident major cardiovascular events.
The aim of this study is to assess the clinical associations of PRL levels in the European Male Ageing Study (EMAS).
EMAS is a prospective, observational cohort of community-dwelling men aged 40-79 years old (mean age 60 ± 11 years old). PRL was available for 2,948 men.
Different parameters were evaluated including the Short Form-36 questionnaire, Becks Depression Inventory, the Adverse Life Events Scale, the Physical Activity Scale for the Elderly, and the EMAS sexual function questionnaire (EMAS-SFQ).
After the adjustment for confounders, PRL levels were inversely related with worsening of sexual function as compared with the previous year, as derived from change in sexual functioning domain of the EMAS-SFQ (adj. r = -0.043; P = 0.029). The strongest correlation (Wald = 6.840; P = 0.009) was observed between lower PRL levels and reduced enjoyment of orgasmic experiences. Furthermore, an inverse relationship between PRL levels and stressful life events or depressive symptoms was observed. Low PRL was also negatively associated with an unhealthy metabolic phenotype as well as with the MetS (Wald = 5.229; P = 0.022). In line with these data, low PRL was associated with a lower level of physical activity and feeling unhealthier.
Low PRL is related to several metabolic, psychological, and sexual unhealthy characteristics in European men. Checking PRL might be useful to stratify men for cardiovascular risk and to encourage appropriate lifestyle changes.
Journal of Sexual Medicine 01/2014; 11:240. · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: L’andropause ou hypogonadisme de survenue tardive est une situation dans laquelle un homme d’âge moyen ou plus âgé présente des taux de testostéronémie bas en conjunction avec une symptomatologie évocatrice d’un authentique hypogonadisme masculin (e.g. diminution de la fonction sexuelle, perte de vitalité, faiblesse musculaire, dépression). Les avis concernant les critères diagnostiques, la prévalence et les stratégies thérapeutiques de l’andropause varient considérablement parmi les experts. Nous revoyons ici les points les plus marquants de la prévalence, des critères diagnostiques et du retentissement sur la santé de l’andropause à partir des données de l’étude européenne des hommes vieillissants (EMAS), étude multi-centrique sur le vieillissement dans une population masculine européenne d’âge moyen ou plus âgée.
[Show abstract][Hide abstract] ABSTRACT: Background: vitamin D deficiency has been associated with an increased risk of mortality, but whether this relationship is causal or linked to co-existent comorbidity and adverse life factors remains uncertain. Our objective was to determine whether endogenous 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (1,25(OH)2D) and parathyroid hormone (PTH) levels predicted all-cause, cardiovascular and cancer mortality independently of health and lifestyle factors.Setting: prospective cohort analysis within the European Male Ageing Study.Participants: 2,816 community-dwelling men aged 40-79 years at baseline.Methods: Cox regression was used to examine the association of all-cause mortality with 25(OH)D, 1,25(OH)2D and PTH; cardiovascular and cancer mortality were modelled using competing-risks regression. Results were expressed as hazard ratios (HR) and 95% confidence intervals (CIs) for Cox models; sub-hazard ratios (SHR) and 95% CIs for competing-risks models.Results: a total of 187 men died during a median of 4.3 years of follow-up. Serum levels of 25(OH)D (per 1 SD decrease: HR = 1.45; 95% CI = 1.16, 1.81) and 1,25(OH)2D (per 1 SD decrease: HR = 1.20; 95% CI = 1.00, 1.44) were associated with an increased risk of all-cause mortality after adjusting for age, centre, smoking, self-reported morbidities, physical activity and functional performance. Only levels of 25(OH)D <25 nmol/l predicted cancer mortality (SHR = 3.33; 95% CI = 1.38, 8.04).Conclusion: lower 25(OH)D and 1,25(OH)2D levels independently predicted all-cause mortality in middle-aged and older European men. Associations with cancer mortality were only observed among men with very low levels of 25(OH)D. These associations were only partially explained by the range of adverse health and lifestyle factors measured here.