Andrea Di Nisio

Multivariate selection drives concordant patterns of pre- and postcopulatory sexual selection in a livebearing fish.

In many species, females mate with multiple partners, meaning that sexual selection on male traits operates across a spectrum that encompasses the competition for mates (that is, before mating) and fertilizations (after mating). Despite being inextricably linked, pre- and postcopulatory sexual selection are typically studied independently, and we know almost nothing about how sexual selection operates across this divide. Here we bridge this knowledge gap using the livebearing fish Poecilia reticulata. We show that both selective episodes, as well as their covariance, explain a significant component of variance in male reproductive fitness. Moreover, linear and nonlinear selection simultaneously act on pre- and postcopulatory traits, and interact to generate multiple phenotypes with similar fitness.

Mutational screening of NR5A1 gene encoding steroidogenic factor 1 in cryptorchidism and male factor infertility and functional analysis of seven undescribed mutations

OBJECTIVE To study the role of NR5A1 in cryptorchidism and male factor infertility. Mutations in NR5A1 have been initially associated with primary adrenal insufficiency and 46,XY gonadal dysgenesis and more recently with less severe phenotypes, including preliminary descriptions in severe forms of male factor infertility. Far less clear is the possible involvement of NR5A1 mutations in cryptorchidism. DESIGN Retrospective cross-sectional cohort study and functional analysis of mutant proteins. SETTING University department. PATIENT(S) Nine hundred fifty-nine subjects, including children with cryptorchidism and adults with different semen phenotypes associated or not associated with a history of cryptorchidism. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Mutation screening of NR5A1 by sequencing all exons. Functional analysis of mutant proteins by transactivation assays of CYP11A1 and CYP17A1 promoters. RESULT(S) We identified seven undescribed and one previously described missense mutation in subjects with severe spermatogenic impairment, without (4/236, 1.7%) and with (3/85, 3.5%) a history of cryptorchidism. Newborns with cryptorchidism carry NR5A1 mutations at low frequency (0.7%), whereas no mutations were found in milder forms of infertility and normozoospermia, irrespective of the presence of cryptorchidism. The mutant proteins showed impaired transactivation of gonadal promoters. A single nucleotide polymorphism (rs1110061; c.437 G→C; p.Gly146Ala) was also associated with more severe forms of spermatogenic impairment with cryptorchidism. CONCLUSION(S) This study, combined with what is already known about NR5A1-associated phenotypes, suggests considering mutations in this gene as a novel genetic cause of more severe forms of male factor infertility, especially when associated with a history of cryptorchidism.

Polymorphism rs2274911 of GPRC6A as a Novel Risk Factor for Testis Failure

CONTEXT The G protein-coupled receptor GPRC6A is an emerging effector with multiple endocrine roles, including stimulation of T production from the testis. Recently, two men with an inactivating mutation (F464Y) of GPRC6A have been identified, and they showed primary testicular failure and deranged spermatogenesis. Furthermore, one of them also reported cryptorchidism at birth. In addition, a polymorphism (rs2274911, Pro91Ser) in GPRC6A is associated with prostate cancer, a typical androgen-sensitive cancer. OBJECTIVE To study the possible association between rs2274911 polymorphism and male fertility and/or cryptorchidism. Design, Patients, Settings: A total of 611 subjects, including 343 infertile patients, 197 normozoospermic controls, and 71 cryptorchid newborns, were retrospectively selected. METHODS Sequencing analysis for rs2274911 polymorphism and F464Y mutation, and serum levels of FSH, LH, and T were assessed. In vitro functional studies for rs2274911 and F464Y were also performed. RESULTS Homozygous subjects for the risk allele A of rs2274911 had a 4.60-fold increased risk of oligozoospermia and 3.52-fold increased risk of cryptorchidism. A significant trend for increased levels of LH in the GA and AA genotypes, compared with GG homozygotes, was detected in men with azoospermia/cryptozoospermia (P for trend = .027), further supporting an association with primary testicular failure. The mutation F464Y was found in one cryptorchid child (one in 71; 1.41%). Functional studies showed that the A allele of rs2274911 and the F464Y substitution were associated with lower exposition of the receptor on the cell membrane and a reduced downstream phosphorylation of ERK1/2 with respect to wild type. CONCLUSION Our results suggest that GPRC6A inactivation or sub-function contributes to reduced exposure to androgens, leading to cryptorchidism during fetal life and/or low sperm production in adulthood.

The rs2274911 polymorphism in GPRC6A gene is associated with insulin resistance in normal weight and obese subjects

Identification of the novel endocrine role of osteocalcin (OC) and its receptor GPRC6A has given rise to a new branch of research in OC/GPRC6A axis related to glucose metabolism. GPRC6A‐ and OC‐deficient mice share features of the metabolic syndrome, in addition to male infertility. Recently, the polymorphism rs2274911 in GPRC6A was shown to be associated with testicular impairment. We aimed to investigate the role of rs2274911 polymorphism in glucose and lipid metabolism in a cohort of normal weight and obese subjects

Regulation of Sclerostin Production in Human Male Osteocytes by Androgens: Experimental and Clinical Evidence.

In this study we aimed to elucidate a possible role of T in the regulation of sclerostin, a glycoprotein secreted by osteocytes known to regulate bone mass. To this end, we evaluated the effect of T stimulation on sclerostin production and gene expression in human cultured osteocytes. In addition, we evaluated serum sclerostin levels in a cohort of 20 hypogonadal male patients, compared with 20 age-matched eugonadal controls. Stimulation with DHT decreased sclerostin expression in cultured osteocytes in a time- and dose-dependent manner. Confirming a direct androgen receptor-mediated effect on sclerostin production, flutamide coincubation and silencing of androgen receptor gene in osteocytes abolished the DHT effects. In addition, hypogonadal patients showed higher serum sclerostin levels with respect to controls (145.87 ± 50.83 pg/mL vs 84.02 ± 32.15 pg/mL; P < .001) and in both probands and controls, serum T levels were negatively correlated with sclerostin (R = -0.664, P = 0.007, and R = -0.447, P = .045, respectively). Finally, multiple stepwise regression analysis showed that T represented the only independent predictor of sclerostin levels. In conclusion, by showing a direct correlation between T and sclerostin, both in vivo and in vitro, this study adds further support to the emerging clinical and experimental studies focusing on sclerostin as a therapeutic target for osteoporosis treatment.

FSH treatment in infertile males candidate to assisted reproduction improved sperm DNA fragmentation and pregnancy rate.

The purpose of this study is to evaluate whether follicle-stimulating hormone treatment improves sperm DNA parameters and pregnancy outcome in infertile male candidates to in-vitro fertilization.Observational study in 166 infertile male partners of couples undergoing in-vitro fertilization. Eighty-four patients were receiving follicle-stimulating hormone treatment (cases) and 82 refused treatment (controls). Semen parameters, sexual hormones, and sperm nucleus (fluorescence in-situ hybridization, acridine orange, TUNEL, and γH2AX) were evaluated at baseline (T0) and after 3 months (T1), when all subjects underwent assisted reproduction techniques. Statistical analysis was performed by analysis of variance.Compared to baseline, cases showed significant improvements in seminal parameters and DNA fragmentation indexes after follicle-stimulating hormone therapy (all P < 0.05), whereas no changes were observed in controls. Within cases, follicle-stimulating hormone treatment allowed to perform intrauterine insemination in 35 patients with a pregnancy rate of 23.2 %. Intracytoplasmic sperm injection was performed in all controls and in 49 patients from cases, with pregnancy rates of 23.2 and 40.8 %, respectively (P < 0.05). After 3 months (T0 vs. T1) of follicle-stimulating hormone therapy, cases with positive outcome had reduced DNA fragmentation index and lower double strand breaks (P < 0.05 and P < 0.001 vs. negative outcome, respectively).In this observational study, we showed that follicle-stimulating hormone treatment improves sperm DNA fragmentation, which in turn leads to increased pregnancy rates in infertile males undergoing in-vitro fertilization. In particular, double strand breaks (measured with γH2AX test) emerged as the most sensible parameter to follicle-stimulating hormone treatment in predicting reproductive outcome.

Heat Sensing Receptor TRPV1 Is a Mediator of Thermotaxis in Human Spermatozoa

The molecular bases of sperm thermotaxis, the temperature-oriented cell motility, are currently under investigation. Thermal perception relies on a subclass of the transient receptor potential [TRP] channels, whose member TRPV1 is acknowledged as the heat sensing receptor. Here we investigated the involvement of TRPV1 in human sperm thermotaxis. We obtained semen samples from 16 normozoospermic subjects attending an infertility survey programme, testis biopsies from 6 patients with testicular germ cell cancer and testis fine needle aspirates from 6 patients with obstructive azoospermia undergoing assisted reproductive technologies. Expression of TRPV1 mRNA was assessed by RT-PCR. Protein expression of TRPV1 was determined by western blot, flow cytometry and immunofluorescence. Sperm motility was assessed by Sperm Class Analyser. Acrosome reaction, apoptosis and intracellular-Ca2+ content were assessed by flow cytometry. We found that TRPV1 mRNA and protein were highly expressed in the testis, in both Sertoli cells and germ-line cells. Moreover, compared to no-gradient controls at 31°C or 37°C (Ctrl 31°C and Ctrl 37°C respectively), sperm migration towards a temperature gradient of 31–37°C (T gradient) in non-capacitated conditions selected a higher number of cells (14,9 ± 4,2×106 cells T gradient vs 5,1± 0,3×106 cells Ctrl 31°C and 5,71±0,74×106 cells Ctrl 37°C; P = 0,039). Capacitation amplified the migrating capability towards the T gradient. Sperms migrated towards the T gradient showed enriched levels of both TRPV1 protein and mRNA. In addition, sperm cells were able to migrate toward a gradient of capsaicin, a specific agonist of TRPV1, whilst capsazepine, a specific agonist of TRPV1, blocked this effect. Finally, capsazepine severely blunted migration towards T gradient without abolishing. These results suggest that TRPV1 may represent a facilitating mediator of sperm thermotaxis.

Directional postcopulatory sexual selection is associated with female sperm storage in Trinidadian guppies

Female sperm storage (FSS) is taxonomically widespread and often associated with intense sperm competition, yet its consequences on postcopulatory sexual selection (PCSS) are poorly known. Theory predicts that FSS will reduce the strength of PCSS, because sperm characteristics favored before and after FSS may be traded‐off, and opportunities for nondirectional PCSS should increase. We explored these questions in the guppy (Poecilia reticulata), by allowing females to mate multiply and by comparing the paternity pattern in two successive broods. Contrary to predictions, the variance in male fertilization success increased after FSS, driven by a change in male paternity share across broods. This change was positively associated with sperm velocity (measured before FSS) but not with the duration of FSS, indirectly suggesting that faster sperm were better in entering female storage organs, rather than in persisting within them. Other male traits, such as male size and orange color, heterozygosity, and relatedness to the female, did not influence paternity after FSS. These results indicate that processes associated with FSS tend to reinforce the strength of PCSS in guppies, rather than weaken it. Further work is necessary to test whether this pattern changes in case of more prolonged FSS.

HPV Prophylactic Vaccination in Males Improves the Clearance of Semen Infection

BACKGROUND Semen represents a reservoir for human papillomavirus (HPV), rising concern in couples eligible for assisted reproduction techniques (ART). Humoral immunity against HPV is considered to protect from reinfection. We investigated the impact of vaccination on virus clearance in a cohort of infertile male patients showing HPV semen infection. METHODS 179 out of 619 infertile patients, showing HPV-DNA detection in semen by FISH analysis,were enrolled. Subjectswere split into 91 vaccine-sensitive (VSPs) and 88 nonvaccine-sensitive patients (NVSPs) by INNO-LiPA. 19 VSPs showed vaccine-type specific seroconvesion at recruitment. All patients underwent specific counselling. 42 seronegative VSPs were randomly assigned to receive quadrivalent vaccination in 6 months, whilst 49 VSPs, 19 seroconverted and 30 seronegative, served as controls. The prevalence of HPV-DNA semen infection and serology was studied in a follow-up of 24 months. RESULTS Compared to seronegative patients, VSP seroconverted at recruitment showed absence of multiple infections and reduced prevalence of HPV semen infection at 12 (P = 0.039), 18 (P = 0.034) and 24 months (P = 0.034) of follow-up. Vaccinated VSP showed improved healing (P = 0.001 at 6 months and P b 0.001 at 12 months vs seroconverted VSP), achieving clearance in 12 months. DISCUSSION Humoral immunity has a major role in healing from HPV infection. Elder ART patients with HPV semen infection may benefit by the union of both specific counselling and available prophylactic vaccination.

Impaired Release of Vitamin D in Dysfunctional Adipose Tissue: New Cues on Vitamin D Supplementation in Obesity

Context Vitamin D accumulates in adipose tissue (AT), and vitamin D deficiency is frequent in obesity. Objective We hypothesize that trafficking of vitamin D is altered in dysfunctional AT. Design, Patients, Settings Fifty-four normal-weight and 67 obese males were recruited in a prospective study and randomly assigned to supplementation with 50 µg/wk 25-hydroxyvitamin-D3 or 150 µg/wk vitamin D3 for 1 year, raising dosage by 50% if vitamin D sufficiency [serum 25-hydroxyvitamin-D3 >50 nmol/L], was not achieved at 6 months; 97 subjects completed the study. Methods Vitamin D3 and 25-hydroxyvitamin-D3 were quantified by HPLC-MS in control and insulin-resistant (IR) 3T3-L1 cells and subcutaneous AT (SAT) from lean and obese subjects, incubated with or without adrenaline; expression of 25-hydroxylase (Cyp27a1), 1α-hydroxylase (Cyp27b1), and vitamin D receptor (Vdr) was analyzed by real-time polymerase chain reaction. Results In IR adipocytes, uptake of D3 and 25-hydroxyvitamin-D3 was higher, but, after adrenaline stimulation, the decrement in D3 and 25-hydroxyvitamin-D3 was stronger in control cells, which also showed increased expression of Cyp27a1 and Cyp27b1 and higher levels of 25-hydroxyvitamin-D3. In SAT from obese subjects, adrenaline-induced release of D3 and 25-hydroxyvitamin-D3 was blunted; in both IR cells and obese SAT, protein expression of β2-adrenergic receptor was reduced. Supplementation with 25-hydroxyvitamin-D3 was more effective in achieving vitamin D sufficiency in obese, but not in normal weight subjects. Conclusion Dysfunctional AT shows a reduced catecholamine-induced release of D3 and 25-hydroxyvitamin-D3 and altered activity of vitamin D-metabolizing enzymes; for these reasons supplementation with 25-hydroxyvitamin-D3 is more effective in obese individuals.