A new LC-MS/MS method for simultaneous and quantitative detection of bisphenol-a and steroids in target tissues: A power tool to characterize the interference of bisphenol-a exposure on steroid levels

Bisphenol A (BPA), an endocrine disruptor, may affect in situ steroidogenesis and alter steroids levels. The present work proposes a liquid chromatography tandem mass spectrometry method to simultaneously quantify BPA, 17β-Estradiol and testosterone in two target tissues: testis and visceral fat mass. Analytes were isolated and lipophilic impurities removed by two serial steps: liquid-liquid and solid phase extraction. All compounds were separated in a single gradient run by Kinetex F5 column and detected via multiple reaction monitoring using a triple quadrupole with a TurboIon electrospray source in both negative and positive modes. The method is selective and very sensitive. In the investigated concentration range, the linearity of the detector response is verified in both tissues. The use of specific SPE cartridges for affinity chromatography purification allows obtaining high percentages of process efficiency (68.0-83.3% for testicular tissue; 63.7-70.7% for visceral fat mass). Good repeatability and reproducibility was observed. The validated method can be efficiently applied for direct biological monitoring in testis and visceral fat mass from mice exposed to BPA. The quantification of compounds in a single assay could be achieved without a loss of sensitivity

Fetal-Perinatal Exposure to Bisphenol-A Affects Quality of Spermatozoa in Adulthood Mouse

Bisphenol-A (BPA) is considered an endocrine disruptor with estrogenic activity. It is described as an environment-polluting industrial chemical whose adverse effects on the male reproductive system depend on the period of exposure (i.e., fetal, prepubertal, or adult life). We exposed male mice to BPA during the fetal-perinatal period (from 10 days post coitum to 31 days post partum) and investigated the impact of this early-life exposure on gamete health in adulthood animals at 78 days of age. Both in control and BPA-exposed mice, viability and motility of spermatozoa, as well as sperm motility acquisition and chromatin condensation of spermatozoa, have been evaluated. Results reveal harmful effect of BPA on viability and motility of sperm cells as well as on chromatin condensation status during epididymal maturation of spermatozoa. In particular, BPA exposure interferes with biochemical mechanism useful to stabilize sperm chromatin condensation, as it interferes with oxidation of thiol groups associated to chromatin

The cannabinoid receptor cb1 stabilizes sperm chromatin condensation status during epididymal transit by promoting disulphide bond formation

The cannabinoid receptor CB1 regulates differentiation of spermatids. We recently characterized spermatozoa from caput epididymis of CB1-knock-out mice and identified a considerable number of sperm cells with chromatin abnormality such as elevated histone content and poorly condensed chromatin. In this paper, we extended our findings and studied the role of CB1 in the epididymal phase of chromatin condensation of spermatozoa by analysis of spermatozoa from caput and cauda epididymis of wild-type and CB1-knock-out mouse in both a homozygous or heterozygous condition. Furthermore, we studied the impact of CB1-gene deletion on histone displacement mechanism by taking into account the hyperacetylation of histone H4 and players of displacement such as Chromodomain Y Like protein (CDYL) and Bromodomain testis-specific protein (BRDT). Our results show that CB1, via local and/or endocrine cell-to-cell signaling, modulates chromatin remodeling mechanisms that orchestrate a nuclear condensation extent of mature spermatozoa. We show that CB1-gene deletion affects the epididymal phase of chromatin condensation by interfering with inter-/intra-protamine disulphide bridges formation, and deranges the efficiency of histone removal by reducing the hyper-acetylation of histone H4. This effect is independent by gene expression of Cdyl and Brdt mRNA. Our results reveal a novel and important role for CB1 in sperm chromatin condensation mechanisms

Characterization of estrogenic activity and site‐specific accumulation of bisphenol‐a in epididymal fat pad: Interfering effects on the endocannabinoid system and temporal progression of germ cells

The objective of this work has been to characterize the estrogenic activity of bisphenol‐A (BPA) and the adverse effects on the endocannabinoid system (ECS) in modulating germ cell progression. Male offspring exposed to BPA during the foetal‐perinatal period at doses below the no‐observed‐adverse‐effect‐level were used to investigate the exposure effects in adulthood. Results showed that BPA accumulates specifically in epididymal fat rather than in abdominal fat and targets testicular expression of 3β‐hydroxysteroid dehydrogenase and cytochrome P450 aromatase, thus promoting sustained increase of estrogens and a decrease of testosterone. The exposure to BPA affects the expression levels of some ECS components, namely type‐1 (CB1) and type‐2 cannabinoid (CB2) receptor and monoacylglycerol‐lipase (MAGL). Furthermore, it affects the temporal progression of germ cells reported to be responsive to ECS and promotes epithelial germ cell exfoliation. In particular, it increases the germ cell content (i.e., spermatogonia while reducing spermatocytes and spermatids), accelerates progression of spermatocytes and spermatids, promotes epithelial detachment of round and condensed spermatids and interferes with expression of cell–cell junction genes (i.e., zonula occcludens protein‐1, vimentin and β‐catenin). Altogether, our study provides evidence that early exposure to BPA produces in adulthood sustained and site‐specific BPA accumulation in epididymal fat, becoming a risk factor for the reproductive endocrine pathways associated to ECS

KISS1R and ANKRD31 Cooperate to Enhance Leydig Cell Gene Expression via the Cytoskeletal-Nucleoskeletal Pathway

Kisspeptins are involved in the regulation of hypothalamic-pituitary-gonadal axis, Leydig cell functions, and testosterone secretion, acting as endogenous ligands of the KISS1 receptor. ANKRD31 protein participates in male fertility, regulating meiotic progression, and epididymal sperm maturation. Here, we show that in Leydig cells, KISS1 receptor and ANKRD31 proteins physically interact; the formation of this protein complex is enhanced by Kisspeptin-10 that also modulates F-actin synthesis, favoring histone acetylation in chromatin and gene expression via the cytoskeletal–nucleoskeletal pathway. Kp/KISS1R system deregulation, expression impairment of cytoskeletal–nucleoskeletal mediators, Leydig gene targets, and the decreased testosterone secretion in Ankrd31−/− testis strongly supported our hypothesis. Furthermore, cytochalasin D treatment subverted the gene expression induction dependent on Kisspeptin-10 action. In conclusion, the current work highlights a novel role for the Kisspeptin-10 in the induction of the cytoskeletal–nucleoskeletal route, downstream a physical interaction between KISS1 receptor and ANKRD31, with gene expression activation as final effect, in Leydig cells

Collapse risk and residual drift performance of steel buildings using post-tensioned MRFs and viscous dampers in near-fault regions

The potential of post-tensioned self-centering moment-resisting frames (SC-MRFs) and viscous dampers to reduce the collapse risk and improve the residual drift performance of steel buildings in near-fault regions is evaluated. For this purpose, a prototype steel building is designed using different seismic-resistant frames, i.e.: moment-resisting frames (MRFs); MRFs with viscous dampers; SC-MRFs; and SC-MRFs with viscous dampers. The frames are modeled in OpenSees where material and geometrical nonlinearities are taken into account as well as stiffness and strength deterioration. A database of 91 near-fault, pulse-like ground motions with varying pulse periods is used to conduct incremental dynamic analysis (IDA), in which each ground motion is scaled until collapse occurs. The probability of collapse and the probability of exceeding different residual story drift threshold values are calculated as a function of the ground motion intensity and the period of the velocity pulse. The results of IDA are then combined with probabilistic seismic hazard analysis models that account for near-fault directivity to assess and compare the collapse risk and the residual drift performance of the frames. The paper highlights the benefit of combining the post-tensioning and supplemental viscous damping technologies in the near-source. In particular, the SC-MRF with viscous dampers is found to achieve significant reductions in collapse risk and probability of exceedance of residual story drift threshold values compared to the MRF. © 2016 Springer Science+Business Media Dordrech

Cytochalasin D restores nuclear size acting on F-actin and IZUMO1 localization in low-quality spermatozoa

In spermatozoa, the nuclear F-actin supports the acroplaxome, a subacrosomal structure involved in the correct exposure of several acrosomal membrane proteins; among them, the glycoprotein IZUMO1 is the major protein involved in sperm-oocyte fusion. Nuclear F-actin is also involved in sperm head shaping and chromosome compartmentalization. To date, few notions regarding the bivalent role of F-actin on sperm chromatin organization and IZUMO1 positioning have been reported. In our work, we characterized subcellular organization of F-actin in human high- and low-quality spermatozoa (A- and B-SPZ), respectively, showing that F-actin over-expression in sperm head of B-SPZ affected IZUMO1 localization. A correct IZUMO1 repositioning following in vitro induction of F-actin depolymerization, by cytochalasin D treatment, occurred. Interestingly, F-actin depolymerization was also associated with a correct acrosome repositioning, thus to favor a proper acrosome reaction onset, with changes in sperm nuclear size parameters and histone acetylation rate reaching high-quality conditions. In conclusion, the current work shows a key role of F-actin in the control of IZUMO1 localization as well as chromatin remodeling and acetylation events

Estrogens and Spermiogenesis: New Insights from Type 1 Cannabinoid Receptor Knockout Mice.

Spermatogenesis is a complex mechanism which allows the production of male gametes; it consists of mitotic, meiotic, and differentiation phases. Spermiogenesis is the terminal differentiation process during which haploid round spermatids undergo several biochemical and morphological changes, including extensive remodelling of chromatin and nuclear shape. Spermiogenesis is under control of endocrine, paracrine, and autocrine factors, like gonadotropins and testosterone.More recently, emerging pieces of evidence are suggesting that, among these factors, estrogens may have a role. To date, this is a matter of debate and concern because of the agonistic and antagonistic estrogenic effects that environmental chemicals may have on animal and human with damaging outcome on fertility. In this review, we summarize data which fuel this debate, with a particular attention to our recent results, obtained using type 1 cannabinoid receptor knockout male mice as animal model