Metabolism of renin-angiotensin and enkephalin in human follicular fluid: An experimental study.

Background: The relationship between the biochemical characteristics of follicular fluid (FF), oocyte quality and embryonic development has not yet been elucidated. We compared samples of FF with a normal metabolic profile against samples with metabolic abnormalities to identify potential predictive biomarkers of reproductive success. Objective: To analyze peptide activity in the FF of women undergoing in vitro fertilization using 3 samples of FF per individual. Materials and Methods: FF samples were obtained by ovum pick-up. Pathological samples were defined as samples of FF obtained from women with a gynecological condition or with infertility. A total of 30 women participated in this study. 3 samples of FF were obtained per individual (90 samples), but 8 samples were excluded because they were hemolyzed. The samples (n = 82 FF) included controls (n = 36, donors without fertility problems), women with endometriosis (n = 15), unexplained infertility (n = 19), and aged > 39 (n = 12). We assessed local encephalinergics: aminopeptidase-N (puromycin sensitive aminopeptidase and neutral endopeptidase; and components of the angiotensin system of the reproductive tract: prolyl-endopeptidase, APN, aspartate-aminopeptidase, and basic-aminopeptidase. Results: No differences were observed in peptide metabolism based on the presence or absence of oocytes in the FF. Women with endometriosis and aged > 39 yr showed alterations in puromycin sensitive aminopeptidase (p = 0.01), aminopeptidase-B (p = 0.01), aspartate-aminopeptidase (p < 0.001) and neutral endopeptidase (p < 0.001). Conclusion: This study reveals alterations in the metabolism of enkephalin and angiotensin in pathological FF, which points to these components as potential diagnostic biomarkers.This work was supported by grants from the University of the Basque Country (UPV/EHU GIU 17/19) and the Gangoiti Barrera Foundation, Spain (Basque Country)

Patterns of Change in Dietary Habits and Physical Activity during Lockdown in Spain Due to the COVID-19 Pandemic

Background: Lockdown due to COVID-19 influenced food habits and lifestyles with potential negative health impact. This study aims to identify patterns of change in eating habits and physical activity during COVID-19 lockdown in Spain and to identify associations with sociodemographic factors and usual habits. Methods: This cross-sectional study included 1155 adults recruited online to answer a 10-section questionnaire. The protocol assessed usual diet by means of a semi-quantitative food frequency questionnaire, usual physical activity (PA) and supplement use, dietary changes, sedentary time, PA, exposure to sunlight, sleep quality, and smoking during confinement. Patterns of dietary change were identified by factor analysis. Factor scores were included in cluster analysis together with change in PA. Results: Six patterns of dietary change were identified that together with PA changes during lockdown defined three clusters of lifestyle change: a cluster less active, a more active cluster, and a third cluster as active as usual. People who were usually less active were more likely to be classified in the cluster that increased physical activity in confinement. Scores of the Healthy Mediterranean-Style dietary pattern were higher in this group. Conclusions: Different patterns of change in lifestyles in confinement suggest the need to tailor support and advice to different population groups.This research received no external funding. J.A.T. was funded by Instituto de Salud Carlos III through the Fondo de Investigacion para la Salud (CIBEROBN CB12/03/30038) which was co-funded by the European Regional Development Fund

Local renin angiotensin system and sperm DNA fragmentation.

[EN] The renin angiotensin system (RAS) appears to influence male fertility at multiple levels. In this work, we analyzed the relationship between the RAS and DNA integrity. Fifty male volunteers were divided into two groups (25 each): control (DNA fragmentation ≤20%) and pathological (DNA fragmentation >20%) cases. Activities of five peptidases controlling RAS were measured fluorometrically: prolyl endopeptidase (which converts angiotensin [A] I and A II to A 1-7), neutral endopeptidase (NEP/CD10: A I to A 1-7), aminopeptidase N (APN/CD13: A III to A IV), aminopeptidase A (A II to A III) and aminopeptidase B (A III to A IV). Angiotensin-converting enzyme (A I to A II), APN/CD13 and NEP/CD10 were also assessed by semiquantitative cytometry and quantitative flow cytometry assays, as were the receptors of all RAS components: A II receptor type 1 (AT1R), A II receptor type 2 (AT2R), A IV receptor (AT4R or insulin-regulated aminopeptidase [IRAP]), (pro)renin receptor (PRR) and A 1-7 receptor or Mas receptor (MasR) None of the enzymes that regulate levels of RAS components, except for APN/CD13 (decrease in fragmented cells), showed significant differences between both groups. Micrographs of RAS receptors revealed no significant differences in immunolabeling patterns between normozoospermic and fragmented cells. Labeling of AT1R (94.3% normozoospermic vs 84.1% fragmented), AT4R (96.2% vs 95.3%) and MasR (97.4% vs 87.2%) was similar between the groups. AT2R (87.4% normozoospermic vs 63.1% fragmented) and PRR (96.4% vs 48.2%) were higher in non-fragmented spermatozoa. These findings suggest that fragmented DNA spermatozoa have a lower capacity to respond to bioactive RAS peptides.This work was supported by grants from the University of the Basque Country (UPV/EHU GIU 17/19) and the Gangoiti Barrera Foundation (Basque Country)

Expression and Localization of Opioid Receptors in Male Germ Cells and the Implication for Mouse Spermatogenesis

The presence of endogenous opioid peptides in different testicular cell types has been extensively characterized and provides evidence for the participation of the opioid system in the regulation of testicular function. However, the exact role of the opioid system during the spermatogenesis has remained controversial since the presence of the mu-, delta-and kappa-opioid receptors in spermatogenic cells was yet to be demonstrated. Through a combination of quantitative real-time PCR, immunofluorescence, immunohistochemistry and flow cytometry approaches, we report for the first time the presence of active mu-, deltaand kappa-opioid receptors in mouse male germ cells. They show an exposition time-dependent response to opioid agonist, hence suggesting their active involvement in spermatogenesis. Our results contribute to understanding the role of the opioid receptors in the spermatogenesis and could help to develop new strategies to employ the opioid system as a biochemical tool for the diagnosis and treatment of male infertility.This work was supported by grants from The Basque Government, University of the Basque Country (UPV/EHU) and Merck Serono. HE was supported by a fellowship from the Gangoti Barrera Foundation; MG was supported by a fellowship from Basque Government (Zabalduz); IM was supported by a fellowship from Basque Government and IU was supported by a fellowship from University of Basque Country (UPV/EHU). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. This work was supported by grants from The Basque Government, University of the Basque Country (UPV/EHU) and Merck Serono. HE was supported by a fellowship from the Gangoti Barrera Foundation; MG was supported by a fellowship from Basque Government (Zabalduz); IM was supported by a fellowship from Basque Government and IU was supported by a fellowship from University of Basque Country (UPV/EHU). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials

Human Sperm Testicular Angiotensin Converting Enzyme Helps Determine Human Embryo Quality

Angiotensin-converting enzyme functions in the male reproductive system, but the extent of its function in reproduction is not fully understood. The primary objective of this work was to investigate the relationship between the testicular isoform of angiotensin-converting enzyme present in human spermatozoa and semen parameters, human embryo quality, and assisted reproduction success. A total of 81 semen samples and 635 embryos from couples undergoing oocyte donation cycles at the IVI Bilbao Clinic were analyzed. Semen parameters, embryos quality, and blastocyst development were examined according to the World Health Organization standards and the Spanish Association of Reproduction Biology Studies criteria. The percentage of testicular angiotensin-converting enzyme-positive spermatozoa and the number of molecules per spermatozoon were analyzed by flow cytometry. Both parameters were inversely correlated with human sperm motility. Higher percentages of testicular angiotensin-converting enzyme-positive spermatozoa together with fewer enzyme molecules per spermatozoon were positively correlated with better embryo quality and development. Our results suggest that embryos with a higher implantation potential come from semen samples with higher percentages of testicular angiotensin-converting enzyme-positive cells and fewer enzyme molecules per spermatozoon. Based on these findings, we propose that testicular angiotensin-converting enzyme could be used to aid embryologists in selecting better semen samples for obtaining high-quality blastocysts during in vitro fertilization procedures.The authors thank Alejandro Diez, Ph.D., technician of the Analytical and High-Resolution Microscopy and Cytometry Laboratory in Biomedicine Service of the University of Basque Country (UPV/EHU), for his technical assistance with samples processing by flow cytometry. This study was supported by grants from the Basque Government (GIC12/173; to MG and IMH) and University of the Basque Country (UPV/EHU) (EHUA14/17; to MG and IUA)

Altered glutamyl-aminopeptidase activity and expression in renal neoplasms

Background: Advances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. 3.4.11.7) is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development. Methods: In this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival. Results: GAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs. Conclusions: This study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms.We wish to thank Arantza Perez (UPV/EHU) for her technical contribution to this study. The authors also thank Ana Abascal, Alicia Esteve and Mar Gonzalez, lab technicians at the Department of Pathology, Cruces University Hospital, for their excellent immunohistochemical work. This work was supported by grants from the Basque Government (IT8-11/13), the University of the Basque Country UPV/EHU (UFI 11/44, EHUA 12/15) and the Gangoiti Barrera Foundation. We want to particularly acknowledge the patients enrolled in this study for their participation and the Basque Biobank for Research-OEHUN for this collaboration