Regulation of Male Fertility by the Renin-Angiotensin System

The renin-angiotensin system (RAS) is a peptidic system known mainly for its roles in the maintenance of blood pressure and electrolyte and fluid homeostasis. However, several tissues and cells have been described to possess an intrinsic RAS that acts locally through different paracrine and autocrine mechanisms. In the male reproductive system, several components of this system have been observed in various organs and tissues, such as the testes, spermatozoa and seminal fluid. Some functions attributed to this local RAS are maintenance of seminal plasma electrolytes, regulation of steroidogenesis and spermatogenesis, and sperm functions. However, their specific actions in these locations are not fully understood. Therefore, a deep knowledge of the functions of the RAS at both the testicular and seminal levels could clarify its roles in male infertility and sperm physiology, and the different RAS elements could be used to design tools enabling the diagnosis and/or treatment of male infertility.This study was supported by grants from the Basque Government (GIC12/173) and University of the Basque Country (UPV/EHU) (EHUA14/17)

(Pro)renin Receptor Is Present in Human Sperm and It Adversely Affects Sperm Fertility Ability

Sperm fertility ability may be modulated by different molecular systems, such as the renin-angiotensin system (RAS). Although renin is one of its most relevant peptides, the presence and role of the (pro)renin receptor (PRR) is completely unknown. We have proved for the first time the existence of PRR and its transcript in human sperm by western blot and RT-PCR. Immunofluorescence studies showed that this receptor is mainly located in the apical region over the acrosome and in the postacrosomal region of the sperm head and along the sperm tail. In addition, this prospective cohort study also proves that semen samples with higher percentages of PRR-positive spermatozoa are associated with poor sperm motility, worse blastocyst development and no-viable blastocysts. Our results provide insight into how PRR play a negative role in sperm physiology that it may condition human embryo quality and development. An in-depth understanding of the role of PRR in sperm fertility can help elucidate its role in male infertility, as well as establish biomarkers for the diagnosis or selection of sperm to use during assisted reproductive techniques.This research was funded by grants from the Basque Government (GIC12/173; to M.G. and I.M.-H.) and University of the Basque Country (UPV/EHU; to M.G. and I.U.-A.) (EHUA14/17)

SPANX-A/D protein subfamily plays a key role in nuclear organisation, metabolism and flagellar motility of human spermatozoa

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes encode a protein family (SPANX-A, -B, -C and -D), whose expression is limited to the testis and spermatozoa in normal tissues and to a wide variety of tumour cells. Present only in hominids, SPANX-A/D is exclusively expressed in post-meiotic spermatids and mature spermatozoa. However, the biological role of the protein family in human spermatozoa is largely unknown. Combining proteomics and molecular approaches, the present work describes the presence of all isoforms of SPANX-A/D in human spermatozoa and novel phosphorylation sites of this protein family. In addition, we identify 307 potential SPANX-A/D interactors related to nuclear envelop, chromatin organisation, metabolism and cilia movement. Specifically, SPANX-A/D interacts with fumarate hydratase and colocalises with both fumarate hydratase and Tektin 1 proteins, involved in meeting energy demands for sperm motility, and with nuclear pore complex nucleoporins. We provide insights into the molecular features of sperm physiology describing for the first time a multifunctional role of SPANX-A/D protein family in nuclear envelope, sperm movement and metabolism, considered key functions for human spermatozoa. SPANX-A/D family members, therefore, might be promising targets for sperm fertility management.Spanish Health Department ISCIII-DT, Basque Government and Danish Medical Research Council. IU-A was supported by a fellowship from the University of the Basque Country (UPV/EHU). IM-H was supported by a fellowship from the Basque Government. I.K. was supported by a grant from the Danish Medical Research Counci

Morphine leads to global genome changes in H3K27me3 levels via a Polycomb Repressive Complex 2 (PRC2) self-regulatory mechanism in mESCs

Background: Environmentally induced epigenetic changes can lead to health problems or disease, but the mechanisms involved remain unclear. Morphine can pass through the placental barrier leading to abnormal embryo development. However, the mechanism by which morphine causes these effects and how they sometimes persist into adulthood is not well known. To unravel the morphine-induced chromatin alterations involved in aberrant embryo development, we explored the role of the H3K27me3/PRC2 repressive complex in gene expression and its transmission across cellular generations in response to morphine. Results: Using mouse embryonic stem cells as a model system, we found that chronic morphine treatment induces a global downregulation of the histone modification H3K27me3. Conversely, ChIP-Seq showed a remarkable increase in H3K27me3 levels at specific genomic sites, particularly promoters, disrupting selective target genes related to embryo development, cell cycle and metabolism. Through a self-regulatory mechanism, morphine downregulated the transcription of PRC2 components responsible for H3K27me3 by enriching high H3K27me3 levels at the promoter region. Downregulation of PRC2 components persisted for at least 48 h (4 cell cycles) following morphine removal, though promoter H3K27me3 levels returned to control levels. Conclusions: Morphine induces targeting of the PRC2 complex to selected promoters, including those of PRC2 components, leading to characteristic changes in gene expression and a global reduction in H3K27me3. Following morphine removal, enhanced promoter H3K27me3 levels revert to normal sooner than global H3K27me3 or PRC2 component transcript levels. We suggest that H3K27me3 is involved in initiating morphine-induced changes in gene expression, but not in their maintenance.This study was supported by grants from the Spanish Health Department ISCIII (DTS 18/00142) and University of the Basque Country. IM was supported by fellowship from Basque Government, and MA and IU were supported by fellowship from the University of the Basque Country (UPV/EHU)

Lifestyle Patterns and Weight Status in Spanish Adults: The ANIBES Study

Limited knowledge is available on lifestyle patterns in Spanish adults. We investigated dietary patterns and possible meaningful clustering of physical activity, sedentary behavior, sleep time, and smoking in Spanish adults aged 18–64 years and their association with obesity. Analysis was based on a subsample (n = 1617) of the cross-sectional ANIBES study in Spain. We performed exploratory factor analysis and subsequent cluster analysis of dietary patterns, physical activity, sedentary behaviors, sleep time, and smoking. Logistic regression analysis was used to explore the association between the cluster solutions and obesity. Factor analysis identified four dietary patterns, “Traditional DP”, “Mediterranean DP”, “Snack DP” and “Dairy-sweet DP”. Dietary patterns, physical activity behaviors, sedentary behaviors, sleep time, and smoking in Spanish adults aggregated into three different clusters of lifestyle patterns: “Mixed diet-physically active-low sedentary lifestyle pattern”, “Not poor diet-low physical activity-low sedentary lifestyle pattern” and “Poor diet-low physical activity-sedentary lifestyle pattern”. A higher proportion of people aged 18–30 years was classified into the “Poor diet-low physical activity-sedentary lifestyle pattern”. The prevalence odds ratio for obesity in men in the “Mixed diet-physically active-low sedentary lifestyle pattern” was significantly lower compared to those in the “Poor diet-low physical activity-sedentary lifestyle pattern”. Those behavior patterns are helpful to identify specific issues in population subgroups and inform intervention strategies. The findings in this study underline the importance of designing and implementing interventions that address multiple health risk practices, considering lifestyle patterns and associated determinants

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

Estudio del Sistema Renina Angiotensina como posible biomarcador para la mejora de los tratamientos de fecundación in vitro

287 p.La infertilidad se ha convertido en un problema médico y social de gran importancia mundial, que afecta al 15% de las parejas en edad reproductiva. El desarrollo de las técnicas de reproducción asistida (TRA) supuso un gran avance en el tratamiento de la infertilidad, no obstante, hay ciertos aspectos de estos tratamientos que continúan sin resolverse. Además, cada vez hay más evidencias que demuestran que las características moleculares espermáticas influyen en la fecundación y en las primeras etapas del desarrollo embrionario. Actualmente, el análisis del semen constituye la única herramienta para evaluar el estatus fértil del varón, sin embargo, presenta una serie de limitaciones, sobre todo en los casos de infertilidad de origen desconocido. Por lo que, el uso de biomarcadores moleculares basados en la fisiología espermática podría complementar los análisis actuales mejorando el diagnóstico y tratamiento de la infertilidad masculina, o prediciendo los resultados de las técnicas de reproducción asistida. El sistema renina angiotensina (RAS) es un sistema molecular que actúa en la fertilidad masculina interviniendo en distintos niveles, como la regulación de la función espermática, lo que supondría que este sistema podría ser usado como diana terapéutica para la mejora de las TRA. A pesar de ello, todavía quedan muchas cuestiones por resolver en cuanto a la presencia y funciones que los distintos elementos del RAS ejercen en los espermatozoides. Esta tesis se ha centrado en describir los receptores de los dos principales péptidos bioactivos de este sistema, el receptor de angiotensina II tipo 2 (AT2R) y el receptor de prorrenina-renina (PRR), así como en estudiar la relación de estos dos receptores y de dos enzimas descritas como esenciales en la función espermática, la enzima convertidora de angiotensina testicular (tACE) y la aminopeptidasa N (APN), con los parámetros evaluados durante los tratamientos de fecundación in vitro. Así, se ha podido determinar que estos componentes se relacionan con los parámetros y etiologías seminales, las tasas de fecundación y la calidad, desarrollo y viabilidad de los embriones obtenidos durante los diferentes tratamientos de fecundación in vitro. Estos resultados confirman que las características moleculares espermáticas contribuyen a la fecundación y al desarrollo embrionario, de forma que estos receptores y enzimas podrían ser considerados biomarcadores de la función y selección espermática.Tesis realizada gracias al Programa Zabaldu

Estudio del Sistema Renina Angiotensina como posible biomarcador para la mejora de los tratamientos de fecundación in vitro

287 p.La infertilidad se ha convertido en un problema médico y social de gran importancia mundial, que afecta al 15% de las parejas en edad reproductiva. El desarrollo de las técnicas de reproducción asistida (TRA) supuso un gran avance en el tratamiento de la infertilidad, no obstante, hay ciertos aspectos de estos tratamientos que continúan sin resolverse. Además, cada vez hay más evidencias que demuestran que las características moleculares espermáticas influyen en la fecundación y en las primeras etapas del desarrollo embrionario. Actualmente, el análisis del semen constituye la única herramienta para evaluar el estatus fértil del varón, sin embargo, presenta una serie de limitaciones, sobre todo en los casos de infertilidad de origen desconocido. Por lo que, el uso de biomarcadores moleculares basados en la fisiología espermática podría complementar los análisis actuales mejorando el diagnóstico y tratamiento de la infertilidad masculina, o prediciendo los resultados de las técnicas de reproducción asistida. El sistema renina angiotensina (RAS) es un sistema molecular que actúa en la fertilidad masculina interviniendo en distintos niveles, como la regulación de la función espermática, lo que supondría que este sistema podría ser usado como diana terapéutica para la mejora de las TRA. A pesar de ello, todavía quedan muchas cuestiones por resolver en cuanto a la presencia y funciones que los distintos elementos del RAS ejercen en los espermatozoides. Esta tesis se ha centrado en describir los receptores de los dos principales péptidos bioactivos de este sistema, el receptor de angiotensina II tipo 2 (AT2R) y el receptor de prorrenina-renina (PRR), así como en estudiar la relación de estos dos receptores y de dos enzimas descritas como esenciales en la función espermática, la enzima convertidora de angiotensina testicular (tACE) y la aminopeptidasa N (APN), con los parámetros evaluados durante los tratamientos de fecundación in vitro. Así, se ha podido determinar que estos componentes se relacionan con los parámetros y etiologías seminales, las tasas de fecundación y la calidad, desarrollo y viabilidad de los embriones obtenidos durante los diferentes tratamientos de fecundación in vitro. Estos resultados confirman que las características moleculares espermáticas contribuyen a la fecundación y al desarrollo embrionario, de forma que estos receptores y enzimas podrían ser considerados biomarcadores de la función y selección espermática.Tesis realizada gracias al Programa Zabaldu

Sex dependent alteration of epigenetic marks after chronic morphine treatment in mice organs

Epigenetic marks may be also affected by several factors, such as age, lifestyle, early life experiences and exposure to chemicals or drugs, such as opioids. Previous studies have focused on how morphine epigenetically regulates different regions of the brain that are implicated in tolerance, dependence and other psychiatric disorders more related to the physio-pathological effects of opioids. Nevertheless, a significant knowledge gap remains regarding the effect of chronic treatment on other organs and biological systems. Therefore, the aim of this work is to increase our knowledge about the impact of chronic morphine exposure on DNA methylation and histone modification levels in each of the organs of male and female model mice in vivo. Our results reveal, for the first time, that chronic morphine treatment induced changes in DNA methylation/hydroxymethylation and histone modification in-vivo at the systemic level, revealing a potential physiological effect on the regulation of gene expression. Notably, morphine-induced epigenetic modification occurs in a sex-dependent manner, revealing the existence of different underlying mechanisms of epigenetic modification in male and female mice.This study was supported by grants from the Spanish Health Department ISCIII (DTS 18/00142) and Research Group University of the Basque Country (GI19/018). IM was supported by fellowship from Basque Government and MA and IU were supported by fellowship from the University of the Basque Country (UPV/EHU)

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