Morfinak eragindako zelulen arteko memoria epigenetikoaren azterketa, bigarren belaunaldiko sekuentziazioaren bitartez

296 p.Epigenetikak, kanpo faktoreek edo inguruneak norbanakoan izan dezaketen eragina aztertzen du. Azken urteetan, zenbait ikerketek erakutsi dutenez, inguruneak eragin zuzena izan dezake norbanako helduaren ezaugarri fisiologikoetan. Hala ere, kanpo faktoreek umekian eta garapen goiztiarrean izan dezaketen eraginari buruz, ezer gutxi deskribatu da. Geneen adierazpenarekin duten elkarteratzea dela eta, histonen aldaketak informazio epigenetikoaren eragile nagusien artean aurkitzen ditugu, eta memoria epigenetikoan paper garrantzitsua izan dezaketela iradoki da. Hala ere, naiz eta histonen aldaketa espezifikoek transkripzio egoerekin erlazioa duten, epe luzean zehar mantendu daitekeen herentziaren mekanismoa ez da oraindik ere argitu.Hau guztia aztertzeko eta kanpo estimulu eredu bezala morfina erabiliz, bigarren belaunaldiko sekuentziazio teknikak erabili dira kromatina eta transkriptomika analisiak burutzeko. Bertatik H3K27me3 marka epigenetiko errepresiogilearen desdoitze orokor bat hauteman dugu, epe luzeko geneen adierazpen aldaketak sortuz, saguen ama zelula enbrionikoetan. Zehazki morfinaren tratamendu kronikoak eragindako efektua CpG irletan ugariak diren sustatzaileetan, inpronta genomikodun geneen metilazioaren erregulazio eremuetan, lncRNA-etan eta elementu errepikakorretan aurkitu ditugu. Aipatzekoa da, H3K27me3-ren eta bere erregulazio konplexuaren (Polycomb repressive complex 2 edo PRC2), auto erregulazio mekanismo bat identifikatu dugula morfinaren presentzian. Horrez gain, morfinak eragindako H3K27me3-ko aldaketek bi gene espezifikotan sortzen dituen adierazpen aldaketak sakonago aztertu ditugu, Bmp4 eta Smchd1 hurrenez hurren, hauek parte hartzen duten prozesuak guztiz eraldatzen direla aurkituz. Bmp4 enbrioi goiztiarren garapenean eta jatorrizko hozi zelulen sorreran parte hartzen duen hazkuntza faktorea da eta morfinaren ondorioz, bi prozesu horien azkartzea eragiten dela hauteman dugu. Bestalde, Smchd1, X kromosomaren inaktibazioaren mantentze lanetan haritzen da eta gure sisteman morfinaren eraginez erreprimituta aurkitu dugu X kromosomaren birraktibazioa azkartuz.Doktoretza tesi honetan lortutako emaitza hauek, kanpo faktoreek sortutako zelulen arteko memoria epigenetikoaren mekanismo molekularrei buruzko informazio baliagarriaz hornitzen gaituzte, belaunaldiz belaunaldiko oinordekotza epigenetikoa ulertzeko oinarri gisa. Hori dela eta, ezagutza hori heredatu daitezkeen aldaketa epigenetikoen identifikaziorako baliagarria litzateke etorkizunean

Morfinak eragindako zelulen arteko memoria epigenetikoaren azterketa, bigarren belaunaldiko sekuentziazioaren bitartez

296 p.Epigenetikak, kanpo faktoreek edo inguruneak norbanakoan izan dezaketen eragina aztertzen du. Azken urteetan, zenbait ikerketek erakutsi dutenez, inguruneak eragin zuzena izan dezake norbanako helduaren ezaugarri fisiologikoetan. Hala ere, kanpo faktoreek umekian eta garapen goiztiarrean izan dezaketen eraginari buruz, ezer gutxi deskribatu da. Geneen adierazpenarekin duten elkarteratzea dela eta, histonen aldaketak informazio epigenetikoaren eragile nagusien artean aurkitzen ditugu, eta memoria epigenetikoan paper garrantzitsua izan dezaketela iradoki da. Hala ere, naiz eta histonen aldaketa espezifikoek transkripzio egoerekin erlazioa duten, epe luzean zehar mantendu daitekeen herentziaren mekanismoa ez da oraindik ere argitu.Hau guztia aztertzeko eta kanpo estimulu eredu bezala morfina erabiliz, bigarren belaunaldiko sekuentziazio teknikak erabili dira kromatina eta transkriptomika analisiak burutzeko. Bertatik H3K27me3 marka epigenetiko errepresiogilearen desdoitze orokor bat hauteman dugu, epe luzeko geneen adierazpen aldaketak sortuz, saguen ama zelula enbrionikoetan. Zehazki morfinaren tratamendu kronikoak eragindako efektua CpG irletan ugariak diren sustatzaileetan, inpronta genomikodun geneen metilazioaren erregulazio eremuetan, lncRNA-etan eta elementu errepikakorretan aurkitu ditugu. Aipatzekoa da, H3K27me3-ren eta bere erregulazio konplexuaren (Polycomb repressive complex 2 edo PRC2), auto erregulazio mekanismo bat identifikatu dugula morfinaren presentzian. Horrez gain, morfinak eragindako H3K27me3-ko aldaketek bi gene espezifikotan sortzen dituen adierazpen aldaketak sakonago aztertu ditugu, Bmp4 eta Smchd1 hurrenez hurren, hauek parte hartzen duten prozesuak guztiz eraldatzen direla aurkituz. Bmp4 enbrioi goiztiarren garapenean eta jatorrizko hozi zelulen sorreran parte hartzen duen hazkuntza faktorea da eta morfinaren ondorioz, bi prozesu horien azkartzea eragiten dela hauteman dugu. Bestalde, Smchd1, X kromosomaren inaktibazioaren mantentze lanetan haritzen da eta gure sisteman morfinaren eraginez erreprimituta aurkitu dugu X kromosomaren birraktibazioa azkartuz.Doktoretza tesi honetan lortutako emaitza hauek, kanpo faktoreek sortutako zelulen arteko memoria epigenetikoaren mekanismo molekularrei buruzko informazio baliagarriaz hornitzen gaituzte, belaunaldiz belaunaldiko oinordekotza epigenetikoa ulertzeko oinarri gisa. Hori dela eta, ezagutza hori heredatu daitezkeen aldaketa epigenetikoen identifikaziorako baliagarria litzateke etorkizunean

(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)

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)

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

The Multifunctional Role of SPANX-A/D Protein Subfamily in the Promotion of Pro-Tumoural Processes in Human Melanoma

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 various tumour cells. SPANX-A/D proteins have been detected in metastatic melanoma cells, but their contribution to cancer development and the underlying molecular mechanisms of skin tumourigenesis remain unknown. Combining functional and proteomic approaches, the present work describes the presence of SPANX-A/D in primary and metastatic human melanoma cells and how it promotes pro-tumoural processes such as cell proliferation, motility and migration. We provide insights into the molecular features of skin tumourigenesis, describing for the first time a multifunctional role of the SPANX-A/D protein family in nuclear function, energy metabolism and cell survival, considered key hallmarks of cancer. A better comprehension of the SPANX-A/D protein subfamily and its molecular mechanisms will help to describe new aspects of tumour cell biology and develop new therapeutic targets and tumour-directed pharmacological drugs for skin tumoursUniversity of the Basque Country (UPV/EHU) (GIU19/018). IU-A is supported by a fellowship from the University of the Basque Country (UPV/EHU). IM-H is supported by a fellowship from the Basque Government

Pirin is a prognostic marker of human melanoma that dampens the proliferation of malignant cells by downregulating JARID1B/KDM5B expression

Originally considered to act as a transcriptional co‑factor, Pirin has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. Here, we have analyzed the diagnostic and prognostic value of Pirin expression in the early stages of melanoma, and its role in the biology of melanocytic cells. Pirin expression was analyzed in a total of 314 melanoma biopsies, correlating this feature with the patient’s clinical course. Moreover, PIR downregulated primary melanocytes were analyzed by RNA sequencing, and the data obtained were validated in human melanoma cell lines overexpressing PIR by functional assays. The immunohistochemistry multivariate analysis revealed that early melanomas with stronger Pirin expression were more than twice as likely to develop metastases during the follow‑up. Transcriptome analysis of PIR downregulated melanocytes showed a dampening of genes involved in the G1/S transition, cell proliferation, and cell migration. In addition, an in silico approach predicted that JARID1B as a potential transcriptional regulator that lies between PIR and its downstream modulated genes, which was corroborated by co‑transfection experiments and functional analysis. Together, the data obtained indicated that Pirin could be a useful marker for the metastatic progression of melanoma and that it participates in the proliferation of melanoma cells by regulating the slow‑cycling JARID1B gene.This project was supported by grants from the Basque Government (KK2017-041 and KK2020-00069 to M.D.B.), the UPV/EHU (GIU17/066 to M.D.B.), H2020-ESCEL JTI (15/01 to M.D.B.) and MINECO (PCIN-2015-241 to M.D.B.). CP holds a predoctoral fellowship from the Basque Government. Part of this project is under European patent No. EP3051291 (EP14796149.4): “Method for diagnosis and prognosis of cutaneous melanoma”, Univer- sity of the Basque Country (UPV/EHU). The authors acknowledge the technical support SGIker resources at the UPV/EHU for the computational calculations, which were carried out in the Arina Informatics Cluster. The authors are grateful to the Basque Biobank for providing the biopsy samples and in particular, to María Jesús Fernández and Arantza Perez Dobaran for their technical support with the immunohistochemistry

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

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)