Nuclear envelope defects cause stem cell dysfunction in premature-aging mice

et al.Nuclear lamina alterations occur in physiological aging and in premature aging syndromes. Because aging is also associated with abnormal stem cell homeostasis, we hypothesize that nuclear envelope alterations could have an important impact on stem cell compartments. To evaluate this hypothesis, we examined the number and functional competence of stem cells in Zmpste24 -null progeroid mice, which exhibit nuclear lamina defects. We show that Zmpste24 deficiency causes an alteration in the number and proliferative capacity of epidermal stem cells. These changes are associated with an aberrant nuclear architecture of bulge cells and an increase in apoptosis of their supporting cells in the hair bulb region. These alterations are rescued in Zmpste24 -/- Lmna+/- mutant mice, which do not manifest progeroid symptoms. We also report that molecular signaling pathways implicated in the regulation of stem cell behavior, such as Wnt and microphthalmia transcription factor, are altered in Zmpste24-/- mice. These findings establish a link between age-related nuclear envelope defects and stem cell dysfunction.This work was supported by grants from Ministerio de Educacion y Ciencia (Spain), Fundación Lilly, Fundación La Caixa, Fundación M. Botín, and the European Union. I. Flores is a Ramón y Cajal senior scientist. M.A. Blasco´s laboratory is funded by the Ministerio de Ciencia y Tecnologia (Spain), the regional government of Madrid, the European Union, and Josef Steiner Cancer Research Award 2003. The Instituto Universitario de Oncología is supported by Obra Social Cajastur.Peer Reviewe

Dynamic localization of SMC5/6 complex proteins during mammalian meiosis and mitosis suggests functions in distinct chromosome processes

et al.Four members of the structural maintenance of chromosome (SMC) protein family have essential functions in chromosome condensation (SMC2/4) and sister-chromatid cohesion (SMC1/3). The SMC5/6 complex has been implicated in chromosome replication, DNA repair and chromosome segregation in somatic cells, but its possible functions during mammalian meiosis are unknown. Here, we show in mouse spermatocytes that SMC5 and SMC6 are located at the central region of the synaptonemal complex from zygotene until diplotene. During late diplotene both proteins load to the chromocenters, where they colocalize with DNA Topoisomerase IIα, and then accumulate at the inner domain of the centromeres during the first and second meiotic divisions. Interestingly, SMC6 and DNA Topoisomerase IIα colocalize at stretched strands that join kinetochores during the metaphase II to anaphase II transition, and both are observed on stretched lagging chromosomes at anaphase II following treatment with Etoposide. During mitosis, SMC6 and DNA Topoisomerase IIα colocalize at the centromeres and chromatid axes. Our results are consistent with the participation of SMC5 and SMC6 in homologous chromosome synapsis during prophase I, chromosome and centromere structure during meiosis I and mitosis and, with DNA Topoisomerase IIα, in regulating centromere cohesion during meiosis II.This work was supported by Ministerio de Economía y Competitividad (Spain) [grant number SAF2011-28842-C02-01 to J.A.S. and SAF2011-25252 to A.M.P.]; a UK-US Fulbright Distinguished Scholar Award; the US National Institutes of Health [grant number HD069458 to P.W.J.; HD33816 to M.A.H.; HD42137 to John Eppig, M.A.H. and J.C.S.]; the Priority Program SPP 1384 ‘Mechanisms of genome haploidization’ (to M.A. and R.J.) from the German Science Foundation.Peer Reviewe

STAG3 is a strong candidate gene for male infertility

Oligo- and azoospermia are severe forms of male infertility. However, known genetic factors account only for a small fraction of the cases. Recently, whole-exome sequencing in a large consanguineous family with inherited premature ovarian failure (POF) identified a homozygous frameshift mutation in the STAG3 gene leading to a premature stop codon. STAG3encodes a meiosis-specific subunit of the cohesin complex, alarge proteinaceous ring with DNA-entrapping ability that ensures sister chromatid cohesion and enables correct synapsis and segregation of homologous chromosomes during meiosis. The pathogenicity of the STAG3 mutations was functionally validated with a loss- of-function mouse model for STAG3 in oogenesis.However,and sincenone of the male members of this family was homozygous for the mutant allele, we only could hypothesized its putative involvement inmale infertility. In this report,we show that male mice devoid of Stag3 display a severe meiotic phenotype that includes a meiotic arrest at zygonema-like shortening of their chromosome axial elements/lateral elements, partial loss of centromeric cohesion at early prophase and maintenance of the ability to initiate but not complete RAD51- and DMC1-mediated double-strand break repair,demonstrating that STAG3 is a crucial cohesin subunit in mammalian gametogenesis and supporting our proposal that STAG3 is a strong candidate gene for human male infertility. © The Author 2014. Published by Oxford University Press. All rights reserved.This work was supported by grant SAF2011-25252 and Junta de Castilla y León (EL and AMP). SC and RAV are supported by the University Paris Diderot-Paris7, the Ligue Nationale contre le Cancer, the Centre National de la Recherche Scientifique (CNRS) and the GIS-Institut des Maladies Rares.Peer Reviewe

Oxysterol-induced soluble endoglin release and its involvement in hypertension

[Background]: Ischemia in the placenta is considered the base of the pathogenesis of preeclampsia, a pregnancy-specific syndrome in which soluble endoglin (sEng) is a prognostic marker and plays a pathogenic role. Here, we investigated the effects of hypoxia and the downstream pathways in the release of sEng. [Methods and Results]: Under hypoxic conditions, the trophoblast-like cell line JAR showed an increase in sEng parallel to an elevated formation of reactive oxygen species. Because reactive oxygen species are related to the formation of oxysterols, we assessed the effect of 22-(R)-hydroxycholesterol, a natural ligand of the liver X receptor (LXR), and the LXR synthetic agonist T0901317. Treatment of JAR cells or human placental explants with 22-(R)-hydroxycholesterol or T0901317 resulted in a clear increase in sEng that was dependent on LXR. These LXR agonists induced an increased matrix metalloproteinase-14 expression and activity and a significant reduction of its endogenous inhibitor, tissue inhibitor of metalloproteinase-3. In addition, mice treated with LXR agonists underwent an increase in the plasma sEng levels, concomitant with an increase in arterial pressure. Moreover, transgenic mice overexpressing sEng displayed high blood pressure. Finally, administration of an endoglin peptide containing the consensus matrix metalloproteinase-14 cleavage site G-L prevented the oxysterol-dependent increase in arterial pressure and sEng levels in mice. [Conclusions]: These studies provide a clue to the involvement of the LXR pathway in sEng release and its pathogenic role in vascular disorders such as preeclampsia. © 2012 American Heart Association, Inc.This work was supported by grants from the Ministerio de Ciencia e Innovación of Spain (SAF2010-61827 to Dr Bernabeu, SAF2011-29244 to Dr Castrillo, and SAF2010-15881 to Dr Lopez-Novoa), Genoma España (MEICA; Dr Bernabeu), Instituto Reina Sofía de Investigación Nefrológica (FRIAT; Dr Lopez-Novoa), Junta de Castilla and Leon (Excellence Group Grant GR-100 to Dr Lopez-Novoa), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; Dr Bernabeu), and Red de Investigación Cooperativa en Enfermedades Renales (REDINREN; Dr Lopez-Novoa). CIBERER and REDINREN are initiatives of the Instituto de Salud Carlos III of Spain supported by European Regional Development Funds (FEDER). The cardiovascular phenotyping unit, including the telemetry equipment, has been acquired with the support of FEDER. Dr Llano is recipient of a Ramón y Cajal Research contract.Peer Reviewe

Nuclear envelope defects cause stem cell dysfunction in premature-aging mice

Nuclear lamina alterations occur in physiological aging and in premature aging syndromes. Because aging is also associated with abnormal stem cell homeostasis, we hypothesize that nuclear envelope alterations could have an important impact on stem cell compartments. To evaluate this hypothesis, we examined the number and functional competence of stem cells in Zmpste24-null progeroid mice, which exhibit nuclear lamina defects. We show that Zmpste24 deficiency causes an alteration in the number and proliferative capacity of epidermal stem cells. These changes are associated with an aberrant nuclear architecture of bulge cells and an increase in apoptosis of their supporting cells in the hair bulb region. These alterations are rescued in Zmpste24−/−Lmna+/− mutant mice, which do not manifest progeroid symptoms. We also report that molecular signaling pathways implicated in the regulation of stem cell behavior, such as Wnt and microphthalmia transcription factor, are altered in Zmpste24−/− mice. These findings establish a link between age-related nuclear envelope defects and stem cell dysfunction

A truncating variant of RAD51B associated with primary ovarian insufficiency provides insights into its meiotic and somatic functions

Primary ovarian insufficiency (POI) causes female infertility by abolishing normal ovarian function. Although its genetic etiology has been extensively investigated, most POI cases remain unexplained. Using whole-exome sequencing, we identified a homozygous variant in RAD51B –(c.92delT) in two sisters with POI. In vitro studies revealed that this variant leads to translation reinitiation at methionine 64. Here, we show that this is a pathogenic hypomorphic variant in a mouse model. Rad51bc.92delT/c.92delT mice exhibited meiotic DNA repair defects due to RAD51 and HSF2BP/BMRE1 accumulation in the chromosome axes leading to a reduction in the number of crossovers. Interestingly, the interaction of RAD51B-c.92delT with RAD51C and with its newly identified interactors RAD51 and HELQ was abrogated or diminished. Repair of mitomycin-C-induced chromosomal aberrations was impaired in RAD51B/Rad51b-c.92delT human and mouse somatic cells in vitro and in explanted mouse bone marrow cells. Accordingly, Rad51b-c.92delT variant reduced replication fork progression of patient-derived lymphoblastoid cell lines and pluripotent reprogramming efficiency of primary mouse embryonic fibroblasts. Finally, Rad51bc.92delT/c.92delT mice displayed increased incidence of pituitary gland hyperplasia. These results provide new mechanistic insights into the role of RAD51B not only in meiosis but in the maintenance of somatic genome stability.This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Grant 2014/14231-0 (to MMF); FAPESP Grant 2013/02162-8, Nucleo de Estudos e Terapia Celular e Molecular (NETCEM), Conselho Nacional de Desenvolvimento Científico e Tecnológico Grant 303002/2016- 6 (to BBM); and FAPESP Grant 2014/50137-5 (to SELA). This work was supported by MINECO (PID2020-120326RB-I00) and by Junta de Castilla y León (CSI239P18 and CSI148P20). NFM, FSS, and MRMH are supported by European Social Fund/JCyLe grants (EDU/310/2015, EDU/556/2019 and EDU/1992/2020). YBC and RSU are funded by a grant from MINECO (BS-2015–073993 and BFU2017-89408-R). Experiments performed at CNIO were supported by grant PID2019-106707-RB to JM, co-sponsored by EU ERDF funds. SM was supported by an international postdoctoral contract “CNIO Friends”. The proteomic analysis was performed in the Proteomics Facility of Centro de Investigación del Cáncer, Salamanca, Grant PRB3(IPT17/0019 -ISCIII-SGEFI/ERDF). CIC-IBMCC is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia cofunded by the Castilla–León autonomous government and the European Regional Development Fund (CLC–2017–01). Veitia’s Lab is supported by the University of Paris and the Centre National de la Recherche Scientifique

PiRNA-associated proteins and retrotransposons are differentially expressed in murine testis and ovary of aryl hydrocarbon receptor deficient mice

Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduction and fertility. However, the mechanisms involved remain mostly unknown. Retrotransposon silencing by Piwi-interacting RNAs (piRNAs) is essential for germ cell maturation and, remarkably, AhR has been identified as a regulator of murine B1-SINE retrotransposons. Here, using littermate AhR+/+ and AhR-/- mice, we report that AhR regulates the general course of spermatogenesis and oogenesis by a mechanism likely to be associated with piRNA-associated proteins, piRNAs and retrotransposons. piRNA-associated proteins MVH and Miwi are upregulated in leptotene to pachytene spermatocytes with a more precocious timing in AhR-/- than in AhR+/+ testes. piRNAs and transcripts from B1-SINE, LINE-1 and IAP retrotransposons increased at these meiotic stages in AhR-null testes. Moreover, B1-SINE transcripts colocalize with MVH and Miwi in leptonema and pachynema spermatocytes. Unexpectedly, AhR-/- males have increased sperm counts, higher sperm functionality and enhanced fertility than AhR+/+ mice. In contrast, piRNA-associated proteins and B1-SINE and IAP-derived transcripts are reduced in adult AhR-/- ovaries. Accordingly, AhR-null female mice have lower numbers of follicles when compared with AhR+/+ mice. Thus, AhR deficiency differentially affects testis and ovary development possibly by a process involving piRNA-associated proteins, piRNAs and transposable elements.Trabajo financiado por: Ministerio de Ciencia e Innovación. Ayuda BFU2011-22678 para Pedro María Fernández Salguero Ministerio de Economía y Competitividad. Ayuda SAF2014-51813-R para Pedro María Fernández Salguero Junta de Extremadura. Ayuda GR15008, para Pedro María Fernández Salguero Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto Carlos III y Ministerio de Economía y Competitividad (RD12/0036/0032). Trabajos de laboratorio de Pedro María Fernández Salguero Ministerio de Economía y Competitividad. Ayuda AGL2013-43211-R, para Fernando Juan Peña Vega Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto Carlos III. Ayuda para Eva María Rico Leo Ministerio de Economía y Competitividad. Ayuda para Francisco Javier González Rico Junta de Extremadura. Ayuda para Eva María Barrasa Ardila Ministerio de Educación, Cultura y Deportes. Beca de Formación de Personal de Investigación, para Nuria Moreno Marín Ministerio de Educación, Cultura y Deportes. Beca FPU13/03991, de Formación de Profesorado Universitario, para Patricia Martín Muñoz Ministerio de Economía y Competitividad. Beca Juan de la Cierva IJCI-2014-21671, para Cristina Ortega Ferrusola Ministerio de Economía y Competitividad, Beca BFU2014-59307-R, para Alberto M. Pendás y Elena Llano Cuadro MEIONet, Junta de Castilla y León y el Programa de Fondeos FEDER, de la Unión EuropeapeerReviewe

Sequential Assembly of Centromeric Proteins in Male Mouse Meiosis

The assembly of the mitotic centromere has been extensively studied in recent years, revealing the sequence and regulation of protein loading to this chromosome domain. However, few studies have analyzed centromere assembly during mammalian meiosis. This study specifically targets this approach on mouse spermatocytes. We have found that during prophase I, the proteins of the chromosomal passenger complex Borealin, INCENP, and Aurora-B load sequentially to the inner centromere before Shugoshin 2 and MCAK. The last proteins to be assembled are the outer kinetochore proteins BubR1 and CENP-E. All these proteins are not detected at the centromere during anaphase/telophase I and are then reloaded during interkinesis. The loading sequence of the analyzed proteins is similar during prophase I and interkinesis. These findings demonstrate that the interkinesis stage, regularly overlooked, is essential for centromere and kinetochore maturation and reorganization previous to the second meiotic division. We also demonstrate that Shugoshin 2 is necessary for the loading of MCAK at the inner centromere, but is dispensable for the loading of the outer kinetochore proteins BubR1 and CENP-E