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Chromatin is a crucial regulator of gene expression and tightly controls development across species. Mutations in only one copy of multiple histone genes were identified in children with developmental disorders characterized by microcephaly, but their mechanistic roles in development remain unclear. Here we focus on dominant mutations affecting histone H4 lysine 91. These H4K91 mutants form aberrant nuclear puncta at specific heterochromatin regions. Mechanistically, H4K91 mutants demonstrate enhanced binding to the histone variant H3.3, and ablation of H3.3 or the H3.3-specific chaperone DAXX diminishes the mutant localization to chromatin. Our functional studies demonstrate that H4K91 mutant expression increases chromatin accessibility, alters developmental gene expression through accelerating pro-neural differentiation, and causes reduced mouse brain size in vivo, reminiscent of the microcephaly phenotypes of patients. Together, our studies unveil a distinct molecular pathogenic mechanism from other known histone mutants, where H4K91 mutants misregulate cell fate during development through abnormal genomic localization.
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Gastrulation is considered the sine qua non of embryogenesis, establishing a multidimensional structure and the spatial coordinates upon which all later developmental events transpire. At this time, the embryo adopts a heavy reliance on glucose metabolism to support rapidly accelerating changes in morphology, proliferation, and differentiation. However, it is currently unknown how this conserved metabolic shift maps onto the three-dimensional landscape of the growing embryo and whether it is spatially linked to the orchestrated cellular and molecular processes necessary for gastrulation. Here we identify that glucose is utilised during mouse gastrulation via distinct metabolic pathways to instruct local and global embryonic morphogenesis, in a cell type and stage-specific manner. Through detailed mechanistic studies and quantitative live imaging of mouse embryos, in parallel with tractable in vitro stem cell differentiation models and embryo-derived tissue explants, we discover that cell fate acquisition and the epithelial-to-mesenchymal transition (EMT) relies on the Hexosamine Biosynthetic Pathway (HBP) branch of glucose metabolism, while newly-formed mesoderm requires glycolysis for correct migration and lateral expansion. This regional and tissue-specific difference in glucose metabolism is coordinated with Fibroblast Growth Factor (FGF) activity, demonstrating that reciprocal crosstalk between metabolism and growth factor signalling is a prerequisite for gastrulation progression. We expect these studies to provide important insights into the function of metabolism in other developmental contexts and may help uncover mechanisms that underpin embryonic lethality, cancer, and congenital disease.
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Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro1-13. Here we show that human pluripotent stem cells can be triggered to self-organize into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast-like and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators and undergoes symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the perigastrulating human embryo14,15 without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie stage16 4-7, offering a reproducible, tractable and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput.
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Linaje de la Célula , Implantación del Embrión , Desarrollo Embrionario , Células Madre Pluripotentes , Femenino , Humanos , Embarazo , Diferenciación Celular , Estratos Germinativos/citología , Estratos Germinativos/enzimología , Células Madre Embrionarias Humanas/citología , Placenta/citología , Células Madre Pluripotentes/citología , Línea Primitiva/citología , Línea Primitiva/embriología , Saco Vitelino/citología , Saco Vitelino/embriologíaRESUMEN
While Mek1/2 and Gsk3ß inhibition ("2i") supports the maintenance of murine embryonic stem cells (ESCs) in a homogenous naïve state, prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential. Additionally, 2i fails to support derivation and culture of fully potent female ESCs. Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin (AlbuMAX) undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture. Mechanistically, lipids in AlbuMAX impact intracellular metabolism including nucleotide biosynthesis, lipid biogenesis, and TCA cycle intermediates, with enhanced expression of DNMT3s that prevent DNA hypomethylation. Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation, which also alleviates X chromosome loss in female ESCs. Importantly, both male and female "all-ESC" mice can be generated from de novo derived ESCs using AlbuMAX-based media. Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.
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Células Madre Embrionarias , Células Madre Embrionarias de Ratones , Masculino , Animales , Femenino , Ratones , Inestabilidad Genómica , Lípidos , ADN/metabolismo , Diferenciación CelularRESUMEN
Histone variants and the associated post-translational modifications that govern the stemness of haematopoietic stem cells (HSCs) and differentiation thereof into progenitors (HSPCs) have not been well defined. H3.3 is a replication-independent H3 histone variant in mammalian systems that is enriched at both H3K4me3- and H3K27me3-marked bivalent genes as well as H3K9me3-marked endogenous retroviral repeats. Here we show that H3.3, but not its chaperone Hira, prevents premature HSC exhaustion and differentiation into granulocyte-macrophage progenitors. H3.3-null HSPCs display reduced expression of stemness and lineage-specific genes with a predominant gain of H3K27me3 marks at their promoter regions. Concomitantly, loss of H3.3 leads to a reduction of H3K9me3 marks at endogenous retroviral repeats, opening up binding sites for the interferon regulatory factor family of transcription factors, allowing the survival of rare, persisting H3.3-null HSCs. We propose a model whereby H3.3 maintains adult HSC stemness by safeguarding the delicate interplay between H3K27me3 and H3K9me3 marks, enforcing chromatin adaptability.
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Cromatina/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Histonas/metabolismo , Mielopoyesis/fisiología , Animales , Linfocitos T CD8-positivos/citología , Proteínas de Ciclo Celular , Línea Celular , Granulocitos/citología , Hematopoyesis/fisiología , Chaperonas de Histonas , Células Endoteliales de la Vena Umbilical Humana , Humanos , Macrófagos/citología , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas/genética , Procesamiento Proteico-Postraduccional/fisiología , Factores de TranscripciónRESUMEN
Mice derived entirely from embryonic stem (ES) cells can be generated through tetraploid complementation. Although XY male ES cell lines are commonly used in this system, occasionally, monosomic XO female mice are produced through spontaneous Y chromosome loss. Here, we describe an efficient method to obtain monosomic XO ES cells by CRISPR-Cas9-mediated deletion of the Y chromosome, allowing generation of female clonal mice by tetraploid complementation. The monosomic XO female mice are viable and able to produce normal male and female offspring. Direct generation of clonal mice in both sexes can significantly accelerate the production of complex genetically modified mouse models.
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Sistemas CRISPR-Cas , Deleción Cromosómica , Células Madre Embrionarias , Infertilidad Masculina , Aberraciones Cromosómicas Sexuales , Trastornos de los Cromosomas Sexuales del Desarrollo Sexual , Cromosoma Y , Animales , Línea Celular , Cromosomas Humanos Y , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Embrión de Mamíferos , Femenino , Técnicas de Genotipaje , Cariotipificación , Masculino , RatonesRESUMEN
OBJECTIVE: To measure the influence of exogenous insulin-like growth factor 1 (IGF1) on follicle growth and maturation in human ovarian cortical xenografts. DESIGN: Xenotransplantation model. SETTING: University-based research laboratory. PATIENTS/ANIMALS: Ovarian tissue was donated with consent and institutional review board approval by brain-dead organ donors or patients undergoing ovarian tissue cryopreservation for fertility preservation. Cortical fragments were transplanted into immunocompromised mice. INTERVENTIONS: Cryopreserved ovarian cortical fragments from four women (aged 19, 25, 33, and 46 years) were transplanted into the gluteus muscle of immunocompromised mice in a fibrin matrix containing endothelial cells that were transduced with lentiviral particles encoding secreted IGF1. Xenografts were recovered after 3, 8, and 14 weeks. In addition, C57/Bl6 mice underwent intraovarian injection of saline or recombinant IGF1 (60 µg), followed by superovulation, analysis of ethynyl-deoxyuridine incorporation, and ribonucleic acid sequencing of the whole ovaries. MAIN OUTCOME MEASURES: For xenografts: follicle count and distribution; antral follicle count; and corpora lutea/albicans count. For mice: follicle count and distribution; oocyte yield, ethynyl-deoxyuridine incorporation (granulosa cell proliferation); and ovarian transcriptomic signature. RESULTS: At 3 weeks, xenografts in the IGF1 condition revealed a decreased percentage of primary follicles and increased percentage of secondary follicles that were concentrated in the preantral subtype; at 8 weeks, an increase in secondary follicles was concentrated in the simple subtype; after 14 weeks, primordial follicles were reduced, and while the number of advanced follicles did not power the experiment to demonstrate significance, antral follicles reduced and corpora lutea increased. Supporting experiments in mice revealed an increase in normal oocytes following intraovarian injection of recombinant IGF1 (60 µg) as well as increased proliferative index among follicles of secondary and preantral stages. Ribonucleic acid sequencing analysis of the whole ovaries following injection of recombinant IGF1 (25 µg) revealed an acute (24 hours) upregulation of transcripts related to steroidogenesis and luteinization. CONCLUSIONS: Exogenous IGF1 advances the pace of growth among primordial, primary, and secondary stage follicles but results in near absence of antral stage follicles in long-term (14 weeks) xenografts. In mice, acute administration of IGF1 promotes follicle advance and increased oocyte yield. The results suggest that while superphysiological IGF1 alone advances the pace of growth among early/preantral follicles, a sustained and/or later-stage influence undermines antral follicle growth/survival or promotes premature luteinization. These findings provide a temporal framework for interpreting follicle growth/mobilization and may be useful in understanding the clinical application of human growth hormone in the context of assisted reproduction.
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Factor I del Crecimiento Similar a la Insulina , Ovario , Animales , Desoxiuridina , Células Endoteliales , Femenino , Xenoinjertos , Humanos , Ratones , Ovario/fisiología , ARN , Trasplante HeterólogoRESUMEN
PURPOSE: Fanconi anemia (FA) genes play important roles in spermatogenesis. In mice, disruption of Fancm impairs male fertility and testicular integrity, but whether FANCM pathogenic variants (PV) similarly affect fertility in men is unknown. Here we characterize a Pakistani family having three infertile brothers, two manifesting oligoasthenospermia and one exhibiting azoospermia, born to first-cousin parents. A homozygous PV in FANCM (c.1946_1958del, p.P648Lfs*16) was found cosegregating with male infertility. Our objective is to validate that FANCM p.P648Lfs*16 is the PV causing infertility in this family. METHODS: Exome and Sanger sequencing were used for PV screening. DNA interstrand crosslink (ICL) sensitivity was assessed in lymphocytes from patients. A mouse model carrying a PV nearly equivalent to that in the patients (FancmΔC/ΔC) was generated, followed by functional analysis in spermatogenesis. RESULTS: The loss-of-function FANCM PV increased ICL sensitivity in lymphocytes of patients and FancmΔC/ΔC spermatogonia. Adult FancmΔC/ΔC mice showed spermatogenic failure, with germ cell loss in 50.2% of testicular tubules and round-spermatid maturation arrest in 43.5% of tubules. In addition, neither bone marrow failure nor cancer/tumor was detected in all the patients or adult FancmΔC/ΔC mice. CONCLUSION: These findings revealed male infertility to be a novel phenotype of human patients with a biallelic FANCM PV.
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ADN Helicasas/genética , Predisposición Genética a la Enfermedad , Infertilidad Masculina/genética , Espermatogénesis/genética , Adulto , Animales , Mutación del Sistema de Lectura , Homocigoto , Humanos , Infertilidad Masculina/patología , Mutación con Pérdida de Función/genética , Masculino , Ratones , Oligospermia/genética , Oligospermia/patología , Linaje , Fenotipo , Testículo/patologíaRESUMEN
Hao Win, Hui Ma and Sajjad Hussain were incorrectly affiliated to 'Department of Radiation Oncology, The Houston Methodist Research Institute, Houston, TX 77030 USA'. These authors should only have been affiliated to 'Hefei National Laboratory for Physical Sciences at Microscale, The First Affiliated Hospital of USTC, USTC-SJH Joint Center for Human Reproduction and Genetics, The CAS Key Laboratory of Innate Immunity and Chronic Diseases, School of Life Sciences, CAS Center for Excellence in Molecular Cell Science, Collaborative Innovation Center of Genetics and Development, University of Science and Technology of China, Hefei 230027, China'. They were also not noted as contributing equally to the paper. Both these errors have now been corrected in the PDF and HTML versions of the paper.
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Three waves of H2AX phosphorylation (γH2AX) have been observed in male meiotic prophase I: the first is ATM-dependent and occurs at leptonema, while the second and third are ATR-dependent, occuring at zygonema and pachynema, respectively. The third wave of H2AX phosphorylation marks and silences unsynapsed chromosomes. Little is known about H2AX phosphorylation expands to chromatin-wide regions in spermatocytes. Here, we report that histone acetyltransferase (HAT) MOF is involved in all three waves of H2AX phosphorylation expansion. Germ cell-specific deletion of Mof in spermatocytes by Stra8-Cre (Mof cKO) caused global loss of H4K16ac. In leptotene and zygotene spermatocytes of cKO mice, the γH2AX signals were observed only along the chromosomal axes, and chromatin-wide H2AX phosphorylation was lost. In almost 40% of early-mid pachytene spermatocytes from Mof cKO mice, γH2AX and MDC1 were detected along the unsynapsed axes of the sex chromosomes, but failed to expand, which consequently caused meiotic sex chromosome inactivation (MSCI) failure. Furthermore, though RAD51 was proficiently recruited to double-strand break (DSB) sites, defects in DSB repair and crossover formation were observed in Mof cKO spermatocytes, indicating that MOF facilitates meiotic DSB repair after RAD51 recruitment. We propose that MOF regulates male meiosis and is involved in the expansion of all three waves of H2AX phosphorylation from the leptotene to pachytene stages, initiated by ATM and ATR, respectively.
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Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Espermatogénesis/fisiología , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN , Femenino , Histona Acetiltransferasas/genética , Masculino , Meiosis , Ratones Noqueados , Ratones Transgénicos , Fase Paquiteno , Fosforilación , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Espermatocitos/citología , Testículo/fisiologíaRESUMEN
Spermatocyte spreading and immunostaining were applied to detect meiotic prophase I progression, homologous chromosome pairing, synapsis and recombination in an azoospermic reciprocal translocation 46, XY, t(5;7;9;13)(5q11;7p11;7p15;9q12;13p12) carrier. Histological examination of the haematoxylin and eosin stained testicular sections revealed reduced germ cells with no spermatids or sperm in the patient. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay showed apoptotic cells in testicular sections of translocation carrier. Immnunofluorescence analysis indicated the presence of an octavalent in all the pachytene spermatocytes analysed in the patient. Meiotic progression was disturbed, as an increase in zygotene (P < 0.001) and decrease in the pachytene spermatocytes (P < 0.001) were observed in the t(5;7;9;13) carrier compared with controls. It was further observed that 93% of octavalents were found partially asynapsed between homologous chromosomes. A significant decrease in the recombination frequency was observed on 5p, 5q, 7q, 9p and 13q in the translocation carrier compared with the reported controls. A significant reduction in XY recombination frequency was also found in the participants. Our results indicated that complex chromosomal rearrangements can impair synaptic integrity of translocated chromosomes, which may reduce chromosomal recombination on translocated as well as non-translocated chromosomes, a phenomenon commonly known as interchromosomal effect.
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Azoospermia/genética , Aberraciones Cromosómicas , Meiosis , Recombinación Genética , Humanos , Hibridación Fluorescente in Situ , Etiquetado Corte-Fin in Situ , Masculino , Persona de Mediana Edad , Translocación GenéticaRESUMEN
Small supernumerary marker chromosomes (sSMCs) are structurally abnormal rare chromosomes, difficult to characterize by karyotyping, and have been associated with minor dysmorphic features, azoospermia, and recurrent miscarriages. However, sSMC with a gonosomal trisomy has never been reported. Spermatocyte spreading and immunostaining were applied to detect meiotic prophase I progression, homologous chromosome pairing, synapsis, and recombination. In all the analyzed spermatocytes of the patient, the extra Y chromosome was not detected while the sSMC was present. The recombination frequency on autosomes was not affected, while the recombination frequencies on XY chromosome was significantly lower in the patient than in the controls. The meiotic prophase I progression was disturbed with significantly increased proportion of zygotene and decreased pachytene spermatocytes in the patients as compared with the controls. These findings highlight the importance of studies on meiotic behaviors in patients with an abnormal chromosomal constitution and provide an important framework for future studies, which may elucidate the impairment caused by sSMC in mammalian meiosis and fertility.
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Cromosomas Humanos X , Cromosomas Humanos Y , Profase Meiótica I/genética , Recombinación Genética , Translocación Genética , Cariotipo XYY , Adulto , Humanos , Infertilidad Masculina/diagnóstico , Infertilidad Masculina/genética , Masculino , Espermatocitos/metabolismoRESUMEN
Embryo implantation is a crucial step in mammalian reproduction. However, little is known regarding the physiological roles of microRNAs in the regulation of embryo implantation. Here we show that a minimum uterine expression of miR-181 is essential for the onset of embryo implantation. Both transient and prolonged transgenic expression of miR-181 led to impaired implantation, which can be rescued by exogenous administration of leukemia inhibitory factor (LIF). Mechanistically, miR-181 is able to directly target LIF and downregulate LIF expression, thereby inhibiting embryo implantation. We also show that miR-181 expression is regulated by the transcriptional factor Emx2, and the Emx2-miR-181 axis plays an important role in regulating embryo implantation. Taken together, these results reveal a novel function of miR-181 in embryo implantation through the regulation of LIF, and also indicate a potential link between miR-181 dysregulation and human embryo implantation defects.
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Implantación del Embrión/genética , Regulación de la Expresión Génica , Factor Inhibidor de Leucemia/genética , MicroARNs/genética , Interferencia de ARN , Animales , Secuencia de Bases , Sitios de Unión , Tasa de Natalidad , Línea Celular , Secuencia de Consenso , Regulación hacia Abajo , Femenino , Proteínas de Homeodominio/metabolismo , Humanos , Factor Inhibidor de Leucemia/química , Ratones , Ratones Transgénicos , MicroARNs/química , Fenotipo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Human infertility affects 10-15% of couples, half of which is attributed to the male partner. Abnormal spermatogenesis is a major cause of male infertility. Characterizing the genes involved in spermatogenesis is fundamental to understand the mechanisms underlying this biological process and in developing treatments for male infertility. Although many genes have been implicated in spermatogenesis, no dedicated bioinformatic resource for spermatogenesis is available. We have developed such a database, SpermatogenesisOnline 1.0 (http://mcg.ustc.edu.cn/sdap1/spermgenes/), using manual curation from 30 233 articles published before 1 May 2012. It provides detailed information for 1666 genes reported to participate in spermatogenesis in 37 organisms. Based on the analysis of these genes, we developed an algorithm, Greed AUC Stepwise (GAS) model, which predicted 762 genes to participate in spermatogenesis (GAS probability >0.5) based on genome-wide transcriptional data in Mus musculus testis from the ArrayExpress database. These predicted and experimentally verified genes were annotated, with several identical spermatogenesis-related GO terms being enriched for both classes. Furthermore, protein-protein interaction analysis indicates direct interactions of predicted genes with the experimentally verified ones, which supports the reliability of GAS. The strategy (manual curation and data mining) used to develop SpermatogenesisOnline 1.0 can be easily extended to other biological processes.
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Bases de Datos Genéticas , Espermatogénesis/genética , Animales , Bovinos , Minería de Datos , Genómica , Humanos , Internet , Masculino , Ratones , Anotación de Secuencia Molecular , Ratas , Transcriptoma , Interfaz Usuario-ComputadorRESUMEN
The muntjacs (Muntiacus, Cervidae) have been extensively studied in terms of chromosomal and karyotypic evolution. However, little is known about their meiotic chromosomes particularly the recombination patterns of homologous chromosomes. We used immunostained surface spreads to visualise synaptonemal complexes (SCs), recombination foci and kinetochores with antibodies against marker proteins. As in other mammals pachytene was the longest stage of meiotic prophase. 39.4% of XY bivalents lacked MLH1 foci compared to less than 0.5% of autosomes. The average number of MLH1 foci per pachytene cell in M. reevesi was 29.8. The distribution of MLH1 foci differed from other mammals. On SCs with one focus, the distribution was more even in M. reevesi than in other mammals; for SCs that have two or more MLH1 foci, usually there was a larger peak in the sub-centromere region than other regions on SC in M. reevesi. Additionally, there was a lower level of interference between foci in M. reevesi than in mouse or human. These observations may suggest that the regulation of homologous recombination in M. reevesi is slightly different from other mammals and will improve our understanding of the regulation of meiotic recombination, with respect to recombination frequency and position.
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Emparejamiento Cromosómico , Ciervo Muntjac/genética , Recombinación Genética , Animales , Mapeo Cromosómico , Cariotipificación , Masculino , Meiosis , Microscopía Fluorescente/métodos , Modelos Genéticos , Ciervo Muntjac/fisiología , Ploidias , Análisis de Secuencia de ADN , Espermatocitos/citología , Testículo/metabolismoRESUMEN
OBJECTIVE: To study the meiotic abnormalities during prophase I in an azoospermic man with t(1;21) reciprocal translocation. DESIGN: Analysis of synapses, recombination, and transcription inactivation in a testicular biopsy sample. SETTING: Research laboratory. PATIENT(S): One azoospermic patient with t(1;21) and five men with normal spermatogenesis. INTERVENTION(S): Immunostaining for SCP3, MLH1, and gamma-H2AX/BRCA1 was performed on biopsy to identify synapses, recombination, and transcriptional inactivation, respectively. MAIN OUTCOME MEASURE(S): Synapses, recombination, and transcriptional inactivation in meiosis I. RESULT(S): The t(1;21) carrier had a larger number of synaptonemal complexes with gaps and a lower rate (46%) of XY pairs with MLH1 foci than the controls (78%). The asynapsed quadrivalents, which were often associated with an XY body (84%), were frequently observed (96%) in pachytene cells of the translocation carrier. The variant histone gamma-H2AX and BRCA1 proteins were found to be located at the asynapsed quadrivalents. CONCLUSION(S): These results suggest that impaired synaptic integrity of translocated chromosomes may affect synapses, recombination frequency of XY pairs, and transcriptional activation of asynapsed areas, and consequently may impair fertility in men.