RESUMO
Embryonic aneuploidy from mis-segregation of chromosomes during meiosis causes pregnancy loss. Proper disjunction of homologous chromosomes requires the mismatch repair (MMR) genes MLH1 and MLH3, essential in mice for fertility. Variants in these genes can increase colorectal cancer risk, yet the reproductive impacts are unclear. To determine if MLH1/3 single nucleotide polymorphisms (SNPs) in human populations could cause reproductive abnormalities, we use computational predictions, yeast two-hybrid assays, and MMR and recombination assays in yeast, selecting nine MLH1 and MLH3 variants to model in mice via genome editing. We identify seven alleles causing reproductive defects in mice including female subfertility and male infertility. Remarkably, in females these alleles cause age-dependent decreases in litter size and increased embryo resorption, likely a consequence of fewer chiasmata that increase univalents at meiotic metaphase I. Our data suggest that hypomorphic alleles of meiotic recombination genes can predispose females to increased incidence of pregnancy loss from gamete aneuploidy.
Assuntos
Aborto Espontâneo/genética , Aneuploidia , Perda do Embrião/genética , Proteína 1 Homóloga a MutL/genética , Proteínas MutL/genética , Aborto Espontâneo/metabolismo , Aborto Espontâneo/fisiopatologia , Alelos , Animais , Troca Genética , Reparo de Erro de Pareamento de DNA , Perda do Embrião/fisiopatologia , Feminino , Recombinação Homóloga , Humanos , Tamanho da Ninhada de Vivíparos , Masculino , Meiose , Camundongos , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL/metabolismo , Gravidez , Reprodução , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Mammalian male fertility relies on complex inter- and intracellular signaling during spermatogenesis. Here we describe three alleles of the widely expressed A-kinase anchoring protein 9 (Akap9) gene, all of which cause gametogenic failure and infertility in the absence of marked somatic phenotypes. Akap9 disruption does not affect spindle nucleation or progression of prophase I of meiosis but does inhibit maturation of Sertoli cells, which continue to express the immaturity markers anti-Mullerian hormone and thyroid hormone receptor alpha in adults and fail to express the maturation marker p27(Kip1). Furthermore, gap and tight junctions essential for blood-testis barrier (BTB) organization are disrupted. Connexin43 (Cx43) and zona occludens-1 are improperly localized in Akap9 mutant testes, and Cx43 fails to compartmentalize germ cells near the BTB. These results identify and support a novel reproductive tissue-specific role for Akap9 in the coordinated regulation of Sertoli cells in the testis.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Células de Sertoli/citologia , Espermatogênese/genética , Proteínas de Ancoragem à Quinase A/genética , Animais , Hormônio Antimülleriano/genética , Hormônio Antimülleriano/metabolismo , Conexina 43/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Junções Comunicantes/ultraestrutura , Masculino , Meiose/genética , Camundongos , Camundongos Mutantes , Proteínas Associadas aos Microtúbulos/genética , Especificidade de Órgãos , Transporte Proteico , Células de Sertoli/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismo , Fuso Acromático/metabolismo , Receptores alfa dos Hormônios Tireóideos/genética , Receptores alfa dos Hormônios Tireóideos/metabolismo , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
The transcriptional regulation of mammalian meiosis is poorly characterized, owing to few genetic and ex vivo models. From a genetic screen, we identify the transcription factor MYBL1 as a male-specific master regulator of several crucial meiotic processes. Spermatocytes bearing a novel separation-of-function allele (Mybl1(repro9)) had subtle defects in autosome synapsis in pachynema, a high incidence of unsynapsed sex chromosomes, incomplete double-strand break repair on synapsed pachytene chromosomes and a lack of crossing over. MYBL1 protein appears in pachynema, and its mutation caused specific alterations in expression of diverse genes, including some translated postmeiotically. These data, coupled with chromatin immunoprecipitation (ChIP-chip) experiments and bioinformatic analysis of promoters, identified direct targets of MYBL1 regulation. The results reveal that MYBL1 is a master regulator of meiotic genes that are involved in multiple processes in spermatocytes, particularly those required for cell cycle progression through pachynema.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Meiose/fisiologia , Proteínas Proto-Oncogênicas c-myb/metabolismo , Espermatócitos/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Quebras de DNA de Cadeia Dupla , Feminino , Perfilação da Expressão Gênica , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Análise em Microsséries , Dados de Sequência Molecular , Mutação , Estágio Paquíteno/fisiologia , Proteínas Proto-Oncogênicas c-myb/genética , Alinhamento de Sequência , Espermatócitos/citologia , Espermatogênese/fisiologia , Transativadores/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
There is increasing evidence that defects in DNA double-strand-break (DSB) repair can cause chromosome instability, which may result in cancer. To identify novel DSB repair genes in mice, we performed a phenotype-driven mutagenesis screen for chromosome instability mutants using a flow cytometric peripheral blood micronucleus assay. Micronucleus levels were used as a quantitative indicator of chromosome damage in vivo. Among offspring derived from males mutagenized with the germline mutagen N-ethyl-N-nitrosourea (ENU), we identified a recessive mutation conferring elevated levels of spontaneous and radiation- or mitomycin C-induced micronuclei. This mutation, named chaos1 (chromosome aberration occurring spontaneously 1), was genetically mapped to a 1.3-Mb interval on chromosome 16 containing Polq, encoding DNA polymerase theta. We identified a nonconservative mutation in the ENU-derived allele, making it a strong candidate for chaos1. POLQ is homologous to Drosophila MUS308, which is essential for normal DNA interstrand crosslink repair and is unique in that it contains both a helicase and a DNA polymerase domain. While cancer susceptibility of chaos1 mutant mice is still under investigation, these data provide a practical paradigm for using a forward genetic approach to discover new potential cancer susceptibility genes using the surrogate biomarker of chromosome instability as a screen.