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1.
Proc Natl Acad Sci U S A ; 120(30): e2219925120, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37459509

RESUMO

Infertility is a heterogeneous condition, with genetic causes thought to underlie a substantial fraction of cases. Genome sequencing is becoming increasingly important for genetic diagnosis of diseases including idiopathic infertility; however, most rare or minor alleles identified in patients are variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of these variants in key fertility genes, we functionally evaluated 11 missense variants in the genes ANKRD31, BRDT, DMC1, EXO1, FKBP6, MCM9, M1AP, MEI1, MSH4 and SEPT12 by generating genome-edited mouse models. Nine variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction (PPI) in the yeast two hybrid (Y2H) assay. Though these genes are essential for normal meiosis or spermiogenesis in mice, only one variant, observed in the MCM9 gene of a male infertility patient, compromised fertility or gametogenesis in the mouse models. To explore the disconnect between predictions and outcomes, we compared pathogenicity calls of missense variants made by ten widely used algorithms to 1) those annotated in ClinVar and 2) those evaluated in mice. All the algorithms performed poorly in terms of predicting the effects of human missense variants modeled in mice. These studies emphasize caution in the genetic diagnoses of infertile patients based primarily on pathogenicity prediction algorithms and emphasize the need for alternative and efficient in vitro or in vivo functional validation models for more effective and accurate VUS description to either pathogenic or benign categories.


Assuntos
Infertilidade Masculina , Mutação de Sentido Incorreto , Humanos , Masculino , Camundongos , Animais , Reprodução , Alelos , Infertilidade Masculina/genética , Modelos Animais de Doenças , Septinas/genética
2.
Nat Commun ; 12(1): 5005, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408140

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/metabolismo
3.
G3 (Bethesda) ; 4(2): 367-72, 2014 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-24362311

RESUMO

Spermiogenesis in mammals is the process by which the newly formed products of meiosis, haploid spermatids, undergo a dramatic morphological transformation from round cells into flagellated spermatozoa. The underlying genetic control of spermiogenesis is complicated and not well-characterized. We have used forward genetic screens in mice to illuminate the mechanisms of spermatozoon development. Here, we report that the oligoasthenoteratospermia in a male-specific infertility mutant (esgd12d) is attributable to disruption of a gene called Iqcg (IQ motif-containing G). The causality of the mutation was confirmed with a targeted null allele. Loss of Iqcg disrupts spermiogenesis such that tail formation either occurs incompletely or breaks apart from the sperm heads. Orthologs are present in diverse species as distant as hemichordates, mollusks, and green algae. Consistent with a conserved role in flagellar formation and/or function, the orthologous Chlamydomonas protein is present in that organism's flagella. Because IQ motif-containing genes typically regulate calmodulin (CaM), which in turn can impact the actin cytoskeleton, these findings suggest a potential role for localized calcium signaling in sperm flagellum morphogenesis.


Assuntos
Proteínas de Membrana/genética , Espermatogênese/genética , Animais , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação
4.
Genetics ; 194(2): 447-57, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23608191

RESUMO

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/metabolismo
5.
Development ; 138(15): 3319-30, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21750041

RESUMO

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 Gênica
6.
BMC Genet ; 11: 106, 2010 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-21118569

RESUMO

BACKGROUND: Forward genetic screens in mice provide an unbiased means to identify genes and other functional genetic elements in the genome. Previously, a large scale ENU mutagenesis screen was conducted to query the functional content of a ~50 Mb region of the mouse genome on proximal Chr 5. The majority of phenotypic mutants recovered were embryonic lethals. RESULTS: We report the high resolution genetic mapping, complementation analyses, and positional cloning of mutations in the target region. The collection of identified alleles include several with known or presumed functions for which no mutant models have been reported (Tbc1d14, Nol14, Tyms, Cad, Fbxl5, Haus3), and mutations in genes we or others previously reported (Tapt1, Rest, Ugdh, Paxip1, Hmx1, Otoe, Nsun7). We also confirmed the causative nature of a homeotic mutation with a targeted allele, mapped a lethal mutation to a large gene desert, and localized a spermiogenesis mutation to a region in which no annotated genes have coding mutations. The mutation in Tbc1d14 provides the first implication of a critical developmental role for RAB-GAP-mediated protein transport in early embryogenesis. CONCLUSION: This collection of alleles contributes to the goal of assigning biological functions to all known genes, as well as identifying novel functional elements that would be missed by reverse genetic approaches.


Assuntos
Mapeamento Cromossômico , Cromossomos/efeitos dos fármacos , Análise Mutacional de DNA , Desenvolvimento Embrionário/genética , Camundongos/genética , Mutação , Animais , Clonagem Molecular , Etilnitrosoureia/toxicidade , Genes Letais , Teste de Complementação Genética , Masculino , Camundongos Endogâmicos C57BL , Deleção de Sequência , Espermatogênese/genética
7.
Dev Biol ; 317(1): 72-82, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18353305

RESUMO

A novel mutation, repro5, was isolated in a forward genetic screen for infertility mutations induced by ENU mutagenesis. Homozygous mutant mice were phenotypically normal but were infertile. Oocytes from mutant females appeared normal, but were severely maturation-defective in that they had reduced ability to progress to metaphase II (MII), and those reaching MII were unable to progress beyond the two pronuclei stage following in vitro fertilization (IVF). Mutant males exhibited defective spermiogenesis, resulting in oligoasthenoteratospermia. Genetic mapping, positional cloning, and complementation studies with a disruption allele led to the identification of a mutation in Brwd1 (Bromodomain and WD repeat domain containing 1) as the causative lesion. Bromodomain-containing proteins typically interact with regions of chromatin containing histones hyperacetylated at lysine residues, a characteristic of chromatin in early spermiogenesis before eventual replacement of histones by the protamines. Previous data indicated that Brwd1 is broadly expressed, encoding a putative transcriptional regulator that is believed to act on chromatin through interactions with the Brg1-dependent SWI/SNF chromatin-remodeling pathway. Brwd1 represents one of a small number of genes whose elimination disrupts gametogenesis in both sexes after the major events of meiotic prophase I have been completed.


Assuntos
Oogênese , Espermatogênese , Animais , Clonagem Molecular , Etilnitrosoureia , Masculino , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese , Oócitos/citologia , Oócitos/metabolismo , Caracteres Sexuais
8.
PLoS Genet ; 3(8): e139, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17784788

RESUMO

Crossing over during meiotic prophase I is required for sexual reproduction in mice and contributes to genome-wide genetic diversity. Here we report on the characterization of an N-ethyl-N-nitrosourea-induced, recessive allele called mei4, which causes sterility in both sexes owing to meiotic defects. In mutant spermatocytes, chromosomes fail to congress properly at the metaphase plate, leading to arrest and apoptosis before the first meiotic division. Mutant oocytes have a similar chromosomal phenotype but in vitro can undergo meiotic divisions and fertilization before arresting. During late meiotic prophase in mei4 mutant males, absence of cyclin dependent kinase 2 and mismatch repair protein association from chromosome cores is correlated with the premature separation of bivalents at diplonema owing to lack of chiasmata. We have identified the causative mutation, a transversion in the 5' splice donor site of exon 1 in the mouse ortholog of Human Enhancer of Invasion 10 (Hei10; also known as Gm288 in mouse and CCNB1IP1 in human), a putative B-type cyclin E3 ubiquitin ligase. Importantly, orthologs of Hei10 are found exclusively in deuterostomes and not in more ancestral protostomes such as yeast, worms, or flies. The cloning and characterization of the mei4 allele of Hei10 demonstrates a novel link between cell cycle regulation and mismatch repair during prophase I.


Assuntos
Proteínas de Ciclo Celular/genética , Troca Genética/genética , Prófase Meiótica I/genética , Mutação , Ubiquitina-Proteína Ligases/genética , Proteínas Adaptadoras de Transdução de Sinal , Alelos , Animais , Pareamento Incorreto de Bases/genética , Bovinos , Proteínas de Ciclo Celular/fisiologia , Quinase 2 Dependente de Ciclina/deficiência , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Recombinação Genética , Ubiquitina-Proteína Ligases/fisiologia
9.
PLoS Biol ; 5(5): e105, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17425408

RESUMO

DMC1 is a meiosis-specific homolog of bacterial RecA and eukaryotic RAD51 that can catalyze homologous DNA strand invasion and D-loop formation in vitro. DMC1-deficient mice and yeast are sterile due to defective meiotic recombination and chromosome synapsis. The authors identified a male dominant sterile allele of Dmc1, Dmc1(Mei11), encoding a missense mutation in the L2 DNA binding domain that abolishes strand invasion activity. Meiosis in male heterozygotes arrests in pachynema, characterized by incomplete chromosome synapsis and no crossing-over. Young heterozygous females have normal litter sizes despite having a decreased oocyte pool, a high incidence of meiosis I abnormalities, and susceptibility to premature ovarian failure. Dmc1(Mei11) exposes a sex difference in recombination in that a significant portion of female oocytes can compensate for DMC1 deficiency to undergo crossing-over and complete gametogenesis. Importantly, these data demonstrate that dominant alleles of meiosis genes can arise and propagate in populations, causing infertility and other reproductive consequences due to meiotic prophase I defects.


Assuntos
Proteínas de Ciclo Celular/genética , Infertilidade Masculina/genética , Meiose/genética , Proteínas Nucleares/genética , Alelos , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Infertilidade Feminina/patologia , Masculino , Camundongos , Oócitos/citologia , Ovário/patologia , Proteínas de Ligação a Fosfato , Recombinação Genética/genética
10.
Biol Reprod ; 69(5): 1615-25, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12855593

RESUMO

The genetic control of mammalian gametogenesis is inadequately characterized because of a lack of mutations causing infertility. To further the discovery of genes required for mammalian gametogenesis, phenotype-driven screens were performed in mice using random chemical mutagenesis of whole animals and embryonic stem cells. Eleven initial mutations are reported here that affect proliferation of germ cells, meiosis, spermiogenesis, and spermiation. Nine of the mutations have been mapped genetically. These preliminary studies provide baselines for estimating the number of genes required for gametogenesis and offer guidance in conducting new genetic screens that will accelerate and optimize mutant discovery. This report demonstrates the efficacy and expediency of mutagenesis to identify new genes required for mammalian gamete development.


Assuntos
Gametogênese/genética , Mutação/genética , Reprodução/genética , Animais , Mapeamento Cromossômico , Etilnitrosoureia/farmacologia , Feminino , Genótipo , Infertilidade/genética , Masculino , Meiose/genética , Camundongos , Mutagênese/efeitos dos fármacos , Testes de Mutagenicidade , Mutagênicos/farmacologia , Oócitos/fisiologia , Fenótipo , Gravidez
11.
Genetics ; 163(3): 1031-40, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12663541

RESUMO

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.


Assuntos
Mapeamento Cromossômico , Cromossomos/genética , Dano ao DNA , Reparo do DNA/genética , Etilnitrosoureia/toxicidade , Camundongos Endogâmicos C3H/genética , Camundongos Endogâmicos C57BL/genética , Mutagênese , Animais , Cromossomos/efeitos dos fármacos , Cruzamentos Genéticos , DNA Polimerase Dirigida por DNA/genética , Feminino , Masculino , Camundongos , Testes para Micronúcleos , Modelos Genéticos , Dados de Sequência Molecular , Mutagênicos/toxicidade , Fenótipo , Transcrição Gênica , DNA Polimerase teta
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