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1.
J Genet Genomics ; 49(11): 1029-1041, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35341968

RESUMO

Meiosis is essential for fertility in sexually reproducing species and this sophisticated process has been extensively studied. Notwithstanding these efforts, key factors involved in meiosis have not been fully characterized. In this study, we investigate the regulatory roles of zinc finger protein 541 (ZFP541) and its interacting protein potassium channel tetramerization domain containing 19 (KCTD19) in spermatogenesis. ZFP541 is expressed from leptotene to the round spermatid stage, while the expression of KCTD19 is initiated in pachytene. Depletion of Zfp541 or Kctd19 leads to infertility in male mice and delays progression from early to mid/late pachynema. In addition, Zfp541-/- spermatocytes show abnormal programmed DNA double-strand break repair, impaired crossover formation and resolution, and asynapsis of the XY chromosomes. ZFP541 interacts with KCTD19, histone deacetylase 1/2 (HDAC1/2), and deoxynucleotidyl transferase terminal-interacting protein 1 (DNTTIP1). Moreover, ZFP541 binds to and activates the expression of genes involved in meiosis and post-meiosis including Kctd19; in turn, KCTD19 promotes the transcriptional activation activity of ZFP541. Taken together, our studies reveal that the ZFP541/KCTD19 signaling complex, acting as a key transcription regulator, plays an indispensable role in male fertility by regulating pachytene progression.


Assuntos
Espermatócitos , Espermatogênese , Masculino , Camundongos , Animais , Espermatogênese/genética , Prófase Meiótica I , Estágio Paquíteno/genética , Meiose/genética , Proteínas Cromossômicas não Histona/genética , Fatores de Transcrição/genética
2.
Cell Rep ; 38(12): 110540, 2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35320728

RESUMO

The DSB machinery, which induces the programmed DNA double-strand breaks (DSBs) in the leptotene and zygotene stages during meiosis, is suppressed before the onset of the pachytene stage. However, the biological significance and underlying mechanisms remain largely unclear. Here, we report that ZFP541 is indispensable for the suppression of DSB formation after mid-pachytene. The deletion of Zfp541 in mice causes the aberrant recruitment of DSB machinery to chromosome axes and generation of massive DSBs in late pachytene and diplotene spermatocytes, leading to meiotic arrest at the diplotene stage. Integrated analysis of single-cell RNA sequencing (scRNA-seq) and chromatin immunoprecipitation (ChIP) sequencing data indicate that ZFP541 predominantly binds to promoters of pre-pachytene genes, including meiotic DSB formation-related genes (e.g., Prdm9 and Mei1) and their upstream activators (e.g., Meiosin and Rxra), and maintains their repression in pachytene spermatocytes. Our results reveal that ZFP541 functions as a transcriptional regulator in pachytene spermatocytes, orchestrating the transcriptome to ensure meiosis progression.


Assuntos
Prófase Meiótica I , Espermatócitos , Animais , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Histona-Lisina N-Metiltransferase/metabolismo , Masculino , Meiose , Camundongos , Estágio Paquíteno , Espermatócitos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Proc Natl Acad Sci U S A ; 119(3)2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-35022236

RESUMO

The fidelity of a signaling pathway depends on its tight regulation in space and time. Extracellular signal-regulated kinase (ERK) controls wide-ranging cellular processes to promote organismal development and tissue homeostasis. ERK activation depends on a reversible dual phosphorylation on the TEY motif in its active site by ERK kinase (MEK) and dephosphorylation by DUSPs (dual specificity phosphatases). LIP-1, a DUSP6/7 homolog, was proposed to function as an ERK (MPK-1) DUSP in the Caenorhabditis elegans germline primarily because of its phenotype, which morphologically mimics that of a RAS/let-60 gain-of-function mutant (i.e., small oocyte phenotype). Our investigations, however, reveal that loss of lip-1 does not lead to an increase in MPK-1 activity in vivo. Instead, we show that loss of lip-1 leads to 1) a decrease in MPK-1 phosphorylation, 2) lower MPK-1 substrate phosphorylation, 3) phenocopy of mpk-1 reduction-of-function (rather than gain-of-function) allele, and 4) a failure to rescue mpk-1-dependent germline or fertility defects. Moreover, using diverse genetic mutants, we show that the small oocyte phenotype does not correlate with increased ectopic MPK-1 activity and that ectopic increase in MPK-1 phosphorylation does not necessarily result in a small oocyte phenotype. Together, these data demonstrate that LIP-1 does not function as an MPK-1 DUSP in the C. elegans germline. Our results caution against overinterpretation of the mechanistic underpinnings of orthologous phenotypes, since they may be a result of independent mechanisms, and provide a framework for characterizing the distinct molecular targets through which LIP-1 may mediate its several germline functions.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Proteínas de Ciclo Celular/metabolismo , Células Germinativas/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proliferação de Células , Ativação Enzimática , Mutação/genética , Oócitos/citologia , Oócitos/metabolismo , Estágio Paquíteno , Fenótipo , Fosforilação , Proteínas Tirosina Fosfatases/genética , Especificidade por Substrato , Complexo Sinaptonêmico/metabolismo , Temperatura
4.
Plant Cell ; 34(1): 433-454, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-34718750

RESUMO

Plant growth and fertility strongly depend on environmental conditions such as temperature. Remarkably, temperature also influences meiotic recombination and thus, the current climate change will affect the genetic make-up of plants. To better understand the effects of temperature on meiosis, we followed male meiocytes in Arabidopsis thaliana by live cell imaging under three temperature regimes: at 21°C; at heat shock conditions of 30°C and 34°C; after an acclimatization phase of 1 week at 30°C. This work led to a cytological framework of meiotic progression at elevated temperature. We determined that an increase from 21°C to 30°C speeds up meiosis with specific phases being more amenable to heat than others. An acclimatization phase often moderated this effect. A sudden increase to 34°C promoted a faster progression of early prophase compared to 21°C. However, the phase in which cross-overs mature was prolonged at 34°C. Since mutants involved in the recombination pathway largely did not show the extension of this phase at 34°C, we conclude that the delay is recombination-dependent. Further analysis also revealed the involvement of the ATAXIA TELANGIECTASIA MUTATED kinase in this prolongation, indicating the existence of a pachytene checkpoint in plants, yet in a specialized form.


Assuntos
Arabidopsis/fisiologia , Pontos de Checagem do Ciclo Celular , Resposta ao Choque Térmico/fisiologia , Estágio Paquíteno , Meiose
5.
Toxicology ; 467: 153061, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-34936917

RESUMO

Deca-brominated diphenyl ether (BDE-209) is a common flame retardant utilized in electronic products, textiles, furniture, and upholstery materials. Environmental BDE-209 exposure results in spermatogenesis disorder, because of the characteristics of bioaccumulation, persistence, and probably toxicity. Meiotic prophase I is a crucial phase during spermatogenesis which is a key influential factor of normal sperm production. However, the effects of BDE-209 on meiotic prophase I during spermatogenesis are poorly understood. The present study aimed to evaluate whether BDE-209 exposure impairs meiotic prophase I during spermatogenesis of spermatocytes. We validated the effects of BDE-209 on alternations of meiotic prophase I in Balb/c male mice. Firstly, we analyzed sperm quality in cauda epididymis with decreasing sperm count, increasing abnormal sperm, and male reproductive dysfunction after exposure to BDE-209. Then, reactive oxygen species (ROS) and malondialdehyde (MDA) levels in testis and GC-2spd cells were significant increased after treated with BDE-209. Furthermore, we found that meiotic prophase I arrest at early-pachytene stage during spermatogenesis with increasing of DSBs damage and trimethylated histone H3 at lysine-4 (H3K4me3) in spermatocytes exposed to BDE-209. Finally, we conducted homologous recombination (HR) analyses to identify the progression of meiosis. The recombination markers, including DMC1 and RAD51, and crossover marker MLH1 were decreased during spermatogenesis after exposure to BDE-209. Collectively, our data indicated that BDE-209 has detrimental impacts on meiotic prophase I of spermatocytes in mice.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Éteres Difenil Halogenados/toxicidade , Estágio Paquíteno/efeitos dos fármacos , Espermatócitos/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Retardadores de Chama , Histonas/genética , Histonas/metabolismo , Masculino , Metilação , Camundongos Endogâmicos BALB C , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Processamento de Proteína Pós-Traducional , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Contagem de Espermatozoides , Espermatócitos/metabolismo , Espermatócitos/patologia , Testículo/metabolismo , Testículo/patologia
6.
Cell Rep ; 37(11): 110110, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34910909

RESUMO

Mechanisms driving the prolonged meiotic prophase I in mammals are poorly understood. RNA helicase YTHDC2 is critical for mitosis to meiosis transition. However, YTHDC2 is highly expressed in pachytene cells. Here we identify an essential role for YTHDC2 in meiotic progression. Specifically, YTHDC2 deficiency causes microtubule-dependent telomere clustering and apoptosis at the pachytene stage of prophase I. Depletion of YTHDC2 results in a massively dysregulated transcriptome in pachytene cells, with a tendency toward upregulation of genes normally expressed in mitotic germ cells and downregulation of meiotic transcripts. Dysregulation does not correlate with m6A status, and YTHDC2-bound mRNAs are enriched in genes upregulated in mutant germ cells, revealing that YTHDC2 primarily targets mRNAs for degradation. Furthermore, altered transcripts in mutant pachytene cells encode microtubule network proteins. Our results demonstrate that YTHDC2 regulates the pachytene stage by perpetuating a meiotic transcriptome and preventing microtubule network changes that could lead to telomere clustering.


Assuntos
Meiose , Microtúbulos/fisiologia , Estágio Paquíteno , RNA Helicases/fisiologia , Espermatócitos/citologia , Telômero , Transcriptoma , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espermatócitos/metabolismo
7.
Nat Commun ; 12(1): 5970, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645830

RESUMO

PIWI-interacting small RNAs (piRNAs) protect the germline genome and are essential for fertility. piRNAs originate from transposable element (TE) RNAs, long non-coding RNAs, or 3´ untranslated regions (3´UTRs) of protein-coding messenger genes, with the last being the least characterized of the three piRNA classes. Here, we demonstrate that the precursors of 3´UTR piRNAs are full-length mRNAs and that post-termination 80S ribosomes guide piRNA production on 3´UTRs in mice and chickens. At the pachytene stage, when other co-translational RNA surveillance pathways are sequestered, piRNA biogenesis degrades mRNAs right after pioneer rounds of translation and fine-tunes protein production from mRNAs. Although 3´UTR piRNA precursor mRNAs code for distinct proteins in mice and chickens, they all harbor embedded TEs and produce piRNAs that cleave TEs. Altogether, we discover a function of the piRNA pathway in fine-tuning protein production and reveal a conserved piRNA biogenesis mechanism that recognizes translating RNAs in amniotes.


Assuntos
Regiões 3' não Traduzidas , Fertilidade/genética , Biossíntese de Proteínas , RNA Interferente Pequeno/genética , Ribossomos/genética , Espermatogênese/genética , Animais , Sequência de Bases , Galinhas , Elementos de DNA Transponíveis , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estágio Paquíteno , RNA Interferente Pequeno/metabolismo , Ribossomos/metabolismo , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
8.
Nat Commun ; 12(1): 4674, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344879

RESUMO

In most organisms, the number and distribution of crossovers that occur during meiosis are tightly controlled. All chromosomes must receive at least one 'obligatory crossover' and crossovers are prevented from occurring near one another by 'crossover interference'. However, the mechanistic basis of this phenomenon of crossover interference has remained mostly mysterious. Using quantitative super-resolution cytogenetics and mathematical modelling, we investigate crossover positioning in the Arabidopsis thaliana wild-type, an over-expressor of the conserved E3 ligase HEI10, and a hei10 heterozygous line. We show that crossover positions can be explained by a predictive, diffusion-mediated coarsening model, in which large, approximately evenly-spaced HEI10 foci grow at the expense of smaller, closely-spaced clusters. We propose this coarsening process explains many aspects of Arabidopsis crossover positioning, including crossover interference. Consistent with this model, we also demonstrate that crossover positioning can be predictably modified in vivo simply by altering HEI10 dosage, with higher and lower dosage leading to weaker and stronger crossover interference, respectively. As HEI10 is a conserved member of the RING finger protein family that functions in the interference-sensitive pathway for crossover formation, we anticipate that similar mechanisms may regulate crossover positioning in diverse eukaryotes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Cromossômicas não Histona/metabolismo , Troca Genética/genética , Meiose/genética , Proteínas de Arabidopsis/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Simulação por Computador , Dosagem de Genes , Estágio Paquíteno/genética , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo
9.
PLoS Genet ; 17(8): e1009753, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34388164

RESUMO

Meiosis is essential for the generation of gametes and sexual reproduction, yet the factors and underlying mechanisms regulating meiotic progression remain largely unknown. Here, we showed that MTL5 translocates into nuclei of spermatocytes during zygotene-pachytene transition and ensures meiosis advances beyond pachytene stage. MTL5 shows strong interactions with MuvB core complex components, a well-known transcriptional complex regulating mitotic progression, and the zygotene-pachytene transition of MTL5 is mediated by its direct interaction with the component LIN9, through MTL5 C-terminal 443-475 residues. Male Mtl5c-mu/c-mu mice expressing the truncated MTL5 (p.Ser445Arg fs*3) that lacks the interaction with LIN9 and is detained in cytoplasm showed male infertility and spermatogenic arrest at pachytene stage, same as that of Mtl5 knockout mice, indicating that the interaction with LIN9 is essential for the nuclear translocation and function of MTL5 during meiosis. Our data demonstrated MTL5 translocates into nuclei during the zygotene-pachytene transition to initiate its function along with the MuvB core complex in pachytene spermatocytes, highlighting a new mechanism regulating the progression of male meiosis.


Assuntos
Meiose/fisiologia , Metalotioneína/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Pareamento Cromossômico/genética , Citoplasma , Proteínas de Ligação a DNA , Fertilidade/genética , Fertilidade/fisiologia , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Prófase Meiótica I/fisiologia , Metalotioneína/genética , Camundongos , Camundongos Endogâmicos C57BL , Estágio Paquíteno/genética , Espermatócitos/fisiologia , Espermatogênese/fisiologia , Testículo , Proteínas Supressoras de Tumor/fisiologia
10.
Nat Commun ; 12(1): 3184, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075040

RESUMO

During spermatogenesis, meiosis is accompanied by a robust alteration in gene expression and chromatin status. However, it remains elusive how the meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identify a protein complex that consists of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulators in mouse meiotic prophase progression. Our genetic study shows that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of the meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses identify that ZFP541 binds to and suppresses a broad range of genes whose function is associated with biological processes of transcriptional regulation and covalent chromatin modification. The present study demonstrates that a germ-cell specific complex that contains ZFP541 and KCTD19 promotes the progression of meiotic prophase towards completion in male mice, and triggers the reconstruction of the transcriptional network and chromatin organization leading to post-meiotic development.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , Estágio Paquíteno/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Espermátides/citologia , Espermatogênese/genética , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas Cromossômicas não Histona/genética , Modelos Animais de Doenças , Feminino , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Oócitos/citologia , Oócitos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA-Seq , Espermátides/metabolismo , Fatores de Transcrição/genética , Transcrição Genética
11.
PLoS Genet ; 17(5): e1009412, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33961623

RESUMO

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Genes Essenciais , Meiose , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Anáfase , Animais , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Pareamento Cromossômico , Sequência Conservada , Dano ao DNA , Evolução Molecular , Fertilidade/genética , Histona Desacetilase 1/metabolismo , Masculino , Prófase Meiótica I , Metáfase , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Estágio Paquíteno , Fenótipo , Espermátides/citologia , Espermatócitos/citologia , Espermatócitos/metabolismo , Testículo/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transgenes
12.
PLoS Genet ; 17(4): e1009485, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33831001

RESUMO

piRNAs are small non-coding RNAs required to maintain genome integrity and preserve RNA homeostasis during male gametogenesis. In murine adult testes, the highest levels of piRNAs are present in the pachytene stage of meiosis, but their mode of action and function remain incompletely understood. We previously reported that BTBD18 binds to 50 pachytene piRNA-producing loci. Here we show that spermatozoa in gene-edited mice lacking a BTBD18 targeted pachytene piRNA cluster on Chr18 have severe sperm head dysmorphology, poor motility, impaired acrosome exocytosis, zona pellucida penetration and are sterile. The mutant phenotype arises from aberrant formation of proacrosomal vesicles, distortion of the trans-Golgi network, and up-regulation of GOLGA2 transcripts and protein associated with acrosome dysgenesis. Collectively, our findings reveal central role of pachytene piRNAs in controlling spermiogenesis and male fertility.


Assuntos
Infertilidade Masculina/genética , RNA Interferente Pequeno/genética , Espermatogênese/genética , Espermatozoides/patologia , Acrossomo/patologia , Animais , Cromossomos/genética , Humanos , Infertilidade Masculina/patologia , Masculino , Meiose/genética , Camundongos , Estágio Paquíteno/genética , Espermátides/crescimento & desenvolvimento , Espermátides/patologia , Testículo/crescimento & desenvolvimento , Testículo/patologia
13.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671248

RESUMO

Cyclin-dependent kinases (CDKs) are crucial regulators of the eukaryotic cell cycle. The critical role of CDK2 in the progression of meiosis was demonstrated in a single mammalian species, the mouse. We used immunocytochemistry to study the localization of CDK2 during meiosis in seven rodent species that possess hetero- and homomorphic male sex chromosomes. To compare the distribution of CDK2 in XY and XX male sex chromosomes, we performed multi-round immunostaining of a number of marker proteins in meiotic chromosomes of the rat and subterranean mole voles. Antibodies to the following proteins were used: RAD51, a member of the double-stranded DNA break repair machinery; MLH1, a component of the DNA mismatch repair system; and SUN1, which is involved in the connection between the meiotic telomeres and nuclear envelope, alongside the synaptic protein SYCP3 and kinetochore marker CREST. Using an enhanced protocol, we were able to assess the distribution of as many as four separate proteins in the same meiotic cell. We showed that during prophase I, CDK2 localizes to telomeric and interstitial regions of autosomes in all species investigated (rat, vole, hamster, subterranean mole voles, and mole rats). In sex bivalents following synaptic specificity, the CDK2 signals were distributed in three different modes. In the XY bivalent in the rat and mole rat, we detected numerous CDK2 signals in asynaptic regions and a single CDK2 focus on synaptic segments, similar to the mouse sex chromosomes. In the mole voles, which have unique XX sex chromosomes in males, CDK2 signals were nevertheless distributed similarly to the rat XY sex chromosomes. In the vole, sex chromosomes did not synapse, but demonstrated CDK2 signals of varying intensity, similar to the rat X and Y chromosomes. In female mole voles, the XX bivalent had CDK2 pattern similar to autosomes of all species. In the hamster, CDK2 signals were revealed in telomeric regions in the short synaptic segment of the sex bivalent. We found that CDK2 signals colocalize with SUN1 and MLH1 signals in meiotic chromosomes in rats and mole voles, similar to the mouse. The difference in CDK2 manifestation at the prophase I sex chromosomes can be considered an example of the rapid chromosome evolution in mammals.


Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Mamíferos/metabolismo , Prófase Meiótica I , Cromossomos Sexuais/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Feminino , Masculino , Modelos Biológicos , Estágio Paquíteno , Ratos , Espermatócitos/metabolismo
14.
PLoS Genet ; 17(2): e1009265, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33635934

RESUMO

Piwi-interacting RNAs (piRNAs) play critical roles in protecting germline genome integrity and promoting normal spermiogenic differentiation. In mammals, there are two populations of piRNAs: pre-pachytene and pachytene. Transposon-rich pre-pachytene piRNAs are expressed in fetal and perinatal germ cells and are required for retrotransposon silencing, whereas transposon-poor pachytene piRNAs are expressed in spermatocytes and round spermatids and regulate mRNA transcript levels. MOV10L1, a germ cell-specific RNA helicase, is essential for the production of both populations of piRNAs. Although the requirement of the RNA helicase domain located in the MOV10L1 C-terminal region for piRNA biogenesis is well known, its large N-terminal region remains mysterious. Here we report a novel Mov10l1 mutation, named yama, in the Mov10l1 N-terminal region. The yama mutation results in a single amino acid substitution V229E. The yama mutation causes meiotic arrest, de-repression of transposable elements, and male sterility because of defects in pre-pachytene piRNA biogenesis. Moreover, restricting the Mov10l1 mutation effects to later stages in germ cell development by combining with a postnatal conditional deletion of a complementing wild-type allele causes absence of pachytene piRNAs, accumulation of piRNA precursors, polar conglomeration of piRNA pathway proteins in spermatocytes, and spermiogenic arrest. Mechanistically, the V229E substitution in MOV10L1 reduces its interaction with PLD6, an endonuclease that generates the 5' ends of piRNA intermediates. Our results uncover an important role for the MOV10L1-PLD6 interaction in piRNA biogenesis throughout male germ cell development.


Assuntos
Infertilidade Masculina/genética , Meiose/genética , Proteínas Mitocondriais/metabolismo , Fosfolipase D/metabolismo , RNA Helicases/metabolismo , RNA Interferente Pequeno/metabolismo , Retroelementos/genética , Espermatogênese/genética , Alelos , Animais , Inativação Gênica , Células Germinativas/metabolismo , Células Germinativas/patologia , Células HEK293 , Humanos , Masculino , Camundongos , Proteínas Mitocondriais/genética , Mutação , Estágio Paquíteno/genética , Fosfolipase D/genética , RNA Helicases/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espermátides/metabolismo , Espermatócitos/metabolismo , Testículo/metabolismo
15.
Proc Natl Acad Sci U S A ; 118(8)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33602822

RESUMO

Meiosis is a specialized cell division that creates haploid germ cells from diploid progenitors. Through differential RNA expression analyses, we previously identified a number of mouse genes that were dramatically elevated in spermatocytes, relative to their very low expression in spermatogonia and somatic organs. Here, we investigated in detail 1700102P08Rik, one of these genes, and independently conclude that it encodes a male germline-specific protein, in agreement with a recent report. We demonstrated that it is essential for pachynema progression in spermatocytes and named it male pachynema-specific (MAPS) protein. Mice lacking Maps (Maps-/- ) suffered from pachytene arrest and spermatocyte death, leading to male infertility, whereas female fertility was not affected. Interestingly, pubertal Maps-/- spermatocytes were arrested at early pachytene stage, accompanied by defects in DNA double-strand break (DSB) repair, crossover formation, and XY body formation. In contrast, adult Maps-/- spermatocytes only exhibited partially defective crossover but nonetheless were delayed or failed in progression from early to mid- and late pachytene stage, resulting in cell death. Furthermore, we report a significant transcriptional dysregulation in autosomes and XY chromosomes in both pubertal and adult Maps-/- pachytene spermatocytes, including failed meiotic sex chromosome inactivation (MSCI). Further experiments revealed that MAPS overexpression in vitro dramatically decreased the ubiquitination levels of cellular proteins. Conversely, in Maps-/- pachytene cells, protein ubiquitination was dramatically increased, likely contributing to the large-scale disruption in gene expression in pachytene cells. Thus, MAPS is a protein essential for pachynema progression in male mice, possibly in mammals in general.


Assuntos
Infertilidade Masculina/patologia , Meiose , Proteínas Nucleares/fisiologia , Estágio Paquíteno , Espermatócitos/patologia , Espermatogênese , Animais , Pareamento Cromossômico , Reparo do DNA , Feminino , Infertilidade Masculina/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cromossomos Sexuais , Espermatócitos/metabolismo
16.
J Vis Exp ; (167)2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33522502

RESUMO

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.


Assuntos
Permeabilidade da Membrana Celular , Separação Celular/métodos , DNA/metabolismo , Espermatócitos/citologia , Espermatogênese , Animais , Benzimidazóis/metabolismo , Sobrevivência Celular , Dissecação , Citometria de Fluxo , Fluorescência , Masculino , Meiose , Camundongos , Estágio Paquíteno , Espalhamento de Radiação , Espermátides/citologia , Coloração e Rotulagem , Testículo/citologia
17.
Nucleic Acids Res ; 49(5): 2609-2628, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33619545

RESUMO

In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation.


Assuntos
Quebras de DNA de Cadeia Dupla , Meiose/genética , ATPases Associadas a Diversas Atividades Celulares/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/fisiologia , Pareamento Cromossômico , Retroalimentação Fisiológica , Gametogênese , Camundongos , Estágio Paquíteno , Cromossomos Sexuais , Transdução de Sinais
18.
Nat Commun ; 12(1): 73, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397987

RESUMO

In the male germ cells of placental mammals, 26-30-nt-long PIWI-interacting RNAs (piRNAs) emerge when spermatocytes enter the pachytene phase of meiosis. In mice, pachytene piRNAs derive from ~100 discrete autosomal loci that produce canonical RNA polymerase II transcripts. These piRNA clusters bear 5' caps and 3' poly(A) tails, and often contain introns that are removed before nuclear export and processing into piRNAs. What marks pachytene piRNA clusters to produce piRNAs, and what confines their expression to the germline? We report that an unusually long first exon (≥ 10 kb) or a long, unspliced transcript correlates with germline-specific transcription and piRNA production. Our integrative analysis of transcriptome, piRNA, and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA clusters. Furthermore, a highly methylated promoter, often containing a low or intermediate level of CG dinucleotides, correlates with germline expression and somatic silencing of pachytene piRNA clusters. Pachytene piRNA precursor transcripts bind THOC1 and THOC2, THO complex subunits known to promote transcriptional elongation and mRNA nuclear export. Together, these features may explain why the major sources of pachytene piRNA clusters specifically generate these unique small RNAs in the male germline of placental mammals.


Assuntos
Epigênese Genética , Éxons/genética , Mamíferos/genética , Estágio Paquíteno/genética , RNA Interferente Pequeno/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Acetilação , Animais , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Histonas/metabolismo , Íntrons/genética , Masculino , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/genética , Testículo/metabolismo , Transcrição Genética
19.
Methods Mol Biol ; 2153: 267-286, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840786

RESUMO

Crossing-over between homologous chromosomes is essential for accurate chromosome segregation at anaphase-I of meiosis. Defective crossing-over is associated with infertility, pregnancy miscarriage, and congenital disease. This chapter presents optimized protocols for the analysis of meiotic crossovers at the cytological level in spermatocytes and oocytes from mouse. The first approach employs immunocytology to detect MLH1, a DNA mismatch-repair protein that specifically marks crossover sites in the pachytene stage of meiotic prophase-I. These immunocytological methods have general utility for the analysis of other recombination steps, such as initiation and DNA strand exchange. The second approach visualizes chiasmata, the points of physical exchange between homologous chromosomes that are present during the diakinesis and metaphase-I stages. Both approaches are readily adaptable to the analysis of crossing over in other vertebrate species.


Assuntos
Troca Genética , Proteína 1 Homóloga a MutL/metabolismo , Oócitos/citologia , Espermatócitos/citologia , Aneuploidia , Animais , Células Cultivadas , Cromossomos de Mamíferos/metabolismo , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Oócitos/metabolismo , Estágio Paquíteno , Espermatócitos/metabolismo
20.
Genes (Basel) ; 12(9)2021 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-34573341

RESUMO

We analyzed the synapsis and recombination between Z and W chromosomes in the oocytes of nine neognath species: domestic chicken Gallus gallus domesticus, grey goose Anser anser, black tern Chlidonias niger, common tern Sterna hirundo, pale martin Riparia diluta, barn swallow Hirundo rustica, European pied flycatcher Ficedula hypoleuca, great tit Parus major and white wagtail Motacilla alba using immunolocalization of SYCP3, the main protein of the lateral elements of the synaptonemal complex, and MLH1, the mismatch repair protein marking mature recombination nodules. In all species examined, homologous synapsis occurs in a short region of variable size at the ends of Z and W chromosomes, where a single recombination nodule is located. The remaining parts of the sex chromosomes undergo synaptic adjustment and synapse non-homologously. In 25% of ZW bivalents of white wagtail, synapsis and recombination also occur at the secondary pairing region, which probably resulted from autosome-sex chromosome translocation. Using FISH with a paint probe specific to the germline-restricted chromosome (GRC) of the pale martin on the oocytes of the pale martin, barn swallow and great tit, we showed that both maternally inherited songbird chromosomes (GRC and W) share common sequences.


Assuntos
Aves/genética , Pareamento Cromossômico/fisiologia , Recombinação Genética , Cromossomos Sexuais , Animais , Galinhas/genética , Feminino , Hibridização in Situ Fluorescente , Proteína 1 Homóloga a MutL/genética , Oócitos/fisiologia , Estágio Paquíteno/genética , Passeriformes/genética
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