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1.
J Agric Food Chem ; 69(40): 11847-11855, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34609142

RESUMO

Estrogen and its analogues are ubiquitous in agricultural environments, with large biological functions of oocyte development. Gap junction intercellular communications (GJICs) are the structural basis in cumulus-oocyte complexes (COCs) and regulate oocyte maturation and developmental material transport through a number of pathways. This study mainly determines the effect and potential mechanism of estrogen (17ß-estradiol) in regulating GJICs in porcine COCs. In our study, 17ß-estradiol increased porcine nuclear maturation in a time-dependent manner. The analysis revealed that 17ß-estradiol upregulated the autophagy in COCs during in vitro maturation. In contrast with the control, 17ß-estradiol decreased GJICs in a time-dependent manner between cumulus cells and oocytes, while it was consistent with the control group at 24 h. Carbenoxolone (CBX) blocks GJICs as a negative control group used in our system. Autophagy inhibitor autophinib decreased oocyte maturation, and the reduced nuclear maturation treated with autophinib was abolished by 17ß-estradiol. Besides, the upregulation effect of autophinib on GJICs and transzonal projections (TZPs) was decreased by 17ß-estradiol. 17ß-Estradiol could reduce serine 368 phosphorylation of connexin 43 (Cx43) protein by autophinib in porcine COCs. These results were dependent upon the MEK/ERK signaling pathway. Furthermore, 17ß-estradiol-induced GJICs and Cx43 phosphorylation were inhibited by autophinib or the MEK/ERK pathway inhibitors (Trametinib and FR 180204), indicating that 17ß-estradiol regulated GJICs through the MEK/ERK signaling pathway. In conclusion, 17ß-estradiol improves the autophagy-mediated nuclear maturation with downregulating GJICs and TZPs in porcine COCs. Such an effect occurs by phosphorylation of Cx43, which was regulated via the MEK/ERK signaling pathway.


Assuntos
Conexina 43 , Sistema de Sinalização das MAP Quinases , Animais , Autofagia , Conexina 43/genética , Conexina 43/metabolismo , Estradiol/metabolismo , Estradiol/farmacologia , Feminino , Junções Comunicantes/metabolismo , Meiose , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Oócitos/metabolismo , Fosforilação , Transdução de Sinais , Suínos
2.
Ecotoxicol Environ Saf ; 225: 112783, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34544023

RESUMO

Sudan I is one of the industry dyes and widely used in cosmetics, wax agent, solvent and textile. Sudan I has multiple toxicity such as carcinogenicity, mutagenicity, genotoxicity and oxidative damage. However, Sudan I has been illegally used as colorant in food products, triggering worldwide attention about food safety. Nevertheless, the toxicity of Sudan I on reproduction, particularly on oocyte maturation is still unclear. In the present study, using mouse in vivo models, we report the toxicity effects of Sudan I on mouse oocyte. The results reflect that Sudan I exposure disrupts spindle organization and chromosomes alignment as well as cortical actin distribution, thus leading to the failure of polar body extrusion. Based on the transcriptome results, it is found that the exposure of Sudan I leads to the change in expression of 764 genes. Moreover, it's further reflected that the damaging effects of Sudan I are mediated by the destruction of mitochondrial functions, which induces the accumulated ROS to stimulate oxidative stress-induced apoptosis. As an endogenous hormone, melatonin within the ovarian follicle plays function on improving oocyte quality and female reproduction by efficiently suppressing oxidative stress. Moreover, melatonin supplementation also improves oocyte quality and increases fertilization rate during in vitro culture. Consistent with these, we find that in vivo supplementation of melatonin efficaciously suppresses mitochondrial dysfunction and the accompanying apoptosis, thus reverses oocyte meiotic deteriorations. Collectively, our results prove the reproduction toxicity of Sudan I for the exposure of Sudan I reduces the oocyte quality, and demonstrate the protective effects of melatonin against Sudan I-induced meiotic deteriorations.


Assuntos
Melatonina , Animais , Apoptose , Feminino , Meiose , Melatonina/metabolismo , Melatonina/farmacologia , Camundongos , Mitocôndrias , Naftóis , Oócitos/metabolismo , Estresse Oxidativo
3.
Nat Methods ; 18(9): 1060-1067, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34480159

RESUMO

N6-methyladenosine (m6A) is the most prevalent modification of messenger RNA in mammals. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels: site, gene and sample. Current approaches address these needs in a limited manner. Here we develop m6A-seq2, relying on multiplexed m6A-immunoprecipitation of barcoded and pooled samples. m6A-seq2 allows a big increase in throughput while reducing technical variability, requirements of input material and cost. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an orthogonal alternative to mass spectrometry-based approaches. Finally, we develop a computational approach for gene-level quantitation of m6A. We demonstrate that using this metric, roughly 30% of the variability in RNA half life in mouse embryonic stem cells can be explained, establishing m6A as a main driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-mediated regulation at three different levels.


Assuntos
Adenosina/análogos & derivados , Estabilidade de RNA/genética , Análise de Sequência de RNA/métodos , Adenosina/análise , Adenosina/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Expressão Gênica , Meia-Vida , Meiose , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/fisiologia , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Leveduras/genética
4.
Nat Commun ; 12(1): 4941, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400639

RESUMO

Plant small RNAs are important regulatory elements that fine-tune gene expression and maintain genome integrity by silencing transposons. Reproductive organs of monocots produce abundant phased, small interfering RNAs (phasiRNAs). The 21-nt reproductive phasiRNAs triggered by miR2118 are highly enriched in pre-meiotic anthers, and have been found in multiple eudicot species, in contrast with prior reports of monocot specificity. The 24-nt reproductive phasiRNAs are triggered by miR2275, and are highly enriched during meiosis in many angiosperms. Here, we report the widespread presence of the 21-nt reproductive phasiRNA pathway in eudicots including canonical and non-canonical microRNA (miRNA) triggers of this pathway. In eudicots, these 21-nt phasiRNAs are enriched in pre-meiotic stages, a spatiotemporal distribution consistent with that of monocots and suggesting a role in anther development. Although this pathway is apparently absent in well-studied eudicot families including the Brassicaceae, Solanaceae and Fabaceae, our work in eudicots supports an earlier singular finding in spruce, a gymnosperm, indicating that the pathway of 21-nt reproductive phasiRNAs emerged in seed plants and was lost in some lineages.


Assuntos
Magnoliopsida/metabolismo , Nucleotídeos/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/metabolismo , Sementes/metabolismo , Fragaria/genética , Fragaria/metabolismo , Regulação da Expressão Gênica de Plantas , Meiose , MicroRNAs/genética , Filogenia , Picea/genética , Proteínas de Plantas/genética , RNA de Cadeia Dupla/metabolismo , Solanaceae/metabolismo , Transcriptoma
5.
Nat Commun ; 12(1): 4802, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376665

RESUMO

During meiosis, gene expression is silenced in aberrantly unsynapsed chromatin and in heterogametic sex chromosomes. Initiation of sex chromosome silencing is disrupted in meiocytes with sex chromosome-autosome translocations. To determine whether this is due to aberrant synapsis or loss of continuity of sex chromosomes, we engineered Caenorhabditis elegans nematodes with non-translocated, bisected X chromosomes. In early meiocytes of mutant males and hermaphrodites, X segments are enriched with euchromatin assembly markers and active RNA polymerase II staining, indicating active transcription. Analysis of RNA-seq data showed that genes from the X chromosome are upregulated in gonads of mutant worms. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, our data indicate that unsynapsed X segments are transcribed. Therefore, our results suggest that sex chromosome chromatin has a unique character that facilitates its meiotic expression when its continuity is lost, regardless of whether or not it is synapsed.


Assuntos
Caenorhabditis elegans/genética , Inativação Gênica , Meiose/genética , Cromossomo X/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Pareamento Cromossômico/genética , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Histonas/metabolismo , Hibridização in Situ Fluorescente , Masculino , Microscopia de Fluorescência , Transcrição Genética
6.
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
7.
N Engl J Med ; 385(8): 707-719, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34347949

RESUMO

BACKGROUND: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility. METHODS: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing. RESULTS: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in PNLDC1: the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying PNLDC1 mutations. CONCLUSIONS: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.).


Assuntos
Azoospermia/genética , Exorribonucleases/genética , Infertilidade Masculina/genética , Meiose/fisiologia , Mutação , RNA Interferente Pequeno/metabolismo , Testículo/patologia , Adulto , Azoospermia/fisiopatologia , Biópsia , Expressão Gênica , Humanos , Masculino , Fenótipo , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/ultraestrutura , Análise de Sequência de RNA , Testículo/metabolismo , Sequenciamento Completo do Exoma
8.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360715

RESUMO

Protein arginine methyltransferase 1 (PRMT1) is a major enzyme responsible for the formation of methylarginine in mammalian cells; however, its function in vivo is not well understood due to its early embryonic lethality in null mice exhibiting spontaneous DNA damage, cell cycle delays, and defects in check point activation. Here, we generated germ cell-specific Prmt1 knock-out (KO) mice to evaluate the function of PRMT1 in spermatogenesis. Our findings demonstrate that PRMT1 is vital for male fertility in mice. Spermatogenesis in Prmt1 KO mice was arrested at the zygotene-like stage of the first meiotic division due to an elevated number of DNA double-strand breaks (DSBs). There was a loss of methylation in meiotic recombination 11 (MRE11), the key endonuclease in MRE11/RAD50/NBS 1 (MRN) complex, resulting in the accumulation of SPO11 protein in DSBs. The ATM-mediated negative feedback control over SPO11 was lost and, consequently, the repair pathway of DSBs was highly affected in PRMT1 deficient male germ cells. Our findings provide a novel insight into the role of PRMT1-mediated asymmetric demethylation in mouse spermatogenesis.


Assuntos
Células Germinativas/enzimologia , Meiose , Proteína-Arginina N-Metiltransferases/metabolismo , Espermatogênese , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína-Arginina N-Metiltransferases/genética
9.
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
10.
Cell Prolif ; 54(10): e13119, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34435400

RESUMO

OBJECTIVES: Histone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood. MATERIALS AND METHODS: HDAC8-targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI-34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ-tubulin dynamics and acetylation level of α-tubulin. RESULTS: We observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ-tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI-34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α-tubulin. CONCLUSIONS: Collectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation.


Assuntos
Histona Desacetilases/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Fuso Acromático/metabolismo , Acetilação/efeitos dos fármacos , Animais , Fenômenos Biológicos/efeitos dos fármacos , Cromossomos/efeitos dos fármacos , Cromossomos/metabolismo , Cromossomos/fisiologia , Feminino , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Meiose/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/fisiologia , Suínos , Tubulina (Proteína)/metabolismo
11.
Nat Struct Mol Biol ; 28(8): 671-680, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34373645

RESUMO

Meiotic cells invoke breast cancer susceptibility gene 2 (BRCA2) to repair programmed double-stranded DNA breaks and accomplish homologous recombination. The meiosis-specific protein MEILB2 facilitates BRCA2 recruitment to meiotic recombination sites. Here, we combine crystallography, biochemical analysis and a mouse meiosis model to reveal a robust architecture that ensures meiotic BRCA2 recruitment. The crystal structure of the MEILB2-BRCA2 complex reveals how two MEILB2 homodimers sandwich two chains of BRCA2 to afford a 4:2 architecture. The sandwich lacks close contact between the two MEILB2 dimers or the two BRCA2 chains. Instead, the two halves of each BRCA2 chain bridge two MEILB2 subunits from different homodimers to form the MEILB2-BRCA2-MEILB2 sandwich. Several identical residues from the two MEILB2 subunits are employed to engage the BRCA2 halves, justifying their strict conservation. Mutational analysis of the interface reveals a synergistic mechanism for MEILB2-BRCA2 recruitment during meiosis. Overall, these studies demonstrate how BRCA2 efficiently localizes in the cell to facilitate meiosis.


Assuntos
Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Meiose/genética , Reparo de DNA por Recombinação/genética , Animais , Proteína BRCA2/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cristalografia por Raios X , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína
12.
Toxicology ; 460: 152884, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34358620

RESUMO

Perfluorodecanoic acid (PFDA) is a member of the perfluoroalkyl substances, which are toxic to organic functions. Recently, it has been found in follicular fluid, seriously interfering with reproduction. Follicular fluid provides the oocyte with necessary resources during the process of oocytes maturation. However, the effects of PFDA on the oocyte need investigation. Our study evaluated the impacts of PFDA on the meiosis and development potential of mouse oocytes by exposing oocytes to PFDA in vitro at 350, 400, and 450 µM concentrations. The results showed that exposure to PFDA resulted in the first meiotic prophase arrest by obstructing the function of the maturation-promoting factor. It also induced the dysfunction of the spindle assembly checkpoint, expedited the progression of the first meiotic process, and increased the risk of aneuploidy. The oocytes treated with PFDA had a broken cytoskeleton which also contributed to meiotic maturation failure. Besides, PFDA exposure caused mitochondria defections, increased the reactive oxygen species level in oocytes, and consequently induced oocyte apoptosis. Moreover, PFDA produced epigenetic modifications in oocytes and increased the frequency of mature oocytes with declined development potential. In summary, our data indicated that PFDA disturbs the meiotic process and induces oocyte quality deterioration.


Assuntos
Ácidos Decanoicos/toxicidade , Fluorcarbonetos/toxicidade , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Relação Dose-Resposta a Droga , Feminino , Humanos , Fator Promotor de Maturação/metabolismo , Meiose/fisiologia , Camundongos , Camundongos Endogâmicos ICR
13.
Elife ; 102021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34342579

RESUMO

Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in synchronized sea star oocytes from prophase I through the first embryonic cleavage. Although we found that protein levels were broadly stable, our analysis reveals that dynamic waves of phosphorylation underlie each meiotic stage. We found that the phosphatase PP2A-B55 is reactivated at the meiosis I/meiosis II (MI/MII) transition, resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI/MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after MI. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 imposes specific phosphoregulated behaviors that distinguish the two meiotic divisions.


Assuntos
Meiose , Oócitos/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Estrelas-do-Mar/genética , Animais , Fosforilação , Estrelas-do-Mar/enzimologia
14.
Nat Struct Mol Biol ; 28(8): 681-693, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34373646

RESUMO

The synaptonemal complex (SC) is a supramolecular protein assembly that mediates synapsis between homologous chromosomes during meiosis. SC elongation along the chromosome length (up to 24 µm) depends on its midline α-fibrous component SYCE2-TEX12. Here, we report X-ray crystal structures of human SYCE2-TEX12 as an individual building block and on assembly within a fibrous lattice. We combine these structures with mutagenesis, biophysics and electron microscopy to reveal the hierarchical mechanism of SYCE2-TEX12 fiber assembly. SYCE2-TEX12's building blocks are 2:2 coiled coils that dimerize into 4:4 hetero-oligomers and interact end-to-end and laterally to form 10-nm fibers that intertwine within 40-nm bundled micrometer-long fibers that define the SC's midline structure. This assembly mechanism bears striking resemblance with intermediate filament proteins vimentin, lamin and keratin. Thus, SYCE2-TEX12 exhibits behavior typical of cytoskeletal proteins to provide an α-fibrous SC backbone that structurally underpins synaptic elongation along meiotic chromosomes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Pareamento Cromossômico/fisiologia , Complexo Sinaptonêmico/metabolismo , Cristalografia por Raios X , Proteínas do Citoesqueleto/metabolismo , Humanos , Queratinas/metabolismo , Laminas/metabolismo , Meiose/fisiologia , Estrutura Quaternária de Proteína , Vimentina/metabolismo
15.
Nat Commun ; 12(1): 4605, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326328

RESUMO

BRCA2 and its interactors are required for meiotic homologous recombination (HR) and fertility. Loss of HSF2BP, a BRCA2 interactor, disrupts HR during spermatogenesis. We test the model postulating that HSF2BP localizes BRCA2 to meiotic HR sites, by solving the crystal structure of the BRCA2 fragment in complex with dimeric armadillo domain (ARM) of HSF2BP and disrupting this interaction in a mouse model. This reveals a repeated 23 amino acid motif in BRCA2, each binding the same conserved surface of one ARM domain. In the complex, two BRCA2 fragments hold together two ARM dimers, through a large interface responsible for the nanomolar affinity - the strongest interaction involving BRCA2 measured so far. Deleting exon 12, encoding the first repeat, from mBrca2 disrupts BRCA2 binding to HSF2BP, but does not phenocopy HSF2BP loss. Thus, results herein suggest that the high-affinity oligomerization-inducing BRCA2-HSF2BP interaction is not required for RAD51 and DMC1 recombinase localization in meiotic HR.


Assuntos
Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Espermatogênese/fisiologia , Animais , Proteína BRCA2/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cristalografia por Raios X/métodos , Feminino , Recombinação Homóloga , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Meiose , Camundongos , Modelos Animais , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência
16.
Theriogenology ; 172: 239-254, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34298284

RESUMO

Meiotic recombination is key to the repair of DNA double-strand break damage, provide a link between homologs for proper chromosome segregation as well as ensure genetic diversity in organisms. Defects in recombination often lead to sterility. The ubiquitously expressed Rad51 and the meiosis-specific DMC1 are two closely related recombinases that catalyze the key strand invasion and exchange step of meiotic recombination. This study cloned and sequenced the coding region of cattle-yak Rad51 and determined its mRNA and protein expression levels, evaluated its molecular and evolutionary relationship as well as evaluated the histo-morphological structure of testes in the yellow cattle, yak and the sterile cattle-yak hybrid. The Rad51 gene was amplified using PCR, cloned and sequenced using testicular cDNA from yak and cattle-yak. Real-time PCR was used to examine the expression levels of Rad51/DMC1 mRNA in the cattle, yak and cattle-yak testis while western blotting, immunofluorescence and immunohistochemistry were used to assess the protein expression and localization of Rad51/DMC1 protein in the testicular tissue sections. The results revealed that the mRNA and protein expression of Rad51 and DMC1 are extremely low in the male cattle-yak testis with a corresponding higher incidence of germ cell apoptosis. There was also thinning of the germinal epithelium possibly due to the depletion of the germ cells leading to the widening of the lumen area of the cattle-yak seminiferous tubule. Our findings provide support for the hypothesis that the low expression of Rad51 and DMC1 may contribute to the male hybrid sterility in the cattle-yak.


Assuntos
Reparo do DNA , Testículo , Animais , Bovinos/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Recombinação Homóloga , Masculino , Meiose , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Testículo/metabolismo
17.
Int J Mol Sci ; 22(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205521

RESUMO

The developmental process of inflorescence and gametophytes is vital for sexual reproduction in rice. Multiple genes and conserved miRNAs have been characterized to regulate the process. The changes of miRNAs expression during the early development of rice inflorescence remain unknown. In this study, the analysis of miRNAs profiles in the early stage of rice inflorescence development identified 671 miRNAs, including 67 known and 44 novel differentially expressed miRNAs (DEMs). Six distinct clusters of miRNAs expression patterns were detected, and Cluster 5 comprised 110 DEMs, including unconserved, rice-specific osa-miR5506. Overexpression of osa-miR5506 caused pleiotropic abnormalities, including over- or under-developed palea, various numbers of floral organs and spikelet indeterminacy. In addition, the defects of ovaries development were frequently characterized by multiple megasporocytes, ovule-free ovary, megasporocyte degenerated and embryo sac degenerated in the transgenic lines. osa-miR5506 targeted REM transcription factor LOC_Os03g11370. Summarily, these results demonstrated that rice-specific osa-miR5506 plays an essential role in the regulation of floral organ number, spikelet determinacy and female gametophyte development in rice.


Assuntos
Inflorescência/crescimento & desenvolvimento , MicroRNAs/metabolismo , Oryza/metabolismo , Óvulo Vegetal/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Meiose , Oryza/genética , Oryza/crescimento & desenvolvimento , Plantas Geneticamente Modificadas
18.
Nucleic Acids Res ; 49(13): 7537-7553, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197600

RESUMO

The synaptonemal complex (SC) is a proteinaceous structure that mediates homolog engagement and genetic recombination during meiosis. In budding yeast, Zip-Mer-Msh (ZMM) proteins promote crossover (CO) formation and initiate SC formation. During SC elongation, the SUMOylated SC component Ecm11 and the Ecm11-interacting protein Gmc2 facilitate the polymerization of Zip1, an SC central region component. Through physical recombination, cytological, and genetic analyses, we found that ecm11 and gmc2 mutants exhibit chromosome-specific defects in meiotic recombination. CO frequencies on a short chromosome (chromosome III) were reduced, whereas CO and non-crossover frequencies on a long chromosome (chromosome VII) were elevated. Further, in ecm11 and gmc2 mutants, more double-strand breaks (DSBs) were formed on a long chromosome during late prophase I, implying that the Ecm11-Gmc2 (EG) complex is involved in the homeostatic regulation of DSB formation. The EG complex may participate in joint molecule (JM) processing and/or double-Holliday junction resolution for ZMM-dependent CO-designated recombination. Absence of the EG complex ameliorated the JM-processing defect in zmm mutants, suggesting a role for the EG complex in suppressing ZMM-independent recombination. Our results suggest that the SC central region functions as a compartment for sequestering recombination-associated proteins to regulate meiosis specificity during recombination.


Assuntos
Proteínas de Ciclo Celular/genética , Troca Genética , Quebras de DNA de Cadeia Dupla , Meiose/genética , Proteínas de Saccharomyces cerevisiae/genética , Complexo Sinaptonêmico/metabolismo , Cromossomos Fúngicos , Replicação do DNA , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Retroalimentação Fisiológica , Deleção de Genes , Recombinação Genética , Saccharomyces cerevisiae/genética , Temperatura , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética
19.
Environ Pollut ; 284: 117508, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34261219

RESUMO

Perfluorooctane sulfonate (PFOS) is a widely used artificial surfactant with potential toxicity to humans and animals. However, little is known about the impact of PFOS on the female germ cell development. Here, we report that PFOS exposure weakens oocyte quality by disturbing oocyte meiotic competency and fertilization ability. Specifically, PFOS exposure impaired cytoskeleton assembly including spindle organization and actin polymerization to cause the oocyte maturation arrest. In addition, PFOS exposure also impaired the mitochondrial dynamics and function, resulting in the increased levels of reactive oxygen species (ROS) and DNA damage as well as generation of apoptosis. Lastly, PFOS exposure compromised the distribution of cortical granules (CGs) and their component ovastacin, leading to the failure of sperm binding and fertilization. Altogether, our study illustrates that oxidative stress-induced apoptosis is a major cause for the deteriorated quality of porcine oocytes exposed to PFOS.


Assuntos
Ácidos Alcanossulfônicos , Meiose , Ácidos Alcanossulfônicos/metabolismo , Ácidos Alcanossulfônicos/toxicidade , Animais , Apoptose , Feminino , Fluorcarbonetos , Humanos , Oócitos/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Suínos
20.
Cell Prolif ; 54(9): e13104, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34323331

RESUMO

OBJECTIVES: RAB14 is a member of small GTPase RAB family which localizes at the endoplasmic reticulum (ER), Golgi apparatus and endosomal compartments. RAB14 acts as molecular switches that shift between a GDP-bound inactive state and a GTP-bound active state and regulates circulation of vesicles between the Golgi and endosomal compartments. In present study, we investigated the roles of RAB14 during oocyte meiotic maturation. MATERIALS AND METHODS: Microinjection with siRNA and exogenous mRNA for knock down and rescue, and immunofluorescence staining, Western blot and real-time RT-PCR were utilized for the study. RESULTS: Our results showed that RAB14 localized in the cytoplasm and accumulated at the cortex during mouse oocyte maturation, and it was also enriched at the spindle periphery. Depletion of RAB14 did not affect polar body extrusion but caused large polar bodies, indicating the failure of asymmetric division. We found that absence of RAB14 did not affect spindle organization but caused the spindle migration defects, and this might be due to the regulation on cytoplasmic actin assembly via the ROCK-cofilin signalling pathway. We also found that RAB14 depletion led to aberrant Golgi apparatus distribution. Exogenous Myc-Rab14 mRNA supplement could significantly rescue these defects caused by Rab14 siRNA injection. CONCLUSIONS: Taken together, our results suggest that RAB14 affects ROCK-cofilin pathway for actin-based spindle migration and Golgi apparatus distribution during mouse oocyte meiotic maturation.


Assuntos
Meiose/fisiologia , Oócitos/metabolismo , Oócitos/fisiologia , Oogênese/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Actinas , Animais , Citoplasma/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Fosforilação/fisiologia , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo
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