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1.
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
2.
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
3.
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
4.
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
5.
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
6.
Exp Cell Res ; 405(2): 112657, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34081985

RESUMO

Checkpoint kinases (Chk) 1/2 are known for DNA damage checkpoint and cell cycle control in somatic cells. According to recent findings, the involvement of Chk1 in oocyte meiotic resumption and Chk2 is regarded as an essential regulator for progression at the post metaphase I stage (MI). In this study, AZD7762 (Chk1/2 inhibitor) and SB218078 (Chk1 inhibitor) were used to uncover the joint roles of Chk1/2 and differentiate the importance of Chk1 and Chk2 during oocyte meiotic maturation. Inhibition of Chk1/2 or Chk1 alone had no significant effect on germinal vesicle breakdown (GVBD) but significantly inhibited the first polar body (PB1). Interestingly, inhibition of Chk1 alone could not increase or completely block the extrusion of PB1 like Chk1/2 inhibition. Also, Chk1/2 inhibition resulted in defective meiotic spindle organization and chromosome condensation both in MI and metaphase II (MII) stages of oocytes. The location of γ-tubulin and Securin were abnormal or missing, while P38 MAPK was activated by Chk1/2 inhibition. Meanwhile, Chk1/2 inhibition reduced the percentage of the second polar body extrusion and pronuclear formation. In conclusion, our results further understand the functions and regulatory mechanism of Chk1/2 during oocyte meiotic maturation.


Assuntos
Cromossomos/metabolismo , Meiose/fisiologia , Metáfase/fisiologia , Oócitos/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Feminino , Camundongos , Securina/metabolismo , Tubulina (Proteína)/metabolismo
7.
Mol Cell Biol ; 41(7): e0037820, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-33941619

RESUMO

In response to nutrient starvation, the budding yeast Saccharomyces cerevisiae abandons mitotic proliferation and embarks on a differentiation process that leads through meiosis to the formation of haploid spores. This process is driven by cascading waves of meiosis-specific-gene expression. The early meiosis-specific genes are repressed during mitotic proliferation by the DNA-binding protein Ume6 in combination with repressors Rpd3 and Sin3. The expression of meiosis-specific transcription factor Ime1 leads to activation of the early meiosis-specific genes. We investigated the stability and promoter occupancy of Ume6 in sporulating cells and determined that it remains bound to early meiosis-specific gene promoters when those genes are activated. Furthermore, we find that the repressor Rpd3 remains associated with Ume6 after the transactivator Ime1 has joined the complex and that the Gcn5 and Tra1 components of the SAGA complex bind to the promoter of IME2 in an Ime1-dependent fashion to induce transcription of the early meiosis-specific genes. Our investigation supports a model whereby Ume6 provides a platform allowing recruitment of both activating and repressing factors to coordinate the expression of the early meiosis-specific genes in Saccharomyces cerevisiae.


Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Meiose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona Desacetilases/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
8.
Commun Biol ; 4(1): 555, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976359

RESUMO

Meiosis is a core feature of eukaryotes that occurs in all major groups, including the early diverging excavates. In this group, meiosis and production of haploid gametes have been described in the pathogenic protist, Trypanosoma brucei, and mating occurs in the salivary glands of the insect vector, the tsetse fly. Here, we searched for intermediate meiotic stages among trypanosomes from tsetse salivary glands. Many different cell types were recovered, including trypanosomes in Meiosis I and gametes. Significantly, we found trypanosomes containing three nuclei with a 1:2:1 ratio of DNA contents. Some of these cells were undergoing cytokinesis, yielding a mononucleate gamete and a binucleate cell with a nuclear DNA content ratio of 1:2. This cell subsequently produced three more gametes in two further rounds of division. Expression of the cell fusion protein HAP2 (GCS1) was not confined to gametes, but also extended to meiotic intermediates. We propose a model whereby the two nuclei resulting from Meiosis I undergo asynchronous Meiosis II divisions with sequential production of haploid gametes.


Assuntos
Células Germinativas/metabolismo , Reprodução/fisiologia , Trypanosoma/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA/genética , Células Germinativas/fisiologia , Meiose/genética , Meiose/fisiologia , Trypanosoma/metabolismo , Trypanosoma/fisiologia , Moscas Tsé-Tsé/genética
9.
Plant Physiol ; 185(4): 1783-1797, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33793950

RESUMO

Meiotic recombination (MR) drives novel combinations of alleles and contributes to genomic diversity in eukaryotes. In this study, we showed that heat stress (36°C-38°C) over the fertile threshold fully abolished crossover formation in Arabidopsis (Arabidopsis thaliana). Cytological and genetic studies in wild-type plants and syn1 and rad51 mutants suggested that heat stress reduces generation of SPO11-dependent double-strand breaks (DSBs). In support, the abundance of recombinase DMC1, which is required for MR-specific DSB repair, was significantly reduced under heat stress. In addition, high temperatures induced disassembly and/or instability of the ASY4- but not the SYN1-mediated chromosome axis. At the same time, the ASY1-associated lateral element of the synaptonemal complex (SC) was partially affected, while the ZYP1-dependent central element of SC was disrupted, indicating that heat stress impairs SC formation. Moreover, expression of genes involved in DSB formation; e.g. SPO11-1, PRD1, 2, and 3 was not impacted; however, recombinase RAD51 and chromosome axis factors ASY3 and ASY4 were significantly downregulated under heat stress. Taken together, these findings revealed that heat stress inhibits MR via compromised DSB formation and homolog synapsis, which are possible downstream effects of the impacted chromosome axis. Our study thus provides evidence shedding light on how increasing environmental temperature influences MR in Arabidopsis.


Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Pareamento Cromossômico/fisiologia , Quebras de DNA de Cadeia Dupla , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Meiose/fisiologia , Pareamento Cromossômico/genética , Variação Genética , Genótipo , Meiose/genética
10.
Biochim Biophys Acta Mol Cell Res ; 1868(7): 119044, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33865884

RESUMO

Cyclin D-CDK4/6 complex mediates the transition from the G1 to S phase in mammalian somatic cells. Meiotic oocytes pass through the G2/M transition and complete the first meiosis to reach maturation at the metaphase of meiosis II without intervening S phase, while Cyclin D-CDK4/6 complex is found to express during meiotic progression. Whether Cyclin D-CDK4/6 complex regulates meiotic cell cycle progression is not known. Here, we found its different role in oocyte meiosis: Cyclin D-CDK4/6 complex served as a regulator of spindle assembly checkpoint (SAC) to prevent aneuploidy in meiosis I. Inhibition of CDK4/6 kinases disrupted spindle assembly, chromosome alignment and kinetochore-microtubule attachments, but unexpectedly accelerated meiotic progression by inactivating SAC, consequently resulting in production of aneuploid oocytes. Further studies showed that the MPF activity decrease before first polar body extrusion was accelerated probably by inactivation of the SAC to promote ubiquitin-mediated cyclin B1 degradation. Taken together, these data reveal a novel role of Cyclin D-CDK4/6 complex in mediating control of the SAC in female meiosis I.


Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Aneuploidia , Animais , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Ciclina B1/metabolismo , Feminino , Meiose/fisiologia , Metáfase/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Oócitos/metabolismo , Corpos Polares/metabolismo , Fuso Acromático/metabolismo
11.
FASEB J ; 35(4): e21460, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33724554

RESUMO

Spermatogenesis is a highly sophisticated process that comprises of mitosis, meiosis, and spermiogenesis. RNF216 (ring finger protein 216), an E3 ubiquitin ligase, has been reported to be essential for spermatogenesis and male fertility in mice. However, the stages affected by Rnf216 deficiency and its underlying molecular pathological mechanisms are still unknown. In this study, we generated Rnf216-deficient mice (Rnf216-/- ) using CRISPR-Cas9 technology. Knockout of Rnf216 led to infertility in male but not female mice. Rnf216 knockout affected the prophase of meiosis I, as no genotypic difference was observed until 12 dpp (days postpartum). Rnf216-/- spermatocytes were incompletely arrested at the zygotene stage and underwent apoptosis at approximately the pachytene stage. The proportion of zygotene spermatocytes was significantly increased, whereas the proportion of pachytene spermatocytes was significantly decreased in Rnf216-/- testes. Nevertheless, there was no significantly genotypic difference in the number of diplotene spermatocytes. We further revealed that the PKA catalytic subunit ß (PRKACB) was significantly increased, which subsequently resulted in elevated PKA activity in testes from adult as well as 9 dpp Rnf216-/- mice. RNF216 interacts with PRKACB and promotes its degradation through the ubiquitin-lysosome pathway. Collectively, our results revealed an important role for RNF216 in regulation of meiosis and PKA stability in the testes.


Assuntos
Meiose/fisiologia , Testículo/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/genética , Feminino , Humanos , Masculino , Camundongos Transgênicos , Espermatócitos/metabolismo , Espermatogênese/fisiologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética
12.
Nat Commun ; 12(1): 1837, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758202

RESUMO

Oocytes are held in meiotic prophase for prolonged periods until hormonal signals trigger meiotic divisions. Key players of M-phase entry are the opposing Cdk1 kinase and PP2A-B55δ phosphatase. In Xenopus, the protein Arpp19, phosphorylated at serine 67 by Greatwall, plays an essential role in inhibiting PP2A-B55δ, promoting Cdk1 activation. Furthermore, Arpp19 has an earlier role in maintaining the prophase arrest through a second serine (S109) phosphorylated by PKA. Prophase release, induced by progesterone, relies on Arpp19 dephosphorylation at S109, owing to an unknown phosphatase. Here, we identified this phosphatase as PP2A-B55δ. In prophase, PKA and PP2A-B55δ are simultaneously active, suggesting the presence of other important targets for both enzymes. The drop in PKA activity induced by progesterone enables PP2A-B55δ to dephosphorylate S109, unlocking the prophase block. Hence, PP2A-B55δ acts critically on Arpp19 on two distinct sites, opposing PKA and Greatwall to orchestrate the prophase release and M-phase entry.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Meiose , Oócitos/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Cromatografia Líquida , Feminino , Meiose/efeitos dos fármacos , Meiose/genética , Meiose/fisiologia , Proteínas Nucleares/metabolismo , Ácido Okadáico/toxicidade , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/genética , Fosforilação , Progesterona/farmacologia , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/isolamento & purificação , Proteínas Recombinantes , Espectrometria de Massas em Tandem , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Xenopus laevis
13.
BMC Plant Biol ; 21(1): 139, 2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33726673

RESUMO

BACKGROUND: In recent years, sugarcane has attracted increasing attention as an energy crop. Wild resources are widely used to improve the narrow genetic base of sugarcane. However, the infertility of F1 hybrids between Saccharum officinarum (S. officinarum) and Erianthus arundinaceus (E. arundinaceus) has hindered sugarcane breeding efforts. To discover the cause of this infertility, we studied the hybridization process from a cytological perspective. RESULTS: We examined the meiotic process of pollen mother cells (PMCs) in three F1 hybrids between S. officinarum and E. arundinaceus. Cytological analysis showed that the male parents, Hainan 92-77 and Hainan 92-105, had normal meiosis. However, the meiosis process in F1 hybrids showed various abnormal phenomena, including lagging chromosomes, micronuclei, uneven segregation, chromosome bridges, and inability to form cell plates. Genomic in situ hybridization (GISH) showed unequal chromatin distribution during cell division. Interestingly, 96.70% of lagging chromosomes were from E. arundinaceus. Furthermore, fluorescence in situ hybridization (FISH) was performed using 45S rDNA and 5S rDNA as probes. Either 45S rDNA or 5S rDNA sites were lost during abnormal meiosis, and results of unequal chromosomal separation were also clearly observed in tetrads. CONCLUSIONS: Using cytogenetic analysis, a large number of meiotic abnormalities were observed in F1. GISH further confirmed that 96.70% of the lagging chromosomes were from E. arundinaceus. Chromosome loss was found by further investigation of repeat sequences. Our findings provide insight into sugarcane chromosome inheritance to aid innovation and utilization in sugarcane germplasm resources.


Assuntos
Meiose/genética , Meiose/fisiologia , Meristema/genética , Poaceae/crescimento & desenvolvimento , Poaceae/genética , Pólen/genética , Saccharum/crescimento & desenvolvimento , Saccharum/genética , Quimera , China , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Genes de Plantas , Variação Genética , Genótipo , Hibridização Genética , Hibridização in Situ Fluorescente , Meristema/crescimento & desenvolvimento , Pólen/crescimento & desenvolvimento
14.
Nat Commun ; 12(1): 1438, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33664246

RESUMO

Germ cells are physically coupled to somatic support cells of the gonad during differentiation, but this coupling must be disrupted when they are mature, freeing them to participate in fertilization. In mammalian females, coupling occurs via specialized filopodia that project from the ovarian follicular granulosa cells to the oocyte. Here, we show that signaling through the epidermal growth factor receptor (EGFR) in the granulosa, which becomes activated at ovulation, uncouples the germ and somatic cells by triggering a massive and temporally synchronized retraction of the filopodia. Although EGFR signaling triggers meiotic maturation of the oocyte, filopodial retraction is independent of the germ cell state, being regulated solely within the somatic compartment, where it requires ERK-dependent calpain-mediated loss of filopodia-oocyte adhesion followed by Arp2/3-mediated filopodial shortening. By uncovering the mechanism regulating germ-soma uncoupling at ovulation, our results open a path to improving oocyte quality in human and animal reproduction.


Assuntos
Adesão Celular/fisiologia , Receptores ErbB/metabolismo , Células da Granulosa/metabolismo , Oócitos/metabolismo , Ovulação/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Calpaína/metabolismo , Comunicação Celular/fisiologia , Células Cultivadas , Feminino , Meiose/fisiologia , Camundongos , Pseudópodes/fisiologia , Transdução de Sinais/fisiologia , Suínos
15.
Toxicology ; 452: 152705, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33548356

RESUMO

Gefitinib is a first-line anti-cancer drug for the treatment of advanced non-small cell lung cancer (NSCLC). It has been reported that gefitinib can generate several drug-related adverse effects, including nausea, peripheral edema, decreased appetite and rash. However, the reproductive toxicity of gefitinib has not been clearly defined until now. Here we assessed the effects of gefitinib on oocyte quality by examining the critical events and molecular changes of oocyte maturation. Gefitinib at 1, 2, 5 or 10 µM concentration was added to culture medium (M2). We found that gefitinib at its median peak concentration of 1 µM did not affect oocyte maturation, but 5 µM gefitinib severely blocked oocyte meiotic progression as indicated by decreased rates of germinal vesicle breakdown (GVBD) and polar body extrusion (PBE). We further showed that gefitinib treatment increased phosphorylation of CDK1 at the site of Try15, inhibited cyclin B1 entry into the nucleus, and disrupted normal spindle assembly, chromosome alignment and mitochondria dynamics, finally leading to the generation of aneuploidy and early apoptosis of oocytes. Our study reported here provides valuable evidence for reproductive toxicity of gefitinib administration employed for the treatment of cancer patients.


Assuntos
Antineoplásicos/toxicidade , Gefitinibe/toxicidade , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Meiose/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Oócitos/metabolismo , Oócitos/patologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Fuso Acromático/patologia
16.
Methods Mol Biol ; 2218: 137-155, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606229

RESUMO

Oocyte production is crucial for sexual reproduction. Recent findings in zebrafish and other established model organisms emphasize that the early steps of oogenesis involve the coordination of simultaneous and tightly sequential processes across cellular compartments and between sister cells. To fully understand the mechanistic framework of these coordinated processes, cellular and morphological analysis in high temporal resolution is required. Here, we provide a protocol for four-dimensional live time-lapse analysis of cultured juvenile zebrafish ovaries. We describe how multiple-stage oocytes can be simultaneously analyzed in single ovaries, and several ovaries can be processed in single experiments. In addition, we detail adequate conditions for quantitative image acquisition. Finally, we demonstrate that using this protocol, we successfully capture rapid meiotic chromosomal movements in early prophase for the first time in zebrafish oocytes, in four dimensions and in vivo. Our protocol expands the use of the zebrafish as a model system to understand germ cell and ovarian development in postembryonic stages.


Assuntos
Cromossomos/fisiologia , Meiose/fisiologia , Oogênese/fisiologia , Ovário/fisiologia , Imagem com Lapso de Tempo/métodos , Peixe-Zebra/fisiologia , Animais , Feminino , Oócitos , Diferenciação Sexual/fisiologia
17.
Methods Mol Biol ; 2218: 157-167, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606230

RESUMO

The polar body, with haploid DNA, is a small cell produced during the meiosis of an oocyte. Here, we describe the detailed procedures for the detection of the second polar body in zebrafish (Danio rerio) embryos after 10 min post fertilization. A polar body can be easily distinguished as a small dot with a DAPI-stained nucleus surrounded by Phalloidin-labeled F-actin in each fertilized zebrafish embryo.


Assuntos
Fertilização/fisiologia , Corpos Polares/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Feminino , Fertilização In Vitro/métodos , Masculino , Meiose/fisiologia , Oócitos/metabolismo , Oócitos/fisiologia , Corpos Polares/metabolismo , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia
18.
PLoS Biol ; 19(1): e3001067, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33406066

RESUMO

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins postfusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 not only imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least 2 mechanisms where the zygotic fate imposed by Mei2 and the cell cycle reentry triggered by Mei3 synergize to prevent zygotic mating.


Assuntos
Ciclo Celular/fisiologia , Fator de Acasalamento/fisiologia , Meiose/fisiologia , Zigoto/fisiologia , Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Proteínas Fúngicas/fisiologia , Genes Fúngicos/fisiologia , Fator de Acasalamento/genética , Fator de Acasalamento/metabolismo , Meiose/genética , Organismos Geneticamente Modificados , Ploidias , Proteínas de Ligação a RNA/fisiologia , Recombinação Genética/fisiologia , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
19.
Exp Cell Res ; 399(2): 112455, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33400935

RESUMO

During meiosis, homologous chromosomes exchange genetic material. This exchange or meiotic recombination is mediated by a proteinaceous scaffold known as the Synaptonemal complex (SC). Any defects in its formation produce failures in meiotic recombination, chromosome segregation and meiosis completion. It has been proposed that DNA repair events that will be resolved by crossover between homologous chromosomes are predetermined by the SC. Hence, structural analysis of the organization of the DNA in the SC could shed light on the process of crossover interference. In this work, we employed an ultrastructural DNA staining technique on mouse testis and followed nuclei of pachytene cells. We observed structures organized similarly to the SCs stained with conventional techniques. These structures, presumably the DNA in the SCs, are delineating the edges of both lateral elements and no staining was observed between them. DNA in the LEs resembles two parallel tracks. However, a bubble-like staining pattern in certain regions of the SC was observed. Furthermore, this staining pattern is found in SCs formed between non-homologous chromosomes, in SCs formed between sister chromatids and in SCs without lateral elements, suggesting that this particular organization of the DNA is determined by the synapsis of the chromosomes despite their lack of homology or the presence of partially formed SCs.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Meiose/fisiologia , Complexo Sinaptonêmico/metabolismo , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromátides/química , Cromátides/metabolismo , Cromátides/ultraestrutura , Pareamento Cromossômico/fisiologia , DNA/química , DNA/ultraestrutura , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Estrutura Quaternária de Proteína , Complexo Sinaptonêmico/fisiologia , Complexo Sinaptonêmico/ultraestrutura
20.
Dev Cell ; 56(1): 22-35.e7, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33278343

RESUMO

Retrotransposon proliferation poses a threat to germline integrity. While retrotransposons must be activated in developing germ cells in order to survive and propagate, how they are selectively activated in the context of meiosis is unclear. We demonstrate that the transcriptional activation of Ty3/Gypsy retrotransposons and host defense are controlled by master meiotic regulators. We show that budding yeast Ty3/Gypsy co-opts binding sites of the essential meiotic transcription factor Ndt80 upstream of the integration site, thereby tightly linking its transcriptional activation to meiotic progression. We also elucidate how yeast cells thwart Ty3/Gypsy proliferation by blocking translation of the retrotransposon mRNA using amyloid-like assemblies of the RNA-binding protein Rim4. In mammals, several inactive Ty3/Gypsy elements are undergoing domestication. We show that mammals utilize equivalent master meiotic regulators (Stra8, Mybl1, Dazl) to regulate Ty3/Gypsy-derived genes in developing gametes. Our findings inform how genes that are evolving from retrotransposons can build upon existing regulatory networks during domestication.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Germinativas/metabolismo , Meiose/genética , Proteínas de Ligação a RNA/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Retroelementos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Ligação a DNA/genética , Evolução Molecular , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Meiose/fisiologia , Camundongos , Gambás/genética , Gambás/metabolismo , Biossíntese de Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/genética , DNA Polimerase Dirigida por RNA/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética
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