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1.
BMC Bioinformatics ; 21(Suppl 14): 369, 2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-32998686

RESUMO

BACKGROUND: Chromosome conformation capture-based methods, especially Hi-C, enable scientists to detect genome-wide chromatin interactions and study the spatial organization of chromatin, which plays important roles in gene expression regulation, DNA replication and repair etc. Thus, developing computational methods to unravel patterns behind the data becomes critical. Existing computational methods focus on intrachromosomal interactions and ignore interchromosomal interactions partly because there is no prior knowledge for interchromosomal interactions and the frequency of interchromosomal interactions is much lower while the search space is much larger. With the development of single-cell technologies, the advent of single-cell Hi-C makes interrogating the spatial structure of chromatin at single-cell resolution possible. It also brings a new type of frequency information, the number of single cells with chromatin interactions between two disjoint chromosome regions. RESULTS: Considering the lack of computational methods on interchromosomal interactions and the unsurprisingly frequent intrachromosomal interactions along the diagonal of a chromatin contact map, we propose a computational method dedicated to analyzing interchromosomal interactions of single-cell Hi-C with this new frequency information. To the best of our knowledge, our proposed tool is the first to identify regions with statistically frequent interchromosomal interactions at single-cell resolution. We demonstrate that the tool utilizing networks and binomial statistical tests can identify interesting structural regions through visualization, comparison and enrichment analysis and it also supports different configurations to provide users with flexibility. CONCLUSIONS: It will be a useful tool for analyzing single-cell Hi-C interchromosomal interactions.


Assuntos
Cromossomos/metabolismo , Análise de Célula Única/métodos , Animais , Cromatina/metabolismo , Fase G1 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Fase S , Zigoto/citologia , Zigoto/metabolismo
2.
Nat Commun ; 11(1): 4486, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900989

RESUMO

Centromeres are epigenetically determined nuclear domains strictly required for chromosome segregation and genome stability. However, the mechanisms regulating centromere and kinetochore chromatin modifications are not known. Here, we demonstrate that LSH is enriched at meiotic kinetochores and its targeted deletion induces centromere instability and abnormal chromosome segregation. Superresolution chromatin analysis resolves LSH at the inner centromere and kinetochores during oocyte meiosis. LSH knockout pachytene oocytes exhibit reduced HDAC2 and DNMT-1. Notably, mutant oocytes show a striking increase in histone H3 phosphorylation at threonine 3 (H3T3ph) and accumulation of major satellite transcripts in both prophase-I and metaphase-I chromosomes. Moreover, knockout oocytes exhibit centromere fusions, ectopic kinetochore formation and abnormal exchange of chromatin fibers between paired bivalents and asynapsed chromosomes. Our results indicate that loss of LSH affects the levels and chromosomal localization of H3T3ph and provide evidence that, by maintaining transcriptionally repressive heterochromatin, LSH may be essential to prevent deleterious meiotic recombination events at repetitive centromeric sequences.


Assuntos
DNA Helicases/metabolismo , Meiose/fisiologia , Oócitos/citologia , Oócitos/metabolismo , Animais , Centrômero/genética , Centrômero/metabolismo , DNA Helicases/deficiência , DNA Helicases/genética , Feminino , Histonas/metabolismo , Cinetocoros/metabolismo , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Transcrição Genética
3.
Nature ; 585(7824): 239-244, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879485

RESUMO

Obligate endosymbiosis, in which distantly related species integrate to form a single replicating individual, represents a major evolutionary transition in individuality1-3. Although such transitions are thought to increase biological complexity1,2,4-6, the evolutionary and developmental steps that lead to integration remain poorly understood. Here we show that obligate endosymbiosis between the bacteria Blochmannia and the hyperdiverse ant tribe Camponotini7-11 originated and also elaborated through radical alterations in embryonic development, as compared to other insects. The Hox genes Abdominal A (abdA) and Ultrabithorax (Ubx)-which, in arthropods, normally function to differentiate abdominal and thoracic segments after they form-were rewired to also regulate germline genes early in development. Consequently, the mRNAs and proteins of these Hox genes are expressed maternally and colocalize at a subcellular level with those of germline genes in the germplasm and three novel locations in the freshly laid egg. Blochmannia bacteria then selectively regulate these mRNAs and proteins to make each of these four locations functionally distinct, creating a system of coordinates in the embryo in which each location performs a different function to integrate Blochmannia into the Camponotini. Finally, we show that the capacity to localize mRNAs and proteins to new locations in the embryo evolved before obligate endosymbiosis and was subsequently co-opted by Blochmannia and Camponotini. This pre-existing molecular capacity converged with a pre-existing ecological mutualism12,13 to facilitate both the horizontal transfer10 and developmental integration of Blochmannia into Camponotini. Therefore, the convergence of pre-existing molecular capacities and ecological interactions-as well as the rewiring of highly conserved gene networks-may be a general feature that facilitates the origin and elaboration of major transitions in individuality.


Assuntos
Formigas/embriologia , Formigas/microbiologia , Bactérias , Evolução Biológica , Regulação da Expressão Gênica no Desenvolvimento/genética , Individualidade , Simbiose/genética , Animais , Formigas/citologia , Formigas/genética , Desenvolvimento Embrionário/genética , Feminino , Genes Homeobox/genética , Herança Materna/genética , Oócitos/citologia , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Nat Commun ; 11(1): 3393, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636388

RESUMO

Meiotic divisions in oocytes are extremely asymmetric and require pre- and post-anaphase-onset phases of spindle migration. The latter induces membrane protrusion that is moulded around the spindle thereby reducing cytoplasmic loss. Here, we find that depleting the NAD biosynthetic enzyme, nicotinamide phosphoribosyl-transferase (Nampt), in mouse oocytes results in markedly longer spindles and compromises asymmetry. By analysing spindle speed in live oocytes, we identify a striking and transient acceleration after anaphase-onset that is severely blunted following Nampt-depletion. Slow-moving midzones of elongated spindles induce cortical furrowing deep within the oocyte before protrusions can form, altogether resulting in larger oocyte fragments being cleaved off. Additionally, we find that Nampt-depletion lowers NAD and ATP levels and that reducing NAD using small molecule Nampt inhibitors also compromises asymmetry. These data show that rapid midzone displacement is critical for extreme asymmetry by delaying furrowing to enable protrusions to form and link metabolic status to asymmetric division.


Assuntos
Anáfase , Citocinas/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Oócitos/citologia , Fuso Acromático , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ciclo Celular , Segregação de Cromossomos , Citoplasma/metabolismo , Citosol/metabolismo , Feminino , Meiose , Camundongos , Microscopia Confocal , NAD/química
5.
J Vis Exp ; (160)2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32658178

RESUMO

In wild animals' conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator's skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.


Assuntos
Oócitos/fisiologia , Manejo de Espécimes/métodos , Animais , Gatos , Criopreservação , Desenvolvimento Embrionário , Feminino , Oócitos/citologia , Gravidez , Técnicas de Reprodução Assistida , Sobrevivência de Tecidos
6.
Nat Commun ; 11(1): 2598, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451402

RESUMO

DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.


Assuntos
Mutação , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reparo de DNA por Recombinação/genética , Aneuploidia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Fertilização , Genes bcl-2 , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Proteínas de Ligação a Fosfato/deficiência , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína X Associada a bcl-2/deficiência , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
7.
Nat Cell Biol ; 22(4): 380-388, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231309

RESUMO

The importance of germline-inherited post-translational histone modifications on priming early mammalian development is just emerging1-4. Histone H3 lysine 9 (H3K9) trimethylation is associated with heterochromatin and gene repression during cell-fate change5, whereas histone H3 lysine 4 (H3K4) trimethylation marks active gene promoters6. Mature oocytes are transcriptionally quiescent and possess remarkably broad domains of H3K4me3 (bdH3K4me3)1,2. It is unknown which factors contribute to the maintenance of the bdH3K4me3 landscape. Lysine-specific demethylase 4A (KDM4A) demethylates H3K9me3 at promoters marked by H3K4me3 in actively transcribing somatic cells7. Here, we report that KDM4A-mediated H3K9me3 demethylation at bdH3K4me3 in oocytes is crucial for normal pre-implantation development and zygotic genome activation after fertilization. The loss of KDM4A in oocytes causes aberrant H3K9me3 spreading over bdH3K4me3, resulting in insufficient transcriptional activation of genes, endogenous retroviral elements and chimeric transcripts initiated from long terminal repeats during zygotic genome activation. The catalytic activity of KDM4A is essential for normal epigenetic reprogramming and pre-implantation development. Hence, KDM4A plays a crucial role in preserving the maternal epigenome integrity required for proper zygotic genome activation and transfer of developmental control to the embryo.


Assuntos
Histona Desmetilases/metabolismo , Histonas/metabolismo , Oócitos/metabolismo , Processamento de Proteína Pós-Traducional , Zigoto/metabolismo , Animais , Implantação do Embrião , Embrião de Mamíferos , Feminino , Fertilização/genética , Heterocromatina/química , Heterocromatina/metabolismo , Histona Desmetilases/genética , Histonas/genética , Masculino , Metáfase , Metilação , Camundongos , Camundongos Knockout , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Transcrição Genética , Zigoto/citologia , Zigoto/crescimento & desenvolvimento
8.
PLoS One ; 15(4): e0231114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32243476

RESUMO

Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 (MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. In this study, we examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation. Although oogenesis progressed in females carrying a dMarf1 loss-of-function allele, the dMarf1 mutant oocytes were found to contain arrested meiotic spindles or disrupted microtubule structures, indicating that the transition from meiosis I to II was compromised in these oocytes. The expression of the full-length dMarf1 transgene, but none of the variants lacking the OST and RRM motifs or the 47 conserved C-terminal residues among insect groups, rescued the meiotic defect in dMarf1 mutant oocytes. Our results indicate that these conserved residues are important for dMarf1 function. Immunoprecipitation of Myc-dMarf1 revealed that several mRNAs are bound to dMarf1. Of those, the protein expression of nanos (nos), but not its mRNA, was affected in the absence of dMarf1. In the control, the expression of Nos protein became downregulated during the late stages of oogenesis, while it remained high in dMarf1 mutant oocytes. We propose that dMarf1 translationally represses nos by binding to its mRNA. Furthermore, the downregulation of Nos induces cycB expression, which in turn activates the CycB/Cdk1 complex at the onset of oocyte maturation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Fusão Celular , Sequência Conservada , Ciclina B , Regulação para Baixo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Feminino , Regulação da Expressão Gênica , Meiose , Proteínas Mutantes/metabolismo , Mutação/genética , Oogênese , Ovário/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Tiorredoxinas/metabolismo
9.
Int J Mol Sci ; 21(7)2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32218212

RESUMO

In the mammalian ovary, the hyaluronan (HA)-rich cumulus extracellular matrix (ECM) organized during the gonadotropin-induced process of oocyte maturation is essential for ovulation of the oocyte-cumulus complex (OCC) and fertilization. Versican is an HA-binding proteoglycan that regulates cell function and ECM assembly. Versican cleavage and function remain to be determined in ovarian follicle. We investigated versican expression in porcine ovarian follicles by real-time (RT)-PCR and western blotting. The aims of the present work were to determine whether 1) versican was produced and cleaved by porcine OCCs during gonadotropin stimulation; 2) these processes were autonomous or required the participation of mural granulosa cells (MGCs); and 3) versican cleavage was involved in the formation or degradation of expanded cumulus ECM. We demonstrate two cleavage products of G1 domain of versican (V1) accumulated in the HA-rich cumulus ECM. One of them, a G1-DPEAAE N-terminal fragment (VG1) of ~70 kDa, was generated from V1 during organization of HA in in vivo and in vitro expanded porcine OCCs. Second, the V1-cleaved DPEAAE-positive form of ~65 kDa was the only species detected in MGCs. No versican cleavage products were detected in OCCs cultured without follicular fluid. In summary, porcine OCCs are autonomous in producing and cleaving V1; the cleaved fragment of ~70 kDa VG1 is specific for formation of the expanded cumulus HA-rich ECM.


Assuntos
Oócitos/metabolismo , Versicanas/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Epitopos/imunologia , Feminino , Oócitos/citologia , Oócitos/imunologia , Suínos , Versicanas/genética
10.
PLoS Genet ; 16(3): e1008676, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32214314

RESUMO

A set of sex chromosomes is required for gametogenesis in both males and females, as represented by sex chromosome disorders causing agametic phenotypes. Although studies using model animals have investigated the functional requirement of sex chromosomes, involvement of these chromosomes in gametogenesis remains elusive. Here, we elicit a germ cell-intrinsic effect of sex chromosomes on oogenesis, using a novel culture system in which oocytes were induced from embryonic stem cells (ESCs) harboring XX, XO or XY. In the culture system, oogenesis using XO and XY ESCs was severely disturbed, with XY ESCs being more strongly affected. The culture system revealed multiple defects in the oogenesis of XO and XY ESCs, such as delayed meiotic entry and progression, and mispairing of the homologous chromosomes. Interestingly, Eif2s3y, a Y-linked gene that promotes proliferation of spermatogonia, had an inhibitory effect on oogenesis. This led us to the concept that male and female gametogenesis appear to be in mutual conflict at an early stage. This study provides a deeper understanding of oogenesis under a sex-reversal condition.


Assuntos
Células Germinativas/metabolismo , Oócitos/metabolismo , Cromossomo X , Cromossomo Y , Animais , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Feminino , Células Germinativas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Endogâmicos , Oócitos/citologia , Oócitos/ultraestrutura , Oogênese
11.
Front Biosci (Schol Ed) ; 12: 116-136, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32114451

RESUMO

Oocyte quality influences early embryonic survival, establishment and maintenance of pregnancy, fetal development and adult diseases. The developmental competence of oocytes is acquired gradually and increases with follicular development. The ability of an oocyte to develop into an embryo depends on, having enough specific information in the form of mRNA or proteins. If this information is insufficient, defects in nuclear or cytoplasmic maturation, or in both processes, may arise and thus affect the in vitro development of fertilized oocytes. The greater developmental competence of oocytes aspirated from larger follicles is reported as compared with smaller follicles. Oocyte developmental competence is greatly correlated with the morphology of the cumulus oocyte complexes (COCs). Apart from morphological or biochemical markers, molecular markers have also been investigated. Until now, no specific markers of oocyte developmental competence could be described for the oocyte developmental competence. To, utilize female germplasm to its maximum, there is a need to enhance developmental competence of lesser competent oocytes derived from the follicles which are not fully grown. The oocyte pre-maturation and maturation conditions affect gene expression not only in the oocyte but till the blastocyst stages too. Strategies have been discussed in this review would be useful to enhance the developmental competence of oocytes.


Assuntos
Oócitos/fisiologia , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Células do Cúmulo/citologia , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário/fisiologia , Feminino , Humanos , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oogênese/fisiologia , Gravidez
12.
Hum Cell ; 33(3): 521-527, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32172344

RESUMO

This study aimed to determine whether fertilization can be obtained by assisted fusion of oocyte and sperm without breaking the oocyte membrane. A total of 79 infertile couples, each with at least one unfertilized oocyte after in vitro fertilization (IVF), were recruited. Sperm collected from the zona pellucida (ZP) were pressed onto the membrane of unfertilized oocytes at either 6 h or 24 h after IVF, a procedure that we designated as assisted sperm fusion insemination (ASFI). The results of ASFI were compared with those obtained in a previous trial on oocytes in which rescue intracytoplasmic sperm injection (ICSI) was performed at 6 h after IVF. Acrosome reaction (AR) rate of sperm bound to ZP, fertilization rate, degeneration rate, and blastocyst formation rate were evaluated. The AR rate of sperm collected from the ZP was significantly higher than that of the motile sperm recovered from around the oocytes but not bound to the ZP after IVF (98.0% vs. 28.6%). ASFI which was performed at 6 h after IVF yielded a mean fertilization rate of 73.4% (58/79), a degeneration rate of 0% (0/79) and a blastocyst formation rate of 60.8% (31/51). Rescue ICSI which was performed at 6 h after IVF yielded a mean fertilization rate of 70.0% (70/100), a degeneration rate of 4% (4/100) and a blastocyst formation rate of 42.4% (25/59). Binding of sperm to the ZP typically results in AR. ASFI with acrosome-reacted sperm collected from the ZP yielded the fertilization rates similar to those obtained with rescue ICSI.


Assuntos
Membrana Celular , Fertilização/fisiologia , Oócitos/citologia , Interações Espermatozoide-Óvulo , Espermatozoides/fisiologia , Zona Pelúcida/metabolismo , Reação Acrossômica , Blastocisto , Feminino , Humanos , Inseminação Artificial/métodos , Masculino , Espermatozoides/metabolismo
14.
Medicine (Baltimore) ; 99(13): e19665, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32221094

RESUMO

The aim of this study was to investigate the relationship between blood lipid level and the parameters of embryo morphology of in vitro fertilization (IVF).A total of 488 patients undergoing conventional IVF were divided into pregnant (n = 286) and nonpregnant (n = 202) groups. Levels of triglycerides (TG), total cholesterol (TC), high-density lipoproteins (HDL), low-density lipoprotein (LDL), lipoprotein (a), lipoprotein (b), and embryo outcomes were studied. Spearman correlation was performed to analyze the correlation between blood lipid levels and embryo quality in pregnant group.The normal fertilization rate and number of good quality embryos were higher than nonpregnant group (P < .05). TG, TC, and LDL levels were negatively correlated with number of normal fertilized oocytes, while TG, TC, and Lp(b) were negatively correlated with number of good quality embryos. TG level was negatively correlated with number of oocytes and cleavage embryos while HDL and Lp(a) were positively correlated with number of oocytes, normal fertilized oocytes and cleavage embryos (P < .05).TG, TC, LDL, and Lp(b) levels had negative correlation with embryo quality, while HDL and Lp(a) had positive correlation with the embryo quality. Our present findings showed blood lipid levels may provide certain reference for the prediction of IVF pregnancy outcome.


Assuntos
Embrião de Mamíferos/citologia , Fertilização In Vitro/métodos , Lipídeos/sangue , Adulto , Índice de Massa Corporal , Feminino , Humanos , Lipoproteínas/sangue , Oócitos/citologia , Triglicerídeos/sangue
15.
Cell Prolif ; 53(3): e12769, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32003502

RESUMO

OBJECTIVES: M-phase phosphoprotein 6 (MPP6) is important for 5.8S pre-rRNA maturation in somatic cells and was screened as a female fertility factor. However, whether MPP6 functions in oocyte meiosis and fertility is not yet known. We aimed to address this. MATERIALS AND METHODS: Mouse oocytes with surrounded nucleus (SN) or non-surrounded nucleus (NSN) were used for all experiments. Peptide nanoparticle-mediated antibody transfection was used to deplete MPP6. Immunofluorescence staining, immunohistochemistry and live tracker staining were used to examine MPP6 localization and characterize phenotypes after control or MPP6 depletion. High-fidelity PCR and fluorescence in situ hybridization (FISH) were used to examine the localization and level of 5.8S rRNAs. Western blot was used to examine the protein level. MPP6-EGFP mRNA microinjection was used to do the rescue. RESULTS: MPP6 was enriched within ovaries and oocytes. MPP6 depletion significantly impeded oocyte meiosis. MPP6 depletion increased 5.8S pre-rRNA. The mRNA levels of MPP6 and 5.8S rRNA decreased within ageing oocytes, and MPP6 mRNA injection partially increased 5.8S rRNA maturation and improved oocyte quality. CONCLUSIONS: MPP6 is required for 5.8S rRNA maturation, meiosis and quality control in mouse oocytes, and MPP6 level might be a marker for oocyte quality.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Oócitos/citologia , RNA Ribossômico 5,8S/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Divisão Celular , Células Cultivadas , Senescência Celular , Feminino , Fertilidade , Fertilização In Vitro , Masculino , Meiose , Camundongos , Camundongos Endogâmicos ICR , Oócitos/metabolismo , Oócitos/ultraestrutura , Proteínas de Ligação a RNA/genética
16.
PLoS One ; 15(2): e0229391, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32092110

RESUMO

Our previous work documented significant advancements in steroid-induced progression of oogenesis, demonstrating that co-treatment of female eels with 11-ketotestosterone (11KT) and estradiol-17ß (E2) successfully induced uptake of vitellogenin by oocytes. Here we evaluate the effects of this steroid co-treatment on subsequent time to ovulation and egg quality in shortfinned eels artificially matured by hypophysation. Co-treatment with 11KT (1 mg) and E2 (0.2 or 2 mg) significantly reduced time to ovulation and therefore, the amount of pituitary homogenate required, without any detrimental effects on gonadosomatic index, oocyte diameter or the total weight of stripped eggs. E2 treatment resulted in promising increases in fertilization rates. These indicators suggest that co-treatment with 11KT and E2 holds promise for future artificial maturation practices in terms of minimising fish handling and stress, and of reducing the need for expensive pituitary preparations.


Assuntos
Anguilla , Estradiol/farmacologia , Oogênese/efeitos dos fármacos , Indução da Ovulação , Testosterona/análogos & derivados , Anguilla/fisiologia , Animais , Feminino , Fertilidade/efeitos dos fármacos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Oogênese/fisiologia , Ovário/citologia , Ovário/efeitos dos fármacos , Indução da Ovulação/métodos , Indução da Ovulação/veterinária , Testosterona/farmacologia
17.
Science ; 367(6482)2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32054698

RESUMO

Sex determination of germ cells is vital to creating the sexual dichotomy of germ cell development, thereby ensuring sexual reproduction. However, the underlying mechanisms remain unclear. Here, we show that ZGLP1, a conserved transcriptional regulator with GATA-like zinc fingers, determines the oogenic fate in mice. ZGLP1 acts downstream of bone morphogenetic protein, but not retinoic acid (RA), and is essential for the oogenic program and meiotic entry. ZGLP1 overexpression induces differentiation of in vitro primordial germ cell-like cells (PGCLCs) into fetal oocytes by activating the oogenic programs repressed by Polycomb activities, whereas RA signaling contributes to oogenic program maturation and PGC program repression. Our findings elucidate the mechanism for mammalian oogenic fate determination, providing a foundation for promoting in vitro gametogenesis and reproductive medicine.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Oócitos/fisiologia , Oogênese/genética , Proteínas Repressoras/fisiologia , Diferenciação Sexual/genética , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Feminino , Feto/citologia , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Oócitos/citologia , Proteínas do Grupo Polycomb/metabolismo , Proteínas Repressoras/genética , Processos de Determinação Sexual , Transdução de Sinais , Fatores de Transcrição/genética , Transcriptoma , Tretinoína/fisiologia
18.
Development ; 147(4)2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32054660

RESUMO

La-related protein 6 (Larp6) is a conserved RNA-binding protein found across eukaryotes that has been suggested to regulate collagen biogenesis, muscle development, ciliogenesis, and various aspects of cell proliferation and migration. Zebrafish have two Larp6 family genes: larp6a and larp6b Viable and fertile single and double homozygous larp6a and larp6b zygotic mutants revealed no defects in muscle structure, and were indistinguishable from heterozygous or wild-type siblings. However, larp6a mutant females produced eggs with chorions that failed to elevate fully and were fragile. Eggs from larp6b single mutant females showed minor chorion defects, but chorions from eggs laid by larp6a;larp6b double mutant females were more defective than those from larp6a single mutants. Electron microscopy revealed defective chorionogenesis during oocyte development. Despite this, maternal zygotic single and double mutants were viable and fertile. Mass spectrometry analysis provided a description of chorion protein composition and revealed significant reductions in a subset of zona pellucida and lectin-type proteins between wild-type and mutant chorions that paralleled the severity of the phenotype. We conclude that Larp6 proteins are required for normal oocyte development, chorion formation and egg activation.


Assuntos
Autoantígenos/genética , Autoantígenos/fisiologia , Córion/fisiologia , Oócitos/fisiologia , Ribonucleoproteínas/genética , Ribonucleoproteínas/fisiologia , Animais , Movimento Celular , Proliferação de Células , Colágeno/fisiologia , Proteínas do Ovo/fisiologia , Feminino , Edição de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Genótipo , Heterozigoto , Homozigoto , Lectinas/fisiologia , Masculino , Mutação , Oócitos/citologia , Oogênese/fisiologia , Fenótipo , Peixe-Zebra , Zona Pelúcida/fisiologia
19.
Nucleic Acids Res ; 48(7): 3525-3541, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32086523

RESUMO

Germ-cell transcription factors control gene networks that regulate oocyte differentiation and primordial follicle formation during early, postnatal mouse oogenesis. Taking advantage of gene-edited mice lacking transcription factors expressed in female germ cells, we analyzed global gene expression profiles in perinatal ovaries from wildtype, FiglaNull, Lhx8Null and Sohlh1Null mice. Figla deficiency dysregulates expression of meiosis-related genes (e.g. Sycp3, Rad51, Ybx2) and a variety of genes (e.g. Nobox, Lhx8, Taf4b, Sohlh1, Sohlh2, Gdf9) associated with oocyte growth and differentiation. The absence of FIGLA significantly impedes meiotic progression, causes DNA damage and results in oocyte apoptosis. Moreover, we find that FIGLA and other transcriptional regulator proteins (e.g. NOBOX, LHX8, SOHLH1, SOHLH2) are co-expressed in the same subset of germ cells in perinatal ovaries and Figla ablation dramatically disrupts KIT, NOBOX, LHX8, SOHLH1 and SOHLH2 abundance. In addition, not only do FIGLA, LHX8 and SOHLH1 cross-regulate each other, they also cooperate by direct interaction with each during early oocyte development and share downstream gene targets. Thus, our findings substantiate a major role for FIGLA, LHX8 and SOHLH1 as multifunctional regulators of networks necessary for oocyte maintenance and differentiation during early folliculogenesis.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Redes Reguladoras de Genes , Proteínas com Homeodomínio LIM/metabolismo , Oócitos/metabolismo , Oogênese/genética , Fatores de Transcrição/metabolismo , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proliferação de Células/genética , Dano ao DNA , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteínas com Homeodomínio LIM/genética , Meiose/genética , Camundongos , Oócitos/citologia , Ovário/metabolismo , Fatores de Transcrição/genética
20.
Dev Growth Differ ; 62(3): 150-157, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32106340

RESUMO

Guaranteeing the sustainability of gametogenesis is a fundamental issue for perpetuating the species. In the mammalian ovary, sustainability is accomplished by keeping a number of oocytes "stocked" in the dormant state. Despite the evident importance of this state, the mechanisms underlying the oocyte dormancy are not fully understood, although it is presumed that both intrinsic and extrinsic factors are involved. Here, we review environmental factors involved in the regulation of oocyte dormancy. Consideration of the environmental factors illustrates the nature of the ovarian compartment, in which primordial follicles reside. This should greatly improve our understanding of the mechanisms and also assist in reconstitution of the dormant state in culture. Accumulating knowledge on the dormant state of oocytes will contribute to a wide range of research in fields such as developmental biology, reproductive biology and regenerative medicine.


Assuntos
Microambiente Celular , Oócitos/citologia , Oócitos/metabolismo , Animais , Humanos
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