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Mammalian centromeres are generally composed of dispersed repeats and the satellites such as α-satellites in human and major/minor satellites in mouse. Transcription of centromeres by RNA polymerase II is evolutionary conserved and critical for kinetochore assembly. In addition, it has been found that the transcribed satellite RNAs can bind DNA repair proteins such as MRE11 and PRKDC, and excessively expressed satellite RNAs could induce genome instability and facilitate tumorigenesis. During the maturation of female oocyte, centromeres are critical for accurate segregation of homologous chromosomes and sister chromatids. However, the dynamics of oocyte centromere transcription and whether it associated with DNA repair proteins are unknown. In this study, we found the transcription of centromeres is active in growing oocytes but it is silenced when oocytes are fully grown. DNA repair proteins like Mlh1, Mre11 and Prkdc are found associated with the minor satellites and this association can be interfered by RNA polymerase II inhibitor α-amanitin. When the growing oocyte is in vitro matured, Mlh1/Mre11/Prkdc foci would release from centromeres to the ooplasm. If the oocytes are treated with Mre11 inhibitor Mirin, the meiosis resumption of growing oocytes with Mre11 foci can be suppressed. These data revealed the dynamic of centromeric transcription in oocytes and its potential association with DNA repair proteins, which provide clues about how oocytes maintain centromere stability and assemble kinetochores.
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In human, endo- or exogeneous factors might alter the cellular composition, the endocrine and inflammatory micro-environments and the metabolic balance in testis. These factors will further impair the testicular spermatogenesis capacity and alter the transcriptome of testis. Conversely, it should be possible that the alteration of the transcriptomes in testes be used as an indicator to evaluate the testicular spermatogenesis capacity and to predict the causing factors. In this study, using the transcriptome data of human testes and whole blood which were collected by the genotype-tissue expression project (GTEx), we analyzed the transcriptome differences in human testes and explored those factors that affecting spermatogenesis. As a result, testes were clustered into five clusters according to their transcriptomic features, and each cluster of testes was evaluated as having different spermatogenesis capacity. High rank genes of each cluster and the differentially expressed genes in lower functional testes were analyzed. Transcripts in whole blood which may be associated with testis function were also analyzed by the correlation test. As a result, factors such as immune response, oxygen transport, thyrotropin, prostaglandin and tridecapeptide neurotensin were found associated with spermatogenesis. These results revealed multiple clues about the spermatogenesis regulation in testis and provided potential targets to improve the fertility of men in clinic.
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Testículo , Transcriptoma , Humanos , Masculino , Testículo/metabolismo , Espermatogénesis/genética , Perfilación de la Expresión GénicaRESUMEN
Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis, are widespread in developed countries and gradually increasing in developing countries. Evidences showed that man with CD has a decrease of serum testosterone, but how IBD take effects on testicular testosterone synthesis is not well elucidated. To investigate the effects of IBD on testis, we analyzed testicular metabolome and transcriptome data of the dextran sulfate sodium (DSS) induced IBD mice. As a result, metabolomic data showed that DSS indeed induced androgen decrease in mouse testis. Correspondingly, androgen synthesis associated genes, especially Lhcgr, were down-regulated in DSS testis. From the metabolomic data, we found vitamin intake associated metabolites vitamin B2 and pyridoxamine were significantly decreased, whereas fatty acid metabolism associated molecules N-lauroylglycine and N-decanoylglycine were increased in DSS testis. In addition, we found 8-hydroxy-deoxyguanosine, a DNA oxidative damage marker, and 8-oxoguanine, a molecule responsible for DNA damage repair, were also changed in DSS testis. Simultaneously, our data also showed that DSS up-regulated the expression of meiosis initiation associated gene Stra8 and oxygen transport associated genes in testis. In summary, these results depicted the complex effects of colitis on testis. These metabolites and transcripts changed in DSS testis could be used as potential targets for IBD treatment or symptom relieve.
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Colitis/genética , Colitis/metabolismo , Testículo/metabolismo , Animales , Colitis/inducido químicamente , Sulfato de Dextran , Modelos Animales de Enfermedad , Masculino , Metaboloma , Ratones Endogámicos ICR , TranscriptomaRESUMEN
Utilizing microinjection to introduce biological molecules such as DNA, mRNA, siRNA, and proteins into the cell is well established to study oocyte maturation and early embryo development in vitro. However, microinjection is an empirical technology. The cellular survival after microinjection is mainly dependent on the operator, and an experienced operator should be trained for a long time, from several months to years. Optimizing the microinjection to be highly efficient and quickly learned should be helpful for new operators and some newly established laboratories. Here, we combined the tip pipette and piezo-assisted micromanipulator to microinject the oocyte and early embryos at different stages of mouse. The results showed that the survival rate after microinjection was more than 85% for cumulus-oocyte complex, germinal vesicle oocyte, two-cell, and four-cell embryos, and close to 100% for MII oocyte and zygotes. The high-rate survival of microinjection can save many experimental samples. Thus, it should be helpful in studying some rare animal models such as aging and conditional gene knockout mice. Furthermore, our protocol is much easier to learn for new operators, who can usually master the method proficiently after several training times. Therefore, we would like to publicly share this experience, which will help some novices master microinjection skillfully and save many laboratory animals.
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Within the development of ovarian follicle, in addition to cell proliferation and differentiation, sophisticated cell-cell cross talks are established among follicular somatic cells such as granulosa cells (GCs) and theca cells. To systematically reveal the cell differentiation and signal transductions in follicular somatic cells, we collected the mouse follicular somatic cells from secondary to ovulatory stage, and analyzed the single cell transcriptomes. Having data filtered and screened, we found 6883 high variable genes in 4888 single cells. Then follicular somatic cells were clustered into 26 cell clusters, including 18 GC clusters, 4 theca endocrine cell (TEC) clusters, and 4 other somatic cell clusters, which include immune cells and Acta2 positive theca externa cells. From our data, we found there was metabolic reprogramming happened during GC differentiation. We also found both Cyp19a1 and Cyp11a1 could be expressed in TECs. We analyzed the expression patterns of genes associated with cell-cell interactions such as steroid hormone receptor genes, insulin signaling genes, and cytokine/transformation growth factor beta associated genes in all cell clusters. Lastly, we clustered the highly variable genes into 300 gene clusters, which could be used to search new genes involved in follicle development. These transcriptomes of follicular somatic cells provide us potential clues to reveal how mammals regulating follicle development and could help us find targets to improve oocyte quality for women with low fertility.
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Comunicación Celular/genética , Expresión Génica/fisiología , Folículo Ovárico/metabolismo , Transducción de Señal , Transcriptoma , Animales , Femenino , Ratones , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR is initiated have not been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short-scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2'-deoxyuridine (EdU). These ssBIRs could only be induced in the fully grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.
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Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Replicación del ADN/fisiología , Animales , Afidicolina/farmacología , Células Cultivadas , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos de Desoxiadenina/farmacología , Didesoxinucleótidos/farmacología , Femenino , Fase G2 , Indoles/farmacología , Ratones , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Oocitos , Cultivo Primario de Células , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/metabolismo , Tetrahidroisoquinolinas/farmacologíaRESUMEN
Cell division consists of nuclear division (mitosis for somatic cells and meiosis for germ cells) and cytoplasmic division (cytokinesis). Embryonic developments are highly programmed, and thus, each cellular event during early embryo development is stable. For mouse embryos, the first time of mitosis is completed about 22 h after fertilization. However, it remains unclear when the embryo completes its first cytokinesis. Here, we microinjected only one cell in the 2-cell stage mouse embryos with mRNA, which encodes green fluorescence protein (GFP). By monitoring the GFP protein transport dynamics between the two cells, we demonstrated that the first time of cytokinesis in mouse embryos is completed about 15 h after mitosis, namely 37 h after fertilization. In addition, our results indicate that the cytoplasmic protein transport between daughter cells is very effective, which relies on microtubules instead of microfilaments in 2-cell mouse embryos. These results should enrich people's understanding of the first cell division and cytoskeleton in mouse embryos and then learn more about the mechanisms of early embryo development in mammals.
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Cruciform DNA is a causing factor of genome instability and chromosomal translocation, however, most studies about cruciform DNA in mammalian cells were based on palindromic sequences containing plasmids and reports about endogenous cruciform DNA are rare. In this study we observed the dynamics of endogenous cruciform DNA in mouse growing oocytes using immunofluorescence labeling method. We found cruciform DNA foci exist in transcription active growing oocytes but not in transcription inactive fully grown oocytes and colocalized with Parp1 but not with DNA damage marker γH2A.X. By analyzing the Genotype-Tissue Expression data, we found cruciform DNA-mediated chromosomal translocation in human spermatocytes is associated with the specific DNA transcription in testis. When inhibiting the transcription with α-amanitin in mouse oocytes, we found oocyte cruciform DNA foci decreased significantly. In summary, we observed the endogenous cruciform DNA in growing oocytes and our results showed that the cruciform DNA formation is transcription-dependent.
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ADN Cruciforme/metabolismo , Oocitos , Transcripción Genética/fisiología , Alfa-Amanitina/efectos adversos , Animales , Técnica del Anticuerpo Fluorescente/métodos , Histonas/metabolismo , Masculino , Ratones , Oocitos/citología , Oocitos/crecimiento & desarrollo , Oogénesis , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Espermatogénesis , Testículo/citología , Testículo/metabolismoRESUMEN
As a gamete, oocyte needs to maintain its genomic integrity and passes this haploid genome to the next generation. However, fully-grown mouse oocyte cannot respond to DNA double-strand breaks (DSBs) effectively and it is also unable to repair them before the meiosis resumption. To compensate for this disadvantage and control the DNA repair events, oocyte needs the cooperation with its surrounding cumulus cells. Recently, evidences have shown that nuclear actin filament formation plays roles in cellular DNA DSB repair. To explore whether these nuclear actin filaments are formed in the DNA-damaged oocytes, here, we labeled the filament actins in denuded oocytes (DOs) and cumulus-enclosed oocytes (CEOs). We observed that the nuclear actin filaments were formed only in the DNA-damaged CEOs, but not in DOs. Formation of actin filaments in the nucleus was an event downstream to the DNA damage response. Our data also showed that the removal of cumulus cells led to a reduction in the nuclear actin filaments in oocytes. Knocking down of the Adcy1 gene in cumulus cells did not affect the formation of nuclear actin filaments in oocytes. Notably, we also observed that the nuclear actin filaments in CEOs could be induced by inhibition of gap junctions. From our results, it was confirmed that DNA DSBs induce the nuclear actin filament formation in oocyte and which is controlled by the cumulus cells.
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Citoesqueleto de Actina/metabolismo , Células del Cúmulo/citología , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Oocitos/citología , Citoesqueleto de Actina/genética , Adenilil Ciclasas/genética , Animales , Femenino , Uniones Comunicantes/metabolismo , Ratones , Ratones Endogámicos ICR , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions.
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Células del Cúmulo/metabolismo , ADN/metabolismo , Oocitos/metabolismo , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Carbenoxolona/farmacología , Células del Cúmulo/citología , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , ADN/química , Roturas del ADN de Doble Cadena/efectos de los fármacos , Femenino , Uniones Comunicantes/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Meiosis/efectos de los fármacos , Metafase , Ratones Endogámicos ICR , Oocitos/citología , Oocitos/efectos de los fármacos , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Prior to the mechanization of agriculture and labor-intensive tasks, humans used donkeys (Equus africanus asinus) for farm work and packing. However, as mechanization increased, donkeys have been increasingly raised for meat, milk, and fur in China. To maintain the development of the donkey industry, breeding programs should focus on traits related to these new uses. Compared to conventional marker-assisted breeding plans, genome- and transcriptome-based selection methods are more efficient and effective. To analyze the coding genes of the donkey genome, we assembled the transcriptome of donkey white blood cells de novo. Using transcriptomic deep-sequencing data, we identified 264,714 distinct donkey unigenes and predicted 38,949 protein fragments. We annotated the donkey unigenes by BLAST searches against the non-redundant (NR) protein database. We also compared the donkey protein sequences with those of the horse (E. caballus) and wild horse (E. przewalskii), and linked the donkey protein fragments with mammalian phenotypes. As the outer ear size of donkeys and horses are obviously different, we compared the outer ear size-associated proteins in donkeys and horses. We identified three ear size-associated proteins, HIC1, PRKRA, and KMT2A, with sequence differences among the donkey, horse, and wild horse loci. Since the donkey genome sequence has not been released, the de novo assembled donkey transcriptome is helpful for preliminary investigations of donkey cultivars and for genetic improvement.