RESUMEN
Self-renewal of spermatogonial stem cells is vital to lifelong production of male gametes and thus fertility. However, the underlying mechanisms remain enigmatic. Here, we show that DOT1L, the sole H3K79 methyltransferase, is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L fail to maintain spermatogonial stem cells, characterized by a sequential loss of germ cells from spermatogonia to spermatids and ultimately a Sertoli cell only syndrome. Inhibition of DOT1L reduces the stem cell activity after transplantation. DOT1L promotes expression of the fate-determining HoxC transcription factors in spermatogonial stem cells. Furthermore, H3K79me2 accumulates at HoxC9 and HoxC10 genes. Our findings identify an essential function for DOT1L in adult stem cells and provide an epigenetic paradigm for regulation of spermatogonial stem cells.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Espermatogonias , Células Madre , Animales , Diferenciación Celular , Masculino , Ratones , Espermatogonias/citología , Espermatogonias/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
Maternal aging can impair the quality and decrease the developmental competence of ovulated oocytes. In this study, compromised germinal vesicle breakdown (GVBD) was found in aged mice oocytes. Furthermore, we observed increased reactive oxygen species (ROS) and mitochondrial Ca2+ levels, along with reduced mitochondrial temperature in aged oocytes. Maternal aging also changed the crotonylation level in oocytes. Forkhead box O3 (FoxO3a), a member of the forkhead protein family involved in the regulation of cell survival and life span reached a peak level in the metaphase II stage. Compared with a younger group, FoxO3a expression increased in aged oocytes. Intracellular localization of FoxO3a changed from the cytoplasm to chromatin in response to aging. The expression of the upstream regulator nicotinamide-phosphoribosyltransferase (Nampt) peaked in the GVBD stage. Moreover, Nampt expression was increased in aged oocytes, and more intense staining of Nampt was found in aged mice ovary. To further study the role of Nampt in mitochondrial function, specific agonist P7C3 and inhibitor FK866 were applied to aged oocytes, and FK866 significantly decreased adenosine triphosphate and mitochondrial membrane potential. In conclusion, mitochondrial dysfunction in aged oocytes was associated with elevated FoxO3a, and suppression of Nampt could further impair mitochondrial function.
Asunto(s)
Proteína Forkhead Box O3/metabolismo , Mitocondrias , Oocitos , Animales , Femenino , Potencial de la Membrana Mitocondrial , Metafase , Ratones , Mitocondrias/metabolismo , Oocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
More than a decade ago, the ENCODE and NIH Epigenomics Roadmap consortia organized large multilaboratory efforts to profile the epigenomes of >110 different mammalian somatic cell types. This generated valuable publicly accessible datasets that are being mined to reveal genome-wide patterns of a variety of different epigenetic parameters. This consortia approach facilitated the powerful and comprehensive multiparametric integrative analysis of the epigenomes in each cell type. However, no germ cell types were included among the cell types characterized by either of these consortia. Thus, comprehensive epigenetic profiling data are not generally available for the most evolutionarily important cells, male and female germ cells. We discuss the need for reproductive biologists to generate similar multiparametric epigenomic profiling datasets for both male and female germ cells at different developmental stages and summarize our recent effort to derive such data for mammalian spermatogonial stem cells and progenitor spermatogonia.
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Células Madre Germinales Adultas/metabolismo , Epigenoma , Epigenómica , Óvulo/crecimiento & desarrollo , Espermatozoides/crecimiento & desarrollo , Animales , Diferenciación Celular , Epigénesis Genética , Femenino , Masculino , Mamíferos , Espermatogonias/crecimiento & desarrolloRESUMEN
Precise separation of spermatogonial stem cells (SSCs) from progenitor spermatogonia that lack stem cell activity and are committed to differentiation remains a challenge. To distinguish between these spermatogonial subtypes, we identified genes that exhibited bimodal mRNA levels at the single-cell level among undifferentiated spermatogonia from Postnatal Day 6 mouse testes, including Tspan8, Epha2, and Pvr, each of which encode cell surface proteins useful for cell selection. Transplantation studies provided definitive evidence that a TSPAN8-high subpopulation is enriched for SSCs. RNA-seq analyses identified genes differentially expressed between TSPAN8-high and -low subpopulations that clustered into multiple biological pathways potentially involved in SSC renewal or differentiation, respectively. Methyl-seq analysis identified hypomethylated domains in the promoters of these genes in both subpopulations that colocalized with peaks of histone modifications defined by ChIP-seq analysis. Taken together, these results demonstrate functional heterogeneity among mouse undifferentiated spermatogonia and point to key biological characteristics that distinguish SSCs from progenitor spermatogonia.
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Células Madre Germinales Adultas/citología , Testículo/citología , Tetraspaninas/metabolismo , Células Madre Germinales Adultas/metabolismo , Animales , Biomarcadores/metabolismo , Ciclo Celular/fisiología , Perfilación de la Expresión Génica , Masculino , Ratones , Receptor EphA2/genética , Receptor EphA2/metabolismo , Espermatogénesis , Testículo/metabolismo , Tetraspaninas/genéticaRESUMEN
This study was conducted to determine the impact of vitrification on the expression of genes regulating pluripotency and apoptosis in mouse morulae. The morulae were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution without freezing (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In vitro development was evaluated by morphology and assessed by the blastocyst rate and the blastocyst total cell number. Gene expression in morulae and blastocysts was assessed by quantitative Real Time-PCR (qRT-PCR) and western blot. The results showed that at morulae stage, the POU class 5 homeobox1 (Oct-4) and B-cell lymphoma2 (Bcl2) mRNA levels of vitrification group were significantly lower (P < 0.05) than those of control. Strikingly, the p53 mRNA level was significantly higher in vitrification group. However, the Oct-4, Bcl2 and p53 mRNA levels in mouse blastocysts were not statistically different. Furthermore, western blot results showed that there was no significant difference in Oct-4, Bcl2 and p53 expression at protein level in mouse morulae among three groups. Additionally, the blastocyst rate (96.67%-100.00%) and the average cell number of blastocysts (89.67-92.33) were similar between all groups. The data demonstrate that vitrification transiently changes the mRNA expression of several key genes in mouse morulae regulating early embryo development but does not affect embryo developmental potential in vitro.
Asunto(s)
Blastocisto/fisiología , Desarrollo Embrionario/fisiología , Mórula/fisiología , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/biosíntesis , Proteína p53 Supresora de Tumor/genética , Vitrificación , Animales , Apoptosis/genética , Recuento de Células , Criopreservación/métodos , Femenino , Congelación , Expresión Génica , Ratones , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , ARN Mensajero/genética , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
BACKGROUND: An easy and user friendly protocol that produces consistent results will facilitate the commercial application of embryo vitrification technology in the field. OBJECTIVE: This study was designed to develop a simple and efficient vitrification, in-straw dilution and direct transfer method for bovine embryos. METHODS: After being vitrified and in-straw thawed, in vivo-derived and in vitro-produced bovine embryos were subjected to in vitro culture or embryo transplantation. RESULTS: There were no significant differences (P > 0.05) in survival rates (100.0% vs. 93.9%) and expansion rates (93.8% vs. 87.5%) between in vivo-derived and in vitro-produced blastocysts after vitrification and in-straw dilution. And there was also no significant difference (P > 0.05) in conception rates (56.5% vs. 58.8%) after ET between cryopreserved and fresh in vivo-derived blastocysts. CONCLUSION: Vitrification using EG-based vitrification solution and in-straw dilution with PBS-based diluent is a simple and efficient method for cryopreservation and direct transfer of bovine embryos.
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Criopreservación/veterinaria , Transferencia de Embrión/veterinaria , Fertilización In Vitro/veterinaria , Vitrificación , Animales , Blastocisto/ultraestructura , Bovinos , Criopreservación/métodos , Transferencia de Embrión/métodos , Fertilización , Fertilización In Vitro/métodosRESUMEN
Aberrant male germline development can lead to the formation of seminoma, a testicular germ cell tumor. Seminomas are biologically similar to primordial germ cells (PGCs) and many bear an isochromosome 12p [i(12p)] with two additional copies of the short arm of chromosome 12. By mapping seminoma transcriptomes and open chromatin landscape onto a normal human male germline trajectory, we find that seminoma resembles premigratory/migratory PGCs; however, it exhibits enhanced germline and pluripotency programs and upregulation of genes involved in apoptosis, angiogenesis, and MAPK/ERK pathways. Using pluripotent stem cell-derived PGCs from Pallister-Killian syndrome patients mosaic for i(12p), we model seminoma and identify gene dosage effects that may contribute to transformation. As murine seminoma models do not exist, our analyses provide critical insights into genetic, cellular, and signaling programs driving seminoma transformation, and the in vitro platform developed herein permits evaluation of additional signals required for seminoma tumorigenesis.
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Epigénesis Genética , Células Germinativas , Seminoma , Neoplasias Testiculares , Humanos , Seminoma/genética , Seminoma/patología , Seminoma/metabolismo , Masculino , Células Germinativas/metabolismo , Neoplasias Testiculares/genética , Neoplasias Testiculares/patología , Neoplasias Testiculares/metabolismo , Transcripción Genética , Regulación Neoplásica de la Expresión Génica , Transcriptoma/genéticaRESUMEN
The continuous regeneration of spermatogonial stem cells (SSCs) underpins spermatogenesis and lifelong male fertility, but the developmental origins of the SSC pool remain unclear. Here, we document that hnRNPU is essential for establishing the SSC pool. In male mice, conditional loss of hnRNPU in prospermatogonia (ProSG) arrests spermatogenesis and results in sterility. hnRNPU-deficient ProSG fails to differentiate and migrate to the basement membrane to establish SSC pool in infancy. Moreover, hnRNPU deletion leads to the accumulation of ProSG and disrupts the process of T1-ProSG to T2-ProSG transition. Single-cell transcriptional analyses reveal that germ cells are in a mitotically quiescent state and lose their unique identity upon hnRNPU depletion. We further show that hnRNPU could bind to Vrk1, Slx4, and Dazl transcripts that have been identified to suffer aberrant alternative splicing in hnRNPU-deficient testes. These observations offer important insights into SSC pool establishment and may have translational implications for male fertility.
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Espermatogénesis , Espermatogonias , Animales , Masculino , Ratones , Células Madre Germinales Adultas/metabolismo , Empalme Alternativo/genética , Diferenciación Celular , Espermatogénesis/genética , Espermatogonias/metabolismo , Espermatogonias/citología , Células Madre/metabolismo , Células Madre/citología , Testículo/metabolismo , Testículo/citología , Ribonucleoproteína Heterogénea-Nuclear Grupo U/metabolismoRESUMEN
The final data-generation step of genome-wide profiling of any epigenetic parameter typically involves DNA deep sequencing which yields large datasets that must then be computationally analyzed both individually and collectively to comprehensively describe the epigenetic programming that dictates cell fate and function. Here, we describe computational pipelines for analysis of bulk mepigenomic profiling data, including whole-genome bisulfite sequencing (WGBS) to detect DNA methylation patterns, chromatin immunoprecipitation-sequencing (ChIP-seq) to detect genomic patterns of either specific histone modifications or bound transcription factors, the assay for transposase-accessible chromatin-sequencing (ATAC-seq) to detect genomic patterns of chromatin accessibility, and high-throughput chromosome conformation capture-sequencing (Hi-C-seq) to detect 3-dimensional interactions among distant genomic regions. In addition, we describe Chromatin State Discovery and Characterization (ChromHMM) methodology to integrate data from these individual analyses, plus that from RNA-seq analysis of gene expression, to obtain the most comprehensive overall assessment of epigenetic programming associated with gene expression.
Asunto(s)
Cromatina , Epigenómica , Epigenómica/métodos , Cromatina/genética , Secuenciación de Inmunoprecipitación de Cromatina , Análisis de Secuencia de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Epigénesis Genética , Células MadreRESUMEN
Epigenomics encompasses analyses of a variety of different epigenetic parameters which, collectively, make up the epigenetic programming that dictates cell fate and function. Here, protocols are provided for four different epigenomic methods including whole-genome bisulfite sequencing (WGBS) to assess DNA methylation patterns, chromatin immunoprecipitation-sequencing (ChIP-seq) to assess genomic patterns of either specific histone modifications or bound transcription factors, the assay for transposase-accessible chromatin-sequencing (ATAC-seq) to assess genomic patterns of chromatin accessibility, and high-throughput chromosome conformation capture-sequencing (Hi-C-seq) to assess three-dimensional interactions among distant genomic regions, plus computational methodology to integrate data from those four methodologies using Chromatin State Discovery and Characterization (ChromHMM) to obtain the most comprehensive overall assessment of epigenetic programming.
Asunto(s)
Cromatina , Epigenómica , Epigenómica/métodos , Análisis de Secuencia de ADN/métodos , Cromatina/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Epigénesis Genética , Células MadreRESUMEN
Reconstitution of germ cell fate from pluripotent stem cells provides an opportunity to understand the molecular underpinnings of germ cell development. Here, we established robust methods for induced pluripotent stem cell (iPSC) culture in the common marmoset (Callithrix jacchus [cj]), allowing stable propagation in an undifferentiated state. Notably, iPSCs cultured on a feeder layer in the presence of a WNT signaling inhibitor upregulated genes related to ubiquitin-dependent protein catabolic processes and enter a permissive state that enables differentiation into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs in vivo. Induction of cjPGCLCs is accompanied by transient upregulation of mesodermal genes, culminating in the establishment of a primate-specific germline transcriptional network. Moreover, cjPGCLCs can be expanded in monolayer while retaining the germline state. Upon co-culture with mouse testicular somatic cells, these cells acquire an early prospermatogonia-like phenotype. Our findings provide a framework for understanding and reconstituting marmoset germ cell development in vitro, thus providing a comparative tool and foundation for a preclinical modeling of human in vitro gametogenesis.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Animales , Ratones , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Callithrix , Diferenciación Celular , Células Madre Pluripotentes/metabolismo , Células Germinativas/metabolismoRESUMEN
Development of the adrenal cortex, a vital endocrine organ, originates in the adrenogonadal primordium, a common progenitor for both the adrenocortical and gonadal lineages in rodents. In contrast, we find that in humans and cynomolgus monkeys, the adrenocortical lineage originates in a temporally and spatially distinct fashion from the gonadal lineage, arising earlier and more anteriorly within the coelomic epithelium. The adrenal primordium arises from adrenogenic coelomic epithelium via an epithelial-to-mesenchymal transition, which then progresses into the steroidogenic fetal zone via both direct and indirect routes. Notably, we find that adrenocortical and gonadal lineages exhibit distinct HOX codes, suggesting distinct anterior-posterior regionalization. Together, our assessment of the early divergence of these lineages provides a molecular framework for understanding human adrenal and gonadal disorders.
RESUMEN
The mechanisms leading to adrenal cortex development and steroid synthesis in humans remain poorly understood due to the paucity of model systems. Herein, we recapitulate human fetal adrenal cortex specification processes through stepwise induction of human-induced pluripotent stem cells through posterior intermediate mesoderm-like and adrenocortical progenitor-like states to ultimately generate fetal zone adrenal-cortex-like cells (FZLCs), as evidenced by histomorphological, ultrastructural, and transcriptome features and adrenocorticotropic hormone (ACTH)-independent Δ5 steroid biosynthesis. Furthermore, FZLC generation is promoted by SHH and inhibited by NOTCH, ACTIVIN, and WNT signaling, and steroid synthesis is amplified by ACTH/PKA signaling and blocked by inhibitors of Δ5 steroid synthesis enzymes. Finally, NR5A1 promotes FZLC survival and steroidogenesis. Together, these findings provide a framework for understanding and reconstituting human adrenocortical development in vitro, paving the way for cell-based therapies of adrenal insufficiency.
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Corteza Suprarrenal , Células Madre Pluripotentes Inducidas , Humanos , Vía de Señalización Wnt , Hormona Adrenocorticotrópica , EsteroidesRESUMEN
Translation of stem cell therapies to the clinic will be most successful following optimization of efficacy and safety in appropriate preclinical model systems. Among available models, nonhuman primates (NHPs) provide the most accurate recapitulation of human anatomy, physiology, genetics and epigenetics. Here, we show that baboon pluripotent cells (PSCs) recapitulate key molecular features of human PSCs with greater accuracy than that found in PSCs from non-primate species such as mice. Specifically, baboon and human PSCs exhibit greater conservation of gene expression patterns, higher sequence and structural homology among pluripotency factors, more equivalent genome-wide patterns of histone and DNA methylation modifications, and similar maintenance of bivalent programming of developmental genes than that found between human and non-primate PSCs.
RESUMEN
In the early fetal stage, the gonads are bipotent and only later become the ovary or testis, depending on the genetic sex. Despite many studies examining how sex determination occurs from biopotential gonads, the spatial and temporal organization of bipotential gonads and their progenitors is poorly understood. Here, using lineage tracing in mice, we find that the gonads originate from a T+ primitive streak through WT1+ posterior intermediate mesoderm and appear to share origins anteriorly with the adrenal glands and posteriorly with the metanephric mesenchyme. Comparative single-cell transcriptomic analyses in mouse and cynomolgus monkey embryos reveal the convergence of the lineage trajectory and genetic programs accompanying the specification of biopotential gonadal progenitor cells. This process involves sustained expression of epithelial genes and upregulation of mesenchymal genes, thereby conferring an epithelial-mesenchymal hybrid state. Our study provides key resources for understanding early gonadogenesis in mice and primates.
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Células Madre Adultas/metabolismo , Gónadas/fisiología , Animales , Diferenciación Celular , Macaca fascicularis , Masculino , RatonesRESUMEN
Spermatogonial stem cells (SSCs) both self-renew and give rise to progenitors that initiate spermatogenic differentiation in the mammalian testis. Questions remain regarding the extent to which the SSC and progenitor states are functionally distinct. Here we provide the first multiparametric integrative analysis of mammalian germ cell epigenomes comparable with that done for >100 somatic cell types by the ENCODE Project. Differentially expressed genes distinguishing SSC- and progenitor-enriched spermatogonia showed distinct histone modification patterns, particularly for H3K27ac and H3K27me3. Motif analysis predicted transcription factors that may regulate spermatogonial subtype-specific fate, and immunohistochemistry and gene-specific chromatin immunoprecipitation analyses confirmed subtype-specific differences in target gene binding of a subset of these factors. Taken together, these results show that SSCs and progenitors display distinct epigenetic profiling consistent with these spermatogonial subtypes being differentially programmed to either self-renew and maintain regenerative capacity as SSCs or lose regenerative capacity and initiate lineage commitment as progenitors.
RESUMEN
Spermatogenesis is a complex and dynamic cellular differentiation process critical to male reproduction and sustained by spermatogonial stem cells (SSCs). Although patterns of gene expression have been described for aggregates of certain spermatogenic cell types, the full continuum of gene expression patterns underlying ongoing spermatogenesis in steady state was previously unclear. Here, we catalog single-cell transcriptomes for >62,000 individual spermatogenic cells from immature (postnatal day 6) and adult male mice and adult men. This allowed us to resolve SSC and progenitor spermatogonia, elucidate the full range of gene expression changes during male meiosis and spermiogenesis, and derive unique gene expression signatures for multiple mouse and human spermatogenic cell types and/or subtypes. These transcriptome datasets provide an information-rich resource for studies of SSCs, male meiosis, testicular cancer, male infertility, or contraceptive development, as well as a gene expression roadmap to be emulated in efforts to achieve spermatogenesis in vitro.
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Mamíferos/genética , Análisis de la Célula Individual , Espermátides/citología , Espermatogénesis/genética , Espermatogonias/citología , Transcriptoma/genética , Adulto , Envejecimiento/genética , Animales , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Haploidia , Humanos , Masculino , Meiosis , Ratones Endogámicos C57BL , Transducción de Señal , Espermátides/metabolismo , Espermatogonias/metabolismo , Células Madre/citología , Células Madre/metabolismo , Testículo/citologíaRESUMEN
Our study examined the in vivo chimeric and survival capacities of chimeras created by injecting tetraploid embryonic stem cells (ESCs) expressing green fluorescent protein (GFP) into diploid embryos. At 3.5 days post-coitum (dpc) and 4.5 dpc, the tetraploid ESCs were able to contribute to the inner cell mass (ICM) just as diploid ESCs tagged with GFP. At 6.5 dpc, 8.0 dpc and 10.5 dpc, the tetraploid ESCs manifested in the same location as the diploid ESCs. The GFP cells in the extraembryonic tissues and fetuses of tetraploid ESC chimeras were tetraploid as determined by fluorescence activated cell sorting (FACS). Furthermore, tetraploid ESCs contributed to the development of the placenta, embryolemma and umbilical cord at 13.5 dpc and 16.5 dpc; however, very less GFP cells were found in the fetuses of tetraploid ESC chimeras. We further found that the proliferation of tetraploid ESCs was slower than that of diploid ESCs. In addition, the relative mRNA expression in the three germ layers and the trophoblast was abnormal in the EBs of tetraploid ESCs compared with diploid ESCs. In short, slower proliferation and abnormal differentiation potential of tetraploid ESCs might be two of the reasons for their poor survival and chimeric capacities.
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Quimerismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Desarrollo Fetal/genética , Organogénesis/genética , Tetraploidía , Blastómeros/citología , Blastómeros/metabolismo , Diferenciación Celular , Línea Celular , Proliferación Celular/genética , Diploidia , Expresión Génica , Genes Reporteros , CariotipoRESUMEN
This study was conducted to investigate the effects of spermatozoa cryopreservation on DNA demethylation in mouse zygotes. Global methylation was studied in zygotes fertilized with cryopreserved sperm by immunostaining, and relative transcript abundance of Tet3, a key gene responsible for zygotic DNA demethylation, was examined by real-time quantitative polymerase chain reaction. Fresh sperm group served as control. Results indicated spermatozoa cryopreservation decreased fertilization rate (68.2% vs. 86.9%; P < 0.01) and delayed pronuclear formation (P < 0.05), compared with the control group. The percentages of embryos developed to cleavage and blastocyst stages in the freezing group (52.9% and 66.8%, respectively) were lower (P < 0.01 and P < 0.05, respectively) than those of the control group (83.4% and 81.1%, respectively). Furthermore, embryos obtained from cryopreserved sperm had higher relative methylation levels (P < 0.05) and less Tet3 mRNA concentrations (P < 0.01) in advanced pronuclear stages. Hence, we reported that spermatozoa cryopreservation disturbed the Tet3-mediated DNA demethylation progression in the zygotic paternal genome, which could be detrimental to the development of early mouse embryos, and most of the differences observed might be explained by delayed fertilization when using cryopreserved sperm.
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Criopreservación/métodos , Preservación de Semen/métodos , Espermatozoides/fisiología , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dioxigenasas , Técnicas de Cultivo de Embriones , Fertilización In Vitro , Regulación del Desarrollo de la Expresión Génica/fisiología , Masculino , Ratones , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , CigotoRESUMEN
BACKGROUND: This study was conducted to investigate effect of exogenous melatonin on the development of mouse mature oocytes after cryopreservation. RESULTS: First, mouse metaphase II (MII) oocytes were vitrified in the open-pulled straws (OPS). After warming, they were cultured for 1 h in M2 medium containing melatonin at different concentrations (0, 10(-9), 10(-7), 10(-5), 10(-3) mol/L). Then the oocytes were used to detect reactive oxygen species (ROS) and glutathione (GSH) levels (fluorescence microscopy), and the developmental potential after parthenogenetic activation. The experimental results showed that the ROS level and cleavage rate in 10(-3) mol/L melatonin group was significantly lower than that in melatonin-free group (control). The GSH levels and blastocyst rates in all melatonin-treated groups were similar to that in control. Based on the above results, we detected the expression of gene Hsp90aa1, Hsf1, Hspa1b, Nrf2 and Bcl-x1 with qRT-PCR in oocytes treated with 10(-7), or 10(-3) mol/L melatonin and untreated control. After warming and culture for 1 h, the oocytes showed higher Hsp90aa1 expression in 10(-7) mol/L melatonin-treated group than in the control (P < 0.05); the Hsf1, Hsp90aa1 and Bcl-x1 expression were significantly decreased in 10(-3) mol/L melatonin-treated group when compared to the control. Based on the above results and previous research, we detected the development of vitrified-warmed oocytes treated with either 10(-7) or 0 mol/L melatonin by in vitro fertilization. No difference was observed between them. CONCLUSIONS: Our results indicate that the supplementation of melatonin (10(-9) to 10(-3) mol/L) in culture medium and incubation for 1 h did not improve the subsequent developmental potential of vitrified-warmed mouse MII oocytes, even if there were alteration in gene expression.