RESUMO
Both endogenous and exogenous factors can induce DNA double-strand breaks (DSBs) in oocytes, which is a potential risk for human-assisted reproductive technology as well as animal nuclear transfer. Here we used bleomycin (BLM) and laser micro-beam dissection (LMD) to induce DNA DSBs in germinal vesicle (GV) stage oocytes and compared the germinal vesicle breakdown (GVBD) rates and first polar body extrusion (PBE) rates between DNA DSB oocytes and untreated oocytes. Employing live cell imaging and immunofluorescence labeling, we observed the dynamics of DNA fragments during oocyte maturation. We also determined the cyclin B1 expression pattern in oocytes to analyze spindle assembly checkpoint (SAC) activity in DNA DSB oocytes. We used parthenogenetic activation to determine if the DNA DSB oocytes could be activated. As a result, we found that the BLM- or LMD-induced DSB oocytes showed lower GVBD rates and took a longer time to undergo GVBD compared with untreated oocytes. PBE was also delayed in DSB oocytes, but once GVBD had occurred, PBE was not affected, even in oocytes with severe DSBs. Compared with control oocytes, the DSB oocytes showed higher SAC activity, as indicated by less Ccnb1-GFP degradation during metaphase I to anaphase I transition. Parthenogenetic activation could activate the metaphase to interphase transition in the DNA DSB mature oocytes, but many oocytes contained multiple pronuclei or numerous micronuclei. These data suggest that DNA damage inhibits or delays the G2/M transition, but once GVBD occurs, DNA-damaged oocytes can complete chromosome separation and polar body extrusion even under a higher SAC activity, causing the formation of numerous micronuclei in early embryos.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Meiose/fisiologia , Oócitos/fisiologia , Animais , Bleomicina/farmacologia , Segregação de Cromossomos/efeitos dos fármacos , Segregação de Cromossomos/fisiologia , Imunofluorescência , Microdissecção e Captura a Laser , Camundongos , Microinjeções , Partenogênese/fisiologia , Corpos Polares/efeitos dos fármacos , Corpos Polares/fisiologia , Fatores de Tempo , Imagem com Lapso de TempoRESUMO
It is well accepted that oocyte meiotic resumption is mainly regulated by the maturation-promoting factor (MPF), which is composed of cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDC2). Maturation-promoting factor activity is regulated by the expression level of CCNB1, phosphorylation of CDC2, and their germinal vesicle (GV) localization. In addition to CCNB1, cyclin O (CCNO) is highly expressed in oocytes, but its biological functions are still not clear. By employing short interfering RNA microinjection of GV-stage oocytes, we found that Ccno knockdown inhibited CDC2 (Tyr15) dephosphorylation and arrested oocytes at the GV stage. To rescue meiotic resumption, cell division cycle 25 B kinase (Cdc25b) and Ccnb1 were overexpressed in the Ccno knockdown oocytes. Unexpectedly, we found that Ccno knockdown did not affect CDC25B entry into the GV, and overexpression of CDC25B was not able to rescue resumption of oocyte meiosis. However, GV breakdown (GVBD) was significantly increased after overexpression of Ccnb1 in Ccno knockdown oocytes, indicating that GVBD block caused by cyclin O knockdown can be rescued by cyclin B1 overexpression. We thus conclude that cyclin O, as an upstream regulator of MPF, plays an important role in oocyte meiotic resumption in mouse oocytes.
Assuntos
Ciclinas/metabolismo , Meiose/genética , Oócitos/metabolismo , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclinas/genética , Feminino , Fator Promotor de Maturação/genética , Fator Promotor de Maturação/metabolismo , Mesotelina , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Microinjeções , Oócitos/citologia , Fosforilação , RNA Interferente PequenoRESUMO
In mouse ovarian follicles, granulosa cells but not oocytes take up glucose to provide the oocyte with nourishments for energy metabolism. Diabetes-induced hyperglycemia or glucose absorption inefficiency consistently causes granulosa cell apoptosis and further exerts a series of negative impacts on oocytes including reduced meiosis resumption rate, low oocyte quality and preimplantation embryo degeneration. Here we compared the transcriptome of mouse oocytes from genetically derived NOD diabetic mice or chemically induced STZ diabetic mice with that of corresponding normal mice. Differentially expressed genes were extracted from the two diabetic models. Gene set enrichment analysis showed that genes associated with metabolic and developmental processes were differentially expressed in oocytes from both models of diabetes. In addition, NOD diabetes also affected the expression of genes associated with ovulation, cell cycle progression, and preimplantation embryo development. Notably, Dnmt1 expression was significantly down-regulated, but Mbd3 expression was up-regulated in diabetic mouse oocytes. Our data not only revealed the mechanisms by which diabetes affects oocyte quality and preimplantation embryo development, but also linked epigenetic hereditary factors with metabolic disorders in germ cells.
