The Effects Of Cited2Level On Oocyte Quality And Embryo Development In Human And Mouse

Cited2, a CBP/p300interacting transcriptional modulator that is crucial for embryonic development. However, little is known about its exact role in oocyte maturation and early embryo development. The purpose of this study was to gain insight into the functions of Cited2during oocyte maturation and pre-implantation embryonic development.During mouse ovary development, the expression of Cited2protein was lowest in0d ovary, significantly increased in4d ovary and highst in8d ovary. Immunohistochemical analysis showed that Cited2protein expression was highest in mouse oocytes at GV stage, and enriched in the germinal vesicle. Furthermore, we also found that Cited2expression was higher in non-surrounded-nucleolus (NSN) GV oocytes, which did not develop past the2-cell stage after fertilization, than in surrounded-nucleolus (SN) oocytes, which developed into normal embryos. Moreover, Cited2expression was higher in in-vitro aged mouse oocyte then the fresh ooyte. To determine the role of Cited2protein in oocyte maturation, we examined the effect of reducing Cited2protein by siRNA s, and the effect of over-expressing a GFP-tagged version of Cited2on oocytes. Reduction of Cited2in mouse GV oocytes accelerated oocyte maturation, whereas over-expression of GFP-Cited2inhibited oocyte maturation by GV arrest and the inhibition of MI progression to MII. Moreover, the unhealthy GV mouse oocyte showed higher Cited2protein levels than healthy GV oocyte.The levels of Cited2protein and mRNA gradually declined with embryo development, which was consistent with earlier reports that Cited2mRNA in the oocyte of maternal origin. Injection of zygotes with GFP-Cited2mRNA led to developmental potentials, including a developmental block at the2-cell stage, inhibition of embryo cleavage, and low rates of blastocyst formation. The level of p21wif/cip1was remarkable increased in embryo with GFP-Cited2overexpression. As the Cited2level was closely related to the quality of mouse oocyte, we was investigated the relationship of Cited2level and the quality of human and anmimal oocytes.In our study, the level of Cited2in aged porcine oocyte was also higher than the fresh porcine oocyte. H2O2and cycloheximide (CHX) were used to artificially promote or inhibit porcine oocyte aging, respectively during in vitro culture. The results demonstrated that incubation of porcine MII oocytes with H2O2led to a high proportion of fragmented oocytes and high level of ROS, while the CHX could inhibit both the increase of fragmentation and ROS level in aged oocytes as well as oocytes with H2O2treatment. A qRT-PCR analysis revealed that level of Cited2and Bim were also markedly increased in aged porcine oocytes with or without H2O2treatment, while level of PPARy was only increased in aged porcine oocytes with H2O2treatment.Using Western immunoblot technology, the levels of Cited2(relative to internal control β-actin levels) in the CCs surrounding oocytes derived from IVF patients were analyzed. The patients were then divided into two groups based on whether the Cited2/p-actin levels in their CCs were above or below the mean level detected for all patients. The oocytes derived from the group of patients whose cumulus cells showed lower Cited2expression levels displayed higher fertilization, transferable embryo, and implantation rates. Moreover, the patients in this group were more likely to have a successful pregnancy outcome. Along with a lower basal follicle-stimulating hormone (FSH) level, active caspase-3protein levels were elevated in the CCs obtained from patients in the group with the higher Cited2levels. A qRT-PCR analysis revealed that a glucose metabolism-related gene (phosphoenolpyruvate carboxykinase1, PCK1) and the progesterone receptor (PR) gene were also markedly increased in CCs derived from patients with the higher Cited2levels of CCs. Furthermore, the glucose content in CCs derived from patients with higher Cited2levels was significantly higher than that in patients with lower Cited2levels. Similarly, the ISCI patients show the same results.In conclusion, our findings suggest that:(1) High Cited2levels inhibited mouse oocyte maturation and embryo development.(2) The Cited2level was closely related to the quality of mouse oocyte.(3) Cited2protein level in CCs can be used as a biomarker to predict embryo quality and clinical outcome. This study allows further elucidation of the role of Cited2in human COCs development and provides new insight for assisted reproduction technology.