O-207 zp1 mutation affects oocyte energy metabolism and leads to oocyte apoptosis

Abstract Study question How does the zp1 variation leads to apoptosis oocytes in antral follicular and infertility in females? Summary answer Mutant zp1 induces abnormal pyruvate transport leading to the accumulation of reactive oxygen species (ROS), which activates the mitochondria-mediated apoptosis pathway leading to oocytes apoptosis. What is known already Previous research reported a zp1 mutation in human infertility, and we develop a homologous rat strain to investigate the pathogenic effect. The ovaries of homozygous rat posses growing and fully grown oocytes, the oocytes completely lack a zona pellucida. Only 1-2 eggs were recovered from oviducts of per superovulated homozygous rat, and were not surrounded by a zona pellucida thus lost their fertilization capacity in vitro.Further studies showed that the homozygous female rats not only lacked ZP but also TZPs (an important communication bridge between granulosa cells and oocytes), and the caval follicles showed obvious apoptotic signals. Study design, size, duration In this study, 10 Sprague-Dawley rats were used at 8 weeks of age (5 wild-type and 5 mutant respectively). Participants/materials, setting, methods We used CRISPR/Cas 9-mediated genomic editing technology to develop a rat model carrying the homologous 8bp deleted mutation in Zp1 gene(Zp1mt/mt). The rats were sacrificed by cervical vertebra dislocation and their ovaries were isolated immediately. Then using bulk RNA sequencing of ovaries from Zp1mt/mt and wild-type female rats to identify the differential expression gene between the two groups.The differentially expressed genes enriched from KEGG were verified by qRT-PCR and in vitro functional experiments. Main results and the role of chance Total of 293 DEGs were obtained, of which 144 were up-regulated and 149 were down-regulated. The results of biological process showed that up-regulated genes were mainly involved in redox reaction and negative regulation of cell migration.Down-regulated genes are mainly involved in the follicular growth and development,and related to oocyte energy metabolism. In order to verify the DEG results obtained from RNA sequencing, the differentially expressed gene Gcgr, Plcβ2, Aox3 and Hpgd enriched from KEGG in cell metabolism were verified by qRT-PCR. The results showed that the differentially expressed gene results were consistent with the expression trend of sequencing data.The results showed that there was no significant difference in the expression of Pkm in granulosa cells, while there was a significant difference in the expression of Pdk4 in Zp1mt/mt oocytes. For ROS detection, 52 eggs of 4 Zp1mt/mt rats and 48 eggs of 6 wide-type rats were analyzed for reactive oxygen species. The results showed that the IOD of mutant eggs was significantly higher than that of wild-type,indicating that ROS content in mutant eggs was significantly increased, which was statistically significant.The detection of apoptotic protein expression in eggs shows that the apoptosis probability of Zp1mt/mt eggs is significantly higher than that ofwild-type. Limitations, reasons for caution This study provides more experimental basis and data support for the involvement of ZP in oogenesis, but the effect of ZP deficiency on the dynamic transcriptional regulation of developing oocytes-granulosa cells and the specific molecular mechanism remain to be further explored. Wider implications of the findings The work provides key insights into the crucial features of the transcriptional regulation in Zp1 mutant animal ovaries, and offers important clues for exploring the potential mechanism of abnormal oocyte development due to ZP deficiency. Trial registration number none