Effects And Mechanisms Of A Novel Mutation In Basonuclin 1 Gene On Premature Ovarian Insufficiency

Part Ⅰ Preliminary Functional Studies of a Novel Mutation in Basonuclin 1Objective:To conduct preliminary functional studies of a novel mutation gene,Basonuclin 1(BNC1)gene mutation,which is identified from premature ovarian insufficiency patients.Materials and Methods:Immunofluorescence was used to detect the expression of BNC1 protein in human ovary tissues and human oocytes.RT-qPCR was used to detect the expression of BNC1 in mouse ovaries and mouse oocytes.BNC1 wild type and mutant vectors were constructed and transfected in vitro,and BNCI protein localization was observed by fluorescence microscopy.BNCI knockdown by small interfering RNA(siRNA)in human oocytes were constructed to study the effect of BNCI knockdown on human oocytes development.Premature ovarian insufficiency mice models were constructed by chemotherapy drugs,to sudy the BNCI gene expression in the ovaries with premature ovarian insufficiency.Results:BNC1 protein is expressed in human ovary tissues,especially in oocytes.BNCI is also expressed in mouse ovaries,mouse testis and mouse oocytes,while the expression in the oocytes is the highest.The wild-type BNC1 protein was observed to locate in the nucleus,while the mutant BNCI protein exhibited impaired nuclear transportation capability.MI oocytes of the BNCI siRNA groups exhibited a higher percentage of oocyte degradation than that of the control group,and a lower percentage of first polar body extrusion and maturity to MII oocytes.The BMP15 and p-AKT gene expression were significantly decreased in BNCI siRNA groups.The BNCI expression is significantly decreased in the ovaries of adriamycin induced POI mice model.Conclusion:BNC1 gene mutation may be associated with premature ovarian insufficiency,which has never been reported before.BNC1 dysfunction leads to impaired protein nuclear localization and disturbed oocyte meiotic maturation.Part Ⅱ The Effects of Basonuclin 1 Mutation on Premature Ovarian InsufficiencyObjective:To investigate the effects of Bnc1 mutation on the occurrence of premature ovarian insufficiency in animal model.Materials and Methods:Mice with the targeted Bnc1 mutation were generated using homologous recombination.Serum and ovary tissue were collected from wild-type(Bnc1+/+),heterozygous(Bnc1+/tr),and homozygous(Bnc1tr/tr)mice at different time points.Bnc1 expression in the ovary was investigated by immunohistochemistry.Growth curve,fertility,estrous cycle,gross morphology and weight of the ovary were observed in the wild-type,heterozygous and homozygous mouse.The serum levels of reproductive hormones(FSH,LH,E2)were determined by radioimmunoassay in the three genotype groups at different weeks of age.By comparing and analyzing the ovarian sections of the three groups and performing follicle counts,number and distribution of different development stages of follicles(primordial follicles,primary follicles,secondary follicles,and antral follicles)were assessed.And whether Bnc1 targeted mutation had a dose-effect were investigated.The subcellular structure of oocytes in the three groups were observed by electron microscopy.Results:The expression of Bnc1 protein in the ovary of homozygous mice was significantly lower than that in the wild-type mice.There was no significant difference in body weight between wild-type and heterozygous mice at each age.The fertility of the heterozygous mice was significantly reduced,showing a significant reduction in litter size,and homozygous mice exhibited infertility.The ovarian size and ovarian/body weight ratio of heterozygous mice were significantly smaller than those of wild-type,and the ovarian size and ovarian/body weight ratio of the homozygous type were significantly smaller than those of heterozygous and wild-type.Compared with wild-type mice,heterozygous mice had significantly lower serum E2 levels,significantly higher FSH,and significantly higher LH at 36 weeks old.Homozygous mice had significantly lower serum E2 levels and significantly higher serum FSH than heterozygous and wild-type mice at 36 weeks old.The estrus cycle of Bncl truncated mutant mice was changed,with shortened proestrus and prolonged metestrus.The primordial follicles,primary follicles,secondary follicles,and antral follicles of the heterozygous mice were significantly less than those of the wild-type,and the primordial follicles,primary follicles,secondary follicles,and antral follicles of the homozygous mice were significantly less than those of the heterozygous mice,respectively.There were no obvious morphological differences in the zona pellucida,granulosa cells,mitochondria,endoplasmic reticulum,Golgi,ribosome and other organelles of the three genotype mouse ovaries,while the homozygous ovary exhibited significantly excessed lipid droplets accumulation in the oocytes.Conclusions:Bnc1 mutation resulted in reduced fertility,altered estrous cycle,significantly increased FSH,significantly decreased E2,premature ovarian atrophy,significant reduction in the number of follicles,marked lipid deposition in oocytes,and premature ovarian insufficiency.Part Ⅲ The Mechanisms Involved in Basonuclin 1 Role in Premature Ovarian InsufficiencyObjective:To investigate the molecular mechanism of abnormal oocyte development and premature ovarian insufficiency caused by Bnc1 mutation.Materials and Methods:Gene expression profiles of ovary from wild-type and homozygous Bnc1 targeted mutation mice at 8 weeks of age were analyzed by RNA-seq.Bioinformatics analysis such as Go analysis,Pathway analysis,gene interaction network analysis,and gene co-expression network analysis of differentially expressed genes in transcriptomics were carried out to identify the possible upstream and downstream regulatory genes.RT-qPCR was conducted to validate differentially expressed genes.ChIP-seq was carried out of wild-type mouse ovary to explore the direct downstream genes of Bnc1.RNA-seq and Chip-seq combination analysis and ChIP verification were performed to find the potential downstream target moiecuies of Bnc1.Results:Fold change>2 and FDR<0.05 were used as differential gene expression criteria.It was found that the ovaries of 8-week-old homozygous Bncl targeted mutants have 232 genes up-regulated and 291 genes down-regulated compared to wild-type mice.Go analysis found that most of the differentially expressed genes participate in the biological process such as lipid metabolism,cholesterol metabolism,steroid hormone metaboiism,and cell differentiation.Pathway analysis revealed that most of of the differentially expressed genes involved in biological pathways such as lipid synthesis,ovarian steroid hormone synthesis,fat digestion,and fat absorption signaling pathways.Chip-seq detected 1323 peak-related genes directly regulated by Bnc1.The 523 differentially expressed genes obtained from the transcriptome and the coexpression network of ovarian genes were analyzed in combination with the 1,323 peak related genes obtained from Chip-seq,and 20 overlapping genes such as Scarb1,Itpr2,Slc5a8 were obtained.Combined with literature review,ChIP validation and qPCR validation,it was confirmed that Bncl protein can bind to Scarbl gene sequence and directly regulate Scarbl gene expression.Conclusions:Bnc1 target mutation directly leading to a significant decrease in Scarb1 expression may be the cause of the occurrence of increased accumulation of lipid droplets in oocytes,abnormal oocyte development,reduced fertility,and premature ovarian insufficiency.