The Effect Of Transcription Factor LHX8 On The Development Of Primary Follicles And The Preliminary Study On The Regulation Of Zp3 Gene Expression

Objective:Premature ovarian failure(POF)refers to ovarian failure caused by the exhaustion of follicles in the ovaries before the age of 40.The clinical manifestations are primary or secondary amenorrhea,accompanied by perimenopausal symptoms.As the reproductive organ of female mammals,the ovary has the functions of producing mature follicles and secreting estrogen.Follicles are its basic structural and functional units,consisting of oocytes,granulosa cells and membrane cells.Follicle development mainly begins with primordial follicles,undergoes primary follicles,secondary follicles,and ovarian follicles that eventually mature and ovulate.The stage in which follicles develop from primordial follicles to secondary follicles is called the precavity or non-gonadotropin-dependent stage,which mainly relies on germ cell-related transcription factors to play an important regulatory role,such as FIGLA,SOHLH1,LHX8,etc.are involved in regulating the precavity the development of follicles.LHX8,as a transcription factor related to germ cell development,is a member of the LIM homeobox transcription factor family.The LIM domain is composed of two special zinc fingers and is located at the N-terminus of the homology domain,enabling the LIM-HD protein to Divide or divide the way to interact with other transcription factors.It is reported in the literature that there are no primordial follicles in the ovary of Lhx8^-/-female mice,the primordial follicle assembly is abnormal,oocytes cannot grow,the germ cells are lost after birth,and the ovaries shrink after adulthood.A large number of germ cell-specific gene expression disorder and reproductive dys function.Germ cell development is a continuous process.In different stages of follicular development,the same transcription factor can play different regulatory roles.Extensive knock-out experiments can only explore the role of transcription factor LHX8 in the assembly process of the original follicles.LHX8expression runs through at various stages of follicular development,the mechanism of action in subsequent follicular development has not been reported in the literature.Zp1,Zp2,and Zp3 are members of the zona pellucida gene family in mice.Zp1,Zp2,and Zp3 form zona pellucida proteins through coding and translation,which play a very important role in oocyte development,fertilization,and implantation.It is reported in the literature that in the early stages of follicle development,FIGLA can directly regulate the expression of Zp1,Zp2,Zp3 genes,SOHLH1 can transcriptionally regulate the expression of Zp1,Zp3 genes,and indirectly regulate the expression of Zp2 genes.During the primary follicle stage,SOHLH1 protein gradually disappeared,and ZP1,ZP2,and ZP3 proteins were expressed in primary follicles until ovulation.It is reported in the literature that the analysis of the results of Lhx8's extensive knockout of neonatal female mice showed that there are differences in the expression of key genes in oocyte development such as Zp2 and Zp3.There is no significant difference in the expression of Zp1 gene.After verification in the primary follicles,it is speculated that LHX8 may Directly or indirectly regulate the expression of Zp2 and Zp3.According to bioinformatics prediction,it is found that there is an LHX8 transcription binding site in the promoter sequence of Zp3 gene,so it is speculated that LHX8 may directly regulate the expression of Zp3 gene.In this study,the primary follicles were isolated and cultured in vitro,the microinjection of siRNA was used,and the effects of the transcription factor LHX8 on the development of primary follicles and the regulation of Zp3 gene expression were initially investigated through morphological observation and molecular biology experiments.Methods:In this study,the Lhx8-pcDNA3.0 eukaryotic expression plasmid was constructed to verify the efficiency of Lhx8-siRNA interference,by comparing the blank control group,the negative control group,and the microinjected Lhx8-siRNA group,the primary follicles were cultured in vitro,and the follicles were observed to develop from primary to secondary Morphological changes and time-point changes at the same level,through q RT-PCR experiments and Western blot experiments to detect the expression levels of m RNA and protein of primary follicular development related genes before and after Lhx8 gene silencing,through Ch IP experiments to find the main LHX8 on the Zp3 gene promoter sequence Binding site.Results:1.Successfully constructed Lhx8-pcDNA3.0 eukaryotic expression plasmid,and co-transfected HEK293T cells with Lhx8-siRNA to obtain Lhx8-mus-972 with the highest interference efficiency and 95.3%inhibition rate.2.The q RT-PCR and Western blot experiments showed that Lhx8 m RNA and protein expression decreased after Lhx8 gene silencing,indicating that microinjection of Lhx8-siRNA can effectively silence Lhx8 gene in primary follicles.3.By cultivating primary follicles in vitro,the negative control group compared with the blank control group showed that the microinjection operation did not affect the growth and development of the primary follicles.Both groups began to develop gradually at 2 days,and developed into secondary follicles after 5 days,Lhx8-Compared with the negative control group,the siRNA group:after Lhx8 gene silencing,the development of primary follicles is slow or even temporarily arrested,and the proliferation of granulosa cells is blocked.After microinjection,development resumes at 6 days,and it can develop into secondary follicles at 7 days.4.The results of q RT-PCR and Western blot showed that in the primary follicles,after the Lhx8 gene was silenced,the expression levels of Zp2 and Zp3 m RNA were significantly down-regulated,and the expression levels of ZP2 and ZP3 protein were significantly down-regulated.5.The luciferase activity of the experimental group co-transfected with the LHX8 eukaryotic expression plasmid and the Zp3 promoter region luciferase plasmid was higher than that of the control group.The results of the Ch IP experiment showed that the transcription factor LHX8 can bind to the upstream of the transcription start site of Zp3(-1663bp,-1556bp),(-1305bp,-1298bp),(-1256bp,-1251bp),(-1047bp,-1040bp)domains,and(-1305bp,-1298bp)(CTAATTAA)sites have the strongest binding effect.Conclusion:1.Lhx8 is a key gene for follicle development from primary to secondary.2.The transcription factor LHX8 can directly activate the transcription of Zp3,(-1305bp,-1298bp)(CTAATTAA)is the main specific binding site.