Screening Of Follicle-Stimulating Hormone(FSH) Does-dependengt Expression Genes

The follicle-stimulating hormone(FSH)is glycoprotein produced by the anterior lobe of the pituitary gland under gonadotropin releasing hormone pulsatile stimulation.It is involved in the regulation of several essential reproductive processes such as gametogenesis,follicular growth and ovulation.The concentration of FSH is critical to the initiation and cycles of follicular development,the time of follicular maturation and the number of mature follicles.FSH is widely used in livestock production such as estrus synchronization,superovulation,estrus induction and treatment of ovarian disease.However,the production of FSH inland have many problems in different sources,refining purity and uneve quality between batches.At the same time,the current method for determing the bioactivity of FSH has the insufficient including the cumbersome operation,high cost and poor accuracy.Therefore,it is an important significance to stabling a convenient,fast and effective method for detection of FSH bioactivity,used for improving the efficiency and safety of FSH in livestock breeding.So the first study is to screen the cells through the stimulation of FSH,then choose the granulosa cells for the treatment by 40IU/L.Secondly,the transcriptome sequence analyzes the changes of gene expression in granulosa cells by FSH treatment.Real-time quantitative PCR is used to validate and screen out the genes with significant changes in expression.Finally,screen the FSH does-dependent expression of FSH through the different does FSH stimulation,which is foundation for establishing the detection system of FSH biological activity.The main results were as follows:1.FSH stimulated sensitive cell selectionThis study selects the ovcarian cell lines including CAOV-3,ES-2,NIH:OVCAR-3,OVCAR-3 and SKOV-3 and granulosa cells.Use semi-quantitative PCR to detect the gene expression in each cell by FSH treated 40IU/L for 48h.The results shows that granulose cells have high sensitivity to FSH stimulation.2.The selection of FSH up-and down-regulated genesThis study screens the gene which were differentially regulated between control(-FSH)and FSH-treated granulose cell cultures by the transcriptome sequence.There are 85 genes up regulated and 105 genes down regulated in FSH-treated granulosa cells compared with control cell.We choose seventeen genes including MTHFD2,PAG1,LHCGR,CTSL,TSHZI,PAFAH1B2,SDC1,IGF1,MESDC2,ALG2,DCTPP1,SAT2,CCDC71,HSD3B1,ADAM10,MRPL22 and STAR from these genes.Analysis the results of Real-time quantitative PCR and then use T-text.We find the gene expression between FSH-treated granulosa cells and control cells have significant differences(P<0.001),including LHCGR,PAFAH1B2,IGF1,CCDC71,HSD3B1 and STAR.The gene expression between FSH-treated granulosa cells and control cells have significant differences(P<0.01),including PAG1,SDC1,SAT2,ADAM10,MRPL22.The gene expression between FSH-treated granulosa cells and control cells have significant differences(P<0.05),including MTHFD2,CTSL,TSHZ1,DCTPPl.The last genes including MESDC2 and ALG2 have no significant differences in ene expression between FSH-treated granulosa cells and control cells.3.The selection of FSH does dependent genesReal-time quantitative PCR results of gene LHCGR,PAFAH1B2,IGF1,CCDC71,HSD3B1 and STAR by FSH does stimulation(0,20,40,60,80,100IU/L)show that the gene expression of PAFAH1B2,IGF1,CCDC71,HSD3B1 and STAR have no linear relationship with the increase dose of FSH.However,the LHCGR is the gene which FSH does dependent.Its expression of LHCGR have a linear trend relationship with the increase dose of FSH.We let the fold expression of LHCGR as ordinate and the dose of FSH as abscissa to make scatter plot.The trendline show R2 is close to 1,indicating a linear correlation between fold expression of LHCGR and the dose of FSH.In conclusion,the research find the gene that its fold expression have a linear trend relationship with the increase dose of FSH by Real-time quantitative PCR.The results provide a basic data for further understanding of detecting the biological activity of follicle stimulating hormone.