Regulation And Mechanism Of Growth Factors As Well As Epigenetic Modifications In Bovine Follicular Development

Although several hundred thousand original oocytes exist in embryonic period of mammals, there is only a few to hundreds of generations, and it is very low in utilization of oocytes. In antral follicle, the mechanism of development and technology of maturation in vitro are well studied. However, the technology of maturation of preantral follicle in vitro is difficulty and focus in the world, because it is inconclusive of the mechanism of development. In order to clarify the mechanism of preantral follicular development, promote the process of developmental biology, improve the techniques of maturation of preantral follicle in vitro and superovulation of young animals, and improve the utilization of follicles. In present study, Q-PCR, West-blot, CHIP, BSP and gene immunization were used, to elucidate whether epigenetic modifications such as DNA methylation and histone acetylation were involved in follicular development; to study the regulation of GDF9 and BMP15 in follicular development via the expression profile during folliculogenesis and epigenetic modifications; to explore the regulation and mechanism of INHa in follicular development via the expression profile during folliculogenesis and epigenetic modifications such as DNA methylation and histone acetylation; to investigate the regulation of SS in follicular development via transfection bGCs and immunization female mice with SS plasmid. The main results are as follows:1. Study the epigenetic regulation of DNA methylation and histone acetylation in bovine follicular developmentTo evaluate whether epigenetic modifications including DNA methylation and histone acetylation were involved in regulating bovine follicular development. Q-PCR analysis was utilized to determine expressions of several enzymes of DNA methylation and histone acetylation; to test expressions of DNMTs, HDACs, and HATs after treatments with inhibitors of DNA methylation and histone deacetylation; and oocyte maturing rate was also investigated after treatments with inhibitors. The results are as follows:(1) DNA methylation and histone acetylation were involved in regulating bovine follicular development. DNA methyltransferase DNMT1, DNMT3a, DNMT3b, and histone acetyltransferase HDAC1 and histone deacetylase HAT1 were triggered with follicular development (P<0.05), HDAC2 increased only at 2-5mm stage (P<0.05). However, expression levels of HAT, HDACs and DNMTs reduced in large follicles (diameter>8 mm). Differential expressions of epigenetic modified enzymes suggested that epigenetic switches were involved in regulating folliculogenesis.(2) DNA methylation and histone acetylation were involved in oocyte maturation. Inhibitors of 5-aza-dc and/or NaB enhanced oocyte maturing rate (P<0.05), by down-regulation of transcription of DNMT1, DNMT3b, HDAC1 and HDAC2 (P<0.05).2. Explore the regulation of GDF9 and BMP15 in bovine follicular developmentExpression profiles of GDF9 and BMP 15 genes in different follicular development stages were evaluated by Q-PCR and West-blot, the follicular development stages were divided into five groups according to the follicular diameter (preantral follicle:<0.2mm; small:0.2-2mm; medium:2-5mm; large:5-8mm; ovum:>8mm); Expressions of GDF9 and BMP 15 were investigated after treatments with inhibitors of DNA methylation and histone deacetylation.The results are as follows:(1) Transcriptions of GDF9 and BMP 15 were differential expression during follicular development. GDF9 and BMP 15 expression were up-regulated at 0.2-2 mm and 5-8 mm stage (P<0.05). Differential expressions of GDF9 and BMP 15 suggested the regulation in the non-steroid-dependent phase, ovulation and luteal formation during follicular growth.(2) Proteins of GDF9 and BMP 15 were differential expression during follicular development. The expression of BMP 15 and GDF9 increased with follicular growth (P<0.05), but decreased at>8 mm stage (P<0.05).(3) Inhibitors of 5-aza-dc and/or NaB activated expressions of GDF9 and BMP15 in COCs (P<0.05), by down-regulation of transcriptions of DNMT1, DNMT3b, HDAC1 and HDAC2 (P<0.05). 3. Investigate the regulation and mechanism of INHαin bovine follicular developmentExpression profile of INHa gene in different follicular development stages was evaluated by Q-PCR and West-blot; regulation of inhibitors in INHa gene was tested; the center promoter of INHa was determined by relative activity; the activity of fully methylated promoter and mutating the p300-binding site in 1294 luc promoter were tested. The mechanism of histone acetylation in promoter was explored by CHIP; methylation status of the CpG island within INHa proximal promoter at different stage of follicle (small:0.2-2mm; medium:2-5mm; large:5-8mm; ovum:>8mm) was determined by BSP. The results are as follows:(1) Transcription and translation of INHa were differential expression during follicular development. Transcription of INHa was up-regulated at 2-5mm, and peaked at>8 mm stage (P<0.05). INHa protein increased followed with follicular development (P<0.05). Differential expression of INHa suggested the important role in response of FSH stimulation, negative feedback regulation of the expression of GDF9 and BMP 15, the formation and ovulation of the dominant follicle during follicular growth.(2) Promoter was the target of DNA methylation and histone acetylation to modify INHa gene expression.The center promoter of INHa was 1294bp (-1017bp～+277bp), the activity of pGL-1294 activated significantly (P<0.05), the expression of INHa increased (P<0.05) after suppressing DNA methylation and histone deacetylation by inhibitors of 5-aza and/or NaB. The activity of methylated promoter reduced (P<0.05).(3) DNA methylation suppress activation of AP2a transcription factor in-907～-804 and-345～-197 within INHa promoter. Relative binding of AP2a to-907～-804 and-345～-197 increased after suppressing DNA methylation (P<0.05).(4) Histone acetylation was involved in regulation of P300 transcription factor in-345～-197 within INHa promoter. After suppressing histone deacetylation, the levels of H3 acetylation in-907～-804 and-345～-197 increased (P<0.05), and relative binding of P300 to-345～-197 increased (P<0.05).(5) P300 as bridge or stand was involved in regulation of-345～-197 by histone acetylation. After site-directed mutation of p300 binding sites, INHa promoter regulatory activity induced (P<0.05), the levels of H3 acetylation in-345～-197 increased (P<0.05), relative binding of P300 to-345～-197 increased (P<0.05).(6) Methylation levels of CpG island within INHαproximal promoter was not correlative with INHa expression during bovine follicle development.27 CpG sites of CpG island were hypomethylated or virtually unmethylated in all follicles from four different sizes (P>0.05).4. Investigate the regulation of SS in follicular developmentTo study the role of SS in follicular development, pGM-CSF/SS plasmid containing SS was used to transfection bovine GCs and immunization female mice. The results are as follows:(1) SS was involved in inhibition of follicular development, via induced apoptosis of GC. Overexpression in bovine GCs, induced expression of Bax and P53 which were pro-apoptosis genes (P<0.05), reduced expression of Bcl-2 which was anti-apoptosis gene (P<0.05), down-regulated E2 level (P<0.05), induced apoptosis of GCs (P<0.05) and repressed follicular development.(2) Immunization with pGM-CSF/SS plasmid could improve reproductive ability of female mice. Immunization with pGM-CSF/SS plasmid could induce secretion of E2 (P<0.05), shorten estrous cycle of female mice (P<0.05).