Study On The Regulation Proliferation Of CPEB1 Gene In Goat Ovarian Granulosa Cells

Ovary is the main reproductive organ in female animals,and follicular granulosa cell plays an extremely important role in maintaining reproductive function.The proliferation and differentiation of granulosa cells can affect ovarian function directly from development of the primordial follicle to the formation of corpus luteum after ovulation.Oocytes in the body have been living in follicular fluid before ovulation,follicular fluid plays an important role on oocyte development,and the basis of the follicular fluid is comprised of plasma exudate and granular cell secretion.Follicular granulosa cells were considered as an optimal cellular model to study animal reproductive physiology.In this study,CPEB1 related to goat cell proliferation was screened out on the basis of transcriptome sequencing.Nantong(in Jiangsu Province)native goat breed was used as research animal.eukaryotic expression vector,pcDNA3.1-CPEB1,and small molecular RNA interference vector,siCPEBl,were constructed.CPEB1 was overexpressed and downregulated of in goat ovarian granulosa cells,Respectively,the levels of mRNA of related genes were tested by qRT-PCR,and Western Blot was used in this study to detect the levels of related gene protein expression in order to explore its biological function in the process of goat ovarian granulosa cell proliferation.The main results were as follows:1.Clone Goat CPEB1 and build the eukaryotic expression pcDNA3.1-CPEB1 and small molecular RNA interference vector siCPEBl:Successfully use nested PCR cloned out CDS area of CPEB1 and clone it to the carrier pcDNA3.1,the eukaryotic expression vector pcDNA3,1-CPEB1 was constructed successfully,and identify it correct by digestion and sequencing;Successfully synthesize siRNA of CPEB1 including siCPEBl-1,siCPEBl-2,siCPEB1-3 and siCPEBl-NC.The disrupting activity between siCPEBl-3 and CPEB1 in ovarian granulosa cells from adult ewes and young ewes.2.Respectively overexpressed and knocked-down levels of mRNA expression of the gene in goat ovarian granulosa cells,and test the level of related gene mRNA expression by qRT-PCR and use Western Blot to detect the level of related protein expression:(1)When transfected eukaryotic expression vector pcDNA3.1-CPEB1 get into follicular granulosa cells,the level of CPEB1 mRNA expression increased about 20 times in adult ewes group and about 16 times in the young ewe group.Meanwhile the mRNA expression level of proliferation related genes CYCLIN B1,CDK1 and C-MOS in adult ewes group raised about 10,17,10 times respectively;The mRNA expression level of proliferation related CYCLIN B1,CDKl and C-MOS in young ewes group raised about 11,12,7 times respectively.The level of CPEB1 protein expression increased by about 1.7 times in adult ewes group and about 2 times in young ewes group,While the protein expression level of proliferation related CYCLIN B1,CDK1 raised about 1.7,1.6 times in adult ewes group and about 2,1.5 times in young ewes group.It indicated that overexpressed gene CPEB1 can increase proliferation related CDK1,CYCLIN B1 and C-MOS and promote the proliferation of the goat ovarian granulosa cells effectively.(2)When transfected small molecular RNA interference carrier siCPEBl get into follicular granulosa cells,the mRNA expression level of gene CPEB1 declined about 80%both in adult and young ewes group.Meanwhile the mRNA expression level of proliferation related CYCLIN B1,CDK1 and C-MOS in adult ewes group declined about 50%,50%,65%respectively;The mRNA expression level of proliferation related genes CYCLIN B1,CDK1 and C-MOS in young ewes group declined about 40%,80%and 40%respectively.The level of CPEB1 protein expression declined about 55%in adult ewes group and about 65%in young ewes group,While the protein expression level of proliferation related CYCLIN B1,CDK1 declined about 35%,40%in adult ewes group and about 60%,35%in young ewes group.It showed that knocked-down gene CPEB1 can decline proliferation related CDK1,CYCLIN B1 and C-MOS and inhibit the proliferation of the goat ovarian granulosa cells effectively.