| The mammalian early folliculogenesis including the formation of primordial follicles and the subsequent to the primary follicle, secondary follicle conversion, TGF beta signaling pathway plays an important role in this process. TGFβ-SMAD pathway is composed of extracellular ligand, cell surface specific receptor, intracellular Smad signal transduction molecules.Cascade close contact formation between them,will eventually make extracellular signal transduction into nucleus and regulate the transcription of target genes, causing biological effect. TGFβ1 and TGFβRâ… as core factors in TGFβ-SMAD pathway and plays an important role in the signal transduction, involved in the regulation of follicular maturation,participated in the two-way signal communication between granulosa cells and follicular cavity cell. Currently, it was found that the ovarian tissue-specific promoter were OSP-1. OSP-1 is a feasible regulatory element to study the roles of exogenous gene in procine granulose cells.To study the affect of TGFβ1 and TGFβRâ… genes in porcine follicular development,we designed the following experiments:(1) The ovarian follicle were collected from 3 Meishan and 3 Duroc sows and classified by diameter. The relative expression amout of TGF β/SMAD pathway genes(THBS1, DCN, LTBP1, TGF beta 1, beta 2, beta 3, TGFβRâ… , TGFβRâ…¡, SMAD2, SMAD3, SMAD4) in S, M1, M2, L follicle of Meishan and Duroc sows were detected by Real-time PCR method. The results showed that: TGF beta 1 beta 2, beta 3, THBS1, LTBP1, DCN, TGFβRâ… , TGFβRâ…¡and SMAD2, SMAD3, SMAD4 were differentially expressed in secondary follicles of Mei Shan and Duroc. Especially the expression of TGFβ1, TGFβ2 and TGFβRâ… in S, M1, M2 and L follicle of Meishan were significantly higher than in Duroc. The results suggested that TGFβ-SMAD pathway may be involved in the process of follicular development in pig.(2) The OSP-1 promoter was amplified according to rat OSP-1 sequence and inserted into the vector pc DNA3.1(+)to replace CMV promoter, following with Zs Green sequence as a reporter. The recombinant plasmids were transfected into porcine granulosa cells, Hela cells and myocytes; the fluorescence in cells was observed under microscope, the expression of Zs Green m RNA in different cells was detected by realtime-RT-PCR. The results showed that: Zs Green gene was expressed highly in ovarian granulosa cells and Hela cells very lowly in myocytes. Which indicated that OSP-1 promoter could promote the expression of exogenous gene efficiently in porcine granulosa cells.(3) According to the TGFβRâ… gene sequence, we designed five pairs of primers sh RNA interference primers. We inserted OSP1 promoter into the p PLL3.7 vertor to replace CMV promoter, constructed TGFβRâ… interference vectors. The recombinant plasmids were transfected into porcine granulosa cells, the expression of TGFβRâ… m RNA in porcine granulosa cells was detected by realtime-RT-PCR. We detected the effect of optimum interference vector and add the TGFβ1 cell growth factor on porcine granulose cell proliferation and cell apoptosis. The results showed that: after sequencing confirmed, the five interference vectors contained designed sequences, it indicated interference expression vector was constructed successfully. Analysis of the quantity expression of TGFβRâ… gene in porcine granulosa cells, there was a significant difference between the interference vector with OSP1 promoter and the interference vector with CMV promoter. The efficiency of interference vector with osp1 were 64%, 56%, 61%, 62%, 78%. which of p OSP1-TGFβRâ… -sh5 was the optimum. p OSP1-TGFβRâ… -sh5 interference vector makes the cell proliferation rate decreased very significantly(P<0.01), adding TGFβ1 makes the cell proliferation rate decreased significantly(P<0.05). At the same time, the optimum interference vector and adding TGFβ1 were promoted cell apoptosis(P<0.01).(4) The pig TGFβ1 and TGFβRâ… genes were selected to study the association between its polymorphism and born alive number. According to the sequence of pig TGFβ1 and TGFβRâ… gene, two pairs of primers were designed to detect SNPs of intron 6 of TGFβ1 and intron 7 of TGFβRâ… by PCR-SSCP and DNA sequencing. The results showed that the PCR products of TGFβ1 and TGFβRâ… had polymprphism.For TGFβ1 gene, two genotypes(CC and TT),and one single nucleotide mutation(Câ†'T) was detected in two pig breeds, and there were no significantly relationship(P>0.05) between two genotypes and reproductive traits of NBA. For TGFβRâ… gene,three genotypes(AA, AG and GG), and one single nucleotide mutation(Aâ†'G) was detected in large white pig, and there were significantly higher relationship(P<0.01) between three genotypes and reproductive traits of NBA.Two genotypes(AA and GG) was detected in landrace pig, and there were no significantly relationship(P>0.05) between two genotypes and reproductive traits of NBA. Therefore, these results have suggested that the TGFβRâ… is a strong candidate gene for selecting productive traits in pigs.In summary, our study expression patterns of genes of TGFβ-SMAD signaling pathway in Meishan and Duroc follicle of different sizes, analyzed the TGFβRâ… and TGFβ1 gene’s regulation in procine granulose cells, polymorphism of the TGFβ1 and TGFβRâ… and its relationship with number born alive in pigs. The result provide a theoretical basis and foundation for further study on the function of TGF β/SMAD signaling pathway during the follicle development, the influence of TGFβRâ… gene in porcine granulosa cell, and the molecular mechanism of interspecific differences in swine ovulation rate. |
PDF Full Text Request