The Effect Of HSP 27 On Apoptosis Of Ovarian Granulosa Cells In Tibetan Sheep

The HSP 27 gene can indirectly affect follicle development and ovulation process by regulating the apoptosis of granulosa cells,thereby affect the reproductive traits of Tibetan sheep.In order to study the role of HSP 27 gene in apoptosis of Tibetan sheep ovarian granulosa cells,this test firstly detected the expression localization of HSP 27 protein in Tibetan sheep ovarian tissues,followed by interfering with and overexpressing HSP 27 gene in granulosa cells,and analyzed the effect of HSP 27 gene on the expression of related genes and cell apoptosis in Tibetan sheep ovarian granulosa cells,finally detected the effect of HSP 27 gene on the secretion of estradiol and progesterone by granulosa cells.Real-time PCR was used to detect the relative expression of genes related to apoptosis and hormone secretion.The results show that:（1）By immunohistochemizing the ovarian tissue of Tibetan sheep,it was found that in Tibetan sheep ovarian tissue,HSP 27 protein was mainly expressed in the granulosa cells layer of follicles of different sizes and reproductive epithelium.（2）Constructed HSP 27 overexpression and interference vectors of Tibetan sheep and transfected vectors into granulosa cells for expression,the results showed that the expression of HSP 27 gene in the HSP 27 overexpression group was significantly higher than that in the control group（NC no-load group）（P<0.01）,and the expression of HSP 27 gene in the interference group（HSP 27-sh RNA2 group）was significantly lower than that in the control group（NC no-load group）（P<0.01）.This illustrates that successful constructed of HSP 27 overexpression vector and interference vector,and successful expressed in Tibetan sheep granulosa cells.（3）Compared with the control group（NC no-load group）,overexpression of HSP 27 gene could significantly inhibit the apoptosis rate of Tibetan sheep ovarian granulosa cells（P<0.01）,interference with HSP 27 gene could significantly promote apoptosis of granulosa cells（P<0.05）,compared with the control group（NC no-load group）,overexpression of HSP 27 gene could significantly downregulate the expression of Bax gene（P<0.01）,there was a tendency to downregulate the expression of the Bcl-2 gene（P=0.053）by interfering with the HSP 27 gene（HSP27-sh RNA group）.The results showed that HSP 27 gene exerts an anti-apoptotic effect by inhibiting the expression of the Bax gene.（4）Compared with the control group（NC no-load group）,it was shown that overexpressed of HSP 27 gene could promote the secretion level of estradiol（P<0.05）and progesterone（P<0.01）of GCs,and promoted the secretion level of Erβ（P<0.01）,3β-HSD（P<0.01）and CYP19A1（P<0.05）of GCs,interfered with the expression of HSP 27 gene had a tendency to inhibit the E₂ secretion level of GCs（P=0.052）,and significantly inhibited the relative expression of Erβgene（P<0.01）.The results showed that overexpressed HSP 27 gene could increase the secretion level of estradiol and progesterone in Tibetan sheep ovarian GCs by promoting the expression of Erβ,3β-HSD and CYP19A1 genes.（5）When overexpressed and interfered with the HSP 27 gene in GCs,the transcriptome sequencing results showed that the overexpression of HSP 27 gene could inhibit the expression of related genes that promote apoptosis in GCs,interference with HSP 27 gene could downregulate the expression of ND2 and COX1genes in the oxidative phosphorylation pathway thereby affect apoptosis.And 11different genes related to follicle development were screened according to the sequencing results:EFNA2,IL2RA,LAMB1,APOA1,CTNND2,ADRB1,NOG,ADCY4,SLC6A12,GABRB3 and RXRA.In summary,under the conditions of this test,overexpression of HSP 27 gene could inhibit the apoptosis of Tibetan sheep ovarian GCs and promote the secretion of estradiol and progesterone by GCs;interference with the HSP 27 gene could promote apoptosis of granulocytes and had a tendency to inhibit the level of E₂ secretion in granulocytes.