| Granulocyte(Granule cells),GCs apoptosis is an important cause of follicular atresia,and apoptosis is a process of strict control of multiple genes.In the female mammalian ovaries,follicular atresia is a cyclical process,which is not very clear about its molecular mechanisms.Recent studies have shown that reproductive hormones play an important role in follicular developmental,FSH can prevent the apoptosis of granulosa cells to inhibit follicular atresia.In this paper,the yak was used as the research object,and the FSH receptor was regulated by q RTPCR,immunohistochemistry,ELISA and Western-blot.FSH was used to investigate regulate FSH receptors and the effect of FSH on FOXO3 a and apoptosis-related gene of Fas,Fas L,Bax,Bcl-2,Bim,BCL-xl,Caspase-6,Caspase-3,Akt and c-fos m RNA and protein expression levels.The mechanism of action of FSH on the regulation of apoptosis by FOXO pathway was analyzed from the death receptor pathway,endoplasmic reticulum pathway and mitochondrial pathway.The regulation mechanism of FSH on growth factor secretion was analyzed by analyzing the expression level of related growth factor of b FGF,EGF,LIF,TNF-?.The expression of related genes of c-fos,Akt,Oct-4,NF-k B and apoptotic genes Fas,Bax,Bcl-2 and Caspase-3 on MAPK pathway regulated by growth factor was discussed.The mechanism of FSH on the regulation of growth factor secretion was analyzed.Based on the above research,the molecular mechanism of FSH regulated GCs was discussed.Research indicates,Immunohistochemical assays found that granulosa cells specifically express FSHR.Fluorescence quantitative results showed that FSHR m RNA was the highest expression at 500ng/mL FSH in 6h,and the expression level was about 48.25.The WB results showed that the expression of FSHR was the highest at 6 h,and the expression of FSHR was higher in the 100ng/mL FSH group.The expression of FSHR was up to 45.32 when add 500ng/mL FSH for 6h.The results of FSHR-induced FSHR showed that the expression of FSHR m RNA was the highest when adding 500ng/mL FSH to the culture medium for 6h.FSHinduced downstream c AMP expression showed that c AMP had a maximum expression of 154.218 mol/mL when supplemented with 100ng/mL FSH for 3h.When the dosage of 500ng/mL FSH was 3h,the expression of c AMP was higher,and the expression level of FSH was about 2.5 times at 100ng/mL FSH for 3h.10,100,200,500ng/mL FSH 6h after c AMP expression did not change and generally low.The results of FOXO3 a showed that Foxo3 a was expressed in the whole medium and 500ng/mL.At different time points,Foxo3 a was expressed in cytoplasm and nucleus at 60 min and 120 min.180min,300 min and 360 min were mainly expressed in the cytoplasm,no expression in the nucleus.The results of Foxo3a(Ser253)protein test showed that: Foxo3a(Ser253)can be expressed mainly in the cytoplasm,no expression in the nucleus when add 500ng/mL FSH to the complete medium.Foxo3a(Ser253)did not complete finish nucleus out when 500 ng/mL FSH 60 min,although it was partially expressed in the cytoplasm.But did not complete the nuclear translocation of nuclear protein,expression is weak.The expression of cytoplasm and nucleus was stronger at 120 min.When Foxo3a(Ser253)undergo a complete nuclear transfer at 3h,it is fully expressed in the cytoplasm but is weakly expressed.Through repeated validation,240 min experimental group there are nuclear and nuclear in the two results,indicating that at 4h Foxo3a(Ser253)expression is unstable.At 5h,there was a nuclear expression,all expressed in the cytoplasm,most of the nuclei were not expressed,complete the nuclear process,and strong expression in the cytoplasm.At 6h Foxo3a(Ser253)was completely expressed in the cytoplasm of granulocytes containing serum-free,500 ng/mL FSH complete medium,and no expression was found in the nucleus.The expressed is strong and stable.The mechanism of FSH on Foxo3 a,Foxo4,Bim,BCL-xl,Fas,Fas L,Bax,Bcl-2 and Caspase-3.The results showed that: the expression of Foxo3 a was significantly up-regulated when added 500ng/mL FSH,the expression of Foxo4 was down-regulated;Foxo3a and Foxo4 ratio was almost 6:1.The expression of Caspase-6 and Caspase-3 was up-regulated by Foxo3 a and Bax,Bcl-2,BCL-xl,Fas L and Fas.The ratio of Bim and Bcl-x L is about 1:1,and the ratio of Bax and Bc L-2 is about 1:1.100ng/mL FSH could increase Foxo3 a,decrease Foxo4 expression and decrease the expression of Bax and Bcl-2,but the down-regulation effect was not significant in 500ng/mL experimental group.There was no significant effect on Bim and Bcl-x L.The ratio of Bc L-x L to Bim was almost 2:1,and the ratio of Bax and Bc L-2 was almost 1.247: 1.Fas L was significantly decreased,500ng/mL FSH treatment group had the lowest expression,Fas was the lowest in 500ng/mL FSH treatment group.Growth factor expression test results show that: the expression of LIF,b FGF,EGF and TNF-? protein in cell supernatant was detected by the treatment of 500 ng / mL FSH for 3h,6h and 24 h respectively.LIF,b FGF,EGF and TNF-? protein expression between the no significant difference,compared with the serum did not add FSH experimental group have a more substantial increase in the trend.The expression of each growth factor protein showed that the 24 h treatment group was generally higher than the 6h treatment group.FGF had the highest secretion at 24 h,but less than LIF protein secretion.EGF had the highest secretion at 24 h.TNF-? had the lowest secretion at 6 h,3h has the highest secretion value.Based on the above results,we selected 500ng/mL FSH treatment 24 h experimental group for the best promotion of growth factor secretion in the experimental group for subsequent experiments.The expressions of c-fos,Akt,NF-k B,Oct-4 and Caspase-6 were detected in the 24 h experimental group by 500 ng/mL FSH.The results showed that the expression of REF and Akt were downregulated and up-regulated the expression of Oct-4 and c-fos in LIF,EGF and b FGF protein secretion.In addition,the expression of FSHR was enhanced by adding 500ng/mL FSH for 6h to GCs in vitro,and the expression of c AMP was up-regulated at 100ng/mL FSH for 3h.Foxo3 a began to undergo nuclear transfer at 3h,all transferred to the cytoplasm,and inhibited until 6h,at 4h when there is unstable expression,5h has an enhanced expression,6h all out of the nucleus and stable expression in the cytoplasm.In Foxo3 a Nuclear translocation can significantly downregulate death receptor pathway,mitochondrial pathway and Caspase-related gene expression.The expression of LIF,b FGF,EGF and TNF-? was downregulated by 500ng/mLFSH for 24 h,and down-regulated the expression of REF,Akt and Caspase-6,up-regulated Oct-4 and c-fos.It is play a major regulatory role on the endoplasmic reticulum path.In this study,we determined the optimal dose and time of FSH-acting granulosa cells,and clarified the effect of different treatment groups on the expression of Foxo3 a under FSH combined serum.It was found that FSH might inhibit the expression of Foxo3 a protein by inhibiting the expression of Foxo3 a protein Granulocyte apoptosis,promote cell proliferation gene expression,promote growth factor secretion.The results provide a theoretical basis for further revealing the mechanism of FSH in follicular granulosa cells during oocyte maturation and early embryonic development,and contribute to the improvement of in vitro development of yak embryos. |
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