| Brain-derived neurotrophic factor(BDNF)is a member of the neurotrophic factor family,which is expressed not only in the nervous system,but also in non-nervous system.It has been confirmed that BDNF and its receptor,tyrosine kinase receptor B(TrkB),were expressed in the ovaries of chickens,mice,rats,cows,pigs and human beings,and the regions of BDNF expression were changed with the different stages of follicular development.Previous studies have shown that BDNF plays an important role in the interaction between the two types of follicular cells,granulosa cells and oocytes,possibly as a paracrine factor to regulate the development of follicles and oocytes by binding to its receptors.BDNF was involved in regulating growth of ovarian follicles,maturation of the oocytes,and development of the early embryo through its receptor,TrkB.However,until now,it is still unclear as to how BDNF influences proliferation and secretion of steroid hormone of bovine granulosa cells.In this paper,we would study these and the main contents and results as follows: 1.Identification of bovine granulosa cells and the expression of BDNF and its receptor TrkBIn order to determine whether the isolated cells were bovine granulosa cells and expressed BDNF and its receptor TrkB,immunofluorescence technology was used to detect the expression of FSHR in the isolated cells.The results showed that FSHR was expressed in the isolated cells,with the percentage of positive cells calculated at a mean percentage of 95.3±1.1%.In this study,immunofluorescence,Western blot and ELISA technology were used to detect the expression of BDNF and its receptor TrkB in bovine granulosa cells.The results showed that both BDNF and TrkB were expressed in bovine granulosa cells,and BDNF could be synthesized and released by bovine granulosa cells.2.Effects of BDNF on proliferation and secretion of steroid hormone of bovine granulosa cellsTo determine whether BDNF could affect proliferation and secretion of steroid hormone in bovine granulosa cells,CCK8 and ELISA technology were used to detect the cell viability,progesterone and estradiol levels in bovine granulosa cells.The cells were treated with different concentrations of BDNF for 12,24 and 48 hours.The results showed that after 24 and 48 hours of BDNF treatment,the cell activity of 100 ng/mL group increased significantly(p<0.01);after 24 hours of treatment,the progesterone secretion of 100 ng/mL group increased significantly(p<0.01);after 48 hours of treatment,the progesterone secretion of 100 ng/mL increased(p<0.05);however,BDNF had no effect on the secretion of estradiol.Then,we examined the numbers and cell cycle of bovine granulosa cells after 24 hours of BDNF treatment.The results showed that the number of cells and the percentage of S phase in the 100 ng/mL group increased significantly(p<0.01).3.Effects of siBDNF on proliferation and secretion of steroid hormone of bovine granulosa cellssiRNA interference technology was used to reduce the expression of endogenous BDNF.Then the proliferation and secretion of steroid hormone of bovine granulosa cells were detected.The results showed that the cell viability and percentage of S-phase were decreased significantly(p<0.01),and the secretion of progesterone decreased(p<0.05),while the secretion of estradiol in bovine granulosa cells were no difference(p>0.05).Furthermore,using real-time fluorescence quantitative PCR and Western blot,we found that siBDNF decreased the expression of CCNA1,CCND1,CCNE2,CDK1 and StAR in bovine granulosa cells at the mRNA levels(p<0.05),and the expression of HSD3B1 was decreased significantly(p<0.01).At the protein levels,siBDNF also decreased the expression of CCNA1,CCNE2 and CDK1 in bovine granulosa cells(p<0.05),and decreased significantly the expression of CCND1,HSD3B1 and STAR(p<0.01).4.Effects of BDNF on proliferation and secretion of steroid hormone of bovine granulosa cells via TrkBIn order to determine whether BDNF could affect the proliferation and progesterone secretion of bovine granulosa cells through TrkB receptor,bovine granulosa cells were treated with BDNF alone or combined with K252 a.The results showed that the BDNF+K252a group,compared with BDNF group,decreased significantly the cell viability and the percentage of S-phase(p<0.01),and also the progesterone secretion(p < 0.01).Furthermore,using real-time quantitative PCR and Western blot,we found that the expression of CCNA1,CCND1,CCNE2,CDK1 and HSD3B1 in bovine granulosa cells in BDNF + K252 a group was lower than that in BDNF group at the mRNA levels(p<0.05),while the expression of StAR was significantly lower(p < 0.01).At the protein levels,the expression of CCNA1,CCND1,CDK1 and HSD3B1 of bovine granulosa cells in BDNF+K252a group,compared with BDNF group,were decreased(p < 0.05),while the expression of CCNE2 and StAR were decreased significantly(p < 0.01).5.Effects of BDNF on proliferation and secretion of steroid hormone of bovine granulosa cells via PI3K/AKT signal pathwayIn order to confirm whether BDNF could activate the PI3K/AKT signal pathway in bovine granulosa cells,the phosphorylation level of AKT was detected by Western blot after the cells were treated respectively for 0,5,15,30,60 and 120 minutes by BDNF.The results showed that the phosphorylation level of AKT increased after treatment for 5 and 30 minutes(p<0.05);the phosphorylation level of AKT increased significantly after treatment for 15 minutes(p<0.01);while there was no significant difference after treatment for 0,60 and 120 minutes(p>0.05).In order to confirm whether BDNF could activate PI3K/AKT signal pathway through TrkB receptor,bovine granulosa cells were treated with TrkB inhibitor(K252a)and PI3 K inhibitor(LY294002)separately or together with BDNF,and then treated with BDNF for 15 minutes to detect the phosphorylation level of AKT.The results showed that the phosphorylation level of AKT in BDNF+K252a group was lower than that in BDNF group(p<0.05),and that in BDNF+LY294002 group was significantly lower than that in BDNF group(p<0.01).In order to confirm whether BDNF could affect the proliferation and progesterone secretion of bovine granulosa cells by activating the PI3K/AKT signal pathway,the proliferation and progesterone secretion of bovine granulosa cells were detected by using the PI3 K inhibitor(LY294002).The results showed that the cell viability of BDNF+LY294002 group,compared with BDNF group,was decreased(p<0.05),the percentage of S-phase also was decreased significantly(p<0.01),and the secretion of progesterone was decreased(p<0.05).Furthermore,using real-time fluorescence quantitative PCR and Western blot,we found that the expression of CCNA1,CCND1,CCNE2,CDK1 and HSD3B1 in bovine granulosa cells of BDNF+LY294002 group were lower than that of BDNF group at the mRNA levels(p<0.05),while the expression of StAR was significantly lower(p<0.01).At the protein levels,the expression of CCNA1,CCND1,CDK1 and HSD3B1 in bovine granulosa cells in BDNF+LY294002 group,compared with BDNF group,were decreased(p<0.05),while the expression of CCNE2 and StAR were decreased significantly(p < 0.01).6.Effects of BDNF on proliferation and secretion of steroid hormone of bovine granulosa cells via MAPK/ERK1/2 signal pathwayIn order to confirm whether BDNF could activate the MAPK/ERK1/2 signal pathway in bovine granulosa cells,the phosphorylation level of ERK1/2 was detected by Western blot after the cells were treated respectively for 0,5,15,30,60 and 120 minutes by BDNF.The results showed that the phosphorylation level of ERK1/2 increased after treatment for 15 and 60 minutes(p<0.05);the phosphorylation level of ERK1/2 increased significantly after treatment for 30 minutes(p<0.01);while there was no significant difference after treatment for 0,5 and 120 minutes(p>0.05).In order to confirm whether BDNF could activate MAPK/ERK1/2 signal pathway through TrkB receptor,bovine granulosa cells were treated with TrkB inhibitor(K252a)and MAPK inhibitor(PD98059)separately or together with BDNF,and then treated with BDNF for 30 minutes to detect the phosphorylation level of ERK1/2.The results showed that the phosphorylation level of ERK1/2 in BDNF+K252a group was lower than that in BDNF group(p<0.05),and that in BDNF+PD98059 group was significantly lower than that in BDNF group(p<0.01).In order to confirm whether BDNF could affect the proliferation and progesterone secretion of bovine granulosa cells by activating MAPK/ERK1/2 signal pathway,the proliferation and progesterone secretion of bovine granulosa cells were detected by using the MAPK inhibitor(PD98059).The results showed that the cell viability of BDNF+PD98059 group,compared with BDNF group,was decreased(p<0.05),the percentage of S-phase was significantly decreased(p < 0.01),and the secretion of progesterone was not significantly different(p>0.05).Furthermore,using real-time fluorescence quantitative PCR and Western blot,we found that the mRNA expression of CCNA1 and CCNE2 were significantly lower in BDNF+PD98059 group than that in BDNF group(p<0.01),while the expression of CCND1,CDK1,HSD3B1 and STAR were not significantly different(p>0.05).At the protein levels,the expression of CCNA1 and CCNE2 in bovine follicular granulosa cells of BDNF + PD98059 group was significantly lower than that of BDNF group(p < 0.05).In conclusion,we are the first to show that BDNF promoted the expression of CCNA1,CCND1,CCNE2 and CDK1 by activating the TrkB-PI3K-AKT signal pathway,and then promoted the proliferation of bovine granulosa cells.Moreover,BDNF also promoted the expression of HSD3B1 and STAR by activating the TrkB-PI3K-AKT signal pathway,and then promoted the secretion of progesterone in bovine granulosa cells.In addition,BDNF also promoted the expression of CCNA1 and CCNE 2 by activating TrkB-MAPK-ERK1/2 signal pathway,and then promoted the proliferation of bovine granulosa cells.However,MAPK-ERK1/2 signal pathway was not involved in the regulation of secretion of progesterone after bovine granulosa cells were treated by BDNF.This study provides a new theoretical basis for the effects of BDNF/TrkB signal pathway on the biological function of bovine granulosa cells,oocyte maturation and follicular development,and a new way to the exploration of molecular regulatory mechanisms. |
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