| The ovary has an important function as a reproductive organ in female mammals.Steroid hormones produced by the ovary are extremely important for maintaining normal ovarian function,including involvement in biological processes such as follicle development,oocyte maturation and ovulation.Studies have shown that nerve growth factor(NGF)plays a key role in regulating ovarian function,from ovarian follicle assembly,primordial follicle activation,follicle growth and development,to steroid hormone secretion,oocyte maturation,ovulation and corpus luteum formation.Normal ovarian function and follicular development depend on the synergistic and complementary effects of these growth factors and their corresponding receptors.Dysregulation of these intraovarian growth factors can lead to infertility or ovarian diseases such as reduced ovarian reserve,polycystic ovary syndrome,endometriosis,and even ovarian cancer.m~6A modifications,the most common internal modifications in eukaryotic m RNAs,are involved in many processes in the female reproductive system.Although studies related to m~6A modification have been increasing in recent years,it is not clear whether NGF is involved in the regulation of testosterone synthesis in porcine follicular membrane cells and the relationship with m~6A modification.Therefore,this study selected porcine follicular theca cells as the research object to explore the effect of NGF on testosterone synthesis in porcine follicular theca cells in vitro,and further elaborated the mediating role of METTL14 in this process.The results are as follows:1.Effects of NGF on testosterone synthesis in porcine follicular theca cellsPorcine follicular theca cells were treated with 0ng/m L,50ng/m L,100 ng/m L and200ng/m L NGF for 24 h,and CCK8 cell detection results showed that cell viability was significantly increased at 100 ng/m L(P<0.05).After 100 ng/m L NGF was added into the treated cells,testosterone ELISA kit was used to detect the level of testosterone in the culture medium.The results showed that NGF could significantly increase the level of testosterone secretion(P<0.01).The m RNA and protein expression levels of genes related to testosterone synthesis were detected,the results showed that NGF could significantly promote the expression of genes related to testosterone synthesis,such as CYP17A1,HSD3B1,STAR(P<0.05).The expression of endogenous NGF in porcine follicular theca cells was decreased by si RNA interference technique,and the level of testosterone secretion in cell culture medium was significantly decreased(P<0.05).It was found that si NGF could significantly inhibit the expression of testosterone synthesis-related genes CYP17A1,HSD3B1,STAR(P<0.05).This part of the study shows that NGF promotes testosterone synthesis in porcine follicular theca cells.2.The role of METTL14 in the regulation of testosterone synthesis in porcine follicular theca cells by NGFThe detection of genes associated with methylation modification was carried out subsequent to NGF treatment of cells in the follicular theca cells.The results showed that NGF could significantly promote the expression level of methyltransferase METTL14(P<0.01).After si METTL14 and OEMETTL14 treatment,testosterone concentration levels and expression levels of genes related to testosterone synthesis were detected in the medium by si RNA interference technique and construction of overexpression vector.The results showed that after si METTL14 treatment,the testosterone secretion level in medium was significantly down-regulated(P<0.05),and the expression levels of testosterone synthesis-related genes CYP17A1,HSD3B1 and STAR were significantly down-regulated(P<0.05).After OEMETTL14 treatment,testosterone secretion level in medium was significantly up-regulated(P<0.05),and the expression levels of testosterone synthesis-related genes CYP17A1,HSD3B1 and STAR were also significantly up-regulated(P<0.05).To further demonstrate the role of METTL14 in the regulation of testosterone synthesis in porcine follicular theca cells by NGF,si METTL14 was co-treated with NGF,and the levels of testosterone secretion in the culture medium and expression of genes related to testosterone synthesis were detected at the m RNA and protein level.The results showed that the levels of testosterone secretion and the expression levels of testosterone synthesist-related genes CYP17A1,HSD3B1 and STAR in the culture medium of si METTL14+NGF group were significantly higher than si METTL14 group,but still significantly lower than NGF group(P<0.05).This part of the study shows that NGF can affect testosterone synthesis in porcine follicular theca cells through METTL14.3.Mechanism of NGF regulating testosterone synthesis in porcine follicular theca cells through METTL14After constructing the METTL14-mediated differential gene expression profile of porcine follicular theca cells,GO and KEGG enrichment analysis showed that the differential genes were mainly enriched in PI3K/AKT signaling pathway.After si METTL14 and OEMETTL14 treatment,the expression of PI3K/AKT signaling pathway at the protein level was detected.The results showed that METTL14significantly activated the expression of PI3K/AKT signaling pathway.si METTL14was co-treated with NGF to detect the expression of PI3K/AKT signaling pathway at the protein level.The results showed that the expression level of PI3K/AKT signaling pathway in si METTL14+NGF group was significantly higher than si METTL14 group,but still significantly lower than NGF group(P<0.05).In order to further explore the role of PI3K/AKT signaling pathway in the regulation of testosterone synthesis by NGF in porcine follicular theca cells,LY294002,an inhibitor of PI3K/AKT signaling pathway,was co-treated with NGF to detect the level of testosterone secretion in culture medium and the expression of genes related to testosterone synthesis at the m RNA and protein level.The results showed that the levels of testosterone secretion and the expression levels of testosterone synthesist-related genes CYP17A1,HSD3B1 and STAR in LY294002+NGF group were significantly lower than NGF group(P<0.05),and there was no significant difference with control group.This study suggests that the PI3K/AKT signaling pathway plays an important role in the regulation of testosterone synthesis in porcine follicular theca cells by NGF via METTL14.In summary,this study revealed that NGF activates the PI3K/AKT signaling pathway through the methyltransferase METTL14 and thus promotes testosterone synthesis in porcine follicular theca cells.This study further revealed the relationship between NGF and testosterone synthesis in follicular theca cells,which will provide new therapeutic targets for the treatment of female reproduction-related diseases in terms of m~6A methylation modification. |
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