Expression Regulation And Function Of POSTN In Ovine Follicular Granulosa Cells

Regulation of ovarian follicle development is one of the main ways to improve animal reproduction efficiency.In mammalian ovaries,more than 99.9%of the follicles undergo atresia and degeneration during follicular development.Some reports pointed out that follicular atresia is closely associated with the apoptosis of follicular granulosa cells(GCs).However,the regulation mechanism of GCs apoptosis remains incompletely understood.In our previous study,the follicular fluid and GCs in folloice with different diameters in adult ewe after FSH-treated superovulation were analysis based on RNA-seq High-throughput Sequencing and iTRAQ quantitative proteomics,the results showed that the expression of POSTN gradually increased together with the follicle diameter.Therefore,a hypothesis is proposed:FSH and POSTN may have some relevance during follicular development,but the specific regulation mechanism is still unclear.The present study investigated the POSTN expression patterns during follicular development and the reguation mechanism of FSH-POSTN for POSTN expression of FSH-treated follicle GCs.The main results were as follows:1.Clone and Expression analysis of POSTN gene in ovine.In the present study,a 1557 bp Coding sequence of POSTN was successfully cloned from ovine ovary.It was found that POSTN CDs encode a protein consisting of 518 amino acids.The evolutionary tree analysis revealed that the POSTN has close homology with that of Bos mutus and Bos Taurus.The obtained POSTN was expressed in heart,liver,spleen,lung,kidney,muscle,uterus and ovary in ovine,and the expression level was highest in ovary tissue.Moreover,as the diameter of the follicles increased,the expression of POSTN were gradually increased,among which POSTN were highly expressed in GCs of follicle.The POSTN fluorescence signal was distributed throughout the nucleus and cytoplasm of GCs.The changes of reproductive hormones and POSTN in follicular fluid and GCs of follicles with different diameters were further detected by radio-immunoassary.The result showed that the expression changes of POSTN in follicular fluid and GCs were significantly positive correlated with FSH,indicating that POSTN may be involved in the regulation of follicular development and this process may be affected by FSH stimulation.2.FSH regulates POSTN expression of ovine follicular GCs.Treatment of GCs cultured in vitro with 10 ng/mL FSH for 24 h could significantly increase the expression of POSTN in GCs,accelerate cell proliferation,reduce apoptosis,and effectively up-regulate GCs proliferation-related genes PCNA,Ccnd-2 and anti-apoptosis related gene Bcl-2 expression.Moreover,knock down of POSTN in vitro cultured GCs could strikingly weaken the up-regulation of POSTN expression induced by FSH,and inhibit the proliferation and differentiation of GCs,increase GCs apoptosis,down-regulate the expression of GCs proliferation-related genes PCNA,Ccnd-2 and anti-apoptotic gene Bcl-2 and up-regulate the expression of apoptosis-related genes Bim,FasL and Caspase-3.3.FSH regulates POSTN expression through PI3K-AKT-FOXO1 signaling pathway.Detection of multiple signaling pathway involved in FSH,it was found that FSH could up-regulate the expression of POSTN in ovine follicular GCs by activating PI3K,AKT and FOXO1 signals,and at the same time inhibited the apoptosis of GCs.Further analysis of the relationship among PI3K,AKT and FOXO1 found that FSH treatment for 24 h could simultaneously activate the activities of these three kinases.When inhibiting PI3K or AKT,the activity of FOXO1 induced by FSH is blocked,while inhibiting FOXO1,the activities of PI3K and AKT were not affected.PI3K inhibitors could simultaneously block the induction of AKT and FOXO1 by FSH,while AKT inhibitors could inhibit FOXO1 activity but not PI3K activity.Moreover,the expression of POSTN was significantly reduced when AKT was blocked,even if adding 10 ng/mL FSH could not restore up-regulating the transcription of POSTN and rescue GCs apoptosis completely within 24 h.4.FSH regulates the expression of POSTN through modulation of the transcription factor SRF.Detection of the POSTN transcription regulatory region indicated that the upstream region of POSTN contains 1 SRF and 1 JunD binding site from-1 to-387 bp.Further site-directed mutagenesis analysis demonstrated that mutation occurring at region-222?-210 bp could greatly reduce the transcriptional activity of POSTN.At the same time,it was found that 10 ng/mL FSH treat GCs for 24 h could promote the expression of SRF and enhance POSTN transcription.This result was consistent with the ChIP experiment.Therefore,it was speculated that FSH may regulate the expression of POSTN through the transcription factor SRF,suggesting that SRF may be the core factor for FSH to regulate POSTN expression and rescue GCs from apoptosis.In summary,this study investigated the role of POSTN on ovine follicular GCs apoptosis and its molecular regulation mechanism,clarified the relationship between FSH,POSTN,GCs apoptosis and follicular survival,and found FSH-PI3K-AKT-FOXO1 axis regulating POSTN expression in follicular GCs.Our results can help to further elucidate the molecular mechanism of follicle survival/atresia,and provide a new theoretical basis for improving the reproductive efficiency of livestock.