Effects Of NAC On Ovarian Granulosa Cells Of Goats And Expression Regulation Of Reproduction-related Gene

Ovarian granulosa cells(GCs)are directly involved in the regulation of follicle development and are one of the factors that can affect the reproduction efficiency of mammals.The largest population of cells within the follicle are GCs,which have a key role in establishing signal communication and nutrient transmission with the oocyte,secreting estrogen and progesterone to regulate follicular maturation.The small relative molecular weight of N-acetylcysteine(NAC),which readily enters cells and is converted into metabolites capable of stimulating glutathione synthesis,has the effects of improving cell viability,adjusting cell signal transduction,and regulating cell metabolism.A previous study by our group found that feeding Qianbei Ma goat with0.07% NAC decreased oxidative stress and increased the rate of conception at the initial stage of pregnancy in ewes,and sequencing their uterine tissues revealed that NAC is involved in the regulation of important signaling pathways in the reproductive system of ewes.To further explore the molecular mechanism of the reproduction regulation of goat by NAC,in this study,GCs from Qianbei Ma goat were used to examine the effects of NAC on cell viability,proliferation,hormone secretion and m RNA expression in GCs in vitro,and the reproduction related gene RSPO2 was investigated to regulate the gene expression of GCs.The main findings are as follows:1.Growth regulation of ovarian granulosa cells by NAC.GCs from Qianbei Ma goat were successfully isolated and purified,and were cultured with different NAC concentrations in vitro.CCK-8 assay showed that 200 μM NAC had the most significant effect on cell viability when cells were incubated with different concentrations of NAC for 48 h(0,50,100,200,400,800,and 1600 μM)(P < 0.05).Further examination using Ed U assay and ELISA revealed that 200 μM NAC significantly increased the proportion of cells in the S phase of GCs compared with the group cultured without NAC(P < 0.05)and increased the levels of estradiol and progesterone in the culture medium(P > 0.05).2.RNA-seq as used to analyze the effect of NAC on the m RNA expression profile of GCs.Total RNA from GCs cultured with 200 μM NAC versus no NAC medium for48 h was extracted for RNA-seq analysis.The results showed a total of 122 differentially expressed genes(DEGs)in the 200 μM NAC group compared with the control group,of which 51 were up-regulated and 71 were down regulated.GO functional analysis revealed that the DEGs were mainly enriched in cell growth regulation,cell growth,growth regulation,extracellular domain,insulin-like growth factor binding and growth factor binding.KEGG enrichment analysis found that the DEGs were mainly enriched in neuroactive ligand receptor interaction,c AMP signaling pathway,cytokine-cytokine receptor interaction,cell cycle and Wnt signaling pathway.In total,seven genes,i.e.,RSPO2,IGFBP4,HTRA4,SST,SSTR1,WISP1 and DAAM2,were selected as the key genes regulating cell proliferation and hormone secretion based on the combined enrichment results.3.Detection of RSPO2 gene expression in gonadal axis tissues and bioinformatics analysis of RSPO2 protein.The expression of RSPO2 was detected by q RT-PCR in pituitary gland,hypothalamus,ovary,oviduct,uterine horn,and uterine body of the gonadal axis tissue of the Qianbei Ma goat,indicating a regulatory role of RSPO2 in the gonadal axis tissue of the Qianbei Ma goat.Bioinformatic analysis showed that the RSPO2 protein has a signal peptide but no transmembrane region and belongs to an extracellular secreted protein,and its secondary structure is dominated by irregular coils,its tertiary structure is loose,the amino acid sequence shows high similarity between common livestock and human,and the genetic distance is close.4.Effects of RSPO2 overexpression / silencing on ovarian GCs.The effects of overexpressing / silencing RSPO2 on key genes of Wnt signaling pathway,cell proliferation and hormone secretion related genes were determined by q RT-PCR analysis after transfection of RSPO2 overexpression / silencing expression constructs into GCs.The results showed that overexpressing(silencing)GCS increased(decreased)the expression levels of intracellular proliferation related genes PCNA(P < 0.05)and CCND1(P < 0.05),Wnt signaling pathway related genes lgr4(P < 0.05)and CTNNB1(P < 0.05),and hormone secretion related genes CYP19A1(P < 0.05),HSD17B1(P >0.05).It indicated that RSPO2 may regulate granulosa cell proliferation through the Wnt signaling pathway,thereby affecting cell proliferation and hormone secretion.Taken together,200 μM NAC may affect the expression of key genes and thus activate signaling pathways to regulate GCS proliferation and hormone secretion.The above results provide a molecular rationale for NAC as a feed additive to improve follicle development and improve reproductive performance in ewes.