PSV-4 - Effects of asprosin on nuclear sirtuins expression in bovine granulosa cells

Abstract: Asprosin is an adipokine that regulates steroid production in granulosa cells (GC). Sirtuins are a family of NAD+-dependent deacetylases associated with GC function. In the nucleus, sirtuins are epigenetic regulators of gene expression. Although the literature shows that sirtuins and asprosin affect GC steroidogenesis and proliferation, studies are required to investigate whether their actions are linked. The aims of this study are to determine the influence of asprosin on mRNA expression of nuclear sirtuins and their association with steroidogenic regulators in bovine GC. GC were isolated from small antral follicles (1-5mm) of bovine ovaries, cultured in vitro, and treated with or without asprosin (200 ng/mL) in combination with FSH (30 ng/mL) or IGF1 (30 ng/mL). Cells were cultured for 24h and submitted to RNA extraction (n= 5 pools). Relative mRNA abundance of SIRTs 1, 2, 6, 7, aromatase (CYP19A1), IGF1 receptor (IGF1r), and FSH receptor (FSHr) were determined via RT-qPCR using 18S as a housekeeping gene. Data were analyzed via factorial ANOVA with GLM procedures of SAS for Windows (SAS Institute Inc., Cary, NC). Normality was tested using the Shapiro-Wilk test. Data sets that showed non-gaussian distribution (p >0.05) after log-transformation were analyzed via the Kruskal-Wallis test followed by Dunn’s post hoc test using GraphPad Prism 9.2.0. Differences among treatments were considered significant at p< 0.05, and a tendency at 0.05< p< 0.10. Pearson correlation was performed using PROC CORR procedures of SAS for Windows (version 9.4, SAS Institute Inc., Cary, NC). SIRT1 (p< 0.05) mRNA abundance increased after Asprosin treatment in comparison to FSH alone and in combination with asprosin as well as to IGF1 alone. Also, SIRT1 under asprosin treatment showed a tendency (p< 0.06) to be increased in comparison to negative control. Additionally, a very high correlation (r = 0.999/p< 0.05) between CYP19A1 and SIRT1 was observed in the treatment with asprosin. SIRT2 had a tendency (p< 0.08) to increase after treatment with asprosin in comparison to FSH alone. Moreover, SIRT2 has a very high (r = 0.995/p< 0.005) correlation to CYP19A1 after treatment of FSH in combination with asprosin and a significant (r = 0.676/p< 0.05) correlation with IGF1r. Asprosin treatment led to a very high (r = 0.997/p< 0.05) correlation of SIRT6 with FSHr. Also, when asprosin was combined with FSH, a very high ( r = 0.993/p< 0.01) correlation between SIRT6 and CYP19A1 was observed. SIRT7 mRNA relative abundance decreased (p< 0.05) after treatment with IGF1 in comparison to the negative control. No significant correlation was observed between SIRT7 and CYP19A1 or receptors. In summary, asprosin increases expression of SIRT1­ and increases the correlation between SIRTs 1, 2, 6, and aromatase. These results indicate that the actions of asprosin on steroidogenesis involve sirtuins regulation.