PSXII-23 - Sirtuins expression and activity in bovine granulosa cells are regulated by hormones that influence ovarian steroidogenesis

Sirtuins (SIRTs) are a family of seven NAD⁺-dependent histone deacetylases that regulate several biological reactions. How SIRTs regulate ovarian steroidogenesis in cattle remains to be fully unveiled. We hypothesize that SIRTs expression and activity are regulated by hormones that influence steroidogenesis in bovine granulosa cells (GC). Bovine ovaries were collected at an USDA-inspected commercial slaughterhouse and GC were isolated from small antral follicles (1-5 mm on surface diameter). Cells were treated with hormones that regulate ovarian folliculogenesis: FSH, IGF1, fibroblast growth factor (FGF) 2, FGF9, and their combinations. Cells were cultured for 24h for total RNA isolation (n = 6 pools) with miRNeasy microkit (Qiagen) or for 48h for isolation of nuclear and cytoplasmic extracts (n = 3 pools) with EpiQuik Nuclear Extraction Kit (Epigentek) according to the manufacturers’ instructions. Relative mRNA abundance was quantified via qPCR and expressed as 2^-ΔΔCt using the relative comparative threshold cycle (Ct) whereas SIRTs activity in nuclear (SIRTs 1, 6, and 7) and cytoplasmic (SIRTs 2, 4, and 5) extracts was analyzed with the Epigenase Universal SIRT Activity/Inhibition assay kit (Epigentek) following the manufacturer’s instructions. Data were analyzed via ANOVA with GLM procedures of SAS for Windows. In terms of mRNA relative abundance, FSH+IGF1+IGF9 increased mRNA relative expression of SIRTs 2 to 7 in comparison to negative control and of SIRTs 2, 3, 4, 6, and 7 in comparison to FSH+IGF1; FSH+IGF1+IGF2 increased mRNA relative abundance of SIRTs 2 and 6 in comparison to FSH+IGF1; FGF2 alone increased SIRT1 in comparison to negative control (P < 0.05). In term of SIRTs activity, FGF2 alone increased nuclear SIRTs activity in comparison to FSH, IGF1, FSH+IGF1, and FGF9 alone; FSH+IGF1+IGF2 increased cytoplasmic SIRTs activity in comparison to all treatments (P < 0.05). Taken together, our data demonstrate that SIRTs expression and activity in bovine GC are regulated by hormones that influence steroidogenesis.