RESUMO
Glucose uptake increases in canine luteal cells under insulin treatment. We hypothesize that insulin also increases luteal cell steroidogenesis. Dogs underwent elective ovariohysterectomy from days 10-60 post ovulation and their corpora lutea (CL) and blood samples were collected. Deep RNA sequencing determined differentially expressed genes in CL; those related to insulin signaling and steroidogenesis were validated in vivo by qPCR and their respective proteins by Western blotting and immunofluorescence. Next, luteal cell cultures were stimulated with insulin with or without inhibition of MAPK14, MAP2K1 and PI3K. Studied proteins except P450 aromatase showed the same expression pattern of coding genes in vivo. The expression of HSD3B and CYP19A1 was higher in insulin-treated cells (P < 0.005). Following respective pathway blockades, the culture medium had decreased concentrations of progesterone (P4) and 17b-estradiol (E2) (P < 0.01). Our results indicate that insulin increases HSD3B and CYP19A1 expression via MAPK and PI3K, and contributes to the regulation of P4 and E2 production in canine luteal cells.