ABSTRACT
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.
Subject(s)
Insulin/pharmacology , Luteal Cells/drug effects , Steroids/biosynthesis , Animals , Cells, Cultured , Corpus Luteum/drug effects , Corpus Luteum/metabolism , Dogs , Estradiol/metabolism , Female , Glucose/metabolism , Luteal Cells/metabolism , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Progesterone/metabolism , Signal Transduction/drug effectsABSTRACT
Considering the key role of the corpus luteum in the regulation of the canine diestrus, the present study aimed to investigate changes in the luteal transcriptome of pseudopregnant dogs (n = 18) from days (D) 10, 20, 30, 40, 50 and 60 post-ovulation. After RNAsequencing was performed, data was analyzed by resorting to several informatic tools. A total of 3300 genes were differently expressed among all samples (FDR < 0.01). By comparing different time points, enriched biological processes as response to estradiol and lipids (D20 vs D10) and intracellular cholesterol transport (D40 vs D60) were observed. Moreover, LXR/RXR (liver X receptor- retinoid X receptor) signaling appeared as an overrepresented pathway in all comparisons. Thus, the expression of 19 genes involved in intracellular cholesterol availability was further evaluated; most were affected by time (P < 0.05). Adding to the deep transcriptomic analysis, presented data implies the importance of cholesterol regulation in luteal physiology of pseudopregnant dogs.
