Differential expression analysis of components of the insulin signaling cascade in the canine corpus luteum after RNA sequencing

The present study aims to characterize the expression of genes regulated by insulin in the canine corpus luteum (CL) along the diestrus. Hypothesis is that insulin plays a role in the regulation of steroidogenesis and the functions of lutein cells, consequently. Non-pregnant bitches were submitted to ovariosalpingohisterectomy every 10 days between 10 and 60 (n = 4 / group) after ovulation (po). CL collected were used for RNA sequencing (RNA-seq) and validated by real-time PCR, and protein analysis by Western blotting and immunofluorescence. Bioinformatics analysis detected the presence of 34.408 genes in the samples, of which 29.011 genes showed some level of gene expression in diestrus. The temporal gene expression revealed the presence of 5.116 genes differentially expressed at least one comparison between groups and 1.106 genes were not noted in the canine genome. Seven genes directly linked to insulin signaling were selected: the IRS1 (insulin substrate 1 receptor), involved with the onset of insulin signaling, the PI3KR3 (phosphoinositide-3-kinase, regulatory subunit 3 (gamma) and PI3KCG (phosphoinositide-3- kinase , catalytic, gamma polypeptide) that are PI3K isoforms involved in phosphorylation of AKT, the MAPK9 (mitogen-activated protein kinase 9), MAPK13 (mitogen-activated protein kinase 13), and MAPK14 (mitogenactivated protein kinase 14) involved in the cell proliferation and SOCS1 (suppressor of cytokine signaling 1), known as a positive regulator of insulin signaling initiation. The expression of IRS1, MAPK13 and MAP14 was higher in the second half of diestrus, concomitant with decreased expression of steroidogenic enzymes HSD3B and CYP7B1, suggesting a negative regulatory performed by these genes. Rather, PI3KCG, PI3KR3, MAPK9 and SOCS1 expression have maximum values at the beginning of estrus, similar to the CYP19A1 expression, suggesting a positive control. The results corroborate with studies in which the different components involved in the insulin signaling establish a direct interaction with the expression of steroidogenic enzymes, from low molecular weight mediators such as inositolphosphoglycan. After the association of insulin with its receptor, the mediators of this class are generated, internalized and alter the activities of some steroidogenic enzymes and cellular metabolism, in addition to acting in the cleavage of cholesterol side chain resulting in increased production of hormones. These data allow a better understanding of the regulation of luteal physiology in general and more specifically on the regulation of steroidogenesis through insulin signaling.