Perfil da expressão de ISG15 no sangue periférico durante o período de reconhecimento materno da gestação em bovinos

Abstract

The maternal recognition of pregnancy (MRP) in ruminants is a complex event, involving several cellular mechanisms. It is at that moment that the highest embryonic mortality occurs in cattle. The MRP period is characterized by the secretion of interferon tau (IFNT) by the embryo. IFNT is produced and secreted by the trofloblast cells of the embryo, with greater intensity at 18 days of pregnancy. The main function of this protein is to keep the corpus luteum fully active, avoiding luteolysis. Direct action of IFNT on extrauterine tissues raises the expression of interferon-stimulated genes (ISGs). Among the ISGs stimulated by IFNT, the gene stimulated by interferon 15 (ISG15) stands out. Studies have demonstrated endocrine action of IFNT. An elevation in ISG expression levels in blood and luteal cells was observed shortly after signaling by IFNT at the start of gestation in ruminants. Peripheral blood polymorphonuclear cells (PMN) are more sensitive to the IFNT stimulus compared to peripheral blood mononuclear cells (PBMCs). The objectives of this study are: (1) to determine the earliest possible time to identify ISG15 expression in pregnant heifers; (2) establish the cell fraction to determine the early diagnosis of pregnancy with high accuracy in cut heifers; (3) demonstrate the expression profile of ISG15 on days 16 and 18 after artificial insemination (AI). Synchronization of the estrous cycle was performed in 28 heifers. The pregnancy diagnosis was performed on days 29 and 60 after AI and resulted in 10 pregnant heifers, 14 non-pregnant heifers, and 4 non-inseminated heifers (negative control group). Blood was collected from the coccygeal vein after AI (Days 16 and 18). Cellular fractions of PBMC and PMN were collected and stored at -80 ° C until further extraction of RNA, cDNA and qPCR, respectively. The relative expression of ISG15 differed between the PMN and PBMC cell fraction of pregnant heifers (p <0.0001). ISG15 expression in PMN cells on Day 16 differed from heifers and non-inseminated heifers (p <0.04), as well as on Day 18 after AI (p <0.01). However, ISG15 expression in PMN cells on Day 16 did not differ from pregnant heifers and non-pregnant heifers. In PBMC cells, ISG15 expression on Day 16 did not differ from pregnant, non-pregnant and non-inseminated heifers. However, on Day 18 after AI, there is a difference in ISG15 expression of pregnant and non-pregnant heifers (p <0.02). Expression of ISG15 in PMN cells on Days 16 and 18 pregnancy is greater 30 times than expression in PBMC. In addition, the expression of ISG15 in PMN can be used to identify embryonic signaling on Day 16 after insemination in beef heifers. Expression of ISG15 in PMN on Day 18 after AI resulted in 93% accuracy for the diagnosis of pregnancy. We suggest that both cellular fractions (PMN and PBMC) can be used as a tool to identify embryonic signaling on Day 18 of pregnancy in beef heifers.