Effects of different fatty acids on the competence of bovine trophoblast cells and embryos to successfully establish pregnancy

Abstract

The prostaglandin E2 (PGE2) synthesized by maternal endometrium, embryo and trophoblastic cells (TC) has luteotrophic and anti-luteolytic effects favoring maternal fetal recognition. The synthesis of prostaglandin F2a (PGF2a) and PGE2 in endometrial tissue and endometrial cells has been extensively studied, however, such synthesis in TC and embryos was poorly pursued and understood. The use of different fatty acids (FA) can alter the proportion of PGE2 and PGF2± produced in TC and embryos. Some strategies using polyunsaturated FA from the omega 6 family (n-6), such as linoleic (LA) and conjugated linoleic (CLA) acids, are investigated by our research group as potential beneficial strategies for successful pregnancy (FAPESP, grant numbers 2016/02805-4 and 2018/24168-1). We have recently performed, by the group (FAPESP, grant number 2018/24168-1), evaluated the effects of treatment with CLA (cis-9, trans-11 and trans-10, cis-12) at different doses in the TC culture medium. Verified a reduction in the synthesis of PGF2± of all groups treated with CLA (10, 20, 50 and 100 µM) in TC with 24, 48 and 72 hours of culture compared to control (0 µM). A higher PGE2/ PGF2± ratio was also observed in all groups treated with CLA compared to the control; and there were also changes in the abundance of transcripts involved in the biosynthesis of / PGF2± and PGE2. We are currently investigating the effects of CLA on in vitro produced (IVP) bovine embryos, during maturation (IVM) and in vitro culture (IVC). Preliminary data indicate that the use of CLA in vitro culture increases the percentage of hatched blastocysts on the 9th day of culture. Fatty acids have the potential to benefit embryos in recent studies, oxidative stress and DNA methylation are suggested. Based on the promising results obtained recently (FAPESP, grant number 2018/24168-1), we present this proposal with the aim of continuing the previous study by supplementing the same biological models (CT1 and IVP embryos) with the following fatty acids: unconjugated linoleic acid (LA, C18:26, n-6), oleic acid (OA, C18:1, n-9) and a combination of eicosapenoic (EPA, 20:53, n-3) and docosahexaenoic acid (DHA, 22:63, n-3). We validated the protocols to measure the concentration of PGE2 and PGF2a in the culture medium by the ELISA technique and the expression of transcripts involved in the biosynthesis of eicosanoids in CT1 and embryos by PCR. We intend to expand our assessments by investigating the impacts of CLA, LA, OA and EPA + DHA on lipid distribution, oxidative stress and DNA methylation. The hypothesis of this study is that supplementation with CLA, LA, OA or EPA + DHA increases the PGE2/PGF2a ratio and modifies the expression of transcripts involved in eicosanoid biosynthesis and maternal-fetal regognition (IFNT) in CT1 and bovine embryos, in addition to altering the distribution of lipids, decreasing oxidative stress and favoring the level of DNA in the embryos. Thus, it becomes objective to evaluate the effects of supplementation with CLA, LA, OA or EPA + DHA on the PGE2/PGF2a ratio and on the expression of transcripts involved in prostaglandin biosynthesis (PTGER2, PTGER4, PTGES1, PLA2G10, PTGS2, PTGES2, AKR1B1, AKR1C4) and maternal-fetal recognition (IFNT) in CT-1 (Experiment 1); to compare the effects of supplementation with CLA, LA, OA or EPA + DHA on the PGE2/PGF2± ratio, on the expression of transcripts involved in prostaglandin biosynthesis (PTGER2, PTGER4, PTGES1, PLA2G10, PTGS2, PTGES2, AKR1B1, AKR1C4) maternal-fetal recognition (IFNT) and on the methylation level of DNA (DNMT1, DNMT3A, DNMT3B) in IVP bovine embryos at D7 (Experiment 2); to evaluate the effects of CLA, LA, OA or EPA + DHA supplementation on lipid distribution and oxidative stress (measurement of reactive oxygen species, mitochondrial membrane potential and intracellular level of the antioxidant glutathione) in IVP bovine embryos at D7 (Experiment 3). (AU)