114 Pharmacological regulation of PPARγ in bovine embryos alters blastocyst development, cell lineage specification, and transcripts of early placental function

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear transcription factor with reproductive roles that are indispensable for placental development and conceptus elongation. PPARγ transcripts increase markedly during bovine conceptus elongation but functional characterisation of PPARγ in early embryo development is incomplete. These studies aimed to determine roles of PPARγ in bovine blastocyst development and cell lineage specification while further characterising PPARγ transcriptional regulation of early placental function. Embryos were produced in vitro using frozen-thawed sperm and oocytes from abattoir-derived ovaries. Immunocytochemistry was used to determine PPARγ expression and co-localisation with the trophectoderm marker CDX2 at the zygote, 2–4, 8–16 cell, morula (D5), D6, and blastocyst (D7.5) stages. PPARγ was not detected until D6 and D7.5 of development, where 26% (9/34) and 24% (11/46) of embryos showed nuclear and ubiquitous expression of PPARγ, respectively. Among D6 embryos, those expressing PPARγ had a decreased CDX2:total cell ratio (P < 0.05). To determine functional roles of PPARγ, a specific PPARγ agonist, rosiglitazone (Ros), or antagonist, GW9662 (GW), was independently added to embryo culture medium on D5 (1, 50, 100 µM, VC [0.5% DMSO]). A one-way ANOVA using the mixed procedure of SAS and Tukey’s post hoc analyses after testing for normality was conducted to determine effects on blastocyst development (n = 6 reps, >100 embryo per treatment). Addition of Ros 50 µM decreased blastocyst development on D7.5 (19%) compared to VC, 1, and 100 µM treatments, respectively (37, 37, 36%; P < 0.01). The PPARγ antagonist GW (50, 100 µM) also decreased D7.5 blastocyst development (23%, 21%) compared to VC (38%) and 1 µM (30%) treatments (P < 0.01). Following treatment, changes to candidate transcripts in D7.5 blastocysts were quantified by qPCR and significance was determined by t-test for each treatment. GW-treated embryos (1, 50 µM) had decreased expression of PPARγ heterodimers, RXRα and RXRβ (P < 0.05). Additionally, GW treatment decreased SP1 (50 µM, P < 0.01) and NFKB1 (1 µM, P < 0.05) abundance, both of which are key transcripts that direct early placental development. Expression of three developmental control genes (CDX2, OCT4, SOX2) remained consistent among all treatments, suggesting that developmental differences were specific to PPARγ targeted influence rather than embryo quality. These results identify PPARγ as a gene important to bovine blastocyst development with regulatory roles in cell lineage specification and transcripts involved in downstream placental function. The functions of SP1 in open-chromatin remodelling of early embryos and co-expression with PPARγ in binucleated cells of the bovine trophoblast further implicate PPARγ as an important regulator of early embryo development. Collectively, targeted modulation of PPARγ function by environmental manipulation through in vitro embryo culture make PPARγ an attractive candidate for early intervention of embryo developmental competence.