62 Functional characterisation of peroxisome proliferator-activated receptor gamma (PPARγ) in bovine blastocyst development and early trophectoderm formation

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear transcription factor with diverse roles in reproductive processes. PPARs regulate metabolism, cell differentiation, inflammation, and immune tolerance and are common targets of environmental toxicants. Functions of PPARs in human placental development, decidualisation, and implantation have been explored, but roles in bovine preimplantation embryo development and cell lineage specification are not defined. PPARγ transcripts are up-regulated in the elongating bovine conceptus, suggesting a role in pregnancy establishment. PPARG mRNA in bovine embryos may be related to embryo quality, but protein detection in preimplantation embryos has not been conclusively demonstrated. We aimed to characterise PPARγ protein expression and putative functions in bovine preimplantation embryo development and cell lineage specification. Bovine oocytes were recovered from abattoir ovaries and in vitro embryos were produced from frozen-thawed sperm from three different bulls and cultured for 7.5 days in 5% CO₂ and 10% O₂. Embryos were fixed at the zygote (12 h post-insemination, n = 8), 2–4 cell (Day 2, n = 13), 8–16 cell (Day 3, n = 8), morula (Day 5, n = 18), Day 6 (n = 18), and blastocyst (Day 7.5, n = 36) stages and subjected to immunohistochemistry (IHC) for detection of PPARγ. Day 5 morulas and blastocysts were co-stained with SOX2 and CDX2 for detection of inner cell mass and trophectoderm cell lineage specification, respectively. PPARγ was not detected in embryos until Day 6 of development. Nuclear expression was observed in 28% (5/18) of Day 6 embryos and co-localised with SOX2 and CDX2. Few Day 7.5 blastocysts were PPARγ+ (7/36,19%). Among Day 7.5 PPARγ+ embryos, no differences were observed from Day 7.5 PPARγ− embryos in total cell number or proportion of CDX2-positive cells. Day 6 PPARγ+ embryos had fewer total cells compared with PPARγ− (47 vs. 88, respectively) and fewer CDX2 cells in proportion to total cells (28% vs. 56%; P < 0.01). To determine roles of PPARγ in embryo development, the PPARγ agonist rosiglitazone (Ros) (1, 50, 100 µM, VC (0.5% DMSO)) was added to Day 5 embryos. Differences in treatments for blastocyst by embryos cleaved were determined by one-way ANOVA using the mixed procedure of SAS (SAS Institute Inc.) and Tukey’s post hoc analyses after testing for normality (n = 3 reps). Addition of Ros 50 µM decreased blastocyst development on Day 7.5 (13%) compared with VC, Ros 1 and 100 µM treatments (36, 34, and 38, respectively; P < 0.002). No differences were observed among other treatments. Collectively, PPARγ detection on Day 6 followed by dose-dependent suppression of blastocyst development and lower CDX2+ cells in Day 6 PPARγ+ embryos suggest a regulatory role for transition to cell lineage specification, trophectoderm formation, and blastocyst development. Few Day 7.5 embryos were PPARγ+, which may support a requirement for PPARγ down-regulation permissive to blastocyst development. Further functional characterisation of PPARγ may aid in determining embryo origins of trophectoderm formation required for successful pregnancy establishment in cattle.