Relationship between different stages of the corpus luteum and the expression of the peroxisome proliferator-activated receptor γ protein in bovine large lutein cells

Abstract

Lutein cells produce progestins that support pregnancy. Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism. Peroxisome proliferator-activated receptors (PPAR) are transcription factors that are central in the regulation of lipid metabolism. Hence, they may play a role in regulation of the development and regression of the corpus luteum. The present study investigated the expression of PPAR-γ,n during different stages of the corpus luteum. Lutein cells were isolated mechanically from non-pregnant and pregnant heifers on days 5, 12 and 20 of the oestrous cycle (n = 3 for each day). PPAR-γ in single cells was analysed by flow cytometry. PPAR-γ1 and PPAR-γ2 isoforms were distinguished by immunoblotting. The cell cycle of the lutein cells was measured by the flow cytometric quantification of DNA in single cells, using propidium iodide staining after ethanol fixation and RNAse treatment, and by the detection of the proliferating cell nuclear antigen (PCNA). The response of the cells to PPAR-γ agonist 15-deoxy-Δ(12,14) prostaglandin J2 (15dPGJ2, 200 and 490 nmol l-1) with and without changing the cell cycle by the anti-apoptogenic drug aurintricarboxylic acid (ATA, 10 μmol l-1) was used as an in vitro model to study the relationship between the cell cycle and PPAR-γ. The concentration of PPAR-γ per cell from non-pregnant heifers was significantly higher on day 5 (3.40 ± 0.30 fmol) compared with that on day 12 (1.34 ± 0.18 fmol, P < 0.05) and day 20 (0.55 ± 0.2 fmol, P < 0.05). In pregnant heifers, the concentration of PPAR-γ was significantly (P < 0.01) higher than in non-pregnant heifers. A decrease in the PPAR-γ1, isoform relative to PPAR-γ2, was observed in cells on day 12 of the oestrous cycle compared with day 5. The cell cycle (S phase portion in cells on days 5, 12 and 20: 16 ± 4%, 6 ± 4% and 4 ± 3%, respectively) and the portion of cells with PCNA correlated with the amount of PPAR-γ in non-pregnant heifers. ATA promoted the S phase in cells of non-pregnant heifers (day 12) and the endogenous agonist of PPAR-γ, 15 dPGJ2 inhibited the response to ATA in a dose-dependent manner, indicating that PPAR-γ plays a role in the arrest of the cell cycle in lutein cells to maintain their differentiated state.