PPAR expression throughout the oestrous cycle in the bovine endometrium

The Role of Peroxisome Proliferator-Activated Receptors in PGF2α-Induced Luteolysis in the Bovine Corpus Luteum

“…Next, plasma was extracted and stored in sterile 7 mL vials at −20 °C until assay using a radioimmunoassay (RIA) [ 26 ]. The concentration of P 4 (ng/mL) in the collected samples during the selected days of the estrous cycle was as follows: day 0—0.38 ± 0.09 (mean ± SEM); day 2—0.069 ± 0.15; day 5—3.77 ± 0.19; day 8—5.96 ± 0.50; day 12—6.94 ± 0.8; day 15—5.70 ± 0.58; day 17—3.08 ± 0.88; day 19—1.76 ± 0.6; day 21—0.69 ± 0.18, as previously described [ 38 ]. The estrous cycle phase was additionally confirmed post-mortem through macroscopic observation of the ovary and uterine features according to a previous report [ 39 ].…”

“…Corpora lutea (CL) were collected from the same heifers, which have been previously described [ 38 ]. In brief, healthy, normally cycling Holstein/Polish Black and White (75% and 25%, respectively) heifers (aged between 18 and 22 months) were used for the present study.…”

“…The qPCR experiments were performed according to the MIQE guidelines [ 42 ], as previously described [ 38 ]. The ABI 7900 HT sequence detection system (Applied Biosystems, Foster City, CA, USA) was used with the SensiFAST SYBR Hi-ROX Kit (Bioline Reagents, London, UK, BIO-92002).…”

“…The total volume of the reaction was 10 µL and contained 5 µL of SensiFAST SYBR Hi-ROX Master Mix, 1 µL each of forward and reverse primers (0.5 µM) and 3 µL of reverse-transcribed cDNA (10 ng). The primer sequences for determining the mRNA expression of reference and target genes were chosen based on scientific reports, i.e., reference genes: glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) [ 38 , 43 ], beta-actin ( ACTB ) [ 38 , 43 ] and 18S ribosomal RNA ( RN18S1 ) [ 38 , 43 ], and target genes: PPARα ( PPARA ) [ 38 , 43 ], PPARδ ( PPARD ) [ 38 , 43 ], PPARγ ( PPARG ) [ 38 , 43 ], steroidogenic acute regulatory protein ( StAR ) [ 20 ], cytochrome P450 family 11 subfamily A member 1 ( P450scc ) [ 20 ], hydroxy-delta-5-steroid dehydrogenase, 3 β- and steroid delta-isomerase 1 ( HSD3B1 ) [ 20 ], prostaglandin-endoperoxide synthase 2 ( PTGS2 ) [ 44 ], prostaglandin F 2α synthase ( PTGFS ) [ 44 ], tumor necrosis factor α ( TNFα ) [ 19 ], tumor necrosis factor receptor superfamily member 1A ( TNFRSF1A ) [ 19 ], tumor necrosis factor receptor superfamily member 1B ( TNFRSF1B ) [ 19 ] and inducible nitric oxide synthase ( iNOS ) [ 45 ]. All primers were synthesized by Sigma-Aldrich (Custom DNA Oligos, Sigma-Aldrich, Saint Louis, MO, USA).…”

“…Immunostaining was carried out according to a published protocol [ 38 ]. The sections were deparaffinized and rehydrated.…”