Endometrial gene expression in isolated hemoperfused equine uteri

As the importance and awareness of animal welfare are growing constantly, the need for alternatives to animal testing is bigger than ever. The aim of the study was to further validate an existing ex vivo model of the equine uterus on the molecular level. For this purpose, real-time-quantitative PCR (RT-qPCR) was performed for oxytocin receptor (OXTR), progesterone receptor (PGR), oestrogen recep-tor 1 (ESR1) and Caspase 3 (a marker for apoptosis), to investigate changes in the temporal gene expression in the endometrium of isolated hemoperfused equine uteri (n = 12). Biopsy samples were obtained right after slaughter, after transportation to the laboratory, after 4, 5 and 6 h of hemoperfusion and then stored in RNA later at −80°C until analysis. Primers for PGR, Caspase 3 and the house-keeping genes GAPDH and B2M were used from the literature, and primers for OXTR and ESR1 were designed with Primer-BLAST. The average threshold (CT) of each triplet was normalized to GAPDH and B2M and the delta ratios were analysed statistically. There were no significant changes in the expression of Caspase 3 until 5 h of perfusion after which the relative expression increased significantly (p < .05), indicating that there was no notable apoptosis during perfu-sion before but after 5 h. Hormone receptors did not show significant changes in relative gene expression over the time of testing, except for PGR, which had significantly lower expression after 5 h (p < .05). In conclusion, no major changes in the mRNA level of the selected genes have been observed until 6 h of perfusion time, making the ex vivo model applicable for short-term studies on the molecular level.