Study of estradiol mechanisms to stimulate endometrial PGF2α synthesis in cows

In cattle, estradiol (E2) plays an important role in endometrial release of PGF2α which is associated with luteolysis. We hypothesized that estradiol modulates the expression of genes and proteins involved in the cascade of PGF2α synthesis by the endometrium of cows. 52 cyclic and non-lactating cows received 2mg of E2 benzoate (Sincrodiol, Ourofino) and an intravaginal progesterone device (1g; Sincrogest; Ourofino) for 8 days. Cows received 0.5mg of sodium cloprostenol (Sincrocio; Ourofino), intramuscular, 48 hours prior to P4 device removal and a second application at the day of its removal. On day 15 of the estrous cycle (D0; estrus), animals were assigned to receive one of the following treatments: placebo (P; 5ml 50% ethanol; IV), estradiol (E; 5mL 50% ethanol containing 3mg of 17β E2; IV) or control (untreated). Endometrial biopsies were collected at 0h (C; n = 10), 4h (E4 n = 11 and P4 n = 10) or 7h (E7 n = 10 and P7 n = 11). Two endometrial biopsies samples were obtained per cow for analysis of gene expression (qPCR) and localization of target proteins (Immunohistochemistry). Gene expression was analyzed by two-way ANOVA and mean comparison, and immunostaining was analyzed by T test. Expression of PTGS2 gene was lower in endometrium of animals from P7 and E7 groups if compared to P4 and E4 groups, respectively (P < 0.05). The gene expression of PLA2G4 and ESR1 were lower for animals from E4 group if compared to the E7 group (P < 0.05). The abundance of ESR2 gene was lower for cows from E4 and E7 groups if compared to the other groups (P < 0.05). A higher OXTR expression was observed in E4 and E7 groups if compared to the other groups (P < 0.05). PRKCα was down-regulated in endometrium from E7 group if compared to the other groups (P < 0.05). A lower expression of PRKCβ was observed in P7 and E7 groups if compared to the other groups (P < 0.05). The abundance of AKR1B1 and AKR1C4 were lower in E4 and E7 groups if compared to the other groups (P < 0.05). PKCγ protein immunostaining was higher (P < 0.05) in the luminal epithelium (LE) of animals from E4 and E7 groups compared to P4 and P7h groups (P < 0.05), and also higher in the glandular epithelium (GE) of P7 group compared to E7 group. The AKR1B1 protein immunostaining intensity was stronger (P < 0.05) in the LE from E4 and E7 groups if compared to P4 and P7 groups and was also stronger in GE of E4 group if compared to the P4 group. ERα Immunostaining was higher (P < 0.05) in GE of P4 and P7 groups if compared to E4 and E7 groups and also higher in LE of P4 group if compared to E4 group. In conclusion, estradiol administration reduced abundance of most genes related to proteins involved in PGF2α synthesis, except for OXTR. Moreover, the E2 treatment increased immunostaining of the PKCγ and AKR1B1 proteins, and decreased ERα. We speculate that E2 increases these proteins and that the reduced transcripts abundance was observed due to a negative feedback. Thus, protein quantification becomes necessary to determine whether modulation of gene expression by E2 is associated to an increased abundance of these proteins.