The expression of prostaglandin E2 receptors (EP2 and EP4) and the proportion of extracellular matrix in cervix of cyclic bitches and bitches with pyometra

Abstract

Exp. I aimed to determine the proportion of collagen and smooth muscle using Masson’s trichrome staining and Alcian blue staining was used to evaluate the relative distribution of cervical GAGs. The proportion of cervical collagen relative to cervical smooth muscle was higher at estrus compared to anestrus (p ≤ 0.05). It was also higher (p ≤ 0.05) in bitches with open- compared to those with closed-cervix pyometra. Overall, hyaluronan (HA) was the predominant GAG in the canine cervix and there were no differences in GAG composition in the stroma of healthy bitches and in the luminal epithelium and muscle of bitches with pyometra. In the luminal epithelium, HA was higher in estrus than in anestrus (p ≤ 0.05) but not in diestrus (p > 0.05). On the contrary, the combined keratan sulfate (KS) and heparan sulfate (HS) content was higher in anestrus than estrus (p ≤ 0.05). In bitches with pyometra, the combined KS and HS content was significantly lower in open- compared to closed-cervix pyometra (p ≤ 0.05). Collectively, the different profiles of collagen and GAG observed at different stages of the estrous cycle as well as in pyometra suggest that the metabolism of both collagens and GAGs in the canine cervix is associated with hormonal statuses during the estrous cycle and cervical patency of bitches with pathological uterine conditions such as pyometra. Exp. II aimed to investigate the expression of EP4 in the cervices of bitches during estrous cycle and those with pyometra. The expression of EP4 was observed at all the layers and all stages but the differences in EP4 expression either among bitches in different stages of the estrous cycle and between open- and closed-cervix pyometra were limited to only luminal epithelium (LE). In cyclic bitches during estrus and in open-cervix pyometra bitches, significantly higher EP4 expression was found in SE of uterine part than vaginal part. In SE of the uterine part, the expression was higher in the bitches during estrus than in anestrus and diestrus, and higher in the bitches affected by open-cervix than closed-cervix pyometra. The results suggest that regulation of cervical dilation appeared in the uterine part of the cervix. Moreover, EP4 may be involved in stimulating dilation of the cervix in both estrus and open-cervix pyometra bitches. Exp. III aimed to investigate mRNA expressions of EP2, EP4, COX-2, and PGES in the bitch cervix. Two groups of bitches; normal cyclic bitches and bitches with pyometra were studied. RNA extracted from cervical tissue was determined for levels of EP2, EP4, COX-2, and PGES mRNA using a real-time qPCR. Western blot was performed to investigate the protein expression of EP2, EP4, and COX-2There were no differences of EP2, EP4, COX-2, and PGES mRNA expression in the bitch cervix among the stages of the estrous cycle. However, the expression of PGES mRNA was higher in the cervix of bitches with open-cervix than closed-cervix pyometra (P<0.05). However, the differences of protein expression were not observed in both normal cyclic bitches and bitches with pyometra. Our findings suggest that mRNA and protein expression of the enzymes involved in PGE2 synthesis and PGE2 receptors are not influenced by hormonal status during the estrous cycle whereas PGES mRNA expression is likely associated with cervical relaxation in the bitches with pyometra.