br Introduction Prostaglandins are important local mediators
Introduction Prostaglandins are important local mediators in a host of reproductive processes including ovulation, fertilization, embryonic development, luteal regression, and parturition (Ushikubi et al., 2000). Prostaglandin E2 (PGE2) is among the more versatile prostanoids, and its effects appear to be mediated via one of four receptors designated EP1, EP2, EP3 and EP4 which are encoded by separate genes (PTGER1-PTGER4) (Sugimoto and Narumiya, 2007). Although PGE2 has many and diverse effects, in general, EP1 and EP3 induce smooth muscle contraction whereas EP2 and EP4 induce smooth muscle relaxation (Wanggren et al., 2006). Both EP2 and EP4 appear to be G-protein-coupled receptors that activate adenylate cyclase and increase cAMP (Sugimoto and Narumiya, 2007). Prostaglandin E2 receptor type 2 is important to fertility, and EP2−/− female mice have reduced fertility associated with failure of cumulus expansion and fertilization in vivo (Hizaki et al., 1999, Tilley et al., 1999). This defect does not appear to be due to inherent defects in the ovum as in vitro fertilization rates with oocytes from EP2−/− female mice are normal, and the authors hypothesized that the reduced fertility was due to alteration of the cumulus or oviductal microenvironment which was deleterious to fertilization (Tilley et al., 1999). In mice and in horses, both EP2 and EP4 receptors are upregulated in the preovulatory follicle after administration of hCG (Sayasith et al., 2009, Segi et al., 2003), and both are presumed to have a role in ovulation. In addition to its role in ovulation and fertilization, PGE2 also has important roles in regulation of oviductal function. In mares, during early pregnancy, leukotriene receptor agonist transit through the oviduct is dependent upon fertilization and subsequent embryonic development, and unfertilized ova are retained in the oviduct of the mare (Betteridge et al., 1979, Betteridge and Mitchell, 1974, Steffenhagen et al., 1972, Van Niekerk and Gerneke, 1966). Transit of the horse embryo from the oviduct into the uterus appears to be regulated by embryonic secretion of PGE2 (Robinson et al., 2000, Weber et al., 1991a, Weber et al., 1991b, Weber et al., 1992). Production of PGE2 by the early horse embryo has been demonstrated (Weber et al., 1991b), and PGE2 binds the oviduct (Weber et al., 1992) where it appears to induce relaxation of circular smooth muscle (Weber et al., 1995). In rats, PGE2 causes relaxation of the isthmic oviduct and the uterotubal junction (Ortega-Moreno, 1995), and PGE2 stimulates ciliary activity of the oviductal mucosa in rabbits (Verdugo et al., 1980) and in hamsters (Hermoso et al., 2001). Together, these studies suggest that PGE2 may have multiple roles in regulating contractility, ciliary activity as well as the secretory activity of the oviduct in mammals with subsequent effects on fertilization, early embryonic development and gamete or embryo transit. In the current study, gene expression of the PGE2 receptors primarily responsible for smooth muscle relaxation (EP2, EP4) was examined in the horse oviduct. The objectives of the current study were: (1) compare differences in relative amounts of mRNA for EP2 and EP4 in the isthmic and ampullar oviduct from mares during estrus, at Day 5 of diestrus and from prepubertal mares as well as (2) compare immunolocalization of EP2 and EP4 in these regions and estrous cycle stages.
Materials and methods
Discussion Both EP2 and EP4 receptors were expressed in the horse oviduct in epithelial cells, vascular endothelium, smooth muscle and serosa. There was no evidence in the current study for differential regulation of EP2 or EP4 between estrus, diestrus or prior to puberty based upon relative amounts of mRNA and IHC for these receptors which suggests that these receptors may not be regulated by steroid environment or estrous cycle stage in the horse oviduct. Relatively few studies have examined the impact of estrous cycle stage or steroid environment on the expression of PGE2 receptor genes in the oviduct. In the human fallopian tube, expression of EP1, EP2 and EP3 genes was increased by administration of the synthetic progestin, levonorgestel (Wanggren et al., 2008), and EP2 gene expression was decreased after administration of the progesterone receptor antagonist, mifepristone (Wanggren et al., 2006). In cattle, EP2 and EP4 gene expression were greater in the pre-ovulatory phase than during the luteal phase in oviductal epithelial cells recovered from abattoir material (Gabler et al., 2008). In mice, the EP2 receptor has a progesterone-responsive region in the 5′-flanking region of the gene (Tsuchiya et al., 2003), although evidence for differential regulation of EP2 gene expression in the oviduct of mice appears lacking. A number of studies suggest that estradiol and progesterone regulate expression of PGE2 receptors in the uterus. The uterine expression of EP2 was upregulated by progesterone or the combination of progesterone/estradiol in mice (Lim and Dey, 1997) and in rats (Blesson et al., 2012). Together, these studies suggest that expression of EP2 receptor genes is regulated by combinations of progesterone and estradiol; however, the available data do not clearly establish steroid-dependent regulation of EP2 within the oviduct across species.