This study aimed to evaluate the influence of ovarian steroids on equine endometrial epithelial and stromal cells, specifically i) prostaglandin (PG) production in a time-dependent manner, ii) specific PG synthases mRNA transcription and protein expression, and iii) cell proliferation. After passage I, cells were exposed to vehicle, oxytocin (OT, positive control, 10−7 M), progesterone (P4, 10−7 M), 17β estradiol (E2, 10−9 M), or P4+E2 for 12, 24, 48, or 72 h. Following treatment, PG concentration was determined using the direct enzyme immunoassay (EIA) method. Alterations in PG synthases mRNA transcriptions, PG synthases protein expression, and cell proliferation in response to the treatments were determined after 24 h using real-time PCR, western blot, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide respectively. After 24 h, E2 and P4+E2 increased PGE2 and PGF2 α secretion as well as specific prostaglandin-endoperoxide synthase-2 (PTGS2), PGE2 synthases (PGES), and PGF2 α synthases (PGFS) expression in the epithelial cells (P<0.05). Additionally, E2 and P4+E2 increased PTGS2 expression in stromal cells after 24 h (P<0.05). In stromal cells, P4+E2 increased PGE2 production as well as PGES expression after 24 h (P<0.05). Both E2 and P4+E2 increased PGF2 α production by stromal cells after 24 h (P<0.05). Ovarian steroids affected proliferation of stromal and epithelial cells during the 24-h incubation period (P<0.05). We provide evidence that ovarian steroids affect PG production in equine endometrial cells, upregulating PTGS2, PGES, and PGFS expression. Ovarian steroid-stimulated PG production could be an important mechanism occurring in the equine endometrium that is involved in the regulation of the estrous cycle and early pregnancy.
Anna Z Szóstek, António M Galvão, Graça M Ferreira-Dias and Dariusz J Skarzynski
Michiyo Tanikawa, Hwa-Yong Lee, Kikuko Watanabe, Magdalena Majewska, Dariusz J Skarzynski, Soo-Bong Park, Dong-Seok Lee, Choon-Keun Park, Tomas J Acosta and Kiyoshi Okuda
Interleukin-1 (IL1) has been shown to be a potent stimulator of prostaglandin (PG) production in bovine endometrium. The aim of the present study was to determine the cell types in the endometrium (epithelial or stromal cells) responsible for the secretion of PGE2 and PGF2α in response to IL1A, and the intracellular mechanisms of IL1A action. Cultured bovine epithelial and stromal cells were exposed to IL1A or IL1B (0.006–3.0 nM) for 24 h. IL1A and IL1B dose-dependently stimulated PGE2 and PGF2α production in the stromal cells, but not in the epithelial cells. The stimulatory effect of IL1A (0.06–3.0 nM) on PG production was greater than that of IL1B. The stimulatory actions of IL1A on PG production was augmented by supplementing arachidonic acid (AA). When the stromal cells were incubated with IL1A and inhibitors of phospholipase (PL) C or PLA2 (1 μM; anthranilic acid), only PLA2 inhibitor completely stopped the stimulatory action of IL1A on PG production. Moreover, a specific cyclooxygenase-2 (COX2) inhibitor blocked the stimulatory effect of IL1A on PG production. IL1A (0.06 nM) promoted COX2 and microsomal PGE synthase-1 (PGES1) gene and its protein expression. The expression of COX1, PGES2, PGES3, and PGF synthase (PGFS) mRNA was not affected by IL1A in the stromal cells. The overall results indicate that 1) the target of IL1A and IL1B for stimulating both PGE2 and PGF2α production is the stromal cells, 2) IL1A is a far more potent stimulator than IL1B on PG production in stromal cells, 3) the stimulatory effect of IL1A on PG production is mediated via the activation of PLA2 and COX2, and (4) IL1A induced PG production by increasing expressions of COX2 and PGES1 mRNAs and their proteins in bovine stromal cells.
Hwa-Yong Lee, Tomas J Acosta, Michiyo Tanikawa, Ryosuke Sakumoto, Junichi Komiyama, Yukari Tasaki, Mariusz Piskula, Dariusz J Skarzynski, Masafumi Tetsuka and Kiyoshi Okuda
To determine whether glucocorticoids (GCs) play a role in regulating uterine function in cow, the present study examined the expression of mRNA encoding GC receptor (GC-R) α, 11β-hydroxysteroid dehydrogenase (11-HSD) type 1 and type 2, and the activity of 11-HSD1 in bovine endometrial tissue throughout the estrous cycle. We also studied the effects of cortisol on basal, oxytocin (OT)- and tumor necrosis factor-α (TNFα)-stimulated prostaglandin (PG) production. A quantitative real-time PCR analysis revealed that GC-Rα mRNA was expressed more strongly in the mid-luteal stage (days 8–12) than in the other stages. In contrast to GC-Rα mRNA expression, 11-HSD1 mRNA expression was greater in the follicular stage than in the other stages, whereas 11-HSD2 mRNA expression was lowest in the follicular stage. The activity of 11-HSD1 was greater in the follicular stage and estrus than in the other stages and was lowest in the mid-luteal stage. Cortisone was dose-dependently converted to cortisol in the cultured endometrial tissue. Although cortisol did not affect either the basal or OT-stimulated production of PGs in the cultured epithelial cells, the production of PGs stimulated by TNFα in the stromal cells was suppressed by cortisol (P < 0.05). Cortisol suppressed basal prostaglandin (PG)F2α without affecting basal PGE2 production in the stromal cells. The overall results suggest that the level of cortisol is locally regulated in bovine endometrium throughout the estrous cycle by 11-HSD1, and that cortisol could act as a luteoprotective factor by selectively suppressing luteolytic PGF2α production in bovine endometrium.