Effects of the estrous cycle, pregnancy and interferon tau on expression of cyclooxygenase two (COX-2) in ovine endometrium

Abstract

In sheep, the uterus produces luteolytic pulses of prostaglandin F2alpha (PGF) on Days 15 to 16 of estrous cycle to regress the corpus luteum (CL). These PGF pulses are produced by the endometrial lumenal epithelium (LE) and superficial ductal glandular epithelium (sGE) in response to binding of pituitary and/or luteal oxytocin to oxytocin receptors (OTR) and liberation of arachidonic acid, the precursor of PGF. Cyclooxygenase-one (COX-1) and COX-2 are rate-limiting enzymes in PGF synthesis, and COX-2 is the major form expressed in ovine endometrium. During pregnancy recognition, interferon tau (IFNtau), produced by the conceptus trophectoderm, acts in a paracrine manner to suppress development of the endometrial epithelial luteolytic mechanism by inhibiting transcription of estrogen receptor alpha (ERalpha) (directly) and OTR (indirectly) genes. Conflicting studies indicate that IFNtau increases, decreases or has no effect on COX-2 expression in bovine and ovine endometrial cells. In Study One, COX-2 mRNA and protein were detected solely in endometrial LE and sGE of both cyclic and pregnant ewes. During the estrous cycle, COX-2 expression increased from Days 10 to 12 and then decreased to Day 16. During early pregnancy, COX-2 expression increased from Days 10 to 12 and remained higher than in cyclic ewes. In Study Two, intrauterine infusion of recombinant ovine IFNtau in cyclic ewes from Days 11 to 16 post-estrus did not affect COX-2 expression in the endometrial epithelium. These results clearly indicate that IFNtau has no effect on expression of the COX-2 gene in the ovine endometrium. Therefore, antiluteolytic effects of IFNtau are to inhibit ERalpha and OTR gene transcription, thereby preventing endometrial production of luteolytic pulses of PGF. Indeed, expression of COX-2 in the endometrial epithelia as well as conceptus is likely to have a beneficial regulatory role in implantation and development of the conceptus.

Figure 1

Steady-state levels of COX-2 mRNA expression in endometrium of cyclic and pregnant ewes. Total RNA was isolated from endometrium and analyzed by semi-quantitative RT-PCR. Data are presented as LSM relative units (RU) with SE.

Figure 2

In situ hybridization and immunohistochemical analysis of COX-2 expression in the endometrium of cyclic ewes. Cross-sections of ovine endometrium were hybridized with radiolabeled antisense or sense bovine COX-2 cRNA probes. Hybridized sections were digested with ribonuclease A, and protected transcripts were visualized by liquid emulsion autoradiography. Developed slides were counterstained lightly with hematoxylin, and photomicrographs were taken under bright-field (left) or dark-field illumination (middle). Immunoreactive COX-2 protein was detected using rabbit anti-human COX-2 polyclonal IgG (right). The negative IgG control was performed by substituting irrelevant rabbit IgG for primary antibody. The white arrow denotes areas of specific COX-2 mRNA or immunoreactive protein expression. Legend: C, cyclic; LE, lumenal epithelium; S, stroma; sGE, superficial ductal glandular epithelium. Bar = 20 μm.

Figure 3

In situ hybridization and immunohistochemical analysis of COX-2 expression in the endometrium of early pregnant ewes. Cross-sections of the ovine endometrium were hybridized with radiolabeled antisense or sense bovine COX-2 cRNA probe. Hybridized sections were digested with ribonuclease A, and protected transcripts were visualized by liquid emulsion autoradiography. Developed slides were counterstained lightly with hematoxylin, and photomicrographs were taken under bright-field or dark-field illumination (left). Immunoreactive COX-2 protein was detected using rabbit anti-human COX-2 polyclonal IgG and a BioStain Super ABC Kit (right). The white arrow denotes areas of specific COX-2 mRNA or immunoreactive protein expression. Legend: C, conceptus; LE, lumenal epithelium; S, stroma; sGE, superficial ductal glandular epithelium; P, pregnant. Bar = 20 μm.

Figure 4

In situ hybridization and immunohistochemical analysis of COX-2 expression in the ovine endometrium (Study Two). Ewes were ovariectomized on Day 5 of the estrous cycle, treated daily with progesterone, and infused from Days 11 to 15 with either control (CX) proteins or recombinant ovine IFNτ (IFN). On Day 16, ewes were hysterectomized. Cross-sections of the ovine uterus were hybridized with radiolabeled antisense or sense bovine COX-2 cRNA probe. Hybridized sections were digested with ribonuclease A, and protected transcripts were visualized by liquid emulsion autoradiography. Developed slides were counterstained lightly with hematoxylin, and photomicrographs were taken under bright-field or dark-field illumination (left). Immunoreactive COX-2 protein was detected using rabbit anti-human COX-2 polyclonal IgG (right). The negative IgG control was performed by substituting irrelevant rabbit IgG for primary antibodies. The white arrow denotes areas of specific COX-2 mRNA or immunoreactive protein expression. Legend: CX, control; IFN, interferon tau; LE, lumenal epithelium; S, stroma; sGE, superficial ductal glandular epithelium. Bar = 20 μm.