No abstract

Ovine interferon-tau (oIFN-tau) may stabilize endometrial progesterone receptor (PR) and/or inhibit estrogen receptor (ER) gene expression during pregnancy recognition to suppress endometrial oxytocin receptor (OTR) formation and production of luteolytic prostaglandin (PG) F2 alpha pulses. This study determined whether or not oIFN-tau stabilized PR expression in the endometrium during PR down-regulation by continuous exposure to progesterone. Twenty cyclic ewes were bilaterally ovariectomized and fitted with uterine catheters on Day 2 of the estrous cycle (Day 0 = estrus). Ewes were then assigned randomly to be treated, in a 2 x 2 factorial arrangement, with recombinant oIFN-tau (roIFN-tau; 2 x 10(7) antiviral units per ewe per day) or control proteins (6 mg/day) by intrauterine injection from Days 10 to 14, and with daily i.m. injections of 20 mg progesterone from Days 2 to 14 (P) or progesterone from Days 2 to 14 plus 50 micrograms estradiol-17 beta from Days 12 to 14 (P+E). All ewes were hysterectomized on Day 15. Endometrial PR mRNA (p < 0.01) and protein (p < 0.03) were higher in ewes receiving P+E than in those receiving P alone. However, the increase in PR mRNA and protein was not as great in the endometrium of roIFN-tau-treated ewes as compared to controls (p < 0.08, treatment x steroid). In ewes receiving P alone, PR mRNA and immunoreactive PR were localized to stroma and deep glandular epithelium and were not present in endometrial luminal and shallow glandular epithelium. Values for endometrial ER mRNA (p < 0.02) and ER protein (p < 0.01) were greater in controls than in roIFN-tau-treated ewes regardless of steroid treatment. Among controls, ER mRNA and immunoreactive ER protein were present in the luminal and glandular epithelium and were increased in the epithelium and stroma in ewes receiving estrogen. In contrast, endometrial ER mRNA and immunoreactive ER protein were very low or absent in the endometrium of roIFN-tau-treated ewes and were not increased by estrogen. Among controls, endometrial OTR density was greater (p < 0.09) in ewes treated with P+E than in those treated with P alone. In roIFN-tau-treated ewes, endometrial OTR density was lower (p < 0.01) than in the controls. Results indicate that roIFN-tau did not stabilize or prevent autologous down-regulation of PR mRNA or protein expression in the endometrium. However, roIFN-tau did suppress endometrial ER expression and OTR formation in ewes regardless of steroid treatment. The results support the hypothesis that the antiluteolytic effects of oIFN-tau are to suppress endometrial ER gene expression in the endometrial epithelium, thereby inhibiting formation of OTR and production of luteolytic PGF2 alpha pulses.

This study determined the effects of intrauterine injections of recombinant ovine interferon-tau; (roIFN-tau; 2 x 10(7) antiviral units/day) or control proteins (6 mg/day) from day 11 to day 14 post-oestrus = day 0) on endometrial expression of receptors fro oestrogen, progesterone and oxytocin in cyclic ewes. Plasma concentrations of progesterone were greater on day 15 in ewes receiving roIFN-tau compared with control proteins (P < 0.02, treatment x day). Ewes injected with roIFN-tau had lower endometrial levels or oestrogen receptor mRNA (P > 0.10) and protein (P < 0.01) on day 15 compared with ewes receiving control proteins. In situ hybridization analysis indicated that oestrogen receptor mRNA was more abundant in the luminal and glandular epithelium of control ewes compared with roIFN-tau-treated ewes. Immunoreactive oestrogen receptor was also present in the luminal and glandular epithelium of control, but not roIFN-tau-treated ewes. Endometrial levels of progesterone receptor mRNA and protein were not different (P > 0.10) between control and roIFN-tau-treated ewes. In situ hybridization analyses indicated that progesterone receptor mRNA abundance was low in endometrial epithelium and stroma of both control and roIFN-tau-injected ewes. Immunoreactive progesterone receptors were present in the endometrial stroma and epithelium of control ewes, but confined to the stroma of roIFN-tau-treated ewes. Oxytocin receptor density was lower (P < 0.01) in the endometrium of ewes injected with roIFN-tau than control proteins; however, oxytocin receptor affinity was not affected (P > 0.10) by treatment. Concentrations of 13,14-dihydro-15-ketoprostaglandin F2a (PGFM) were not increased by exogenous oxytocin administration in control and roIFN-tau-treated ewes on days 10 or 12 post-oestrus. However, on day 14, control ewes responded to oxytocin with increased plasma concentrations of PGFM, whereas ewes receiving roIFN-tau remained unresponsive to oxytocin. These results indicate that the an tiluteolytic effects of IFN-tau are to prevent increases in endometrial oestrogen receptor MRNA and protein and oxytocin receptor density which abrogates uterine release of prostaglandin F2a during maternal recognition of pregnancy. IFN-tau may inhibit the synthesis of oestrogen receptor mRNA by a transcriptional or post-transcriptional regulatory mechanism to suppress oxytocin receptor formation during early pregnancy in ewes.

Connective tissue growth factor (CTGF) is a growth and chemotactic factor for fibroblasts encoded by an immediate early gene that is transcriptionally activated by transforming growth factor ss. Although the primary translational product of the pig CTGF gene is predicted to be of approximate Mr 38 000, pig uterine luminal flushings (ULF) contained 10- to 20-kDa CTGF proteins that were heparin-binding and mitogenic, whereas 38-kDa CTGF was not apparent. The N-termini of two microheterogeneous forms of 16-kDa CTGF, as well as 18-kDa and 20-kDa forms of CTGF, commenced at, respectively, Cys199, Ala197, Asp186, and Asp186 and did not correspond to intron-exon boundaries in the CTGF gene. Northern blotting revealed a single porcine (p) CTGF transcript of 2.4 kilobases in endometrium from Day 10 to 16 cycling or pregnant pigs. Ten- to twenty-kilodalton pCTGF proteins in ULF were stable for 48 h at 37 degreesC whereas native 38-kDa pCTGF was degraded within 10 min under the same conditions. CTGF-degrading activity in pig ULF was heat-sensitive and concentration- and time-dependent. Ten- to twenty-kilodalton CTGF levels in ULF peaked on Day 16 of the cycle and on Day 12 of pregnancy and were highly correlated with the levels of proteolytic activity for 38-kDa CTGF. Collectively these data suggest that bioactive 10- to 20-kDa CTGF proteins are generated in utero through limited proteolysis of the 38-kDa CTGF primary translational product.

Pregnancy and interferon-tau (IFN tau) upregulate uterine Mx gene expression in ewes; however, the only known role for Mx is in the immune response to viral infection. We hypothesize that Mx functions as a conceptus-induced component of the anti-luteolytic mechanism and/or regulator of endometrial secretion or uterine remodeling during early pregnancy. This study was conducted to determine the effects of early pregnancy on uterine Mx expression in domestic farm species with varied mechanisms of pregnancy recognition. Endometrium from cows, gilts, and mares was collected during the first 20 d of the estrous cycle or pregnancy, and total messenger RNA (mRNA) and protein were analyzed for steady-state levels of Mx mRNA and protein. Northern blot analysis of Mx mRNA detected an approximately 2.5 Kb of mRNA in endometrium from each species. In pregnant cows, steady-state levels of Mx mRNA increased 10-fold (P < 0.05) above levels observed in cyclic cows by d 15 to 18. In cyclic gilts, slot blot analysis indicated that endometrial Mx mRNA levels did not change between d 5 and 18 of the cycle. However, in pregnant gilts, Mx levels tended (P = 0.06) to be elevated two-fold on d 16 only, and in situ hybridization indicated that this increase occurred in the stroma. In mares, Mx mRNA was low, but detectable, and did not change between ovulation (d 0) and d 20, regardless of reproductive status. Western blot analysis revealed multiple immunoreactive Mx protein bands in each species. One band was specific to pregnancy in cows. As in ewes, in situ hybridization analysis indicated that Mx mRNA was strongly expressed in the luminal epithelium, stroma, and myometrium by d 18 in cows. However, on d 14 in gilts, Mx was expressed primarily in the stroma, and on d 14 in mares, low levels of Mx expression were confined largely to the luminal epithelium. The uteruses of cows, gilts, and mares express Mx, and expression is upregulated during pregnancy in cows and gilts--animals whose conceptuses secrete interferons during early pregnancy, but that possess different mechanisms for pregnancy recognition.