Discrepancies were also reported among in vitro studies conducted by different teams, and distinct responses to IFN-т might depend upon the concentration or isoform used. Overall, this reminds us that care must be taken in evaluating observations made in vitro, and that performing in vivo protocols in parallel often offers physiological clarification. Although this may seem obvious, our own experience and examples from other labs prove this notion can often be forgotten. The sole expression of IFN receptors or any type of response to the molecule in vitro does not guarantee that stromal cells are a direct target of IFN-т or that the response obtained really has a physiological counterpart.
The present study lends support to our previous hypothesis built on data obtained with in vitro experiments, that IFN-т stimulates both COX-2 and GM-CSF in the endometrium. However, a few differences were observed. In vitro, IFN-т induced the expression of COX-2 in both epithelial and stromal cells and up-regulated GM-CSF in stromal cells and leukocytes.
The present report confirms the expression of GM-CSF in the LE, SG, DG, endometrial vasculature, and isolated cells in the stroma during the estrous cycle in the cow. GM-CSF is secreted in the uterine lumen because it is detectable in flushings from nonpregnant but particularly from pregnant cows. Accordingly, endometrial GM-CSF is up-regulated during pregnancy in both the ewe and the cow, and the moderate stimulation of GM-CSF in the LE after exposure to IFN-т also fits with this expression pattern. GM-CSF is known to stimulate human and murine placen-to-fetal growth, differentiation and survival, in vitro and in vivo. Moreover, GM-CSF promotes development of in vitro-produced bovine embryos and may up-reg-ulate IFN-т production.
Additionally, PGE2 exhibits functions on immune cells that are not regulated by PGF2a (or PGI2) and that could be critical to maternal tolerance of the con-ceptus. Such functions include the differentiation of leukocytes into cells that are less lytic, more granulated, or that produce growth factors or Th2-type cytokines. It would be interesting to look at the spatio-temporal expression of both PGE synthase and PGF synthase in the endometrium to monitor any regulation in the expression of enzymes downstream of COX-2.
Indeed, levels of PGF2a remain elevated during early pregnancy or in cultured conceptus, just like the expression of COX-2, and luteolysis is nonetheless prevented. It is well known that the pul-satility in PGF2a production, rather than the absolute quantity, is the major factor that triggers luteolysis. Because this pulsatility is itself regulated by pulses of OT and that OTRs are undetectable during early pregnancy, high quantities of endometrial COX-2 and PGF2a do not seem to be a critical issue at this time. On the other hand, PGE2 production is also stimulated during early pregnancy and PGE2 secretion in bovine conceptus from d10P to d19P is increased in vitro.
Because a d16 embryo can be successfully transferred to a cycling cow at d16 (the day that luteolysis is initiated), it has been suggested that IFN-т also has a quick, direct effect on endometrial synthesis of PGs. In the present study, the expression of COX-2 is strongly enhanced in the LE during the pregnancy recognition period and after IFN-т treatment. COX-2 might be involved in opposed physiological events, forcing the female reproductive tract either to return to the cycle or to accommodate the embryo, because both luteolysis/OT treatment and pregnancy recognition/ IFN-т treatment are accompanied by an elevated expression of COX-2 in the LE.
n the present study, the increase in COX-2 expression after treatment with IFN-т was observed only in the ipsi horn of the uterus. The same side-specific effect was reported when studying the regulation of the EP2 receptor. On the other hand, there is no similar difference in the regulation of GM-CSF, and IFN receptors are expressed uniformly in the ipsi and the contralateral horns. Because the antiluteolytic effects of IFN-т are P4 dependent, and because P4 stimulates endometrial synthesis of PGs and is more concentrated in the ipsi horn, it is possible that the effect of IFN-т on the PG axis is favored in the ipsi horn.
In the ewe, endometrial expression of cOx-2 is limited to the LE and SG and is detectable only at peak expression between Day 12 and Day 17 of the cycle or pregnancy. In the cow, staining for COX-2 is observed at all stages, even in the stroma and in DG. Nonetheless, four major resemblances remain between the two species regarding the expression of COXs: first, COX-1 is undetectable in the conceptus; second, COX-2 expression in the conceptus decreases with time; third, endometrial COX-2 is high in the late phase of the estrous cycle; and fourth, endometrial COX-2 is stimulated during early pregnancy. Thus, in both species, COX-2 is high in the endometrium at the time of onset of luteolysis and maximal at the ma-terno-fetal interface during establishment of pregnancy. COX-2 null female mice have reproduction failures at multiple levels.
Expression patterns of COX-2 and GM-CSF reported in the present study fit well with this hypothesis. COX-2 and GM-CSF are strongly expressed during early pregnancy in regions where fusion has not yet occurred, whereas the staining for the two proteins is reduced at sites of attachment (in the hybrid syncytium) as was reported for IFN-t. This supports the view that COX-2 and GM-CSF are important mediators during the elongation and apposition periods, preparing the materno-fetal interface for the adhesion stage. It is possible that COX-2 or GM-CSF levels would be reestablished after d30P when the LE is reconstituted, but this question could not be addressed in the current study.
This study provides the first in vivo demonstration that both COX-2 and GM-CSF are up-regulated during critical steps of the establishment of pregnancy in cattle, and that this stimulation can be mimicked by IFN-t treatment. Indeed, the secretion of IFN-t (between d12P-d28P, peaking at d15P-d19P) by uninucleate trophoblastic cells is concomitant with dramatic cellular modifications that happen to both the uterine epithelium and the conceptus. Early attachment in ruminants is divided in three main stages.