Spatiotemporal patterns of macrophage migration inhibitory factor (Mif) expression in the mouse placenta

Abstract

Background: Macrophage migration inhibitory factor (MIF) has special pro-inflammatory roles, affecting the functions of macrophages and lymphocytes and counter-regulating the effects of glucocorticoids on the immune response. The conspicuous expression of MIF during human implantation and early embryonic development also suggests this factor acts in reproductive functions. The overall goal of this study was to evaluate Mif expression by trophoblast and embryo placental cells during mouse pregnancy.

Methods: Mif was immunolocalized at implantation sites on gestation days (gd) 7.5, 10.5, 13.5 and 17.5. Ectoplacental cones and fetal placentas dissected from the maternal tissues were used for Western blotting and qRT-PCR assays on the same gestation days.

Results: During the post-implantation period (gd7.5), trophoblast giant cells showed strong Mif reactivity. In later placentation phases (gds 10.5-17.5), Mif appeared to be concentrated in the junctional zone and trophoblast giant cells. Mif protein expression increased significantly from gd7.5 to 10.5 (p = 0.005) and from gd7.5 to 13.5 (p = 0.03), remaining at high concentration as gestation proceeded. Higher mRNA expression was found on gd10.5 and was significantly different from gd13.5 (p = 0.048) and 17.5 (p = 0.009).

Conclusions: The up-regulation of Mif on gd10.5 coincides with the stage in which the placenta assumes its three-layered organization (giant cells, spongiotrophoblast and labyrinth zones), fetal blood circulation begins and population of uNK cells reaches high proportions at the maternal counter part of the placenta, suggesting that Mif may play a role in either the placentation or in the adaptation of the differentiated placenta to the uterus or still in gestational immunomodulatory responses. Moreover, it reinforces the possibility of specific activities for Mif at the maternal fetal interface.

Figure 1

Immunolocalization of MIF in implantation sites on days 7.5 (A-C), 10.5 (D-F), 13.5 (G-I) and 17.5 (J-L) of gestation. MIF reactivity (pink to red color) is seen in trophoblast cells and some cells in the mesometrial decidua. Numbers on the left represent gestation days. (ec) Ectoplacental cone; (d) Decidua; (g) Trophoblast giant cells; (j) Junctional zone; (L) Labyrinthine zone; (S and *) Spongiotrophoblast. The boxed areas within low magnifications panels (A, D, G) indicate similar areas of focus in B, E and H. Figures C, F, I and L are negative controls in which the primary antibody was replaced with non-immune serum. Bar in A = 1 mm in C, J and L, 500 μm in A and G, 350 μm in D, I and K, 250 μm in E-F and 150 μm in B and H.

Figure 2

Western blotting of ectoplacental cone (gd7.5) and placental (gds 10.5, 13.5 and 17.5) homogenates. The top panel (A) is a representative Western blot showing the detection of MIF protein at different gestational days (numbers on the top). Equal amounts of total cell lysate protein from each sample were separated by SDS-PAGE and immunodetected by Western blotting using anti-mouse MIF antibody. Equivalence of protein loading was confirmed by Ponceau S staining (lower bands). Panel B shows the MIF level by densitometry, presented as mean ± SD of three samples from three separate experiments. *P < 0.05 in relation to the gd7.5.

Figure 3

PCR analysis of Mif mRNA in ectoplacental cone and placentas at different gestational periods. Panel A. Representative agarose gel photograph showing ethidium bromide-stained RT-PCR products of Mif and cyclophilin genes (internal control, cyclophilin). 1 and 2, 7.5 gd; 3 and 4, 10.5 gd; 5 and 6, 13.5 gd; 7 and 8, 17.5 gd. Panel B. Mif mRNA expression using RT-qPCR. Mif mRNA levels relative to β-actin were measured in all gestational periods indicated using RT-qPCR; the values are given as means ± SEM of three independent experiments. All comparisons were two-tailed. The RT-qPCR results revealed that placentas from gd10.5 show significant up-regulation (*) of Mif mRNA levels compared with gd7.5 (p = 0.054), 13.5 (p = 0.048) and 17.5 (p = 0.009).