Variation in Macrophage Migration Inhibitory Factor [MIF] immunoreactivity during bovine gestation

Abstract

Macrophage Migration Inhibitory Factor (MIF) is a proinflammatory cytokine involved in several aspects of the immune response. MIF appears to play important roles in materno-fetal immuno-tolerance during placental establishment, modulation and growth as studied in epitheliochorial porcine and hemochorial human and mouse placentae. Here we studied the bovine placenta being multiplex, villous and synepitheliochorial with a low degree of invasion, to see if MIF could be involved. Placental tissues sampled from 12 cows at 9 stages of gestation (days 18–250), and endometrial tissues from two non-pregnant animals were processed for immunohistochemistry. Bovine MIF was detected by Western blot using anti-human MIF monoclonal antibodies. An immunoreactive band of approximately 12 kDa confirmed similarities between bovine and human MIFs. Compared to the non-pregnant stage with very faint staining, the caruncular epithelium during pregnancy showed stronger staining for MIF. The intercaruncular epithelium in non-pregnant endometrium showed some reaction apically with increasing intensity at uterine gland openings; in contrast, at day 18 of gestation this staining was markedly increased. During gestation both caruncular and trophoblast epithelium of the placentomes were positive with different intensity in relation to the gestational stage. In the uterine glands, some strongly stained cells were present. The mature binucleated trophoblast giant cells were negative throughout pregnancy. During reestablishment of vascularisation, the vasculature in the caruncular area showed MIF reactivity. While supporting involvement of MIF in different placental types, the spatio-temporal variation in the bovine placenta suggests a regulatory role for MIF mainly in the interhemal barrier and during vascular development.

Introduction

Macrophage Migration Inhibitory Factor (MIF), first demonstrated as a factor capable of inhibiting the random migration of macrophages in vitro, is now recognised as a proinflammatory cytokine involved in several aspects of the immune response [1]. MIF is produced by a number of immune and non-immune cells including macrophages, lymphocytes and fibroblasts as well as those of the endocrine, nervous and reproductive systems [1]. Recent literature has shown that MIF is involved in different reproductive processes mainly in humans and mice [2], [3], [4]. MIF mRNA and protein are highly expressed in the human endometrium throughout the menstrual cycle and in early pregnancy in predecidualized stromal cells [5]. MIF expression in human endometrium was found to increase during the mid-late proliferative phase reaching a maximum around ovulation and decrease in the mid-secretory phase before increasing again in the late secretory phase [6]. In the human placenta, MIF mRNA and protein are highly expressed mainly by the villous and extravillous cytotrophoblast [7]. Placental MIF levels were higher in the very early stages of gestation and declined at late first trimester [8]. In addition, other studies in humans have shown that decreasing levels of maternal serum MIF were associated with first trimester miscarriages [9].

In mice, MIF mRNA is expressed in uteri during the pre-implantation period and throughout the estrous cycle as well as in early embryos [10]. MIF mRNA and protein are also expressed by the fetal placental components with an increase at gestational day 10.5, the stage at which the placenta assumes the three-layered organization and the fetal blood circulation begins [11]. The importance of MIF in early murine gestation is supported by the findings of Bondza et al. [12] who showed that an intraperitoneal injection of recombinant MIF on the day after mating resulted in an enhancement of the pregnancy rate.

All these data suggest that MIF is a key cytokine involved in uterine receptivity and pregnancy in species with a hemochorial type of placenta. Studies in other species with different placental type are limited. In the diffuse folded epitheliochorial placenta of the pig, it was shown that MIF is mainly expressed by trophoblast and maternal epithelium throughout gestation with a dramatic decrease in the maternal epithelium in late gestation, whereas immunostaining in trophoblast remained relatively high [13]. In a recent extensive study in the sheep [14], MIF was characterized and localised in all compartments of the female genital tract as well as in the placenta from early and late stages, but without a more detailed description of placental MIF throughout gestation. In ruminants, the placenta is cotyledonary, villous and epitheliochorial but it is a subtype, namely synepitheliochorial, as the binucleated trophoblast giant cells (TGC) migrate to fuse with one maternal epithelial cell forming a multinucleated hybrid cell [15], [16]. The TGC have high secretory activity and release hormonal products including progesterone, prostacyclins, prostaglandins, placental lactogen and pregnancy specific glycoproteins to the maternal compartment [17], [18], [19]. The invasion does not go beyond the basal lamina of the maternal epithelium and the hormonal products are released close to the maternal circulation. At present there is no evidence in the literature of MIF in the bovine placenta, however MIF has been localised on days 1–3 of the estrus cycle in the luminal epithelium of the bovine endometrium and in uterine glands [20]. The aim of this study is therefore to investigate the expression of MIF in the bovine placenta at various gestational stages from initial placentation to late pregnancy.