Role of leukemia inhibitor factor (LIF) in decidualisation of murine uterine stromal cells in vitro

Abstract

Decidualisation of uterine stromal cells is a prerequisite for implantation of the embryo in mice. Here we have used an in vitro culture system in which stromal cells decidualise as indicated by a number of markers, including an increase in alkaline phosphatase (ALP) activity. The latter was used as a quantitative marker of decidualisation in the presence of low (2%) fetal calf serum. Prostaglandin E(2) (PGE(2)), which is known to induce decidualisation, increased ALP activity, and this effect was blocked in a dose-dependent manner by indomethacin. Leukemia inhibitory factor (LIF) was then examined, but it had no effect on PGE(2) secretion. However, LIF suppressed ALP activity in a dose-dependent manner in the presence of 2% serum, while an inhibitor of LIF that competes for binding to its receptor reversed the effect of LIF and increased ALP activity above the control level. In serum-free cultures, stromal cells differentiated rapidly, and no differences were observed between LIF-treated and untreated cultures. Stromal cells produce LIF during in vitro culture, and this peaked at 48 h. Freshly collected stromal cells from both day-2 and -4 pregnant mice expressed mRNA for the LIF receptor, and the transcript level was higher in cells isolated on day 4. However, no differences were observed in the relative levels of transcripts in cells from day 2 and day 4 after culture, nor were there differences between the LIF-treated cultures and controls. Therefore, in this study, we have shown that LIF suppresses decidualisation of murine uterine stromal cells in the presence of serum, this is not due to the regulation of PGE(2) secretion by stromal cells.

Determining the LIF-sensitive period for implantation using a LIF-receptor antagonist

“…Often, the genetic alteration prevents the survival of null mice to adulthood and thus studies on reproductive physiology are hampered, reviewed in Salamonsen et al (2001). Previous work has centred upon identifying possible targets of LIF during early pregnancy from comparisons between wild-type and Lif deficient mice (Fouladi-Nashta et al 2004, 2005, 2008, Sherwin et al 2004. However, this neither permits the study of the temporal and spatial influence of LIF, particularly during pregnancy nor does it address the possibility that the phenotype may partly arise from the gene deletion construct causing -cis or -trans effects on adjacent genes.…”

“…Experiments have shown that functional inactivation of LIFR by hLIF-05 is required for the arrest of murine rod differentiation in vitro (Neophytou et al 1997) and previous work in our laboratory has shown that LIF induced effects can be antagonised by hLIF-05 in cultured murine uterine stromal cells (Fouladi-Nashta et al 2004, 2008. However, this antagonist has not been used previously in vivo.…”

“…Alexa Fluor Invitrogen) or biotin conjugated secondary anti-goat, -rabbit or -rat IgG (7 mg/ml; Vector Labs, Oxford, UK). The LIFR antagonist, hLIF-05, was characterised and previously described (Hudson et al 1996, Fouladi-Nashta et al 2004, 2008.…”

Egr1 Protein Acts Downstream of Estrogen-Leukemia Inhibitory Factor (LIF)-STAT3 Pathway and Plays a Role during Implantation through Targeting Wnt4

“…It has been reported that LIF can enhance decidualization of endometrial stromal cells in humans (16). In vivo experiments showed that polyethylene glycol (PEG)-conjugated LIF antagonist treatment reduced the decidual area of implantation site (15).…”

“…Conditional knock-out or transient suppression of STAT3 results in implantation failure, which suggests the critical role of STAT3 during implantation (11)(12)(13)(14). As a pleiotropic cytokine, LIF is also essential for mouse and human decidualization (15,16). However, the direct target gene of LIF-STAT3 pathway in mouse uterus during implantation remains poorly understood.…”