Practical Engineer

Leptin and leukemia inhibitory factor (LIF) have been implicated as important mediators of implantation. The present study was designed to investigate whether leptin can directly regulate the expression of LIF and its receptor (LIF-R) in human endometrial cells and/or whether leptin-induced effects are linked to, or regulated in part by IL-1 signaling. Primary endometrial cells and endometrial epithelial cell lines (HES and Ishikawa cells) were cultured for 24-48 h in a medium containing insulin (5 microg/ml) and leptin (3, 10, and 62 nm) or IL-1beta (0.6, 3, and 10 nm) in the presence or absence of cytokines and/or receptor antagonists. The endpoints included phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the relative levels of LIF, LIF-R, IL-1beta, IL-1 receptor antagonist (IL-1Ra) and IL-1 receptor type I (IL-1R tI) as determined by ELISA or Western blotting techniques. Leptin treatment increases the level of phosphorylated STAT3, LIF-R, and LIF. Leptin also increases the levels of IL-1 ligand, receptor, and antagonist as was previously reported. Blockade of OB-R with antibodies or with a specific OB-R inhibitor (leptin peptide antagonist-2) abrogated leptin-induced effects, suggesting that leptin binding to its receptor activates Janus kinase 2/STAT3 signaling. Treatment of endometrial cells with IL-1beta also results in elevated levels of LIF-R. Interestingly, the inhibition of IL-1R tI with a specific antibody or with IL-1Ra negatively affects both leptin-induced and IL-1-induced effects on LIF-R levels. Abnormal endometrial LIF expression has been associated with human infertility and leptin has profound effects on the levels of LIF, IL-1, and their cognate receptors in vitro. Thus, it is tempting to speculate that leptin's role in vivo could include the regulation of other key cytokines to be fundamental to endometrial receptivity during implantation (i.e. LIF and IL-1).

Leptin is essential for mouse reproduction, but the exact roles it serves are yet to be determined. Treatment of cultured endometrial cells with leptin increases the level of beta3-integrin, IL-1, leukemia inhibitory factor, and their corresponding receptors. These leptin-induced effects are eliminated by inhibitors of leptin receptor (OB-R) signaling. Herein the impact of blocking leptin/OB-R signaling in the mouse endometrium was assessed. Intrauterine injection of either leptin peptide antagonists (LPA-1 or -2) or OB-R antibody on d 3 of pregnancy impaired mouse implantation in comparison to intrauterine injection of scrambled peptides (LPA-Sc) or species-matched IgGs. Significant reduction in the number of implantation sites and uterine horns with implanted embryos was found after intrauterine injection of LPA-1 (1 of 22) vs. LPA-1Sc (11 of 15) and LPA-2 (3 of 17) vs. LPA-2Sc (14 of 16). The impact of disruption of leptin signaling on the endometrial expression of several molecules in pregnant mice was assessed by Western blot, immunohistochemistry, and confocal microscopy. Disruption of leptin signaling resulted in a significant reduction of IL-1 receptor type I, leukemia inhibitory factor, vascular endothelial growth factor receptor 2, and beta3-integrin levels. The levels of colony stimulating factor-1 receptor and OB-R were unaltered after treatment with LPAs compared with controls. Expression of OB-R protein was pregnancy dependent and found only in glandular epithelium after implantation occurred. Our findings support previous observations that leptin signaling is critical to the implantation process and suggest that molecules downstream of leptin-activated receptor may serve obligatory roles in endometrial receptivity and successful implantation.