Leukemia inhibitory factor enhances endometrial stromal cell decidualization in humans and mice

Abstract

Adequate differentiation or decidualization of endometrial stromal cells (ESC) is critical for successful pregnancy in humans and rodents. Here, we investigated the role of leukemia inhibitory factor (LIF) in human and murine decidualization. Ex vivo human (H) ESC decidualization was induced by estrogen (E, 10(-8) M) plus medroxyprogesterone acetate (MPA, 10(-7) M). Exogenous LIF (≥50 ng/ml) induced STAT3 phosphorylation in non-decidualized and decidualized HESC and enhanced E+MPA-induced decidualization (measured by PRL secretion, P<0.05). LIF mRNA in HESC was down-regulated by decidualization treatment (E+MPA) whereas LIF receptor (R) mRNA was up-regulated, suggesting that the decidualization stimulus 'primed' HESC for LIF action, but that factors not present in our in vitro model were required to induce LIF expression. Ex vivo first trimester decidual biopsies secreted >100 pg/mg G-CSF, IL6, IL8, and MCP1. Decidualized HESC secreted IL6, IL8, IL15 and MCP1. LIF (50 ng/ml) up-regulated IL6 and IL15 (P<0.05) secretion in decidualized HESC compared to 0.5 ng/ml LIF. In murine endometrium, LIF and LIFR immunolocalized to decidualized stromal cells on day 5 of gestation (day 0 = day of plug detection). Western blotting confirmed that LIF and the LIFR were up-regulated in intra-implantation sites compared to inter-implantation sites on Day 5 of gestation. To determine the role of LIF during in vivo murine decidualization, intra-peritoneal injections of a long-acting LIF antagonist (PEGLA; 900 or 1200 µg) were given just post-attachment, during the initiation of decidualization on day 4. PEGLA treatment reduced implantation site decidual area (P<0.05) and desmin staining immuno-intensity (P<0.05) compared to control on day 6 of gestation. This study demonstrated that LIF was an important regulator of decidualization in humans and mice and data provides insight into the processes underlying decidualization, which are important for understanding implantation and placentation.

Figure 1. LIF and LIFR immunolocalization in human endometrial biopsies.

A. LIF immunolocalized to glandular epithelium (*), endothelial cells (a) and decidualized stromal cells (arrows) during the mid-secretory phase. B. LIF immunolocalized to decidual cells (arrows), endothelial cells (a) and glandular epithelium (*) in 1^st trimester decidua. C. LIFR immunolocalized to glandular epithelium (*) and decidualized stromal cells (arrows) during the mid-secretory phase. D. LIFR immunolocalized to decidual cells (arrows) in 1^st trimester decidua. Representative photomicrographs from n = 3. Scale, 100 µm; Insert, negative (IgG) control; a, spiral arterioles.

Figure 2. LIF and LIFR mRNA expression in non-decidualized (media control [C] and estradiol [E] treated) and decidualized (E plus medroxy-progesterone acetate [MPA] treated) HESC determined by semi-quantative reverse-transcription PCR.

18 s ribosomal RNA was used as loading control. LIF expression was downregulated by E+MPA, whilst LIFR expression was stimulated by both E and particularly E+MPA. Representative gel of n = 5.

Figure 3. Leukemia inhibitory factor (LIF) activated STAT3 in HESC.

Cells were treated with LIF for 15 min and with a LIF antagonist (LA) for 30 min prior to the addition of LIF. A. Representative immunoblot for pSTAT3 and total STAT3 in cell lysates from non-decidualized HESC (n = 3). B. Representative immunoblot for pSTAT3 and total STAT3 in cell lysates from decidualized HESC (n = 3).

Figure 4. Exogenous LIF enhanced estradiol (E) + medroxy-progesterone acetate (MPA)-induced in vitro decidualization (measured by PRL secretion) in HESC.

A. LIF treatment significantly (*; p<0.05) promoted E+MPA-induced decidualization in HESC compared to E+MPA-treated control on D14 (n = 5). B. Co-incubation of E+MPA plus LIF and LIF antagonist (LA) significantly (*; p<0.05) inhibited HESC decidualization compared to HESC treated with E+MPA plus LIF (n = 3). Treatments: E 10⁻⁸ M; MPA 10⁻⁷ M.

Figure 5. LIF (50 ng/ml) enhanced IL6 and IL15 secretion by in vitro decidualized HSEC.

A. Cytokine secretion by first trimester decidual biopsies (n = 3). B. Cytokine secretion by HESC decidualized with estradiol (E) and medroxy-progesterone acetate (MPA) (n = 5). C. LIF treatment (50 ng/ml) significantly (*; compared to 0.5 ng/ml; p<0.05) induced IL6 secretion by decidualized HESC (n = 5). D. LIF treatment (50 ng/ml) had no significant effect (compared to 0.5 ng/ml; p = 0.222) on IL8 secretion by decidualized HESC (n = 5). E. LIF treatment (50 ng/ml) significantly (*; compared to 0.5 ng/ml; p<0.05) induced IL15 secretion by decidualized HESC (n = 5). F. LIF treatment (50 ng/ml) had no significant effect (compared to 0.5 ng/ml; p = 0.222) on MCP1 secretion by decidualized HESC (n = 5).

Figure 6. LIF and LIFR immunolocalized to decidualizing stromal cells on Day 5 of gestation in mice.

A–D. LIF immunolocalized strongly to the decidua (d) and also to the luminal epithelium (le). E–H. LIFR immunolocalized strongly to the decidua. Bottom row, negative controls to sections above. The same negative is shown for both antibodies as the primary antibodies shown were both raised in goat. n = 3; m. myometrium; s, stroma.

Figure 7. LIF and LIFR protein in murine inter- and intra-implantation sites.

A. LIF protein in cellular extracts quantified by ELISA (inter- and intra-implantation sites from n = 3 independent animals). B. LIFR protein in cellular extracts quantified by Western blot (inter- and intra-implantation sites from n = 3 independent animals).

Figure 8. In vivo LIF inhibition impaired decidualization in mice.

A–B. PEG (control) implantation sites (A; n = 5) had larger decidual areas (identified by desmin staining, brown) than PEGLA-treated implantation sites (B; n = 4). C. Graphical representation of the decidual area (identified by area of desmin staining) in implantation sites on D6. D–E. PEG control treated implantation sites (D; n = 5) showed stronger staining intensity of desmin (brown, arrows) than PEGLA-treated implantation sites (E; n = 4). F. Graphical representation of desmin staining intensity in decidual cells on D6. *. Significant difference between treatments, p<0.05; Scale 2.0 mm (A&B); 500 µm (D&E).