Spatiotemporal functions of leukemia inhibitory factor in embryo attachment and implantation chamber formation

RNA-seq analyses using embryos and uterine luminal layers collected from Lif-floxed, Lif uterine epithelial-specific KO (eKO), and recombinant Lif (rLif)-treated Lif eKO females on 8pm of day 4 of pregnancy. Lif-loxP/loxP, Ltf-iCre, and Pgr-Cre mice were used in this study. Lactoferrin (Ltf) is expressed in the uterine epithelium, whereas progesterone receptor (Pgr) is expressed in the entire uterus (i.e., epithelium, stroma, and myometrium). Lif-loxP/loxP females were crossed with Ltf-iCre and Pgr-Cre males to generate mice with deletion of Lif in the epithelium (Lif eKO mice) or the whole uterus (Lif uKO mice), respectively. The day when the vaginal plug was detected was considered day 1 of pregnancy. Pregnant mice were euthanized by cervical dislocation on designated days of pregnancy for the evaluation of pregnancy phenotypes and sample collection. On day 4, one uterine horn was flushed with saline to confirm the presence of blastocysts. To determine the function of Lif, female mice received rLif (20 µg/head, i.p.) at 10 am and 6 pm of day 4 of pregnancy or pseudopregnancy. For RNA-seq analyses, mice were dissected 2 h after the last injection. Uterine tissues were collected from Lif-floxed, Lif eKO, and rLif-treated Lif eKO mice at 8 pm on day 4. One side of the uterine horn was flushed with PBS to collect blastocysts and confirm pregnancy. The other side of the uterine horn was snap-frozen for cryosectioning. Laser microdissection was performed to collect luminal layers.