Preparation of mammalian uterus for embryo implantation requires a precise sequence of cell proliferation. In rodent uterus, estradiol stimulates proliferation of epithelial cells. Progesterone operates as a molecular switch and redirects proliferation to the stroma by down-regulating glycogen synthase kinase-3β (GSK-3β) and stimulating β-catenin accumulation in the periluminal stromal cells. In this study, the WNT signal involved in the progesterone-dependent proliferative switch was investigated. Transcripts of four candidate Wnt genes were measured in the uteri from ovariectomized (OVX) rats, progesterone-pretreated (3 days of progesterone, 2mg/daily) rats, and progesterone-pretreated rats given a single dose (0.2µg) of estradiol. The spatial distribution of the WNT proteins was determined in the uteri after the same treatments. Wnt5a increased in response to progesterone and the protein emerged in the periluminal stromal cells of progesterone-pretreated rat uteri. To investigate whether WNT5A was required for proliferation, uterine stromal cell lines were stimulated with progesterone (1µM) and fibroblast growth factor (FGF, 50ng/mL). Proliferating stromal cells expressed a two-fold increase in WNT5A protein at 12h post stimulation. Stimulated stromal cells were cultured with actinomycin D (25µg/mL) to inhibit new RNA synthesis. Relative Wnt5a expression increased at 4 and 6 h of culture, suggesting that progesterone plus FGF preferentially increased Wnt5a mRNA stability. Knockdown of Wnt5a in uterine stromal cell lines inhibited stromal cell proliferation and decreased Wnt5a mRNA. The results indicate that progesterone initiates and synchronizes uterine stromal cell proliferation by increasing WNT5A expression and signaling.

Progesterone pretreatment of ovariectomized rat uteri increases the number of synchronously proliferating stromal cells in response to estradiol 17-β. To identify the signals involved in stimulating synchronous proliferation, sexually mature ovariectomized rats were injected with progesterone (2 mg) for 3 consecutive days. Estradiol 17-β (0.2 μg) was administered to initiate cell cycle entry. Uterine samples were removed at various times after hormone administration and changes in wingless (Wnt) pathway effectors and gene targets were identified by microarray. Progesterone pretreatment decreased glycogen synthase kinase-3β (GSK-3β) and increased expression of T-cell factor/lymphoid enhancer factor (TCF/LEF). GSK-3β protein decreased markedly in the uterine stroma of progesterone-pretreated uteri with the concomitant appearance of β-catenin in these stromal cells. Translocation of β-catenin from the cytosol to the nuclei in progesterone-pretreated stromal cells was stimulated in response to estradiol. β-Catenin binding to TCF/LEF increased (P<0.05) in progesterone-pretreated uteri in response to estradiol. Progesterone stimulated the expression of the Wnt target gene urokinase plasminogen activator receptor (uPA-R) in the periluminal uterine stromal cells. The expression of uPA-R increased in progesterone-pretreated stromal cells in response to estradiol administration. Together, the results indicate that progesterone initiates Wnt signaling in the uterine stroma by down-regulating GSK-3β. However, nuclear translocation of β-catenin and sufficient complex formation with TCF/LEF to activate stromal cell cycle entry requires estradiol. Stimulation of a uterine stromal cell line to proliferate and differentiate resulted in β-catenin accumulation, suggesting that endocrine-dependent Wnt signaling controls proliferation and differentiation (decidualization).