FIGURE 1.

Transient β₂-AR activation at implantation window caused aberrant embryo spacing. A, dominant expression of β₂-AR mRNA during preimplantation uteri. D4M, day 4 morning; D4N, day 4 night; NEG, negative control; POS, positive control. B, β₂-AR localization in the day 4 mouse uterus showed intense expression at myometrium and weak expression at epithelium. FITC-labeled β₂-AR antibody is in green, and propidium iodide-labeled nuclei are in red. Le, luminal epithelium; Ge, glandular epithelium; Lm, longitudinal muscular layer; Cm, circular muscular layer. Scale bars, 200 μm. C, the day 5 (D5) implantation site (IS) number is comparable after different adrenergic agonist (ago)/antagonist (ant) treatment on day 4 (each treatment when compared with vehicle group, p > 0.05, error bars represent S.E., one-way analysis of variance). D, β-AR agonist and β₂-AR-selective agonist (when compared with vehicle, *, p = 0.0044 and ***, p < 0.00001. rank sum test) administration on day 4 (D4) caused aberrant embryo spacing on day 5. E, normal timing of implantation initiation on day 4 at 12:00 a.m. in both vehicle-treated and β₂-AR agonist-treated groups. F, the number of implantation sites was comparable on days 5 and 6 after β₂-AR agonist (agon) treatment on day 4 (p = 0.9461, t test). G, representative pictures of normal and crowded implantation sites by the blue dye method (days 5 and 6) and tissue section (day 8). Each blue band indicates an implantation site. The bracket shows crowded implantation sites. anta, antagonist; M, mesometrial pole; AM, anti-mesometrial pole. H, genetic ablation of β-AR or pharmacological blockade by β₂-AR antagonist (butoxamine or propranolol) reversed the abnormal embryo spacing by β₂-AR agonist (Salbutamol) (***, p < 0.00001, rank sum test). Numbers above/within bars indicate number of mice examined. For D and H, numbers above the bars indicate the number of mice with crowded implantation sites per total mice examined. All error bars represent S.E.