Uterine Rbpj is required for embryonic-uterine orientation and decidual remodeling via Notch pathway-independent and -dependent mechanisms

Abstract

Coordinated uterine-embryonic axis formation and decidual remodeling are hallmarks of mammalian post-implantation embryo development. Embryonic-uterine orientation is determined at initial implantation and synchronized with decidual development. However, the molecular mechanisms controlling these events remain elusive despite its discovery a long time ago. In the present study, we found that uterine-specific deletion of Rbpj, the nuclear transducer of Notch signaling, resulted in abnormal embryonic-uterine orientation and decidual patterning at post-implantation stages, leading to substantial embryo loss. We further revealed that prior to embryo attachment, Rbpj confers on-time uterine lumen shape transformation via physically interacting with uterine estrogen receptor (ERα) in a Notch pathway-independent manner, which is essential for the initial establishment of embryo orientation in alignment with uterine axis. While at post-implantation stages, Rbpj directly regulates the expression of uterine matrix metalloproteinase in a Notch pathway-dependent manner, which is required for normal post-implantation decidual remodeling. These results demonstrate that uterine Rbpj is essential for normal embryo development via instructing the initial embryonic-uterine orientation and ensuring normal decidual patterning in a stage-specific manner. Our data also substantiate the concept that normal mammalian embryonic-uterine orientation requires proper guidance from developmentally controlled uterine signaling.

Uterine RAC1 via Pak1-ERM signaling directs normal luminal epithelial integrity conducive to on-time embryo implantation in mice

“…In situ hybridization was performed as previously described. 64,65 Mouse-specific cRNA probes for Rac1, Areg, Muc1, Ltf, Lif, Hoxa-10 and Wnt5a were used for hybridization. Cryosections hybridized with sense probes served as negative controls.…”

Polycomb subunit BMI1 determines uterine progesterone responsiveness essential for normal embryo implantation

“…Mice that failed to recover any embryos were excluded from the statistical analysis. Mouse blood samples were collected on day 4 in the morning and serum P4 as well as E2 levels were measured by radioimmunoassay (40). For P4 treatment, each mouse was subcutaneously injected with 2 mg in 100 μl of sesame oil at 9:00 am for 3 consecutive days starting on day 3 until the pregnant mice were sacrificed for analysis (41).…”

“…Tissues were cut into 1-to 3-mm pieces, and then were fixed in 3% glutaraldehyde and 2.0% formaldehyde in cacodylate buffer with 2 mM CaCl 2 , washed, postfixed in 1% osmium tetroxide (OsO 4 ), and embedded in EMbed812 (Electron Microscopy Sciences) for ultrastructural analysis under a Hitachi H-7600 transmission electron microscope (Hitachi High-Technologies America, Inc.) (40).…”

“…Quantitative real-time PCR was performed as described previously (40). Total RNA was extracted from uterine tissues or cells using TRIzol reagent (Invitrogen) following the manufacturer's protocol.…”

“…Co-IP assays were performed as previously described (40). Anti-HA mouse mAb (agarose conjugated) (20 μl, Abmart, m20013), anti-flag M2 Affinity Gel (20 μl, MilliporeSigma, A2220), anti-BMI1, anti-PR, anti-Myc, and His-Select Nickel Affinity Gel (25 μl, MilliporeSigma, P6611) were used.…”