Localization of parathyroid hormone-related protein in the preimplantation mouse embryo is associated with events of blastocyst hatching.

PURPOSE: To determine the pattern of expression of parathyroid hormone-related protein (PTHrP) and its receptor, parathyroid hormone receptor 1 (PTHR1), in mouse embryos in different stages of preimplantation development. METHODS: Embryos were cultured from the pronuclear zygote stage and harvested as 2-cell, 4-cell and 8-cell embryos, morulae and blastocysts. RT-PCR was carried out on mRNAs of these and of trophoblast outgrowths for detection of PTHrP and PTHR1. Whole mounted embryos intact or stripped of zonae pellucidae were immunofluorescently stained for PTHrP and PTH receptor and observed with confocal microscopy. RESULTS: PTHrP mRNA was present in the pronuclear zygote, not present in 2-cell, 4-cell and uncompacted 8-cell embryos, present in the 8-cell compacting embryo, and not detected in 16-cell morulae or blastocysts. The mRNA was present in trophoblasts growing on fibronectin beds. mRNA for PTHR1 was detected in the pronuclear zygote, then undetected until the compacted 8-cell stage and thereafter. PTH receptor protein was observed in 2-cell embryos, morulae and in the inner cell mass and trophectoderm of blastocysts. PTHrP was observed dispersed in the cytoplasm of 2-cell, 4-cell and uncompacted 8-cell embryos, and in distinct foci near the nuclei of morulae. In blastocysts, PTHrP appeared on the apical surface of only trophoblast cells which had extruded from the zona pellucida. Fully hatched blastocysts expressed the protein on the apical side of all trophoblasts. When morulae were prematurely stripped of their zonae, PTHrP was observed on the embryos' outer surface. CONCLUSIONS: PTHrP protein is expressed throughout early embryo development, and its receptor PTHR1 is expressed from the morula stage. Embryo hatching is associated with translocation of PTHrP to the apical plasma membrane of trophoblasts. PTHrP may thus have autocrine effects on the developing blastocyst.

The antifibrotic drug halofuginone inhibits proliferation and collagen production by human leiomyoma and myometrial smooth muscle cells.

OBJECTIVE: To investigate the effects of the antifibrotic drug halofuginone on extracellular matrix production, cell proliferation, and apoptosis of cultured myometrial and leiomyoma smooth muscle cells. DESIGN: Comparative and controlled experimental research study. SETTING: University research laboratory. PATIENT(S): Leiomyoma and myometrial tissues were obtained from eight different patients at the time of elective hysterectomy. MAIN OUTCOME MEASURE(S): The effects of halofuginone on cell proliferation were assessed by tritiated thymidine uptake assays and cell count assays. Effects on TGFbeta1, collagen type I, and collagen type III mRNA levels were assessed by quantitative real-time polymerase chain reaction. Effects on apoptosis were assayed using a chemiluminescent assay to measure changes in caspase 3 and 7. RESULT(S): Halofuginone inhibited cell proliferation of both leiomyoma and autologous myometrial cells in a dose-dependent manner by inhibiting DNA synthesis within 24 hours and later inducing apoptosis (as measured by increased caspase 3/7) by 48-72 hours. Halofuginone also significantly reduced collagen type I (alpha1) and collagen type III (alpha1) mRNA levels, as well as the profibrotic factor TGFbeta1 mRNA levels in both cell types. CONCLUSION(S): These results provide evidence to support the use of the antifibrotic drug halofuginone as a novel drug treatment for uterine leiomyomas.