PARENT SESSION
SLIDE SESSION 12: IMPLANTATION & EARLY DEVELOPMENT
Chairs: Richard Leach, Carol Brenner, Jeff Reese (Trainee)
Univ Ottawa-Monpetit 202
1:30 PM-3:30 PM
250
PARATHYROID HORMONE-RELATED PROTEIN (PTHrP) AND INDIAN HEDGEHOG ARE EXPRESSED DURING EARLY DEVELOPMENT IN THE MOUSE EMBRYO AND INTERACT WITH EACH OTHER TO STIMULATE MOUSE BLASTOCYST OUTGROWTH IN VITRO.
Erbach, Gregory1, Manning, Peter2, Charnock-Jones, Stephen3, Biggers, John4, Nowak, Romana1, 1 2 3 4
ABSTRACT- We have reported in previous studies that PTHrP is produced by mouse blastocysts and that it stimulates blastocyst outgrowth in vitro. PTHrP has been shown to be part of the Hedgehog protein signaling pathway. The goals of this study were: 1) to determine at what stage of preimplantation development mouse embryos express PTHrP, PTHrP receptor, and hedgehog proteins; and 2) to test the effects of sonic hedgehog (SHH) protein on blastocyst outgrowth in vitro. Mouse embryos were collected as fertilized one cell zygotes, 2-cells, 4-cells, uncompacted 8-cells, compacting 8-cells, morulae, expanded and hatched blastocysts, and after undergoing outgrowth for 3 days in vitro. Embryos were processed to RNA for RT-PCR analysis or were fixed for confocal microscopy. RT-PCR showed that mouse embryos did not express SHH mRNA. Indian HH mRNA was expressed only in mouse blastocysts after 3 days of outgrowth in vitro. PTHrP mRNA was expressed in one-cell zygotes. The mRNA then disappeared, was transiently expressed in compacting 8-cell embryos, disappeared again and was not expressed until the blastocysts were undergoing outgrowth. PTHrP receptor mRNA was expressed in one-cell zygotes, disappeared and then was expressed in all stages from compacting 8-cell to blastocysts undergoing outgrowth. Confocal microscopy for PTHrP protein showed that PTHrP was present in embryos up to the morula stage as small, intensely stained packets located near the nuclei. In blastocysts, PTHrP protein was rapidly translocated to the apical surface of the trophectoderm cells during hatching. PTHrP receptor was expressed on the surface of embryos from the 8-cell stage onward. In the second study, we tested the effects of recombinant SHH protein on blastocyst outgrowth in vitro. The doses tested were 0.1, 1.0 and 10.0 ng/ml. Mouse blastocysts were treated for 72 hrs, the area of outgrowth measured, and then the blastocysts were fixed for confocal staining. SHH protein caused a 100-120% increase in trophoblast cell outgrowth when compared to controls. Immunostaining for PTHrP showed that blastocysts treated with SHH had stronger immunoreactivity for PTHrP suggesting that SHH might act to increase outgrowth by stimulating PTHrP. These results support the hypothesis that PTHrP is part of the Hedgehog protein signaling pathway during early development.
KEY WORDS: parathyroid hormone-related protein, hedgehog protein, blastocyst, embryo