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a Department of Obstetrics and Gynecology, Brigham&Women's Hospital,
b Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
The goals of this study were 1) to compare the effects of transforming growth factor-ß (TGF-ß) and parathyroid hormone-related protein (PTHrP) on mouse blastocyst attachment and outgrowth in vitro, 2) to determine whether TGF-ß acts through a mechanism involving PTHrP, 3) to examine effects of PTHrP on preimplantation mouse embryo development, and 4) to determine the pattern of expression of PTHrP protein in the uterus of the mouse during early gestation. In the first set of experiments, hatched blastocysts were placed in fibronectin-coated wells. Cultures were treated with PTHrP or TGF-ß1 and assessed at 24, 48, and 72 h for attachment and surface area of blastocyst outgrowth. Results showed that both PTHrP and TGF-ß1 increased blastocyst outgrowth significantly. A PTHrP-neutralizing antibody blocked the stimulatory effect of both PTHrP and TGF-ß1, suggesting that TGF-ß1 acts to increase endogenous production of PTHrP by the blastocyst. Immunoassay of conditioned medium from blastocysts treated with either TGF-ß1 or PTHrP 1–34 confirmed a 3- to 4-fold increase in levels of PTHrP 1–141. In the second series of experiments, pronuclear zygotes were cultured in various concentrations of PTHrP for 96 h. Blastocysts then were subjected to differential fluorescent staining of inner cell mass and trophectoderm cells. Treatment of mouse embryos with the various concentrations of PTHrP altered neither the number developing to the blastocyst stage nor the number of inner cell mass or trophectoderm cells in the resulting blastocysts. In the third experiment, pregnant mice were killed at Days 3, 4, 5, 6, and 7 of gestation, and uterine horns were processed for immunohistochemistry. Uterine sections were stained with antibodies to PTHrP, desmin, and laminin. On Days 3, 4, and 5, uterine luminal and glandular epithelial cells stained intensely for PTHrP, while stromal cells were negative. By Days 6 and 7, decidualized stromal cells stained positively for PTHrP, desmin, and laminin. These results support the hypothesis that TGF-ß and PTHrP play an important role in the process of implantation.
2 Correspondence: Romana Nowak, Brigham&Women's Hospital, 221 Longwood Ave., Boston, MA 02115. FAX: 617 566 7980.
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