Abstract
Pregnancy in mice and rats is associated with the production of a large family of hormones/cytokines related to prolactin (PRL). The hormones/cytokines are hypothesized to coordinate maternal and fetal adaptations to pregnancy. In this study, PRL-like protein-J (PLP-J, also known as PRL family 3, subfamily c, member 1 (Prl3c1)) is shown to be a product of the uterine decidua and a regulator of postimplantation intrauterine events. PLP-J-specific antibodies and a series of recombinant PLP-J proteins were generated and used to investigate PLP-J expression and as ligands for investigating biological targets. Decidual PLP-J migrates as a 29-kDa protein and localizes to a band of decidual cells surrounding the trophoblast cell layer on gestation day 8.5. PLP-J ligands specifically bound in situ to the surrounding uterine stromal cells and vasculature within the decidua of gestation day 8.5 implantation sites. We then investigated the in vitro actions of PLP-J on uterine stromal cells and endothelial cells. PLP-J specifically interacted with both cell populations. PLP-J promoted uterine stromal cell proliferation and inhibited endothelial cell proliferation. We determined that PLP-J does not interact with PRL receptors. Instead, PLP-J interacts with heparin-containing molecules, including syndecan-1, which is expressed in gestation day 8.5 pregnant uteri, as well as in uterine stromal cells and endothelial cells. The restricted expression of PLP-J and its specific interactions with uterine stromal cells and endothelial cells suggests that it acts locally and regulates decidual cell development and the endometrial vasculature.
Highlights
Purified mFLAG-PLP-J consisted of three spe- PLP-J Interacts with Heparin—Based on the heparin-depencies with molecular masses ranging from ϳ31 to 37 kDa
PLP-J Interacts with Syndecans—Syndecans are a family of four transmembrane proteins possessing heparan sulfate sugar chains associated with their extracellular domains (60, 61)
Uterine decidua, uterine stromal cells, and endothelial cells were assessed for their expression of syndecan family members by Northern blotting (Fig. 6A)
Summary
Holtzman rats were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). The animals were housed in an environmentally controlled facility, with lights on from 0600 to 2000 h and were allowed free access to food and water. Holtzman rats were obtained from Harlan Sprague-Dawley Inc. The animals were housed in an environmentally controlled facility, with lights on from 0600 to 2000 h and were allowed free access to food and water. Tissue dissections were performed as previously detailed (40). Conceptuses with associated uteri were removed on specific days of gestation. Tissues were frozen in dry ice-cooled heptane and stored at Ϫ80 °C until used for in situ hybridization, immunohistochemistry, and in situ ligand binding or were frozen in liquid nitrogen and stored at Ϫ80 °C for subsequent RNA and protein analyses. The presence of sperm in the vaginal smear was designated as day 0.5 of pregnancy. New Zealand White rabbits were obtained from Myrtle’s Rabbitry (Thompsons Station, TN) and used for antibody production.
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