Effect of proline rich 15-deficiency on trophoblast viability and survival

<div><p>Deviations from the normal program of gene expression during early pregnancy can lead to early embryonic loss as well as dysfunctional placentation, which can cause significant morbidity and mortality. Proline rich 15 (PRR15) is a low molecular weight nuclear protein expressed by the trophoblast during early gestation. Lentivirus-mediated knockdown of <i>PRR15</i> mRNA in ovine trophectoderm led to demise of the embryo by gestational day 15, providing compelling evidence that <i>PRR15</i> expression is critical during this precarious window of development. Our objective was to determine the effect of <i>PRR15</i> knockdown on trophoblast gene expression, proliferation, and survival. The first-trimester human trophoblast cell line, ACH-3P, was infected with control lentivirus or a lentivirus expressing a short hairpin (sh)RNA to target <i>PRR15</i> mRNA for degradation, resulting in a 68% reduction in <i>PRR15</i> mRNA. Microarray analysis of these cell lines revealed differential expression of genes related to cancer, focal adhesion, and p53 signaling. These changes included significant up-regulation of <i>GDF15</i>, a cytokine increased in pregnancies with preeclampsia. Viability and proliferation decreased in PRR15<i>-</i>deficient cells, which was consistent with down-regulation of cell cycle-related genes <i>CCND1</i> and <i>CDK6</i> and an up-regulation of <i>CCNG2</i> and <i>CDKN1A</i> in the PRR15-deficient cells. <i>TNFSF10</i>, a tumor necrosis factor superfamily member known to induce apoptosis increased significantly in the PRR15-deficient cells. Migration through a basement membrane matrix decreased and an increased population of apoptotic cells was present when treated with shRNA to target <i>PRR15</i>. These results suggest that PRR15 enhances trophoblast viability and survival during early implantation and placentation.</p></div

Placentome morphology and fetal weight.

<p><b>A</b>) Testosterone propionate treatment (TP) decreased the number of type A placentomes, and increased type C and type D placentomes collected at gestational day 90. <b>B</b>) TP treatment did not affect placental weight while <b>C</b>) female fetuses from TP ewes had significantly reduced body weight at gestational day 90 compared to female fetuses from controls. * Indicates P≤0.06</p

Androgen Receptor and Histone Lysine Demethylases in Ovine Placenta

<div><p>Sex steroid hormones regulate developmental programming in many tissues, including programming gene expression during prenatal development. While estradiol is known to regulate placentation, little is known about the role of testosterone and androgen signaling in placental development despite the fact that testosterone rises in maternal circulation during pregnancy and in placenta-induced pregnancy disorders. We investigated the role of testosterone in placental gene expression, and focused on androgen receptor (AR). Prenatal androgenization decreased global DNA methylation in gestational day 90 placentomes, and increased placental expression of AR as well as genes involved in epigenetic regulation, angiogenesis, and growth. As AR complexes with histone lysine demethylases (KDMs) to regulate AR target genes in human cancers, we also investigated if the same mechanism is present in the ovine placenta. AR co-immunoprecipitated with KDM1A and KDM4D in sheep placentomes, and AR-KDM1A complexes were recruited to a half-site for androgen response element (ARE) in the promoter region of <i>VEGFA</i>. Androgenized ewes also had increased cotyledonary VEGFA. Finally, in human first trimester placental samples KDM1A and KDM4D immunolocalized to the syncytiotrophoblast, with nuclear KDM1A and KDM4D immunostaining also present in the villous stroma. In conclusion, placental androgen signaling, possibly through AR-KDM complex recruitment to AREs, regulates placental VEGFA expression. AR and KDMs are also present in first trimester human placenta. Androgens appear to be an important regulator of trophoblast differentiation and placental development, and aberrant androgen signaling may contribute to the development of placental disorders.</p></div

Immunolocalization of KDM1A and KDM4D in first trimester human placenta samples.

<p>KDM1A immunostaining also localized to the nuclei in the syncytium and cells in the villous stroma at 11.5 weeks of gestation. KDM4D immunolocalization was similar to KDM1A, with nuclear immunostaining present in the syncytium and in the villous stroma at 11.5 weeks of gestation. White scale bar = 20μm for 20X and 40X. Insert is a representative image from control slides.</p