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

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

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

Real time PCR results of AR target-genes known to regulate trophoblast differentiation and proliferation.

<p><i>IGF2</i> increased in type D placentomes from TP treated ewes compared to type A placentomes from control or TP treated ewes. <i>IGFBP1</i> and <i>IGFBP2</i> both decreased in caruncle tissue from type A placentomes from TP treated ewes compared to controls. <i>IGFBP2</i> also decreased in type A and D placentomes from TP treated ewes compared to control type A placentomes. <i>CYP19</i> increased in type D placentomes form TP treated ewes compared to type A placentomes from controls. TP treated ewes had increased <i>VEGFA</i> in type D placentomes compared to type A placentomes from control ewe. TP treated ewes had increased VEGFA monomer in type A placentome cotyledon tissue. Different letters indicate statistical difference of P<0.05.</p

Interaction of KDMs with AR.

<p><b>A</b>) Immunoprecipitation of AR, KDM1A, and KDM4D from placentome protein isolated from control and TP treated ewes followed by Western blot detection of co-immunoprecipitated protein. Anti-rabbit IP represents pull down by secondary antibody used for Western blot immunolabeling. Antibody preabsorption with blocking peptide was used as a negative control. <b>B</b>) PCR of an ARE promoter region of <i>VEGFA</i> from ChIP samples. IP, immunoprecipitate; ChIP, chromatin immunoprecipitation; ARE, primers spanning androgen response element in promoter region</p

Differential levels of epigenetic regulators with TP treatment.

<p>Analysis of relative mRNA levels for genes regulating histone and DNA methylation and imprinting in ovine placentome tissue from real time PCR. <i>KDM4C</i> was decreased in type A placentomes from TP treated ewes compared to controls. <i>H19</i> was increased in cotyledon tissue from type A placentomes in TP treated ewes compared to controls. <i>DNMT1</i> increased in TP treated ewe type A cotyledon tissue compared to controls. DNMT1 increased in TP treated ewe type A placentomes (P<0.001). Different letters indicate statistical difference of P<0.05.</p

Immunolocalization of AR, KDM1A, and KDM4D in type A placentomes from control ewes at GD90.

<p>Inserts represent control IHC straining. Immunolocalization of AR was present in the trophoblasts of the villous epithelium with primarily nuclear staining (arrow). KDM1A immunolocalized to the nucleus of the trophoblasts in the villous epithelium (arrow), while KDM4D immunostaining was prominent in the apical surface of maternal uterine epithelium (arrow). Images were taken at 20X maginification.</p

Real time PCR and representative Western blot depicting placentome mRNA and protein levels of AR, KDM1A and KDM4D in control and TP treated ewes.

<p>TP treatment did not alter mRNA for <i>AR</i>, <i>KDM1A</i>, or <i>KDM4D</i>. AR was increased in type A caruncle tissue from TP treated ewes compared to controls. No difference was found in KDM1A, though KDM4D increased in type A placentomes from TP treated ewes compared to controls. Placentome values represent average of cotyledon and caruncle quantified mRNA levels in type A cotyledons. Different letters indicate statistical difference of P<0.05.</p