Imprinted genes play a key role in regulating mammalian placental and embryonic development. Here, we generated glutaminyl-peptide cyclotransferase-knockout (Qpct^-/-) mice utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) platform and identified Qpct as a novel anti-angiogenic factor in regulating mouse placentation. Compared with Qpct^+/+ mice, placentae and embryos (Qpct^-/+ and Qpct^-/-) showed significant overgrowth at embryonic Day 12.5 (E12.5), E15.5, and E18.5. Using single-cell transcriptome analysis of 32 309 cells from Qpct^+/+ and Qpct^-/- mouse placentae, we identified 13 cell clusters via single-nucleus RNA sequencing (snRNA-seq) (8880 Qpct^+/+ and 13 577 Qpct^-/- cells) and 20 cell clusters via single-cell RNA sequencing (scRNA-seq) (6567 Qpct^+/+ and 3285 Qpct^-/- cells). Furthermore, we observed a global up-regulation of pro-angiogenic genes in the Qpct^-/- background. Immunohistochemistry assays revealed a notable increase in the number of blood vessels in the decidual and labyrinthine layers of E15.5 Qpct^-/+ and Qpct^-/- mice. Moreover, the elevation of multiple pairs of ligand-receptor interactions was observed in decidual cells, endothelial cells, and macrophages, promoting angiogenesis and inflammatory response. Our findings indicate that loss of maternal Qpct leads to altered phenotypic characteristics of placentae and embryos and promotes angiogenesis in murine placentae.
Animals ; Female ; Pregnancy ; Mice ; Placentation/genetics* ; Single-Cell Analysis ; Gene Expression Profiling ; Mice, Knockout ; Transcriptome ; Placenta/blood supply* ; Neovascularization, Pathologic/genetics* ; Genomic Imprinting ; Single-Cell Gene Expression Analysis ; Angiogenesis

Trophoblast cells serve as the foundation for placental development. We analyzed published multiomics sequencing data and found that trophoblast cells highly expressed RRS1 compared to primitive endoderm and epiblast. We used HTR-8/SVneo cells for further investigation, and Western blot and immunofluorescence staining confirmed that HTR-8/SVneo cells highly expressed RRS1. RRS1 was successfully knocked down in HTR-8/SVneo cells using siRNA. Using IncuCyte S3 live-cell analysis system based on continuous live-cell imaging and real-time data, we observed that proliferation, migration, and invasion abilities were all significantly decreased in RRS1-knockdown cells. RNA-seq revealed that knockdown of RRS1 affected the gene transcription, and upregulated pathways in extracellular matrix organization, DNA damage response, and intrinsic apoptotic signaling, downregulated pathways in embryo implantation, trophoblast cell migration, and wound healing. Differentially expressed genes were enriched in diseases related to placental development. Consistent with these findings, human chorionic villus samples collected from spontaneous abortion cases exhibited significantly reduced RRS1 expression compared to normal controls. Our results highlight the functional importance of RRS1 in human trophoblasts and suggest that its deficiency contributes to early pregnancy loss.
Humans ; Trophoblasts/physiology* ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Female ; Pregnancy ; Abortion, Spontaneous/metabolism* ; Cell Line ; Placentation/genetics*

The present study aims to investigate the effects of soluble endoglin (sEng) on invasive ability of cultured cytotrophoblasts of first trimester of pregnancy. Cytotrophoblasts of normal 6 to 8-week pregnancy were cultured by trypsin digestion method, and were incubated with cell culture medium without (control group) and with 10 μg/L sEng (sEng group), respectively for 24 h. The invasive ability was determined by transwell invasion assay, and expressions of MMP-2, MMP-9 mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The results showed that the invasive ability of cytotrophoblasts in sEng group was lower than that in control group (P < 0.05). Compared with control group, the expressions of MMP-2 and MMP-9 mRNA and protein of cytotrophoblasts were significantly lower (P < 0.05). In conclusion, sEng may participate in the genesis of preeclampsia by affecting the invasive ability of cytotrophoblasts through regulation of the expression of MMP-2 and MMP-9.
Antigens, CD ; pharmacology ; Cell Movement ; drug effects ; physiology ; Cells, Cultured ; Endoglin ; Female ; Humans ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Placentation ; physiology ; Pre-Eclampsia ; physiopathology ; Pregnancy ; Pregnancy Trimester, First ; RNA, Messenger ; genetics ; metabolism ; Receptors, Cell Surface ; Trophoblasts ; cytology

Primate placentation involves a series of cell proliferation, immigration and apoptosis which account for the progressive tissue remodelling at the implantation site. p53 is an important proto-oncogene involved in the regulation of cell-cycle and apoptosis. To study the effect of RU486 on apoptosis and expression of p53 at the fetal-maternal interface, the location of apoptotic cells and expression of p53 were examined using in situ 3'-end labeling method, immunohistochemistry and Western blot assay at the fetal-maternal interface of normal and RU486 treated rhesus monkey. Western blot analysis showed the specificity of the anti-human antibody used with the monkey tissue. In the placental villi, the apoptotic nuclei were observed mainly in the syncytiotrophoblast and part of the cytotrophoblast within the cell column; p53 protein was detected mainly in the cytotrophoblast. In the endometrium, positive signals for apoptosis and p53 were detected in some stromal cells. After two days of mifepristone treatment, the apoptotic cells increased significantly in both placental villi and endometrium. In the villi, the increased apoptotic nuclei were mainly localized to the cytotrophoblast. At the same time, p53-positive nuclei also increased in both villous cytotrophoblast cells and endometrial stromal cells, after the treatment of RU486. These results suggest that apoptosis and expression of p53 are essential in regulating trophoblastic homeostasis by controlling its proliferation in normal placenta, whereas the up-regulation of p53 protein may play an important role in apoptosis that happens at the fetal-maternal interface induced by RU486.
Abortifacient Agents, Steroidal ; pharmacology ; Animals ; Apoptosis ; drug effects ; Chorionic Villi ; pathology ; Female ; Macaca mulatta ; Mifepristone ; pharmacology ; Placentation ; drug effects ; physiology ; Pregnancy ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics