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Z. Zhu, L. Cheng, Z. Tsui, S. Hakomori, and B. A. Fenderson

Summary. The glycolipids of nonpregnant and pregnant rabbit endometrium were characterized using a combination of biochemical and immunochemical techniques. Quantitative analyses indicated a 70% decline in acidic glycolipid (ganglioside) content during early pregnancy (day 6), and a 2·5-fold increase in neutral glycolipid content during later pregnancy (day 26). The major gangliosides of rabbit endometrium were identified by thin-layer chromatography as GM3 and GD3, with minor amounts of GM1, GD1 a and GT1b. The major neutral glycolipids were identified similarly as globo-series structures Gb3 and Gb4. Monoclonal antibodies (mAbs) directed to glycolipid antigens permitted the detection of additional glycolipid species, including sialylated, sulfated and fucosylated lacto-series structures. Difucosyl Ley structure (defined by mAb AH-6) and sulfated-galactosyl structure (defined by mAb VESP 6·2) were identified by indirect immunofluorescence along the luminal surface of the endometrium during the implantation period. Rapid changes in the glycolipid composition of endometrial cells during early pregnancy may facilitate embryo adhesion and trophectoderm outgrowth during implantation.

Keywords: endometrium; glycolipids; pregnancy; immunohistochemistry; rabbit

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Liang Wu, Honghui Zhou, Haiyan Lin, Jianguo Qi, Cheng Zhu, Zhiying Gao, and Hongmei Wang

Until recently, the molecular pathogenesis of preeclampsia (PE) remained largely unknown. Reports have shown that circulating microRNAs (miRNAs) are promising novel biomarkers for cancer, pregnancy, tissue injury, and other conditions. The objective of this study was to identify differentially expressed miRNAs in plasma from severe preeclamptic pregnancies compared with plasma from normal pregnancies. By mature miRNA microarray analysis, 15 miRNAs, including 13 up- and two downregulated miRNAs, were screened to be differentially expressed in plasma from women with severe PE (sPE). Seven miRNAs, namely miR-24, miR-26a, miR-103, miR-130b, miR-181a, miR-342-3p, and miR-574-5p, were validated to be elevated in plasma from severe preeclamptic pregnancies by real-time quantitative stem-loop RT-PCR analysis. Gene ontology and pathway enrichment analyses revealed that these miRNAs were involved in specific biological process categories (including regulation of metabolic processes, regulation of transcription, and cell cycle) and signaling pathways (including the MAP kinase signaling pathway, the transforming growth factor-β signaling pathway, and pathways in cancer metastasis). This study presents, for the first time, the differential expression profile of circulating miRNAs in sPE patients. The seven elevated circulating miRNAs may play critical roles in the pathogenesis of sPE, and one or more of them may become potential markers for diagnosing sPE.

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Dong Zhang, Shen Yin, Man-Xi Jiang, Wei Ma, Yi Hou, Cheng-Guang Liang, Ling-Zhu Yu, Wei-Hua Wang, and Qing-Yuan Sun

The present study was designed to investigate the localization and function of cytoplasmic dynein (dynein) during mouse oocyte meiosis and its relationship with two major spindle checkpoint proteins, mitotic arrest-deficient (Mad) 1 and Mad2. Oocytes at various stages during the first meiosis were fixed and immunostained for dynein, Mad1, Mad2, kinetochores, microtubules, and chromosomes. Some oocytes were treated with nocodazole before examination. Anti-dynein antibody was injected into the oocytes at germinal vesicle (GV) stage before the examination of its effects on meiotic progression or Mad1 and Mad2 localization. Results showed that dynein was present in the oocytes at various stages from GV to metaphase II and the locations of Mad1 and Mad2 were associated with dynein’s movement. Both Mad1 and Mad2 had two existing states: one existed in the cytoplasm (cytoplasmic Mad1 or cytoplasmic Mad2), which did not bind to kinetochores, while the other bound to kinetochores (kinetochore Mad1 or kinetochore Mad2). The equilibrium between the two states varied during meiosis and/or in response to the changes of the connection between microtubules and kinetochores. Cytoplasmic Mad1 and Mad2 recruited to chromosomes when the connection between microtubules and chromosomes was destroyed. Inhibition of dynein interferes with cytoplasmic Mad1 and Mad2 transportation from chromosomes to spindle poles, thus inhibits checkpoint silence and delays anaphase onset. These results indicate that dynein may play a role in spindle checkpoint inactivation.

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Ru Zheng, Yue Li, Huiying Sun, Xiaoyin Lu, Bao-Fa Sun, Rui Wang, Lina Cui, Cheng Zhu, Hai-Yan Lin, and Hongmei Wang

The syncytiotrophoblast (STB) plays a key role in maintaining the function of the placenta during human pregnancy. However, the molecular network that orchestrates STB development remains elusive. The aim of this study was to obtain broad and deep insight into human STB formation via transcriptomics. We adopted RNA sequencing (RNA-Seq) to investigate genes and isoforms involved in forskolin (FSK)-induced fusion of BeWo cells. BeWo cells were treated with 50 μM FSK or dimethyl sulfoxide (DMSO) as a vehicle control for 24 and 48 h, and the mRNAs at 0, 24 and 48 h were sequenced. We detected 28,633 expressed genes and identified 1902 differentially expressed genes (DEGs) after FSK treatment for 24 and 48 h. Among the 1902 DEGs, 461 were increased and 395 were decreased at 24 h, whereas 879 were upregulated and 763 were downregulated at 48 h. When the 856 DEGs identified at 24 h were traced individually at 48 h, they separated into 6 dynamic patterns via a K-means algorithm, and most were enriched in down–even and up–even patterns. Moreover, the gene ontology (GO) terms syncytium formation, cell junction assembly, cell fate commitment, calcium ion transport, regulation of epithelial cell differentiation and cell morphogenesis involved in differentiation were clustered, and the MAPK pathway was most significantly regulated. Analyses of alternative splicing isoforms detected 123,200 isoforms, of which 1376 were differentially expressed. The present deep analysis of the RNA-Seq data of BeWo cell fusion provides important clues for understanding the mechanisms underlying human STB formation.

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Wen-Lin Chang, Qing Yang, Hui Zhang, Hai-Yan Lin, Zhi Zhou, Xiaoyin Lu, Cheng Zhu, Li-Qun Xue, and Hongmei Wang

Placenta-specific protein 1 (PLAC1), a placenta-specific gene, is known to be involved in the development of placenta in both humans and mice. However, the precise role of PLAC1 in placental trophoblast function remains unclear. In this study, the localization of PLAC1 in human placental tissues and its physiological significance in trophoblast invasion and migration are investigated by technical studies including real-time RT-PCR, in situ hybridization, immunohistochemistry, and functional studies by utilizing cell invasion and migration assays in the trophoblast cell line HTR8/SVneo as well as the primary inducing extravillous trophoblasts (EVTs). The results show that PLAC1 is mainly detected in the trophoblast columns and syncytiotrophoblast of the first-trimester human placental villi, as well as in the EVTs that invade into the maternal decidua. Knockdown of PLAC1 by RNA interference significantly suppresses the invasion and migration of HTR8/SVneo cells and shortens the distance of the outgrowth of the induced EVTs from the cytotrophoblast column of the explants. All the above data suggests that PLAC1 plays an important role in human placental trophoblast invasion and migration.

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Qian Zhang, Song Yu, Xing Huang, Yi Tan, Cheng Zhu, Yan-Ling Wang, Haibin Wang, Hai-Yan Lin, Jiejun Fu, and Hongmei Wang

Cullin 3 (CUL3), a scaffold protein, assembles a large number of ubiquitin ligase complexes, similar to Skp1-Cullin 1-F-box protein complex. Several genetic models have shown that CUL3 is crucial for early embryonic development. Nevertheless, the role of CUL3 in human trophoblast function remains unclear. In this study, immunostaining revealed that CUL3 was strongly expressed in the villous cytotrophoblasts, the trophoblast column, and the invasive extravillous trophoblasts. Silencing CUL3 significantly inhibited the outgrowth of villous explant ex vivo and decreased invasion and migration of trophoblast HTR8/SVneo cells. Furthermore, CUL3 siRNA decreased pro-MMP9 activity and increased the levels of TIMP1 and 2. We also found that the level of CUL3 in the placental villi from pre-eclamptic patients was significantly lower as compared to that from their gestational age-matched controls. Moreover, in the lentiviral-mediated placenta-specific CUL3 knockdown mice, lack of CUL3 resulted in less invasive trophoblast cells in the maternal decidua. Taken together, these results suggest an essential role for CUL3 in the invasion and migration of trophoblast cells, and dysregulation of its expression may be associated with the onset of pre-eclampsia.

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Pei-Li Wu, Jing-Wen Zhu, Cheng Zeng, Xin Li, Qing Xue, and Hui-Xia Yang

In brief

Insufficient trophoblast invasion at the maternal–fetal interface contributes to abortion-prone pregnancy. Our study shows that decreased levels of IGFBP7 in unexplained recurrent spontaneous abortion (URSA) trophoblast cells inhibit MMP2 and Slug expression as well as trophoblast invasion, suggesting that IGFBP7 should be considered a potential therapeutic protein target in URSA.


Insufficient trophoblast invasion at the maternal–fetal interface contributes to abortion-prone pregnancy. Cyclosporine A (CsA) can exert therapeutic effects on URSA by promoting trophoblast invasion. A previous study showed decreased expression of insulin-like growth factor-binding protein 7 (IGFBP7) in the sera of recurrent spontaneous abortion patients. However, the role of IGFBP7 in URSA remains unknown. The aim of this study was to determine whether IGFBP7 modulates trophoblast invasion in URSA and the underlying molecular mechanisms. We found that IGFBP7 was expressed at lower levels in villous specimens from URSA patients. Manipulating IGFBP7 expression significantly affected the MMP2 and Slug expression in HTR-8/SVneo cells as well as trophoblast invasion in vitro. Inactivation of IGF-1R by IGFBP7 was observed, and IGF-1R inhibition increased the IGFBP7-induced MMP2 and Slug expression in HTR-8/SVneo cells. Moreover, the level of c-Jun was significantly upregulated in the URSA group. Silencing IGFBP7 increased the binding of downstream c-Jun to the MMP2 and Slug promoter regions in HTR-8/SVneo cells, thus suppressing transcription. In addition, increased expression of IGFBP7 in HTR-8/SVneo cells was observed upon CsA treatment. Knockdown of IGFBP7 inhibited the CsA-enhanced MMP2 and Slug expression in HTR-8/SVneo cells. Our results suggest that in normal pregnancy, IGFBP7 induces MMP2 and Slug expression via the IGF-1R-mediated c-Jun signaling pathway, thereby promoting trophoblast invasion. IGFBP7 depletion in URSA inhibits MMP2 and Slug expression as well as trophoblast invasion. Moreover, IGFBP7 participates in CsA-induced trophoblast invasion, suggesting that IGFBP7 is a potential therapeutic target for URSA.

Free access

Yue Li, Ru Zheng, Rui Wang, Xiaoyin Lu, Cheng Zhu, Hai-Yan Lin, Hongmei Wang, Xiaoguang Yu, and Jiejun Fu

The placenta has numerous functions, such as transporting oxygen and nutrients and building the immune tolerance of the fetus. Cell fusion is an essential process for placental development and maturation. In human placental development, mononucleated cytotrophoblast (CTB) cells can fuse to form a multinucleated syncytiotrophoblast (STB), which is the outermost layer of the placenta. Nephrin is a transmembrane protein that belongs to the Ig superfamily. Previous studies have shown that nephrin contributes to the fusion of myoblasts into myotubes in zebrafish and mice, presenting a functional conservation with its Drosophila ortholog sticks and stones. However, whether nephrin is involved in trophoblast syncytialization remains unclear. In this study, we report that nephrin was localized predominantly in the CTB cells and STB of human placenta villi from first trimester to term pregnancy. Using a spontaneous fusion model of primary CTB cells, the expression of nephrin was found to be increased during trophoblast cell fusion. Moreover, the spontaneous syncytialization and the expression of syncytin 2, connexin 43, and human chorionic gonadotropin beta were significantly inhibited by nephrin-specific siRNAs. The above results demonstrate that nephrin plays an important role in trophoblast syncytialization.

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Li-Jun Huo, Cheng-Guang Liang, Ling-Zhu Yu, Zhi-Sheng Zhong, Zeng-Ming Yang, Heng-Yu Fan, Da-Yuan Chen, and Qing-Yuan Sun

The present study investigated the subcellular localization of inducible nitric oxide synthase (iNOS) during mouse oocyte meiotic maturation and fertilization using confocal microscopy, and further studied the roles of iNOS-derived NO in oocyte maturation by using an iNOS-specific inhibitor aminoguanidine (AG) and iNOS antibody microinjection. In germinal vesicle-stage oocytes, iNOS immunoreactivity was mainly localized in the germinal vesicle. Shortly after germinal vesicle breakdown, the iNOS immunoreactivity accumulated around the condensed chromosomes. At metaphase I and metaphase II, with the organization of chromosomes to the equatorial plate, iNOS immunoreactivity was concentrated around the aligned chromosomes, putatively the position of the metaphase spindle. The accumulation of iNOS immunoreactivity could not be detected at anaphase I and anaphase II. However, at telophase I and telophase II, the staining of iNOS was concentrated in the region between the separating chromosomes/chromatids. Furthermore, the staining of iNOS also accumulated in the male and female pronuclei in fertilized eggs. Germinal vesicle breakdown and the first polar body emission of the oocytes were significantly blocked by the iNOS-specific inhibitor AG in a dose-dependent manner. The germinal vesicle breakdown in oocytes injected with iNOS antibody was also inhibited. We found that the phosphorylation of mitogen-activated protein kinase in oocytes after germinal vesicle breakdown was inhibited by AG treatment. The control oocytes extruded a normal first polar body, while the AG-treated oocytes exhibited an elongated protrusion or no elongated protrusion. The results of confocal microscopy showed that the AG-treated oocytes were arrested at anaphase I–telophase I. Our results suggest that the iNOS-derived NO pathway plays important roles in mouse oocyte meiotic maturation, especially in germinal vesicle breakdown and the anaphase–telophase transition.

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Wen-Min Cheng, Lei An, Zhong-Hong Wu, Yu-Bo Zhu, Jing-Hao Liu, Hong-Mei Gao, Xi-He Li, Shi-Jun Zheng, Dong-Bao Chen, and Jian-Hui Tian

We recently reported that electrical activation followed by secondary chemical activation greatly enhanced the developmental competence of in vitro matured porcine oocytes fertilized by intracytoplasmic sperm injection (ICSI). We hypothesized that sperm treatment with disulfide bond reducing agents will enhance the development competence of porcine embryos produced by this ICSI procedure. We examined the effects of glutathione (GSH), dithiothreitol (DTT), GSH or DTT in combination with heparin on sperm DNA structure, paternal chromosomal integrity, pronuclear formation, and developmental competence of in vitro matured porcine oocytes after ICSI. Acridine orange staining and flow cytometry based sperm chromatin structure assay were used to determine sperm DNA integrity by calculating the cells outside the main population (COMP αT). No differences were observed in COMP αT values among GSH-treated and control groups. COMP αT values in GSH-treated groups were significantly lower than that in DTT-treated groups. Following ICSI, GSH treatments did not significantly alter paternal chromosomal integrity. Paternal chromosomal integrity in sperm treated with DTT plus or minus heparin was also the lowest among all groups. GSH-treated sperm yielded the highest rates of normal fertilization and blastocyst formation, which were significantly higher than that of control and DTT-treated groups. The majority of blastocysts derived from control and GSH-treated spermatozoa were diploid, whereas blastocysts derived from DTT-treated spermatozoa were haploid. In conclusion, sperm treatment with GSH enhanced the developmental capacity of porcine embryos produced by our optimized ICSI procedure.