Yujing Cao

A novel class of tRNA-derived small RNAs extremely enriched in mature mouse sperm

Author(s): Peng, Hongying; Shi, Junchao; Zhang, Ying; Zhang, He; Liao, Shangying; Li, Wei; Lei, Li; Han, Chunsheng; Ning, Lina; Cao, Yujing; Zhou, Qi; Chen, Qi; Duan, Enkui

Embryo-uterine cross-talk during implantation: The role of Wnt signaling

During mammalian pregnancy, it has been demonstrated that the quality of embryo implantation determines the quality of ongoing pregnancy and fetal development. Recent studies have provided increasing evidence that differential Wnt signaling plays diverse roles in multiple peri-implantation events. This review focuses on recent progress on various aspects of Wnt signaling in preimplantation embryo development, blastocyst activation for implantation and uterine decidualization. Future studies with conditional deletion of Wnt family members are hoped to provide deeper insight on the pathophysiological significance of Wnt proteins on early pregnancy events.

Aquaporin3 is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration.

In the journey from the male to female reproductive tract, mammalian sperm experience a natural osmotic decrease (e.g., in mouse, from ∼415 mOsm in the cauda epididymis to ∼310 mOsm in the uterine cavity). Sperm have evolved to utilize this hypotonic exposure for motility activation, meanwhile efficiently silence the negative impact of hypotonic cell swelling. Previous physiological and pharmacological studies have shown that ion channel-controlled water influx/efflux is actively involved in the process of sperm volume regulation; however, no specific sperm proteins have been found responsible for this rapid osmoadaptation. Here, we report that aquaporin3 (AQP3) is a sperm water channel in mice and humans. Aqp3-deficient sperm show normal motility activation in response to hypotonicity but display increased vulnerability to hypotonic cell swelling, characterized by increased tail bending after entering uterus. The sperm defect is a result of impaired sperm volume regulation and progressive cell swelling in response to physiological hypotonic stress during male-female reproductive tract transition. Time-lapse imaging revealed that the cell volume expansion begins at cytoplasmic droplet, forcing the tail to angulate and form a hairpin-like structure due to mechanical membrane stretch. The tail deformation hampered sperm migration into oviduct, resulting in impaired fertilization and reduced male fertility. These data suggest AQP3 as an essential membrane pathway for sperm regulatory volume decrease (RVD) that balances the “trade-off” between sperm motility and cell swelling upon physiological hypotonicity, thereby optimizing postcopulatory sperm behavior.

NASA-Approved Rotary Bioreactor Enhances Proliferation of Human Epidermal Stem Cells and Supports Formation of 3D Epidermis-Like Structure

The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a “stimulatory” environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.

Enrichment of putative human epidermal stem cells based on cell size and collagen type IV adhesiveness

The enrichment and identification of human epidermal stem cells (EpSCs) are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rapidly adherent keratinocytes into three populations that range in size from 5–7 μm (population A), to 7–9 μm (population B), to ≥9 μm (population C) in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of β1-integrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency (CFE). When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to β1-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.

Inhibition of the Beta-Catenin Signaling Pathway in Blastocyst and Uterus During the Window of Implantation in Mice

Abstract Beta-catenin, the mammalian homolog of Drosophila armadillo protein, was first identified as a cadherin-associated protein at cell-cell junctions. Another function of beta-catenin is the transduction of cytosolic signals to the nucleus in a variety of cellular contexts, which usually are elicited by the active form of beta-catenin. The aim of the present study was to examine the potential role of active beta-catenin in the mouse embryo and uterus during embryo implantation. Active beta-catenin was detected differentially in mouse embryos and uteri during the peri-implantation period. Aberrant activation of beta-catenin by LiCl, a well-known glycogen synthase kinase-3 inhibitor, significantly inhibited blastocyst hatching and subsequent adhesion and outgrowth on fibronectin. Results obtained from pseudopregnant and implantation-delayed mice imply an important role for implanting blastocysts in the temporal and spatial changes of active beta-catenin in the uterus during the window of implantation. Collectively, these results suggest that the beta-catenin signaling pathway is inhibited in both blastocyst and uterus during the window of implantation, which may represent a new mechanism to synchronize the development of preimplantation embryos and differentiation of the uterus during this process.

Navigating the site for embryo implantation: biomechanical and molecular regulation of intrauterine embryo distribution.

The distribution of intrauterine embryo implantation site(s) in most mammalian species shows remarkably constant patterns: in monotocous species such as humans, an embryo tends to implant in the uterine fundus; in polytocous species such as rodents, embryos implant evenly along the uterine horns. These long-time evolved patterns bear great biological significance because disruption of these patterns can have adverse effects on pregnancies. However, lack of suitable models and in vivo monitoring techniques has impeded the progress in understanding the mechanisms of intrauterine embryo distribution. These obstacles are being overcome by genetically engineered mouse models and newly developed high-resolution ultrasound. It has been revealed that intrauterine embryo distribution involves multiple events including uterine sensing of an embryo, fine-tuned uterine peristaltic movements, time-controlled uterine fluid reabsorption and uterine luminal closure, as well as embryo orientation. Diverse molecular factors, such as steroid hormone signaling, lipid signaling, adrenergic signaling, developmental genes, ion/water channels, and potentially embryonic signaling are actively involved in intrauterine embryo distribution. This review covers the biomechanical and molecular aspects of intrauterine embryo distribution (embryo spacing at the longitudinal axis and embryo orientation at the vertical axis), as well as its pathophysiological roles in human reproductive medicine. Future progress requires multi-disciplinary research efforts that will integrate in vivo animal models, clinical cases, physiologically relevant in vitro models, and biomechanical/computational modeling. Understanding the mechanisms for intrauterine embryo distribution could potentially lead to development of therapeutics for treating related conditions in reproductive medicine.

The Cytokine Gene CXCL14 Restricts Human Trophoblast Cell Invasion by Suppressing Gelatinase Activity

Well-controlled trophoblast invasion into uterine decidua is a critical process for the normal development of placenta, which is tightly regulated by various factors produced within the trophoblast-endometrial microenvironment. CXCL14 is involved in tumor growth and metastasis, and its expression in placenta is temporally regulated during pregnancy. However, the role of CXCL14 in trophoblast function during human pregnancy is not clear. In this study, by using RT-PCR through human pregnancy, we found that CXCL14 was selectively expressed at early but not late pregnancy. Immunostaining revealed that CXCL14 proteins were strongly expressed in villous cytotrophoblasts and moderately in decidualized stromal cells but very weakly in syncytiotrophoblasts and extravillous trophoblasts. The effect of CXCL14 on trophoblast invasion were examined by using human villous explants cultured on Matrigel and further proved by invasion and migration assay of primary trophoblast cells and trophoblast cell line HTR-8/SVneo. Our data showed that CXCL14 significantly inhibited outgrowth of villous explant in vitro; this effect is due to suppression of trophoblast invasion and migration through regulating matrix metalloproteinases activities, whereas the trophoblast proliferation was not affected. Moreover, because a receptor for CXCL14 has not been identified, we performed further cell-specific CXCL14 binding activities with regard to different cell types within the maternal-fetal interface. Our data revealed that CXCL14 could specifically bind to trophoblast cells but not decidual cells from the maternal-fetal interface. These results suggest that CXCL14 plays an important role in regulating trophoblast invasion through an autocrine/paracrine manner during early pregnancy.

Transient β2-Adrenoceptor Activation Confers Pregnancy Loss by Disrupting Embryo Spacing at Implantation

Pregnancy loss is a serious social and medical issue, with one important cause associated with aberrant embryo implantation during early pregnancy. However, whether and how the process of embryo implantation is affected by environmental factors such as stress-induced sympathetic activation remained elusive. Here we report an unexpected, transient effect of β2-adrenoreceptor (β2-AR) activation (day 4 postcoitus) in disrupting embryo spacing at implantation, leading to substantially increased midterm pregnancy loss. The abnormal embryo spacing could be prevented by pretreatment of β2-AR antagonist or genetic ablation of β-AR. Similar β2-AR activation at day 5 postcoitus, when implantation sites have been established, did not affect embryo spacing or pregnancy outcome, indicating that the adverse effect of β2-AR activation is limited to the preimplantation period before embryo attachment. In vitro and in vivo studies demonstrated that the transient β2-AR activation abolished normal preimplantation uterine contractility without adversely affecting blastocyst quality. The contractility inhibition is mediated by activation of the cAMP-PKA pathway and accompanied by specific down-regulation of lpa3, a gene previously found to be critical for uterine contraction and embryo spacing. These results indicated that normal uterine contraction-mediated correct intrauterine embryo distribution is crucial for successful ongoing pregnancy. Abnormal β2-AR activation at early pregnancy provided a molecular clue in explaining how maternal stress at early stages could adversely affect the pregnancy outcome.

Enrichment and characterization of mouse putative epidermal stem cells

Epidermis, a continuously renewing tissue, is maintained by stem cells that proliferate and replenish worn out or damaged cells in the tissue during life. Cultured epidermal stem cells have great potential in scientific research and clinical application. However, isolating a pure and viable population of epidermal stem cells and culturing them has been challenging. In this study, putative epidermal stem cells of mouse were isolated by combining Hoechst 33342 and propidium iodide staining with fluorescence‐activated cell sorting. Molecular markers expression pattern analysis showed that cytokeratin 14, integrin β1 and p63 are expressed in the sorted putative stem cells, but not active β‐catenin, nestin and involucrin. Our results provide further supporting data that mouse putative epidermal stem cells could be successfully isolated by combining Hoechst dye staining with fluorescence‐activated cell sorting and cultured in vitro. The cultured mouse putative epidermal stem cells could be used as a potent tool for studying stem cell biology and testing stem cell therapy.