Site specificity of blastocyst hatching significantly influences pregnancy outcomes in mice.

Blastocysts hatch from the zona pellucida (ZP) to enable implantation into the uterine endometrial epithelium, but little is known regarding the effect of hatching sites on pregnancy outcomes. Murine hatching embryos were categorized into five groups based on initial trophectoderm projection (TEP)/ZP position corresponding to the inner cell mass center. In blastocysts (3.5 dpc) post-12 hours in vitro culture, TEP rates of A-site (44.4%) and B-site (38.6%) embryos were higher than those of C-site (12.5%) and D-site (3.1%) embryos, while the O-site (1.4%) was the lowest (P < .05). Post-ET A-site (55.6%) and B-site (65.6%) birth rates were higher than those of C-site embryos (21.3%) and controls (P < .05). Furthermore, live birth rate of B-site embryos remained higher than C-site embryos (68.8% vs 31.3%; P < .05) when both were transferred into the same recipients. Different TEP site blastocysts exhibited different implantation competences: the implantation rate of C-site embryos was lower than that of both A- and B-site groups (67.7% vs 84.3% and 83.2%, respectively; P < .05) at 2 days post-ET. C-site embryos also had a distinctly higher ratio of developmental defects (47.5%) than A- and B-site embryos (22.5% and 14.6%, respectively), with implantation failure mainly associated with poor birth rate, a finding corroborated by differential gene expression analysis such as LIF, LIFR, and S100a9. Surprisingly, acidified Tyrode's solution (AAH)-treated B-site blastocysts had a significantly increased birth rate (77.1%) than C-site (55.3%) and controls (43.4%). Site specificity and differential gene expression during embryo hatching can be applied in ART screening. More importantly, assisted hatching by AAH is effective and feasible for improving pregnancy and term development, particularly at the B-site, for humans and in animal husbandry.

The flavonoid luteolin suppresses infantile hemangioma by targeting FZD6 in the Wnt pathway.

Infantile hemangioma is the most common vascular tumor of childhood. It is characterized by clinical expansion of endothelial cells and promoted by angiogenic factors. Luteolin is a flavonoid compound that carries anti-cancer and anti-angiogenesis properties. The study aimed to investigate the effect of luteolin in treating infantile hemangioma. We first tested the effect of luteolin on cell proliferative potential and VEGFA expression in hemangioma-derived stem cells (HemSCs). We then examined the efficacy of systemic application of luteolin in a murine hemangioma model. We then identified the downstream factor regulated by luteolin in HemSCs and validated its causative relationship with knock-down method in both in vitro and in vivo models. We also investigated the protein expression change of this targeting factor in proliferating hemangiomas. Luteolin inhibited HemSC growth and suppressed VEGF-A expression in a dose-dependent manner. Luteolin inhibited microvessel formation and de novo vasculogenesis in the murine model. FZD6 was induced by luteolin and exerted the anti-angiogenesis effect in our tumor models. Lastly, FZD6 level was repressed in the clinical tissues of human proliferating hemangiomas. Luteolin is a promising new agent to treat infantile hemangioma. Targeting the Wnt pathway may represent a potential therapeutic strategic to inhibit angiogenesis in proliferating hemangiomas.

Long noncoding RNA LINC00342 promotes growth of infantile hemangioma by sponging miR-3619-5p from HDGF.

Infantile hemangiomas (IH) are a type of benign vascular neoplasm that may cause permanent scarring. Hemangioma-derived endothelial cells (HemECs) are commonly used as an in vitro model to study IH. Long noncoding RNA is a type of RNA transcript longer than 200 nucleotides that does not encode any protein. LINC00342 was discovered to regulate proliferation and apoptosis in nonsmall cell lung cancer. However, the role of LINC00342 in IH has never been reported before. Expressions of LINC00342 and miR-3619-5p were detected in proliferating versus normal skin tissues. Colony formation and Cell-Couting Kit 8 assays were carried out to study the effects on cell proliferation after knockdown and overexpression of LINC00342, respectively. Meanwhile caspase-3 activity and nucleosomal fragmentation assay were applied to detect cell apoptosis. Micro-RNA binding sites on LINC00342 and hepatoma-derived growth factor (HDGF) were predicted and confirmed via dual-luciferase reporter assay. Biotin RNA pulldown assay was used to verify the direct binding between RNA molecules. LINC00342 enhanced proliferation and inhibited apoptosis in HemECs. MiR-3619-5p targeted both LINC00342 and HDGF, where LINC00342 sponged miR-3619-5p and positively regulated HDGF. HDGF knockdown rescued the effects of LINC00342 on HemECs. The LINC00342-miR-3619-5p-HDGF signaling pathway could regulate cell proliferation and apoptosis in HemECs.NEW & NOTEWORTHY The role of LINC00342 in infantile hemangiomas has not yet been elucidated. This paper highlights the regulatory role of LINC00342 in cell proliferation and apoptosis in hemangioma-derived endothelial cells and the underlying molecular mechanisms. The findings would provide potential target for treatment of infantile hemangiomas.

Evaluation of deoxynivalenol-induced toxic effects on mouse endometrial stromal cells: Cell apoptosis and cell cycle.

Deoxynivalenol (DON) is a type B trichothecene mycotoxin which has toxic effects on humans and animals. Although DON has been studied in various cell types for its cytotoxicity, there is litter information about the effects of DON on mouse endometrial stromal cells (ESCs). Thus, in this study, we investigated the toxic effects of DON on mouse ESCs and its possible mechanisms. DON inhibited the cell viability in a dose- and time-dependent manner. TUNEL assay results showed that DON caused apoptosis and TUNEL-positive cells increased with increasing DON concentrations in mouse ESCs. Western blot showed that DON significantly increased the expression levels of apoptosis-related protein including Caspase-9, Caspase-3, poly (ADP-ribose) polymerase (PARP) and the ratio of Bax/Bcl-2. After DON treatment, the expression levels of cell cycle-related protein including p38/p-p38, Cdc25C/p-Cdc25C, Cdc2/p-Cdc2 and cyclinB1 were significantly decreased and immunoprecipitation analysis showed that cyclinB1-Cdc2 complex was significantly decreased. However, the combination of SB203580 (p38 specific inhibitor) and DON treatment significantly reversed the depression of Cdc25C/p-Cdc25C, Cdc2/p-Cdc2, cyclinB1 and cyclinB1-Cdc2 complex. Collectively, these data suggest that DON causes apoptosis via mitochondria apoptosis pathway and induces G2 arrest via p38 MAPK signaling pathway in mouse ESCs.

Exploration of ZEA cytotoxicity to mouse endometrial stromal cells and RNA-seq analysis.

Because zearalenone (ZEA) causes harmful influence to animals and widely exists in the world, the researches on ZEA have never stopped. However, the mechanisms of ZEA on proliferation, cycle, and apoptosis in endometrial stromal cells (ESCs) remain poorly defined. Therefore, the purpose of our study was to explore the effects of ZEA to ESCs and demonstrate them by transcriptomic analysis. The results showed that after ZEA treatment, ESCs appeared numerous adverse reactions, and the phenomena of cell viability decrease, DNA replication block and apoptosis were detected by flow cytometry, Annexin V-FITC/PI double-staining method, TUNEL assay, and so on. Then, RNA-seq approach was adopted to prove the validity of above experiments, as expected, the results from different expression genes, gene ontology terms, and KEGG pathway were all consistent with those. Overall, the results suggested that ZEA could cause a series of reactions by cytotoxicity to mouse ESCs, meanwhile there must be some substances and mechanisms protect cells against cytotoxicity damage.

Cell-free ribosome display and selection of antibodies on arrayed antigens.

In vitro display technology is a powerful tool for discovery and optimisation of novel antibodies. With increasing demands on various binding molecules in proteomics studies, techniques for a large-scale generation of antibodies or antibody fragments are needed. Here, we describe a novel method for parallel generation of different antibody fragments (scFv) by integrating cell-free ribosome display with array technology. We have demonstrated the procedure by successfully isolating scFv antibodies specific to 16 different cancer biomarkers via a single process. Our results provide proof of principle for multiple production of various scFv antibodies simultaneously.

Exploration of Bcl-2 family and caspases-dependent apoptotic signaling pathway in Zearalenone-treated mouse endometrial stromal cells.

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. Although the toxicity of ZEA have been widely studied in a number of cell types, the mechanistic role of ZEA on apoptosis of endometrial stromal cells (ESCs) remains poorly understood. The objective of this study was to determine the effects of ZEA on apoptosis of mouse ESCs and explore the signaling pathway underlying the cytotoxicity of ZEA. The results showed that ZEA treatment caused obvious apoptosis in ESCs as determined by the flow cytometry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Immunoblotting and real-time quantitative polymerase chain reaction (RT-qPCR) revealed that ZEA treatment increased the ratio of Bax/Bcl-2. The enzymatic activity assays revealed that caspases-3 and caspase-9 were activated by ZEA treatment in a dose-dependent manner. In addition, flow cytometry show that the apoptotic percentages of cells pretreated with Z-VAD-FMK and Z-LEHD-FMK were markedly reduced compared to the ZEA-treated cells. Overall, the results suggested that ZEA induced obvious apoptosis in ESCs via a Bcl-2 family and caspases-dependent signaling pathway.

miR-342-3p Suppresses glioblastoma development via targeting CDK6.

BACKGROUND: Glioblastoma is the most malignant primary brain tumor with dysregulated microRNAs affecting development and malignant transformation. METHODS: Gene Expression Omnibus (GEO) dataset (GSE165937) was retrieved, and the differential expressed microRNAs were screened and testified by quantitative real-time PCR (qRT-PCR) in glioblastoma cells. miR-342-3p mimic was transfected into U87MG and U251MG cells. EdU staining, cell counting kit-8, and transwell assay were utilized to evaluate the proliferation, migration, and invasion of glioblastoma. The potential binding sequences between miR-342-3p and CDK6 were predicted and testified by TargetScan and luciferase reporter assay. Relative CDK6 and miR-342-3p expression were detected with qRT-PCR and Western blot. RESULTS: Down-regulated miR-342-3p was observed in both glioblastoma tissues and cell lines. Over-expressed miR-342-3p prohibited glioblastoma cells proliferation, migration, and invasion, which could be rescued by further CDK6 transfection. Mechanically, miR-342-3p could directly bind with CDK6 as testified with luciferase analysis and down-regulated CDK6 expression. CONCLUSION: Down-regulated miR-342-3p may promote glioblastoma cells proliferation, migration, and invasion with up-regulated CDK6, which indicates that miR-342-3p/CDK6 might be a treatment target in glioblastoma development.

PKC inhibitors regulate stem cell self-renewal by regulating H3K27me3 and H3K9me3.

Embryonic stem cell (ESC) research is critical to the scientific community, as their application in regenerative medicine can be widely beneficial. ESCs eventually withdraw from their self-renewal program and subsequently differentiate into specific cell lineages; however, the mechanisms regulating these processes remain unclear. PKC inhibition using 3-[1-[3-(dimethylamino) propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (PKCi) is responsible for the derivation and maintenance of human, rat, and mouse ESCs, but the mechanism by which PKCi maintains stem cell self-renewal is poorly understood. By studying the PKCi stem cell (PKCi-mESC) transcriptome and epigenetic modification, we found the transcriptome of PKCi-mESC differed from 2i stem cells (2i-mESC), with 2010 up-regulated genes and 1784 down-regulated genes. Among them, genes related to core transcription factors, naïve-specific markers, and pluripotency are differentially expressed between the two stem cell lines. We analyzed epigenetic modification of PKCi-mESC and found the distribution of H3K27me3 signal was significantly reduced at transcription start sites (TSSs) throughout the genome and at differentially expressed genes (DEGs). Likewise, the H3K9me3 signal at TSSs throughout the genome was significantly reduced in PKCi-mESC, but the distribution on DEGs is reversed. Kdm4d and Kdm6a knockdown by RNA interference (RNAi) significantly altered the expression of genes related to self-renewal in PKCi-mESC. In conclusion, we revealed PKCi-mESC and 2i-mESC differentially express numerous genes, including stem cell-related genes. Furthermore, PKCi-mESC regulated gene expression through H3K27me3 and H3K9me3 modification, which maintained stem cell self-renewal capacity.

Nucleosome remodeling and deacetylation complex and MBD3 influence mouse embryonic stem cell naïve pluripotency under inhibition of protein kinase C.

The pluripotency of naïve mouse embryonic stem cells (mES) is regulated by multiple signaling pathways, with inhibition of protein kinase C (PKCi) playing a particularly important role in maintaining naïve mES. However, the regulatory function of nucleosome remodeling and deacetylase (NuRD) complex in mES cultured in a PKCi system is unknown. We found that, compared with 2iL-derived mES, PKCi-derived mES showed low mRNA expression of NuRD complex subunits, including MBD3, HDAC1/HDAC2, MTA1, and RbAP46/RbAP48. Western blot showed that PKCi-derived mES expressed lower protein levels of MBD3 and HDAC2 at passage 3, as well as MBD3, HDAC2, and MTA1 at passage 10, indicating that PKCi suppressed NuRD complex expression. Knockdown of MBD3 increased PKCi-derived mES pluripotency by increasing NANOG and OCT4 expression and colony formation. By contrast, overexpression of MBD3 or removal of PKC inhibitor-induced differentiation of mES, results in reduced NANOG, OCT4, and REX1 expression and colony formation, increased differentiation-related gene expression, and differentiation into flat cells. Knockdown of MBD3 in mES upon PKC inhibitor removal partially reversed cell differentiation. Our results show that the regulatory NuRD complex and its MBD3 subunit influence the naïve pluripotency of mES cultured in a PKCi system.

Methyl-CpG-binding domain 3 (Mbd3) is an important regulator for apoptosis in mouse embryonic stem cells.

Methyl-CpG-binding domain 3 (Mbd3) is a core repressor complex component. Although Mbd3 is required for the pluripotency of embryonic stem cells (ES), the role of Mbd3 in mouse ES (mES) cell apoptosis remains undefined. In this study naïve-state mES were derived and maintained in the presence of a selective protein kinase C pathway inhibitor (PKCi; GÓ§6983) to study the function of Mbd3 during mES apoptosis. Mbd3 overexpression in mES decreased the total cell number and viability, and it also dramatically increased the rate of apoptosis. Further investigation of Mbd3 overexpression revealed a 3-fold increase in the proapoptotic/prosurvival protein ratio (Bax/Bcl-2) and elevated RNA expression levels of apoptosis-related genes, including Bim, Trail, Fasl, and caspase 3, with reduced Bcl-2 RNA expression levels. Removal of PKCi from the mES cell culture resulted in upregulated Mbd3 expression and apoptosis, similar to the effects of Mbd3 overexpression. Furthermore, specific knockdown of endogenous Mbd3 partially rescued the mES apoptosis induced by the removal of PKCi, thus increasing the total cell number and viability while decreasing the rate of apoptosis. Additionally, Bax, Bim, Trail, and caspase 3 RNA expression levels were partially reduced, and that of Bcl-2 was partially increased. Our findings support Mbd3 as a pivotal regulator of apoptosis in mES.

Dynamic patterns of H3K4me3, H3K27me3, and Nanog during rabbit embryo development.

Epigenetic modification and expression of key pluripotent factors are critical for development, cell fate determination, and differentiation in early embryos. In this study, we systematically examined the dynamic patterns of histone modifications (H3K4me3 and H3K27me3) and Nanog expression during the development of preimplantation rabbit embryos. Rabbit oocytes, 1-, 2-, 4-, 8-, and 16-cell embryos, morulae, and blastocysts were collected at specific time points following superovulation and assessed for nuclear H3K4me3, H3K27me3, and Nanog expression by immunofluorescence microscopy. The frequency of H3K4me3-positive nuclear staining was highest in oocytes through 4-cell embryos (100%), decreased in 8-cell (97.2%) and 16-cell (94.4%) embryos (P > 0.05), declined dramatically in morulae (86.7%) (1- through 8-cell embryos vs morulae, P < 0.05), and was the lowest in blastocysts (76.2%) (P < 0.05). Nuclear staining of H3K27me3 was negative in oocytes and embryos through the 16-cell stage but was positive in 25.9% of morulae and 34.2% of blastocyst (P < 0.05). Similarly, rabbit oocytes and embryos through the 16-cell stage did not express Nanog, but Nanog was expressed in 24.9% of morulae and 36.5% of blastocysts (P < 0.05). The observed decrease in H3K4me3 and increase in H3K27me3 as development progressed in preimplantation rabbit embryos, together with late Nanog expression, indicates a correlation of these factors with early embryonic cell fate determination and differentiation. Our study provides a specific and dynamic profile of histone modifications and gene expression that will be important for the derivation of rabbit embryonic stem cells and improving rabbit cloning by somatic cell nuclear transfer.

ROCK inhibitor Y-27632 maintains the propagation and characteristics of hair follicle stem cells.

Hair follicle stem cells (HFSCs) are an important source for skin tissue engineering studies and clinical applications. Here, we describe a differential enrichment approach to derive HFSCs from hair follicles of vibrissae and ear skin using the Rho-associated protein kinase (ROCK) inhibitor Y-27632. In the presence of Y-27632, primary cultured hair follicle cells grew in clustered colonies surrounded by keratinocyte-like cells and simultaneously expressed three HFSC markers: CD34, K15, and ITGB1. HFSCs cultured in medium containing Y-27632 were presented at a stable ratio of 30.7%, 34.1%, and 32.9% after passages 5, 10, and 15, respectively. By contrast, in medium containing epidermal growth factor, clustered HFSC colonies disappeared after 6 passages and lacked HFSC marker expression. After withdrawal of Y-27632 from the medium, HFSCs rapidly differentiated into keratinocyte-like cells. Furthermore, HFSCs derived with Y-27632 formed spherical clusters in collagen matrix in vitro, differentiated into keratinocytes and adipose cells under in vitro induction conditions, and cooperated with fetal dermal cells to regenerate hair follicles in vivo 6 weeks after their intracutaneous injection into immune-deficient mice. These findings suggest that Y-27632 maintains the self-renewal and stemness characteristics of HFSCs during primary skin tissue culture followed by enrichment passaging and that HFSCs derived with Y-27632 possess the differentiation potentials important for tissue engineering and other clinical applications.