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1.
Front Cell Dev Biol ; 9: 772625, 2021.
Article in English | MEDLINE | ID: mdl-34957105

ABSTRACT

Sturgeons are among the most ancient linages of actinopterygians. At present, many sturgeon species are critically endangered. Surrogate production could be used as an affordable and a time-efficient method for endangered sturgeons. Our study established a method for identifying and isolating type A spermatogonia from different developmental stages of testes using flow cytometric cell sorting (FCM). Flow cytometric analysis of a whole testicular cell suspension showed several well-distinguished cell populations formed according to different values of light scatter parameters. FCM of these different cell populations was performed directly on glass slides for further immunocytochemistry to identify germ cells. Results showed that the cell population in gate P1 on a flow cytometry plot (with high forward scatter and high side scatter parameter values) contains the highest amount of type A spermatogonia. The sorted cell populations were characterized by expression profiles of 10 germ cell specific genes. The result confirmed that setting up for the P1 gate could precisely sort type A spermatogonia in all tested testicular developmental stages. The P2 gate, which was with lower forward scatter and side scatter values mostly, contained type B spermatogonia at a later maturing stage. Moreover, expressions of plzf, dnd, boule, and kitr were significantly higher in type A spermatogonia than in later developed germ cells. In addition, plzf was firstly found as a reliable marker to identify type A spermatogonia, which filled the gap of identification of spermatogonial stem cells in sterlet. It is expected to increase the efficiency of germ stem cell culture and transplantation with plzf identification. Our study thus first addressed a phenotypic characterization of a pure type A spermatogonia population in sterlet. FCM strategy can improve the production of sturgeons with surrogate broodstock and further the analysis of the cellular and molecular mechanisms of sturgeon germ cell development.

2.
Reprod Toxicol ; 96: 175-184, 2020 09.
Article in English | MEDLINE | ID: mdl-32619501

ABSTRACT

Di-(2-ethylhexyl)-phthalate (DEHP) is a compound widely used as a plasticizer, which can leach from plastics into the environment and thus influence human health. The aim of this study was to analyze whether exposure to an environmentally relevant dose of DEHP during mice fetal development or puberty can cause long-lasting changes detectable month/s after the last exposure. We used a DEHP concentration relevant to a daily human intake of 2.4-3 µg/kg of body weight/day. CD1 outbred mice were treated either in utero or postnatally during puberty and analyzed in adulthood. Analyzing fertility parameters using morphometric, histologic, genomic and proteomic methods we showed that DEHP exposure leads to decreased sperm concentration and quality, in both experimental groups. Moreover, the changes in anogenital distance, seminal vesicle weight, and testicular gene expression suggest a disturbance of androgen signaling in exposed animals. In conclusion, we hereby present, that the prenatal and pubertal exposure to a low dose of DEHP negatively influenced reproductive endpoints in male mice, and some of the effects were persistent until adulthood.


Subject(s)
Diethylhexyl Phthalate/toxicity , Endocrine Disruptors/toxicity , Plasticizers/toxicity , Prenatal Exposure Delayed Effects/chemically induced , Sexual Maturation/drug effects , Anal Canal/anatomy & histology , Anal Canal/drug effects , Animals , Female , Gene Expression Regulation, Developmental/drug effects , Genitalia, Male/anatomy & histology , Genitalia, Male/drug effects , Male , Maternal-Fetal Exchange , Mice, Inbred ICR , Pregnancy , Prenatal Exposure Delayed Effects/genetics , Spermatozoa/drug effects , Testis/anatomy & histology , Testis/drug effects
3.
Cells ; 8(2)2019 01 22.
Article in English | MEDLINE | ID: mdl-30678275

ABSTRACT

In this study, we investigated the influence of metformin (MF) on proliferation and viability of adipose-derived stromal cells isolated from horses (EqASCs). We determined the effect of metformin on cell metabolism in terms of mitochondrial metabolism and oxidative status. Our purpose was to evaluate the metformin effect on cells derived from healthy horses (EqASCHE) and individuals affected by equine metabolic syndrome (EqASCEMS). The cells were treated with 0.5 µM MF for 72 h. The proliferative activity was evaluated based on the measurement of BrdU incorporation during DNA synthesis, as well as population doubling time rate (PDT) and distribution of EqASCs in the cell cycle. The influence of metformin on EqASC viability was determined in relation to apoptosis profile, mitochondrial membrane potential, oxidative stress markers and BAX/BCL-2 mRNA ratio. Further, we were interested in possibility of metformin affecting the Wnt3a signalling pathway and, thus, we determined mRNA and protein level of WNT3A and ß-catenin. Finally, using a two-tailed RT-qPCR method, we investigated the expression of miR-16-5p, miR-21-5p, miR-29a-3p, miR-140-3p and miR-145-5p. Obtained results indicate pro-proliferative and anti-apoptotic effects of metformin on EqASCs. In this study, MF significantly improved proliferation of EqASCs, which manifested in increased synthesis of DNA and lowered PDT value. Additionally, metformin improved metabolism and viability of cells, which correlated with higher mitochondrial membrane potential, reduced apoptosis and increased WNT3A/ß-catenin expression. Metformin modulates the miRNA expression differently in EqASCHE and EqASCEMS. Metformin may be used as a preconditioning agent which stimulates proliferative activity and viability of EqASCs.


Subject(s)
Adipose Tissue/cytology , Cell Separation , Metabolic Syndrome/drug therapy , Metabolic Syndrome/veterinary , Metformin/therapeutic use , Multipotent Stem Cells/cytology , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Horses , Membrane Potential, Mitochondrial/drug effects , Metabolic Syndrome/pathology , Metformin/pharmacology , MicroRNAs/genetics , MicroRNAs/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Oxidation-Reduction , Wnt3A Protein/metabolism , beta Catenin/metabolism
4.
Carcinogenesis ; 39(11): 1359-1367, 2018 12 13.
Article in English | MEDLINE | ID: mdl-30277504

ABSTRACT

MicroRNA (miRNA) profiling represents a promising source of cancer-related biomarkers. miRNA signatures are specific for each cancer type and subgroups of patients with diverse treatment sensitivity. Yet this miRNA potential has not been satisfactorily explored in rectal cancer (RC). The aim of the study was to identify the specific miRNA signature with clinical and therapeutic relevance for RC. Expressions of 2555 miRNA were examined in 20 pairs of rectal tumors and matched non-malignant tissues by 3D-Gene Toray microarray. Candidate miRNAs were validated in an independent cohort of 100 paired rectal tissues and in whole plasma and exosomes of 100 RC patients. To study the association of miRNA profile with therapeutic outcomes, plasma samples were taken repeatedly over a time period of 1 year reflecting thus patients' treatment responses. Finally, the most prominent miRNAs were investigated in vitro for their involvement in cell growth. We identified RC-specific miRNA signature that distinguishes responders from non-responders to adjuvant chemotherapy. A predominant part of identified miRNAs was represented by the members of miR-17/92 cluster. Upregulation of miRNA-17, -18a, -18b, -19a, -19b, -20a, -20b and -106a in tumor was associated with higher risk of tumor relapse and their overexpression in RC cell lines stimulated cellular proliferation. Examination of these miRNAs in plasma exosomes showed that their levels differed between RC patients and healthy controls and correlated with patient's treatment response. miRNAs from miR-17/92 cluster represent a non-invasive biomarker to predict posttreatment prognosis in RC patients.


Subject(s)
Antineoplastic Agents/therapeutic use , MicroRNAs/genetics , Rectal Neoplasms/drug therapy , Rectal Neoplasms/genetics , Biomarkers, Tumor/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , Humans , Rectal Neoplasms/mortality , Treatment Outcome
5.
Oncotarget ; 8(4): 6376-6398, 2017 Jan 24.
Article in English | MEDLINE | ID: mdl-28031527

ABSTRACT

The importance of iron in the growth and progression of tumors has been widely documented. In this report, we show that tumor-initiating cells (TICs), represented by spheres derived from the MCF7 cell line, exhibit higher intracellular labile iron pool, mitochondrial iron accumulation and are more susceptible to iron chelation. TICs also show activation of the IRP/IRE system, leading to higher iron uptake and decrease in iron storage, suggesting that level of properly assembled cytosolic iron-sulfur clusters (FeS) is reduced. This finding is confirmed by lower enzymatic activity of aconitase and FeS cluster biogenesis enzymes, as well as lower levels of reduced glutathione, implying reduced FeS clusters synthesis/utilization in TICs. Importantly, we have identified specific gene signature related to iron metabolism consisting of genes regulating iron uptake, mitochondrial FeS cluster biogenesis and hypoxic response (ABCB10, ACO1, CYBRD1, EPAS1, GLRX5, HEPH, HFE, IREB2, QSOX1 and TFRC). Principal component analysis based on this signature is able to distinguish TICs from cancer cells in vitro and also Leukemia-initiating cells (LICs) from non-LICs in the mouse model of acute promyelocytic leukemia (APL). Majority of the described changes were also recapitulated in an alternative model represented by MCF7 cells resistant to tamoxifen (TAMR) that exhibit features of TICs. Our findings point to the critical importance of redox balance and iron metabolism-related genes and proteins in the context of cancer and TICs that could be potentially used for cancer diagnostics or therapy.


Subject(s)
Breast Neoplasms/genetics , Iron/metabolism , Neoplastic Stem Cells/enzymology , Prostatic Neoplasms/genetics , Transcriptome , Animals , Antineoplastic Agents/pharmacology , Biological Transport , Breast Neoplasms/drug therapy , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Female , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Iron Chelating Agents/pharmacology , Leukemia, Promyelocytic, Acute/enzymology , Leukemia, Promyelocytic, Acute/genetics , MCF-7 Cells , Male , Mice, Transgenic , Mitochondria/enzymology , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Phenotype , Principal Component Analysis , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/pathology , Spheroids, Cellular , Tamoxifen/pharmacology
6.
Sci Rep ; 6: 29023, 2016 07 07.
Article in English | MEDLINE | ID: mdl-27383461

ABSTRACT

Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing.


Subject(s)
Carcinoma/chemistry , Colon/chemistry , Colonic Neoplasms/chemistry , Cryopreservation/methods , Neoplasm Proteins/genetics , Preservation, Biological/methods , RNA, Neoplasm/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Specimen Handling/methods , Carcinoma/surgery , Colonic Neoplasms/surgery , Cryopreservation/instrumentation , DNA Topoisomerases, Type I/genetics , DNA, Neoplasm/genetics , DNA, Ribosomal/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , High-Throughput Screening Assays/instrumentation , High-Throughput Screening Assays/methods , Humans , Neoplasm Proteins/biosynthesis , Nitrogen , Organ Preservation Solutions , Preservation, Biological/instrumentation , Quality Control , RNA, Neoplasm/analysis , RNA, Neoplasm/genetics , RNA, Ribosomal, 18S/genetics , Real-Time Polymerase Chain Reaction/instrumentation , Reproducibility of Results , Specimen Handling/instrumentation , Tissue Fixation/methods
7.
BMC Mol Biol ; 16: 5, 2015 Mar 11.
Article in English | MEDLINE | ID: mdl-25888347

ABSTRACT

BACKGROUND: With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm). RESULTS: For evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted. CONCLUSIONS: We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step.


Subject(s)
Gene Expression Profiling/instrumentation , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Gene Expression Profiling/methods , Healthy Volunteers , Humans , RNA, Messenger/analysis , RNA, Messenger/blood , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , Sensitivity and Specificity
8.
BMC Med Genet ; 15: 17, 2014 Jan 31.
Article in English | MEDLINE | ID: mdl-24484585

ABSTRACT

BACKGROUND: Mismatch repair (MMR) genes are known to be frequently altered in colorectal cancer (CRC). Both genetics and epigenetics modifications seems to be relevant in this phenomenon, however it is still not clear how these two aspects are interconnected. The present study aimed at characterizing of epigenetic and gene expression profiles of MMR genes in sporadic CRC patients from the Czech Republic, a country with one of the highest incidences of this cancer all over Europe. METHODS: Expression levels and CpG promoter methylation status of all MMR genes were evaluated in DNA from tumor and adjacent mucosal samples of 53 incident CRC patients. RESULTS: We have found significantly increased transcription levels in EXO1 gene in tumor tissues (P = 0.05) and significant over-expression of MSH3 gene in colon tumors when compared to adjacent mucosal tissues (P = 0.02). Interestingly, almost all MMR genes were differently expressed when localization of tumors was compared. In particular, colon tumors showed an up-regulation of EXO1, MSH2, MSH3, MSH6, and PMS2 genes in comparison to rectal tumors (P = 0.02). Expression levels of all MMR genes positively correlated between each other. The promoter methylation of MLH1 gene was observed in 9% of CRC tissues only. CONCLUSIONS: In our study, we have observed different pattern of MMR genes expression according to tumor localization. However, a lack of association between methylation in MMR genes and their corresponding expressions was noticed in this study, the relationship between these two aspects is worthy to be analyzed in larger population studies and in pre-malignant stages.


Subject(s)
Colorectal Neoplasms/genetics , DNA Mismatch Repair/genetics , Aged , Colorectal Neoplasms/epidemiology , Colorectal Neoplasms/pathology , Czech Republic/epidemiology , DNA Methylation , Epigenesis, Genetic , Female , Gene Expression Profiling , Humans , Incidence , Male , Microsatellite Instability , Promoter Regions, Genetic/genetics
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