5 ASSESSMENT OF HSP70.1 TRANSCRIPTION LEVELS IN BOVINE EMBRYOS AFTER CUMULUS REMOVAL BY DIFFERENT TECHNIQUES

2006 ◽  
Vol 18 (2) ◽  
pp. 111 ◽  
Author(s):  
A. Reeder ◽  
V. Schutzkus ◽  
J. Wiebelhaus-Finger ◽  
H. Khatib ◽  
R. L. Monson ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
De Novo ◽  
Housekeeping Gene ◽  
Cumulus Cells ◽  
Treated Group ◽  
Specific Gene ◽  
In Vitro Production ◽  
Rna Transcription

Previous results indicated that there was a difference in transcriptional activity depending on the method used to remove the cumulus cells after IVF (Zeringue et al. 2005 LabChip 5, 86-90). However, specific gene expression was not examined and therefore was the goal of the present study. The objective of this study was to compare the transcription levels of the HSP70.1 gene in bovine in vitro production (IVP) embryos that underwent cumulus removal by vortexing, hand stripping, or microfluidic treatment. Quantitative real-time PCR was used to estimate transcription levels of the HSP70.1 gene in bovine IVP zygotes and two-cell embryos. Transcription levels were compared between embryos that underwent three methods of cumulus removal. Presumptive zygotes were harvested at 2 or 24 h after cumulus removal by vortexing, by hand stripping, or by microfluidic means in order to compare the relative embryonic stress of these three treatments. Bovine in vitro embryos were produced by standard means with the only variable being the cumulus removal technique 24 h after fertilization. At 2 and 24 h post-cumulus removal, randomly selected presumptive zygotes were taken out of culture, preserved in RNAlater (Ambion, Inc., Austin, TX, USA) and stored at -20�C. RNA was extracted from single embryos via Qiagen's RNeasy Micro kit (Valencia, CA, USA). RNA was amplified and PCR products were detected with SYBR Green 1 (Applied Biosystems, Foster City, CA, USA) using an Opticon Monitor 3 real-time PCR machine (Bio-Rad Laboratories, Inc., Waltham, MA, USA). The threshold cycle (CT) numbers were determined for the amplified cDNA of the bovine HSP70.1 mRNA and for the housekeeping gene, acidic ribosomal phosphoprotein (PO), used as a reference. Then the amount of HSP70.1 was divided by the amount of PO to obtain a normalized HSP70.1 value expressed as the ratio of HSP70.1/PO. Ratios were analyzed by the GENMOD procedure in SAS (SAS Institute, Inc., Cary, NC, USA) accounting for replicates and treatments. Transcription levels of the HSP70.1 gene did not differ significantly between the microfluidically treated and vortexed groups of zygotes at 2 h post-cumulus removal (P = 0.1032) or between the hand-stripped and vortexed groups (P = 0.7567). In contrast, at 24 h post-cumulus removal, the embryos in the microfluidically treated group showed significantly higher levels of HSP70.1 transcription than the vortexed group (P = 0.0115). The transcription levels did not differ significantly between the hand-stripped and vortexed groups (P = 0.7875). This work strongly suggests that there is de novo RNA transcription in the early embryonic stages of the bovine. In addition to previously described improved developmental kinetics, the use of microfluidics in IVP leads to statistically significant differences in RNA transcription levels of HSP70.1.

195 ALTERED mRNA TRANSCRIPT EXPRESSION OF IN VITRO- VERSUS IN VIVO-MATURED PORCINE OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 210
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
K. M. Whitworth ◽  
W. G. Spollen ◽  
S. M. Blake ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Real Time ◽  
Real Time Pcr ◽  
Genetic Background ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Illumina Genome Analyzer ◽  
Similar Genetic Background

In contrast to oocytes matured in vitro, porcine embryos that result from in vivo maturation and fertilization have a high developmental competence and readily make the transition from oocyte to blastocyst. This observation led us to investigate the transcript profile differences between in vivo- and in vitro-matured porcine oocytes. For the in vivo-matured group, oviducts of 3 gilts of similar genetic background were flushed 2 days after detection of standing oestrus. MII oocytes were collected in pools of 10 and snap frozen in liquid nitrogen for RNA isolation. The in vitro-matured oocytes were obtained by euthanizing 3 gilts, again with a similar genetic background and recovering the ovaries. Follicles (2 to 8 mm in size) were aspirated and oocytes with multiple layers of cumulus cells and uniform cytoplasm were placed in M-199 supplemented with LH, FSH and epidermal growth factor for 42 h. Upon maturation, cumulus cells were stripped and the healthy MII oocytes were collected in pools of 10 and snap frozen. Total RNA was extracted from 3 pools of 10 oocytes for both treatments using an All prep DNA/RNA micro isolation kit (Qiagen, Valencia, CA, USA). Complementary DNA was synthesized using oligo (dT′) primed reverse transcriptase with superscript III (Invitrogen, Carlsbad, CA, USA). Second-strand cDNA was synthesized using DNA polymerase I and sequenced using Illumina Genome Analyzer II. All reads were aligned to a custom-built porcine transcriptome. There were over 18 million reads in the 2 maturation groups that tiled to the 34 433-member transcriptome: 1317 transcripts were detected with a P ≤ 0.1 (Students t-test), a minimum of 7 reads in at least 1 of the treatments and ≥2-fold difference. Real-time PCR was used on selected transcripts. Comparative CT Method was used on an IQ real-time PCR system with the Bio–Rad SYBR green mix. Statistical differences were determined using the Proc general linear model procedure of SAS (SAS Institute Inc., Cary, NC) and means separated with a l.s.d. (P ≤ 0.05). The misrepresented transcripts from the sequencing data were also characterized using the functional annotation tool DAVID. Twelve pathways were overrepresented in the in vitro-matured oocytes (the top 4 are pathways to cancer, spliceosome, cell cycle and ubiquitin-mediated proteolysis). Eight pathways were underrepresented in the in vitro-matured oocytes (the top 4 are cytoskeleton regulation, T-cell receptor signaling pathway, ubiquitin-mediated proteolysis and cell cycle). Eight transcripts were selected for real-time PCR. ZP2 was higher in the in vitro-matured oocytes as determined by both sequencing and real time. ATG4, HSP90, UBAP2 and SOX4 were not different, regardless of assay. SLC7A3, MRPS36 and PDHX2 were not different based on sequencing, but based on real-time MRPS36 and PDHX2, were higher in the in vivo group and SLC7A3 was higher in the in vitro group. In conclusion, there is an abundance of misregulated transcripts and altered pathways in in vitro-matured oocytes. This dataset is a tool that may provide clues to improve the in vitro maturation process so that in vitro-matured oocytes will be more like their in vivo-matured counterparts, thus improving developmental competence. Funded by Food for the 21st Century.

scholarly journals Platelet Microparticles Accelerate Proliferation and Growth of Mesenchymal Stem Cells through Longevity-Related Genes

2021 ◽  
Vol 24 (8) ◽  
pp. 607-614
Author(s):  
Maryam Samareh Salavati Pour ◽  
Fatemeh Hoseinpour Kasgari ◽  
Alireza Farsinejad ◽  
Ahmad Fatemi ◽  
Gholamhossein Hassanshahi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Treated Group ◽  
Population Doubling ◽  
Control Group ◽  
Population Doubling Time ◽  
Longevity Genes

Background: Due to their self-renewal and differentiation ability, the mesenchymal stem cells (MSCs) have been studied extensively. However, the MSCs lifespan is restricted; they undergo several divisions in vitro that cause several alternations in cellular features and relatively lessens their application. Thus, this study was aimed to assess the effect of platelet-derived microparticles (PMPs), a valuable source of proteins, microRNAs (miRNAs), and growth factors, on the expression of hTERT, c-MYC, p16, p53, and p21 as the most important aging and cell longevity genes alongside with population doubling time (PDT) of PMP-treated cells in comparison to a control group. Methods: Umbilical cord MSCs (UC-MSCs) were used in this study, whereby they reached a confluency of 30%. MSCs were treated by PMPs (50 µg/mL), and then, PDT was determined for both groups. Quantitative expression of hTERT, c-MYC, p16, p53, and p21 was examined through quantitative real-time PCR at various intervals (i.e. after five and thirty days as well as freezing-thawing process). Results: Our results demonstrated that the treated group had a shorter PDT in comparison to the control group (P<0.050). The real-Time PCR data also indicated that PMPs were able to remarkably up-regulate hTERT and c-MYC genes expression while down-regulating the expression of p16, p21, and p53 genes (P<0.050), especially following five days of treatment. Conclusion: According to these data, it appears that PMPs are a safe and effective candidate for prolonging the lifespan of UC-MSCs; however, further investigations are needed to corroborate this finding.

163IMPROVEMENT OF IN VITRO PRODUCTION OF PIG EMBRYOS BY SYNCHRONISATION OF OOCYTE MEIOTIC MATURATION WITH CYCLOHEXIMIDE

2004 ◽  
Vol 16 (2) ◽  
pp. 204 ◽  
Author(s):  
J. Ye ◽  
K.H.S. Campbell ◽  
M.R. Luck
Keyword(s):  
Cumulus Cells ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Pre Treatment ◽  
Vitro Production

It is suggested that the relatively high rates of polyspermic fertilization and poor development of pig embryos produced in vitro are caused by asynchronous oocyte maturation. We have recently shown that pre-treatment of pig oocytes with cycloheximide (CHX) is an efficient way of synchronizing their meiotic maturation in vitro. However, it is not known whether this procedure affects fertilization or further development. The present study examined the effects of CHX-synchronised meiotic maturation on subsequent embryo development and the response to FSH. Pig ovaries were collected from a local abattoir. Cumulus-oocyte complexes (COCs) were aspirated from 3–5mm diameter follicles with a translucent appearance and extensive vascularization. COCs were first pre-incubated in defined maturation medium (DM; M199 with Earle’s salts, 25mM HEPES and sodium bicarbonate, 3mM L-glutamine, 0.1% (w/v) BSA, 0.57mM cysteine, 10ngmL−1 EGF, 0.2μgmL−1 pLH, 100μmL−1 penicillin and 0.1mgmL−1 streptomycin) or in DM supplemented with 50ngmL−1 pFSH (DMF) and 5μgmL−1 CHX for 12h. COCs were then further cultured in the same DM without CHX for 24–30h or in DMF for 36h. For controls, COCs were cultured conventionally in DM for 42h or DMF for 48h. After removal of cumulus cells, all cultured oocytes were inseminated with ejaculated sperm at a final concentration of 300000mL−1 for 6h. The IVF medium was modified Tris-buffered medium containing 0.1% BSA, 20μM adenosine and 0.2mM reduced glutathione. Putative embryos were cultured in NCSU23 without glucose but supplemented with 4.5mM Na lactate and 0.33 mM Na pyruvate for 2 days. Cleaved embryos were further cultured in normal NCSU23 for 4 days. IVM and IVF were performed in 5% CO2 in air and IVC in 5% CO2, 5% O2, 90% N2, all at 39°C and 95% RH. Three replicates with DM, with or without CHX, and one with DMF, with or without CHX, were performed with 30–50 oocytes in each replicate. Statistical comparisons were by t-test. The result with DM showed that the rate for normal cleavage at 2 days after insemination of CHX-treated oocytes (40.6±3.8%) was similar to that of controls (40.4±3.5%). However, the proportion developing to healthy blastocysts at Day 6 was significantly higher in the CHX-treated group (16.9±1.2%) than in controls (9.6±1.3%; P&lt;0.05). A significantly higher number of Day 2-cleaved embryos from CHX-treated oocytes developed to the day 6 blastocyst stage compared with controls (44.7±5.0% and 22.3±2.4%, respectively; P&lt;0.05). Supplementation of the basic maturation medium with pFSH increased the rate of cleavage in both CHX-treated oocytes (73.2%) and controls (76.9%) and increased the proportions developing to healthy blastocysts at Day 6 (CHX-treated: 39.0%; control: 11.5%). We conclude that oocytes pre-treated with CHX retain their developmental competence and that meiotic synchronization with CHX improves the efficiency of in vitro production of pig embryos. (Supported by BBSRC 42/S18810.)

260 COMPARISON OF HOUSEKEEPING GENE EXPRESSION IN INDIVIDUAL 8-CELL STAGE MOUSE EMBRYOS PRODUCED UNDER DIFFERENT CONDITIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 246
Author(s):  
A. Baji Gal ◽  
S. Mamo ◽  
S. Bodo ◽  
A. Dinnyes
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Standard Error ◽  
Real Time Pcr ◽  
Housekeeping Gene ◽  
Mouse Embryos ◽  
Cell Stage ◽  
The Mean

Real-time PCR has the potential to accurately quantify the mRNA level of selected genes in single cells and individual pre-implantation-stage embryos. The goal of our study was to examine the variations in gene expression within individual embryos of the same stage and between embryos of the same stage but from different sources. In our study, we determined expression level of the 7 most commonly used housekeeping genes in 8-cell-stage mouse embryos produced under different culture conditions. Messenger RNA of 6 embryos each that was derived in vivo, or cultured in vitro from the zygote stage, or derived from oocytes activated parthenogenetically and developed in vitro were extracted individually followed by reverse transcription into cDNA. Optimized real-time PCR was performed for cytoplasmic beta-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), H2A histone family, member Z (H2afz), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ubiquitin C (Ubc), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and eukaryotic translation elongation factor 1 epsilon 1 (Eef1e1) genes. The results were analyzed, and the percentage standard error of the mean relative expression value was compared for all genes. All 7 genes were presented above the detection limit in all samples. One or two individual embryos showed 2- to 4-fold higher mRNA levels than the average for all genes in the group. The embryos cultured in vitro showed much higher expression levels of H2afz, Ppia, and Eef1e1 genes than those in the in vivo group. The parthenogenetic group was similar to the in vivo group in expression of Actb, H2afz, Hprt, and Eef1e1 genes, but showed significant differences (P &lt; 0.05; Student's t-test) compared to the in vitro group (Table 1). The percent standard error of the mean decreased gradually as the number of samples was increased. The 6 individual embryos in similar groups showed relatively low variability compared to embryos at similar stage but produced in different conditions. Interestingly, the parthenogenetic embryos showed a level of gene expression comparable to that of the in vivo ones, notwithstanding their culture in vitro. In conclusion, morphological observations and similarity in developmental stage alone cannot guarantee the uniformity of embryo samples, and a minimum of 4–6 replicates per treatment is needed. Moreover, we showed that culture condition itself has an effect on housekeeping gene expression, which, if neglected, might result in misinterpretation of data. Table 1.Relative expression values of the different culture groups (mean ±SE; n =6 embryos) This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No. 070246), and Hungarian National Science Fund (OTKA) (Grant No. T046171).

In vitro Anti-Cancer Activity of Oliveria decumbens Vent. Extract, an Endemic Persian Medicinal Plant, on HT-29 Colorectal Cancer Cell Line

2021 ◽  
Author(s):  
Amir Khodavirdipour ◽  
Fatemeh Haddadi ◽  
Hamideh Rouhani Nejad ◽  
Yasoub Shiri ◽  
Veronica Preetha Tilak
Keyword(s):  
Dna Damage ◽  
Real Time ◽  
Dna Fragmentation ◽  
Real Time Pcr ◽  
De Novo ◽  
Ethanolic Extract ◽  
The United States ◽  
Chemotherapeutic Agents ◽  
Colorectal Cancer Cell Line

Introduction: The top 3 causes of death worldwide include heart disease, injury, and cancer; and cancer records the 2nd place as the leading cause of death in the United States of America after cardiovascular diseases and injuries. Cancer can begin and progress in a very highly twisted and complex pattern and follow the multifactorial route. There is only very few research on medicinal properties Oliveria decumbens rare and valuable plant specially on cancer. So, in this study we tried to cover all needs for future in vivo research. Methods: MTT assay has been performed to estimate the cytotoxicity of the ethanolic extract of the plant. Its free radical capacity evaluation was done by DPPH assay. Furthermore, real-time PCR, the wound-healing assay along with a DNA damage test to study DNA fragmentation characteristics. The plants transcriptomic study was performed by NGS de Novo assembly. Result: Oliveria decumbens ethanolic extract showed an Ic50 of 14.39 mu g/ml. The real-time PCR showed that Oliveria decumbens ethanolic extract significantly induced apoptosis by upregulating the bax gene and slight downregulation of bcl2 an anti-apoptosis gene. The NGS de Novo transcriptome analysis discovered 38 genes responsible for secondary metabolite synthesis so far. The remaining genes and reconstruction of the co-expression network of the transcriptome are underway. Conclusion: The outcome of the Scratch-test and DNA fragmentation confirmed the anti-metastatic and DNA damage properties respectively. Based on these findings; Oliveria decumbens ethanolic extract shall be considered as potential anticancer and chemotherapeutic agents which may elucidate in upcoming studies.

227. Eggs with a surprise: the 'sperm-specific' protein SPRASA is also expressed in the oocyte

2008 ◽  
Vol 20 (9) ◽  
pp. 27
Author(s):  
L. W. Chamley ◽  
A. Wagner ◽  
D. Prendergast ◽  
K. J. Woad ◽  
A. N. Shelling
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
In Silico Analysis ◽  
Human Sperm ◽  
Cumulus Cells ◽  
Reproductive Organs ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Bovine Oocytes

SPRASA is a newly identified protein which in silico analysis suggests is not expressed in other tissues. Antibodies reactive with SPRASA have been identified in some infertile men and an antiserum reactive with recombinant SPRASA prevented human sperm binding to hamster oocytes in vitro, indicating an important role in sperm/oocyte recognition. The aim of this study was to investigate the spatial and temporal expression of SPRASA in reproductive and other tissues. Brain, thymus, heart, spleen, kidney, liver and the reproductive organs from duplicate female and male Balb/C mice were collected at several postnatal timepoints. RNA was extracted, reversed transcribed and analysed by quantitative real time PCR for SPRASA expression. Abattoir-derived, in vitro matured, bovine oocytes were examined for SPRASA expression by fluorescent immunochemistry. To examine SPRASA binding sites on oocytes, matured bovine oocytes were exposed to biotinylated recombinant human SPRASA or biotinylated α-lactalbumin (control), then visualised by confocal microscopy using DTAF-conjugated streptavidin. We found SPRASA mRNA was expressed in the reproductive organs of both females and male mice from postnatal day 10. Fluorescent immunochemistry indicated SPRASA was expressed on the oolemmal membrane and in the few cumulus cells remaining attached to zona-intact oocytes. Control preimmune serum did not stain the oocytes or cumulus cells. Recombinant human SPRASA bound to the oolemmal membrane of both zona intact and zona free bovine oocytes. To date the expression of SPRASA has only been reported in the testes/sperm with an additional single EST identified in brain. Our quantitative real-time PCR analysis demonstrated SPRASA is also expressed in the female reproductive organs. This was confirmed by our immunoassays which show oocytes and possibly cumulus cells express SRPASA while the oolemmal membrane has the ability to bind (sperm-derived) SPRASA. That SPRASA expression is restricted sperm and oocytes confirms the likely function of this protein in reproduction.

scholarly journals The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2341
Author(s):  
Normann Steiner ◽  
Karin Jöhrer ◽  
Selina Plewan ◽  
Andrea Brunner-Véber ◽  
Georg Göbel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Tyrosine Kinase Receptor ◽  
Bone Marrow Biopsies ◽  
Flt3 Inhibitors ◽  
Flt3 Expression

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

scholarly journals The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Dong Chen ◽  
Yaqin Wang ◽  
Feiya Yang ◽  
Adili Keranmu ◽  
Qingxin Zhao ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
Expression Pattern ◽  
Real Time Pcr ◽  
Bioinformatic Analysis ◽  
Biological Functions ◽  
Quantitative Real Time Pcr ◽  
Regulatory Effects

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

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