163 Effect of melatonin and its receptors on bovine oocyte maturation and cumulus cell gene expression after heat shock in vitro: preliminary results

2019 ◽  
Vol 31 (1) ◽  
pp. 206 ◽  
Author(s):  
H. Fernandes ◽  
F. C. Castro ◽  
L. Schefer ◽  
D. M. Paschoal ◽  
C. L. V. Leal
Keyword(s):  
Gene Expression ◽  
Antioxidant Enzymes ◽  
Heat Stress ◽  
Heat Shock ◽  
Geometric Mean ◽  
Stress Conditions ◽  
Melatonin Receptors ◽  
Nuclear Maturation

The aim of this study was to assess the effect of melatonin (MLT) during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COC) under normal and heat stress conditions, on oocyte nuclear maturation and expression of genes related to antioxidant, apoptotic, and expansion activities in cumulus cells (CC), and whether effects are mediated by melatonin receptors (MT1, MT2, or both). The COC were submitted to IVM (25-30/group) in TCM-199 supplemented with 3mg mL−1 BSA, 0.5µg mL−1 FSH, 11µg mL−1 sodium pyruvate and 10µg mL−1 gentamicin. Melatonin (10−6 M) was used alone or associated with luzindole (10−6 M; LUZ) or 4-phenyl-2-propionamidotetralin (10−6 M; 4-P-PDOT). Groups were matured under normal conditions for 24h (38.5°C, control, and 38.5°C+MLT) and heat shock (cultured for 14h at 41°C, 41°C+MLT, 41°C+MLT+LUZ, and 41°C+MLT+4-P-PDOT), and then incubated for the remaining 10h at 38.5°C. The CC were collected and evaluated by real-time quantitative PCR for GPX4, SOD1, and SOD2 (antioxidant enzymes); BAX, CASP3, and P53 (apoptosis-related); and HAS1, HAS2, and PTX3 (expansion-related) transcripts. Gene expression data were normalized by the geometric mean of housekeeping genes ACTB, GAPDH, and PPIA. Relative expression levels were calculated by the comparative method of 2−ΔΔCt (Livak and Schmittgen 2001 Methods, 25, 402-408). Maturation rates (metaphase II; MII) were determined by staining oocytes with Hoechst 33342. Data were analysed by ANOVA followed by Tukey’s test to compare effects of treatments on maturation rate (3 replicates/treatment). Significance level was 5% (GraphPrism software, GraphPad Inc., San Diego, CA, USA). After 24h of IVM, the MII rate decreased (P<0.05) for the 41°C group (30±14.1%, 21/70) compared with the control at 38.5°C (76.1±14.7%, 51/67; P<0.05) and addition of MLT to the 41°C group reversed the decrease (41°C+MLT, 55.3±7.1%, 26/47) and was similar to that of the control (P>0.05). When the heat stress with MLT group was associated with MT inhibitors, MII rates decreased relative to control (P<0.05) and were similar to 41°C without MLT and to each other (41°C+MLT+LUZ, 31.6±8.9%, 24/76; and 41°C+MLT+4-P-PDOT, 30±9.0%, 21/70; P>0.05). Addition of MLT did not differ from control without the hormone (38.5°C+MLT, 90.5±6.7%, 38/42; P>0.05). For gene expression in CC, only the 41°C+MLT+LUZ group increased (P<0.05) transcripts for CASP3 compared with control. There was no difference for antioxidants (GPX4, SOD1, and SOD2), expansion (HAS1, HAS2, and PTX3), or apoptotic (BAX and P53) transcripts for any of the groups (P>0.05). In conclusion, MLT reversed the negative effects on nuclear maturation caused by heat stress conditions during IVM; this effect was probably mediated by the MT2 receptor. However, MLT did not influence the antioxidant enzymes, apoptosis, or expansion-related transcripts in CC, but inhibition of MT1/MT2 receptors increased CASP3 transcripts, suggesting a possible role of the receptors on apoptosis in these cells. Further studies are necessary to improve our knowledge on the role of MLT in heat stress during IVM. The authors acknowledge FAPESP for funding (HF-2016/24884-3; CLVL-2015/20379-0).

183 GENE EXPRESSION OF IN VITRO-MATURATED OOCYTES CAN BE MODULATED BY FOLLICLE EXOSOMES FROM COWS KEPT UNDER THERMONEUTRAL OR HEAT STRESS CONDITIONS

2017 ◽  
Vol 29 (1) ◽  
pp. 200 ◽  
Author(s):  
F. M. Dalanezi ◽  
F. C. Destro ◽  
R. A. Ferrazza ◽  
H. D. Mogollon García ◽  
F. F. Franchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Cell Viability ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Stress Conditions ◽  
Standard Medium ◽  
Humidity Index ◽  
Lower Expression

There are several intrafollicular agents that have the ability to interfere with the metabolism and development of the oocyte, among these we highlight the exosomes (EXO). Thus, the aim of this study was to evaluate the capacity of EXO extracted from the follicular fluid of cows kept under thermoneutral or heat stress conditions to modulate oocyte maturation in vitro. Twenty-four Holstein cows were subjected to the following treatments for 14 days: heat stress (HS; n = 12), 38°C, 60% RH, temperature-humidity index = 88; and thermo-neutral (TN; n = 12), 24°C, 60% RH, temperature-humidity index = 71. Cows had their follicles aspirated when their diameter reached 9 to 12 mm; all follicles with this diameter were aspirated. All follicular fluid aspirated from cows subjected to HS or TN was pooled forming the groups (HS and TN). The EXO were obtained by ultracentrifugation of follicular fluid (120,000 × g for 70 min at 4°C, twice) and had their presence confirmed by transmission electron microscopy. Bos indicus cumulus-oocyte complexes (COC) collected from ovaries obtained in commercial slaughterhouse, were pooled in groups of 20 COC and randomly subjected to 1 of the following treatments: Control, matured in standard medium (TCM 199, supplemented with Earle’s salts, glutamine, NaHCO3, pyruvate, FSH, and amikacin); HS-EXO, matured in standard medium added with 10 µL of a solution of follicular EXO from HS cows; and TN-EXO, matured in standard medium added with 10 µL of a solution of follicular EXO from TN cows. The procedures were repeated 4 times, always with 20 COC per treatment in each replica. After 22 h of maturation, COC were recovered and the expression of genes related to apoptosis protection (BCL2), cell viability (STAT3), cell maintenance (RPL15), oocyte competence (BMP15), oxidative stress (CPT1B), cumulus cell expansion (HAS2), cell cycle (CDCA8), and heat stress protection (HSF1) were assessed. Oocyte genes were differentially expressed according to the source of EXO. Groups were statistically analysed using ANOVA and Tukey tests. All genes, except CPT1B, showed lower expression in TN-EXO oocytes when compared with control and HS-EXO (P < 0.05). CPT1B showed a higher expression in HS-EXO oocytes (P < 0.05). The results showed that the addition of EXO from exogenous follicles can modulate the expression of oocytes genes related to cell viability and survival. The lower expression of these genes in TN-EXO suggested that the EXO obtained in TN conditions attenuate several genes related to the oocytes maturation and viability. Surprisingly, the control oocytes showed a similar gene expression pattern of the HS-EXO. In conclusion, EXO derived from follicular fluid of cows submitted to TN or HS conditions can modulate the gene expression of oocytes matured in vitro. These results open new perspectives for the use of theses EXO as a tool to increase the efficiency of in vitro oocyte maturation. Financial support from FAPESP #12/18297–7.

In Silico analysis of CCGAC and CATGTG cis-regulatory elements across genomes reveals their roles in gene regulation under stress

2021 ◽  
Vol 22 ◽  
Author(s):  
Sneha Lata Bhadouriya ◽  
Abhishek Suresh ◽  
Himanshu Gupta ◽  
Sandhya Mehrotra ◽  
Divya Gupta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Functional Analysis ◽  
Heat Stress ◽  
Statistical Analysis ◽  
In Silico Analysis ◽  
Regulatory Elements ◽  
Stress Conditions ◽  
Base Pairs ◽  
Inducible Genes

Background: Plant yield closely depends on its environment and is negatively affected by abiotic stress conditions like drought, salinity, heat, and cold. Analysis of the stress-inducible genes in Arabidopsis has previously shown that CCGAC and CATGTG play a crucial role in controlling the gene expression through the binding of DREB/CBF and NAC TFs under various stress conditions, mainly drought and salinity. Methods: The pattern of these motifs is conserved, which has been analyzed in this study to find the mechanism of gene expression through spacer specificity, inter motif distance preference, functional analysis, and statistical analysis for four different plants, namely Oryza sativa, Triticum aestivum, Arabidopsis thaliana, and Glycine max. Results: The spacer frequency analysis has shown a preference for particular spacer lengths among four genomes. The spacer specificity at all the spacer lengths which predicts dominance of particular base pairs over others, was analyzed to find the preference of the sequences in the flanking region. Functional analysis on stress-regulated genes for saline, osmotic, and heat stress clearly shows that these motif frequencies with inter motif distance (0-30) in the promoter region of Arabidopsis are highest in genes which are upregulated by saline and osmotic stress and downregulated by heat stress. Conclusion: Microarray data were analyzed to confirm the role of both motifs in stress response pathways. Transcription factors seem to prefer larger motif size with repeated CCGAC and CATGTG elements. The common preference for one spacer was further validated through Box and Whisker’s statistical analysis.

Regulation of heat-inducible HSPA1A gene expression during maternal-to-embryo transition and in response to heat in in vitro-produced bovine embryos

2017 ◽  
Vol 29 (9) ◽  
pp. 1868 ◽  
Author(s):  
Jean-Marc Lelièvre ◽  
Nathalie Peynot ◽  
Sylvie Ruffini ◽  
Ludivine Laffont ◽  
Daniel Le Bourhis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Heat Shock ◽  
Transcriptional Activation ◽  
Luciferase Activity ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Nuclei ◽  
Cell Stage

In in vitro-produced (IVP) bovine embryos, a burst in transcriptional activation of the embryonic genome (EGA) occurs at the 8–16-cell stage. To examine transcriptional regulation prior to EGA, notably in response to heat stress, we asked (1) whether the spontaneous expression of a luciferase transgene that is driven by the minimal mouse heat-shock protein 1b (hspa1b) gene promoter paralleled that of HSPA1A during EGA in IVP bovine embryo and (2) whether expression of the endogenous heat-inducible iHSPA group member HSPA1A gene and the hspa1b/luciferase transgene were induced by heat stress (HS) prior to EGA. Using two culture systems, we showed that luciferase activity levels rose during the 40-h long EGA-associated cell cycle. In contrast, iHSPA proteins were abundant in matured oocytes and in blastomeres from the two-cell to the 16-cell stages. However, normalised results detected a rise in the level of HSPA1A and luciferase mRNA during EGA, when transcription was required for their protein expression. Prior to EGA, HS-induced premature luciferase activity and transgene expression were clearly inhibited. We could not, however, establish whether this was also true for HSPA1A expression because of the decay of the abundant maternal transcripts prior to EGA. In bovine embryos, heat-induced expression of hspa1b/luciferase, and most likely of HSPA1A, was therefore strictly dependent on EGA. The level of the heat-shock transcription factor 1 molecules that were found in cell nuclei during embryonic development correlated better with the embryo’s capacity for heat-shock response than with EGA-associated gene expression.

scholarly journals A Novel Mechanism for Cross-Adaptation between Heat and Altitude Acclimation: The Role of Heat Shock Protein 90

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Roy M. Salgado ◽  
Ailish C. White ◽  
Suzanne M. Schneider ◽  
Christine M. Mermier
Keyword(s):  
Heat Stress ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 90 ◽  
Heat Acclimation ◽  
Functional Roles ◽  
Hypoxic Environment ◽  
Client Proteins

Heat shock protein 90 (HSP90) is a member of a family of molecular chaperone proteins which can be upregulated by various stressors including heat stress leading to increases in HSP90 protein expression. Its primary functions include (1) renaturing and denaturing of damaged proteins caused by heat stress and (2) interacting with client proteins to induce cell signaling for gene expression. The latter function is of interest because, in cancer cells, HSP90 has been reported to interact with the transcription hypoxic-inducible factor 1α (HIF1α). In a normoxic environment, HIF1α is degraded and therefore has limited physiological function. In contrast, in a hypoxic environment, stabilized HIF1α acts to promote erythropoiesis and angiogenesis. Since HSP90 interacts with HIF1α, and HSP90 can be upregulated from heat acclimation in humans, we present a proposal that heat acclimation can mimic molecular adaptations to those of altitude exposure. Specifically, we propose that heat acclimation increases HSP90 which then stabilizes HIF1α in a normoxic environment. This has many implications since HIF1α regulates red blood cell and vasculature formation. In this paper we will discuss (1) the functional roles of HSP90 and HIF1α, (2) the interaction between HSP90 and other client proteins including HIF1α, and (3) results from in vitro studies that may suggest how the relationship between HSP90 and HIF1α might be applied to individuals preparing to make altitude sojourns.

Gene expression, oocyte nuclear maturation and developmental competence of bovine oocytes and embryos produced after in vivo and in vitro heat shock

Zygote ◽  
2016 ◽  
Vol 24 (5) ◽  
pp. 748-759 ◽  
Author(s):  
Krishna C. Pavani ◽  
Erica Baron ◽  
Pedro Correia ◽  
Joana Lourenço ◽  
Bruno Filipe Bettencourt ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Embryo Development ◽  
Target Genes ◽  
Housekeeping Genes ◽  
Early Embryo ◽  
Developmental Competence ◽  
Nuclear Maturation

SummaryThree assays were performed. In assay 1, oocytes harvested during the winter months were subjected to kinetic heat shock by stressing the oocytes at 39.5°C (HS1) or at 40.5°C (HS2) for either 6, 12, 18 or 24 h and then matured at control temperature (38.5°C). The nuclear maturation rates (NMR) of all oocytes were recorded after 24 h. In assay 2, oocytes collected year-round maturated, were implanted via in vitro fertilization (IVF) and developed for 9 days. Gene expression analysis was performed on target genes (Cx43, CDH1, DNMT1, HSPA14) with reference to the two housekeeping genes (GAPDH and SDHA) in embryos. Similarly, in assay 3, genetic analysis was performed on the embryos produced from heat-stressed oocytes (from HS1 and HS2). In assay 1, the duration of heat stress resulted in a significant decline in NMR (P < 0.05) with HS1 for maturated oocytes at 86.4 ± 4.3; 65.5 ± 0.7; 51.3 ± 0.9; 38.1 ± 1.9 and 36.3 ± 0.9, for control, 6 h, 12 h, 18 h and 24 h, respectively. For assays 2 and 3, results demonstrated that DNMT1, Cx43 and HSPA14 were down-regulated in the embryos produced in the warm with respect to the cold months (P < 0.05). A constant up- and down-regulation of DNMT1 and HSPA14 genes were observed in both HS-treated samples. Also, an inconsistent pattern of gene expression was observed in Cx43 and CDH1 genes (P < 0.05). Targeted gene expression was aberrant in embryo development, which can provide evidence on early embryo arrest and slowed embryo development.

scholarly journals Exploring the Crosstalk between Hydrostatic Pressure and Adipokines: An In Vitro Study on Human Osteoarthritic Chondrocytes

2021 ◽  
Vol 22 (5) ◽  
pp. 2745
Author(s):  
Sara Cheleschi ◽  
Sara Tenti ◽  
Marcella Barbarino ◽  
Stefano Giannotti ◽  
Francesca Bellisai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antioxidant Enzymes ◽  
Hydrostatic Pressure ◽  
Cyclin D1 ◽  
Mirna Gene ◽  
In Vitro Study ◽  
B Cell Lymphoma ◽  
Superoxide Anion Production

Obesity is a risk factor for osteoarthritis (OA) development and progression due to an altered biomechanical stress on cartilage and an increased release of inflammatory adipokines from adipose tissue. Evidence suggests an interplay between loading and adipokines in chondrocytes metabolism modulation. We investigated the role of loading, as hydrostatic pressure (HP), in regulating visfatin-induced effects in human OA chondrocytes. Chondrocytes were stimulated with visfatin (24 h) and exposed to high continuous HP (24 MPa, 3 h) in the presence of visfatin inhibitor (FK866, 4 h pre-incubation). Apoptosis and oxidative stress were detected by cytometry, B-cell lymphoma (BCL)2, metalloproteinases (MMPs), type II collagen (Col2a1), antioxidant enzymes, miRNA, cyclin D1 expressions by real-time PCR, and β-catenin protein by western blot. HP exposure or visfatin stimulus significantly induced apoptosis, superoxide anion production, and MMP-3, -13, antioxidant enzymes, and miRNA gene expression, while reducing Col2a1 and BCL2 mRNA. Both stimuli significantly reduced β-catenin protein and increased cyclin D1 gene expression. HP exposure exacerbated visfatin-induced effects, which were counteracted by FK866 pre-treatment. Our data underline the complex interplay between loading and visfatin in controlling chondrocytes’ metabolism, contributing to explaining the role of obesity in OA etiopathogenesis, and confirming the importance of controlling body weight for disease treatment.

scholarly journals Heat Shock Protein 70 Improves In Vitro Embryo Yield and Quality from Heat Stressed Bovine Oocytes

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1794
Author(s):  
Konstantina Stamperna ◽  
Themistoklis Giannoulis ◽  
Eleni Dovolou ◽  
Maria Kalemkeridou ◽  
Ioannis Nanas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Cumulus Cells ◽  
Intramolecular Interactions ◽  
Developmental Competence ◽  
Embryo Yield ◽  
Bovine Oocytes

Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.

scholarly journals Cytoprotective Effects of Taurine on Heat-Induced Bovine Mammary Epithelial Cells In Vitro

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 258
Author(s):  
Hui Bai ◽  
Tingting Li ◽  
Yan Yu ◽  
Ningcong Zhou ◽  
Huijuan Kou ◽  
...  
Keyword(s):  
T Cells ◽  
Heat Stress ◽  
Heat Shock ◽  
T Antigen ◽  
Mammary Epithelial ◽  
Epithelial Cell Line ◽  
Heat Shock Factor 1 ◽  
Alveolar Cells ◽  
Bovine Mammary Epithelial Cells

It is a widely known that heat stress induces a reduction in milk production in cows and impairs their overall health. Studies have shown that taurine protects tissues and organs under heat stress. However, there have yet to be studies showing the functions of taurine in mammary alveolar cells-large T antigen (MAC-T) (a bovine mammary epithelial cell line) cells under heat shock. Therefore, different concentrations of taurine (10 mM, 50 mM, and 100 mM) were tested to determine the effects on heat-induced MAC-T cells. The results showed that taurine protected the cells against heat-induced damage as shown by morphological observations in conjunction with suppressed the translocation and expression of heat shock factor 1 (HSF1). Moreover, taurine not only reversed the decline in antioxidase (superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX)) activities but also attenuated the accumulation of malondialdehyde (MDA). Meanwhile, mitochondrial damage (morphology and complex I activity) resulting from heat exposure was mitigated. Taurine also alleviated the rates of cell apoptosis and markedly depressed the mRNA expressions of BCL2 associated X, apoptosis regulator (BAX) and caspase3. Furthermore, compared with the heat stress (HS) group, the protein levels of caspase3 and cleaved caspase3 were decreased in all taurine groups. In summary, taurine improves the antioxidant and anti-apoptosis ability of MAC-T cells thereby alleviates damage of cells due to heat insults.

scholarly journals The Emerging Terbinafine-Resistant Trichophyton Epidemic: What Is the Role of Antifungal Susceptibility Testing?

Dermatology ◽  
2021 ◽  
pp. 1-20
Author(s):  
Julia J. Shen ◽  
Maiken C. Arendrup ◽  
Shyam Verma ◽  
Ditte Marie L. Saunte
Keyword(s):  
Gene Mutation ◽  
Susceptibility Testing ◽  
Geometric Mean ◽  
Antifungal Susceptibility ◽  
Antifungal Susceptibility Testing ◽  
Squalene Epoxidase ◽  
Exclusion Criteria ◽  
Huge Variation

<b><i>Background:</i></b> Dermatophytosis is commonly encountered in the dermatological clinics. The main aetiological agents in dermatophytosis of skin and nails in humans are <i>Trichophyton</i> (<i>T</i>.) <i>rubrum</i>, <i>T. mentagrophytes</i> and <i>T. interdigitale</i> (former <i>T. mentagrophytes-</i>complex). Terbinafine therapy is usually effective in eradicating infections due to these species by inhibiting their squalene epoxidase (SQLE) enzyme, but increasing numbers of clinically resistant cases and mutations in the SQLE gene have been documented recently. Resistance to antimycotics is phenotypically determined by antifungal susceptibility testing (AFST). However, AFST is not routinely performed for dermatophytes and no breakpoints classifying isolates as susceptible or resistant are available, making it difficult to interpret the clinical impact of a minimal inhibitory concentration (MIC). <b><i>Summary:</i></b> PubMed was systematically searched for terbinafine susceptibility testing of dermatophytes on October 20, 2020, by two individual researchers. The inclusion criteria were <i>in vitro</i> terbinafine susceptibility testing of <i>Trichophyton (T.) rubrum</i>, <i>T. mentagrophytes</i> and <i>T. interdigitale</i> with the broth microdilution technique. The exclusion criteria were non-English written papers. Outcomes were reported as MIC range, geometric mean, modal MIC and MIC<sub>50</sub> and MIC<sub>90</sub> in which 50 or 90% of isolates were inhibited, respectively. The reported MICs ranged from &#x3c;0.001 to &#x3e;64 mg/L. The huge variation in MIC is partly explained by the heterogeneity of the <i>Trichophyton</i> isolates, where some originated from routine specimens (wild types) whereas others came from non-responding patients with a known SQLE gene mutation. Another reason for the great variation in MIC is the use of different AFST methods where MIC values are not directly comparable. High MICs were reported particularly in isolates with SQLE gene mutation. The following SQLE alterations were reported: F397L, L393F, L393S, H440Y, F393I, F393V, F415I, F415S, F415V, S443P, A448T, L335F/A448T, S395P/A448T, L393S/A448T, Q408L/A448T, F397L/A448T, I121M/V237I and H440Y/F484Y in terbinafine-resistant isolates.

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