218 GENE AND PROTEIN EXPRESSION OF ANTIOXIDANT ENZYMES IN BOVINE EMBRYOS EXPOSED TO HEAT SHOCK

In a previous study, we reported that 8-cell-stage embryos exposed to a temperature of 41°C for 6 h had significantly increased embryonic mortality and intracellular reactive oxygen species (ROS). There have been some reports that ROS regulates the expression of genes encoding antioxidant enzymes in culture cells. In this study, we investigated the gene and protein expression of antioxidant enzymes in bovine 8-cell-stage embryos exposed to heat shock. In vitro-produced bovine embryos were used for the experiment. Embryos were cultured with CR1aa + 5% FCS at 38.5°C in 5% CO2 and 5% O2. On Day 2 after fertilization, 8-cell-stage embryos were exposed to heat shock at 41°C in 5% CO2 and 5% O2 for 6 h (HS). Eight-cell-stage embryos cultured at 38.5°C in 5% CO2 and 5% O2 were sampled at the same collection time as controls. After HS, 20 embryos were immediately collected for gene expression analysis. Expression of heat shock protein 70 (HSP70), CuZn-containing superoxide dismutase (SOD), catalase (CAT), and glutathione peroxide (GPx) genes was examined by real-time polymerase chain reaction. Twenty embryos were also collected after 3 h of HS (3 h) and at 18 h after HS (18 h) to evaluate the expression of proteins. Expression of HSP70, SOD, and CAT proteins was examined by Western blotting. Both the gene and protein expression levels of HS groups were normalized to those of the controls to obtain the relative expression levels. All results were analyzed by Student’s t-test. Expression of the HSP70 gene significantly increased in HS embryos (P < 0.05). Expression of the SOD and CAT genes tended to increase in HS embryos (P < 0.07), but there were no significant differences in expression of the GPx gene. There was no significant difference in protein expression in all the antioxidant enzymes in 3-h-sampled embryos. Expression of the HSP70 protein increased significantly in heat-shocked embryos sampled at 18 h (P < 0.05). These results indicate that expression of antioxidant enzymes was not greatly affected in 8-cell-stage embryos exposed to HS. Thus, these results suggest the possibility that the early-stage embryos were stressed and damaged from heat shock because of their poor antioxidative potency. Table 1.Gene and protein expression of embryos This work was supported by KAKENHI [16780209, Grant-in-Aid for Young Scientists (B)].

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

Reproduction Fertility and Development ◽

10.1071/rd15504 ◽

2017 ◽

Vol 29 (9) ◽

pp. 1868 ◽

Cited By ~ 7

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.

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Effect of EPA on Neonatal Pig Sertoli Cells “In Vitro”: A Possible Treatment to Help Maintain Fertility in Pre-Pubertal Boys Undergoing Treatment With Gonado-Toxic Therapies

Frontiers in Endocrinology ◽

10.3389/fendo.2021.694796 ◽

2021 ◽

Vol 12 ◽

Author(s):

Iva Arato ◽

Veronica Ceccarelli ◽

Francesca Mancuso ◽

Catia Bellucci ◽

Cinzia Lilli ◽

...

Keyword(s):

Protein Expression ◽

Sertoli Cells ◽

Fold Change ◽

Control Group ◽

Mrna Levels ◽

Expression Levels ◽

Chemotherapy Drugs ◽

Gene And Protein Expression ◽

Pubertal Boys

The incidence of cancer in pre-pubertal boys has significantly increased and, it has been recognized that the gonado-toxic effect of the cancer treatments may lead to infertility. Here, we have evaluated the effects on porcine neonatal Sertoli cells (SCs) of three commonly used chemotherapy drugs; cisplatin, 4-Hydroperoxycyclophosphamide and doxorubicin. All three drugs induced a statistical reduction of 5-hydroxymethylcytosine in comparison with the control group, performed by Immunofluorescence Analysis. The gene and protein expression levels of GDNF, were significantly down-regulated after treatment to all three chemotherapy drugs comparison with the control group. Specifically, differences in the mRNA levels of GDNF were: 0,8200 ± 0,0440, 0,6400 ± 0,0140, 0,4400 ± 0,0130 fold change at 0.33, 1.66, and 3.33μM cisplatin concentrations, respectively (**p < 0.01 at 0.33 and 1.66 μM vs SCs and ***p < 0.001 at 3.33μM vs SCs); 0,6000 ± 0,0340, 0,4200 ± 0,0130 fold change at 50 and 100 μM of 4-Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7000 ± 0,0340, 0,6200 ± 0,0240, 0,4000 ± 0,0230 fold change at 0.1, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 μM vs SCs and ***p < 0.001 at 1 μM vs SCs). Differences in the protein expression levels of GDNF were: 0,7400 ± 0,0340, 0,2000 ± 0,0240, 0,0400 ± 0,0230 A.U. at 0.33, 1.66, and 3.33μM cisplatin concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7300 ± 0,0340, 0,4000 ± 0,0130 A.U. at 50 and 100 μM of 4- Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,6200 ± 0,0340, 0,4000 ± 0,0240, 0,3800 ± 0,0230 A.U. at 0.l, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 μM vs SCs and ***p < 0.001 at 1 μM vs SCs). Furthermore, we have demonstrated the protective effect of eicosapentaenoic acid on SCs only at the highest concentration of cisplatin, resulting in an increase in both gene and protein expression levels of GDNF (1,3400 ± 0,0280 fold change; **p < 0.01 vs SCs); and of AMH and inhibin B that were significantly recovered with values comparable to the control group. Results from this study, offers the opportunity to develop future therapeutic strategies for male fertility management, especially in pre-pubertal boys.

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142 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN KSOM, CR1AA, OR KSOM/CR1aa

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab142 ◽

2005 ◽

Vol 17 (2) ◽

pp. 221 ◽

Cited By ~ 1

Author(s):

M.R.B. Mello ◽

C.E. Ferguson ◽

A.S. Lima ◽

M.B. Wheeler

Keyword(s):

Embryo Culture ◽

Cumulus Cells ◽

Bovine Embryos ◽

In Vitro Production ◽

Cell Stage ◽

In Vitro Development ◽

Significant Difference ◽

Developmental Rates ◽

Vitro Development

In vitro embryo culture is an important step of in vitro production of bovine embryos. It has been shown that IVF-derived bovine embryos cultured in KSOM or CR1aa have high development rates. In our laboratory, we have observed that 8-cell embryos are morphologically superior when embryos are cultured in KSOM whereas blastocysts are morphologically superior when embryos are cultured in CR1aa. Based on these observations, we hypothesized that development of IVF-derived bovine embryos can be improved by sequential use of these media (KSOM and CR1aa). The aim of this experiment was to compare the in vitro development of bovine embryos cultured in KSOM, CR1aa or KSOM/CR1aa supplemented with BSA at Day 0 and BSA and FBS at Day 3. In order to accomplish the sequential culture, fertilized oocytes where cultured in KSOM to the 8-cell stage and then transferred to CR1aa for further development. Oocytes were purchased from Bomed (Madison, WI, USA), and after 22 hours of maturation were fertilized with frozen-thawed semen for 5 hours at 39°C in 5% CO2. After fertilization, the presumptive zygotes were denuded from cumulus cells by votexing and were randomly allotted to one of 3 treatments: (1) cultured only in KSOM (n = 110), (2) cultured only in CR1aa (n = 102), and (3) cultured in KSOM in the first 3 days and then in CR1aa from Day 3 to Day 9 (n = 110). The embryo culture was carried out in 50-μL droplets of medium that were placed in an airtight modular incubator filled with 5% CO2, 5% O2 and 90% N2. The embryos were evaluated on Days 6 to 9 post insemination. All embryo developmental rates were calculated from presumptive zygotes. The Day 6 morula rates were 52%, 40%, and 47% for KSOM, CR1aa, and KSOM/CR1aa, respectively. The Day 7 blastocyst rates for KSOM (40%), CR1aa (25%), and KSOM/CR1aa (30%) were not significantly different; however, Day 9 hatched blastocyst rates were significantly higher (P < 0.05) for KSOM (22%) compared to CR1aa (9%) but not different from KSOM/CR1aa (14%). Regarding embryo quality, Day 7 transferable embryos rates (Grade 1 and Grade 2) were 35%, 25%, and 30%, respectively for KSOM, CR1aa, and KSOM/CR1aa; however, no significant difference was observed. These results indicate that IVF-derived bovine embryos can develop in KSOM, CR1aa, or KSOM/CR1aa with no significant difference among morula, blastocyst and hatched blastocyst rates. However, the combination of KSOM and CR1aa during in vitro culture did not decrease the morula and blastocyst rates.

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77 EFFECT OF HEAT SHOCK DURING IN VITRO MATURATION ON HETEROCHROMATIN COMPACTION IN BOVINE EMBRYOS AT 4- AND 8-CELL STAGES: PRELIMINARY STUDY

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab77 ◽

2015 ◽

Vol 27 (1) ◽

pp. 132

Author(s):

L. S. A. Camargo ◽

T. Aguirre-Lavin ◽

P. Adenot ◽

T. D. Araujo ◽

E. D. Souza ◽

...

Keyword(s):

Heat Shock ◽

In Vitro Maturation ◽

Gene Activation ◽

Developmental Competence ◽

Bovine Embryos ◽

Cleavage Rate ◽

Cell Stage ◽

Morphological Criteria ◽

Preliminary Study

High temperatures cause several reproductive losses in cattle. Under in vitro conditions, heat shock decreases oocyte developmental competence and influences embryonic gene expression (Gendelman and Roth 2012 Anim. Reprod. Sci. 134, 125–134). This preliminary study aimed to evaluate whether heat shock during oocyte in vitro maturation (IVM) could have any further effect on chromatin remodelling of fertilized embryos at 4- and 8-cell stages, once such modifications are required for the gene activation in bovine embryos. We evaluated the distribution of heterochromatin 1 (HP1β) and of histone H3 trimethylated at lysine 9 (H3K9me3), both reportedly correlated with heterochromatin formation, in 4- and 8-cell stage embryos derived from control (C) and heat-shocked (HS) bovine oocytes. Immature cumulus-oocyte complexes (COC) collected from crossbred cows in Brazil were exposed for 12 h to 38.8°C (C group) or 41.0°C (HS group) followed by 12 h at 38.8°C, totalizing 24 h of IVM at 5% CO2 in air. Oocytes were in vitro fertilized (IVF) with non-sexed sperm and denuded zygotes were in vitro cultured in CR2aa medium at 38.8°C and 5% CO2, 5% O2 and 90% N2. Four- and 8-cell embryos at 44 h post-IVF were fixed in 4% paraformaldehyde and stained with anti-mouse HP1β and anti-rabbit H3K9me3 first antibodies. Immunofluorescence was evaluated by confocal microscopy (Zeiss LSM 700, MIMA platform, INRA) and 3D images processed by ZEN Lite software (Zeiss, Jena, Germany). Three different distribution patterns of fluorescence were identified based on morphological criteria: diffuse, little clusters, and big clusters. Proportions of embryos in every distribution pattern were compared between C and HS groups by Chi-squared test. No difference (P > 0.05) on cleavage rate was found between C and HS groups until 44 h post-fertilization. Embryos at the 4-cell stage from HS group displayed an increased (P < 0.01) proportion of nuclei with H3K9m3 big clusters (44%, n = 7/16 embryos), whereas embryos from C group displayed only few nuclei with this pattern (5%, n = 1/18). At the 8-cell stage, distribution of H3K9m3 was similar (P > 0.05) between C and HS groups. For HP1β, embryos at the 4-cell stage from HS group displayed an increased (P < 0.05) proportion of nuclei with little clusters (81%, n = 13/16 embryos), whereas embryos from C group had low proportion of nuclei with this same pattern (40%, n = 7/18). Mostly 4-cell stage embryos from C group presented the diffuse pattern (61%, n = 11/18 v.18%, n = 3/16 in the HS group; P < 0.05). At the 8-cell stage, some embryos from the C group (31%, n = 5/16) still showed nuclei with diffuse distribution of HP1β, whereas no nucleus with this pattern was found for the HS group. These preliminary data suggest that bovine embryos derived from heat-shocked oocytes can display precocious heterochromatin compaction, represented by the accumulation of H3K9me3 and HP1β at the 4-cell stage, compared with embryos derived from non-heat-shocked oocytes, which may affect embryonic genome activation with consequences for further gene expression.Research was supported by CNPq, FAPEMIG, FAPES and Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73).

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145 CHANGES IN OXYGEN COMSUMPTION RATES OF EMBRYOS IN KOREAN CATTLE

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab145 ◽

2010 ◽

Vol 22 (1) ◽

pp. 231

Author(s):

C. Y. Choe ◽

S. R. Cho ◽

J. K. Son ◽

S. H. Choi ◽

C. Y. Cho ◽

...

Keyword(s):

Oxygen Consumption ◽

Embryo Quality ◽

Blastocyst Stage ◽

Bovine Embryo ◽

Bovine Embryos ◽

Cell Stage ◽

Significant Difference ◽

Morula Stage

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was carried out to identify whether oxygen consumption rates measured in bovine embryos using SECM can be used as a standard criteria to evaluate bovine embryo quality. Oxygen consumption of bovine embryos at various developmental stages was measured and analyzed using SECM and ANOVA analysis, respectively. We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell stage to morula stage), indicating that oxygen consumption reflects the cell number (5.2-7.6 × 1014 mol-1 s-1 v. 1.2-2.4 × 1014 mol-1 s-1, P < 0.05). There was no significant difference between 2-cell-stage embryos and 8-cell-stage embryos. In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos (4.0 × 1014 mol-1 s-1 v. 2.4 × 1014 mol-1 s-1, P < 0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst-stage embryos (P > 0.05). Good-quality embryos with grade 1 or 2 showed significantly higher oxygen consumption than grade 3 or 4 embryos. These results showed that SECM could measure oxygen consumption in bovine embryos and the oxygen consumption could reflect embryonic development stage and embryo quality.

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200 STAGE-SPECIFIC EXPRESSION PATTERNS OF THE INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR DURING BOVINE PRE-IMPLANTATION EMBRYONIC DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab200 ◽

2015 ◽

Vol 27 (1) ◽

pp. 191 ◽

Cited By ~ 1

Author(s):

F. Poppicht ◽

H. Stinshoff ◽

C. Wrenzycki

Keyword(s):

Growth Factor ◽

Embryonic Development ◽

Protein Expression ◽

Mrna Expression ◽

Early Embryonic Development ◽

Insulin Like Growth Factor ◽

Bovine Embryos ◽

Cell Stage ◽

Igf1r Expression

Insulin-like growth factor 1 (IGF1) is a key regulator in early embryonic development, influencing physiological processes and stimulating growth and development (Fowden et al. 2003). Supplementing IGF1 during in vitro culture of bovine embryos improved cleavage and developmental rates while it reduced apoptosis (Byrne et al. 2002). The signal transduction of IGF1 is performed by its binding to the insulin-like growth factor 1 receptor (IGF1R). At the mRNA level, IGF1R is expressed throughout pre-implantation embryonic development and was identified as a potential marker of good quality embryos (Yaseen et al. 2001). However, information on protein level is rare. Therefore, protein expression of the IGF1R during early embryonic development in vitro was analysed in the present study by immunofluorescence staining. Furthermore, the mRNA expression of the IGF1R was investigated by RT-qPCR. In vitro derived embryos of different stages (2-cell, 4-cell, 8-cell, 16-cell stage, morula, blastocyst, and expanded blastocyst) were either directly subjected to immunofluorescence staining or frozen at –80°C for use in RT-qPCR. Staining was performed with a peptide antibody against two peptide sequences of the bovine IGF1R α unit, which was specifically produced. Pixel intensity of immunofluorescence was measured and a mean grey value was calculated using the cellsens® software (Olympus, Hamburg, Germany). Data were analysed by one-way ANOVA followed by a Tukey's test using SigmaStat 3.5 Software (Systat Software GmbH, Erkrath, Germany). The detection of the IGF1R mRNA and protein was possible in all stages of embryonic development beginning at the 2-cell stage up to the expanded blastocyst. The maximal mRNA expression could be observed in 2- and 4-cell embryos. It significantly decreased to the 8-cell stage, followed by an increase up to the expanded blastocyst. The IGF1R protein was mainly localised in the plasma membrane of single blastomeres and also weakly in the cytoplasm. Mean grey values are highest in the 2-cell stage, showing a significant decline up to the 16-cell stage and an increase again until the expanded blastocyst. The mRNA and protein expression showed similar patterns during early embryonic development. IGF1R expression started to increase at the 8-cell stage (mRNA) and 16-cell stage (protein) indicating a link to the maternal-embryonic transition. For the first time, these results show that in bovine embryos, the IGF1R expression is related to the activation of the embryonic genome. We gratefully acknowledge the financial support of the H. Wilhelm Schaumann Foundation (Hamburg, Germany).

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Effects of kiwifruit extracts on colonic gene and protein expression levels in IL-10 gene-deficient mice

British Journal Of Nutrition ◽

10.1017/s0007114511005241 ◽

2011 ◽

Vol 108 (1) ◽

pp. 113-129 ◽

Cited By ~ 11

Author(s):

Shelley J. Edmunds ◽

Nicole C. Roy ◽

Marcus Davy ◽

Janine M. Cooney ◽

Matthew P. G. Barnett ◽

...

Keyword(s):

Protein Expression ◽

Dietary Intervention ◽

Dietary Recommendations ◽

Susceptible Host ◽

Expression Levels ◽

Background Strain ◽

Gene And Protein Expression ◽

Inflammatory Processes

Inflammatory bowel disease (IBD) is a collective term for conditions characterised by chronic inflammation of the gastrointestinal tract involving an inappropriate immune response to commensal micro-organisms in a genetically susceptible host. Previously, aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (A. deliciosa) have demonstrated anti-inflammatory activity usingin vitromodels of IBD. The present study examined whether these kiwifruit extracts (KFE) had immune-modulating effectsin vivoagainst inflammatory processes that are known to be increased in patients with IBD. KFE were used as a dietary intervention in IL-10-gene-deficient (Il10− / −) mice (anin vivomodel of IBD) and the C57BL/6J background strain in a 3 × 2 factorial design. While allIl10− / −mice developed significant colonic inflammation compared with C57BL/6J mice, this was not affected by the inclusion of KFE in the diet. These findings are in direct contrast to our previous study where KFE reduced inflammatory signalling in primary cells isolated fromIl10− / −and C57BL/6J mice. Whole-genome gene and protein expression level profiling indicated that KFE influenced immune signalling pathways and metabolic processes within the colonic tissue; however, the effects were subtle. In particular, expression levels across gene sets related to adaptive immune pathways were significantly reduced using three of the four KFE in C57BL/6J mice. The present study highlights the importance of investigating food components identified by cell-based assays with appropriatein vivomodels before making dietary recommendations, as a food that looks promisingin vitromay not be effectivein vivo.

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