scholarly journals Retinol Improves Development of Bovine Oocytes Compromised by Heat Stress During Maturation

2004 ◽  
Vol 87 (8) ◽  
pp. 2449-2454 ◽  
Author(s):  
J.L. Lawrence ◽  
R.R. Payton ◽  
J.D. Godkin ◽  
A.M. Saxton ◽  
F.N. Schrick ◽  
...  
Keyword(s):  
Heat Stress ◽  
Bovine Oocytes

37 Effect of invivo and invitro heat stress on DNA methylation and DNA hydroxymethylation of bovine oocytes and embryos

2021 ◽  
Vol 33 (2) ◽  
pp. 126
Author(s):  
F. A. Diaz ◽  
E. J. Gutierrez ◽  
B. A. Foster ◽  
P. T. Hardin ◽  
K. R. Bondioli
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Dna Hydroxymethylation ◽  
Early Embryos ◽  
Oocyte Collection ◽  
Maturation Rate ◽  
Bovine Oocytes ◽  
Fluorescence Immunohistochemistry

Reduced reproductive performance is one of the main effects caused by heat stress in cattle. Its negative effects have been observed at the transcriptional, biochemical, morphological, and developmental levels on the oocyte and embryo. There are no studies evaluating the effect of heat stress on the epigenetic profile of bovine oocytes and early embryos. The objective of this study was to evaluate the effect of invivo and invitro heat stress on DNA methylation and DNA hydroxymethylation in bovine MII oocytes, pronuclear, and 2- to 4-cell stage embryos. Seven Bos taurus crossbred nonpregnant, non-lactating beef cows located in Saint Gabriel, Louisiana (30.269746, −91.103357) were used for oocyte collection. Dominant follicle removal was performed 5 days before oocyte collection. Cumulus–oocyte complexes were collected by ovum pickup from follicles >2mm. Samples were collected during the summer (August) and winter (February) (5 collections each). Three treatments were utilised: invivo heat stress (August samples), invitro heat stress (February samples subjected to 41°C during the first 12h of IVM and then to 38.5°C during the next 12h of IVM), and control (February samples IVM at 38.5°C). All oocytes collected per treatment were assigned to 3 developmental stages: MII oocytes, pronuclear, and 2- to 4-cell stage embryos. Embryos were obtained through standard IVF. DNA methylation and DNA hydroxymethylation was assessed by fluorescence immunohistochemistry utilising primary antibodies against 5′-methylcytosine and 5′-hydromethylcytosine and secondary antibodies Alexa Fluor 488 and Alexa Fluor 546, respectively. Samples were visualised with a fluorescence deconvolution microscope, and immunofluorescence data were expressed as corrected relative fluorescence per nucleus. Results were analysed by the Type III test of fixed effects and Tukey media separation utilising the Proc Glimmix of SAS 9.4 (P<0.05). Maturation rate, 2 pronuclei (2PN) rate, cleavage rate, and 2- to 4-cell rate were analysed by Chi-square. There was no difference in maturation rate (88.19±7.57, 82.91±5.18, 94.51±5.04; P=0.2516), 2PN rate (79.34±10.23, 93.75±7.21, 81.74±12.53; P=0.1757), cleavage rate (79.26±2.69, 70.65±7.22, 81.85±16.65; P=0.2388) and 2- to 4-cell rate (69.38±7.83, 81.25±10.34, 61.11±11.69; P=0.4392) between invivo and invitro heat stress compared with control, respectively. No difference was found in DNA methylation (P=0.0537) or DNA hydroxymethylation (P=0.4632) between treatments in MII oocytes. When evaluating the paternal and maternal pronuclei, there was no difference in DNA methylation (P=0.9766; P=0.1954, respectively) or DNA hydroxymethylation (P=0.6440; P=0.1932, respectively) between invivo and invitro heat stress compared with control. Similarly, there was no difference in DNA methylation (P=0.0903) or DNA hydroxymethylation (P=0.2452) between treatments when evaluating the 2- to 4-cell embryos. In conclusion, we detected no effect of invivo or invitro heat stress on MII oocytes and early embryos when evaluating global DNA methylation and hydroxymethylation through fluorescence immunohistochemistry.

scholarly journals Effect of heat stress during in vitro maturation on developmental competence of vitrified bovine oocytes

2017 ◽  
Vol 52 ◽  
pp. 48-51 ◽  
Author(s):  
M Vendrell-Flotats ◽  
N Arcarons ◽  
E Barau ◽  
M López-Béjar ◽  
T Mogas
Keyword(s):  
Heat Stress ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Bovine Oocytes

186 TOTAL RNA AND TRANSCRIPT ABUNDANCE IN HEAT-STRESSED BOVINE OOCYTES AND SURROUNDING CUMULUS

2008 ◽  
Vol 20 (1) ◽  
pp. 172 ◽  
Author(s):  
R. R. Payton ◽  
L. A. Rispoli ◽  
J. L. Edwards
Keyword(s):  
Heat Stress ◽  
Block Design ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Molecular Probes ◽  
Hsp70 Expression ◽  
Developmental Potential ◽  
Total Rna ◽  
Lysis Buffer ◽  
Bovine Oocytes

Previous efforts of our laboratory revealed heat-induced perturbations in bovine oocytes that were coincident with reduced developmental potential. The objective of this study was to examine the effect of heat stress on total RNA and specific transcripts during oocyte maturation. After cumulus–oocyte complex (COC) collection, a subset at the germinal vesicle (GV) stage was denuded. Oocytes and surrounding cumulus were stored in RNA lysis buffer. Remaining COCs were matured for 24 h at 38.5 or 41�C (first 12 h of IVM followed by 38.5�C). At 12 and 24 h of IVM, subsets of COCs were denuded and stored in lysis buffer. Four to eight-cell embryos and blastocysts (developmental controls) derived from control and heat-stressed oocytes were collected at 40.5 and 192 h after IVF, respectively. Total RNA was isolated (PicoPure™, Molecular Devices Corp., Sunnyvale, CA, USA), quantified (RiboGreen�, Molecular Probes, Inc., Eugene, OR, USA), spiked with GFP cRNA (for normalization), and reverse transcribed with random primers. Real-time PCR was performed in triplicate using 0.1 oocyte or embryo equivalents or 100 pg cumulus RNA for analysis of BMP15, GDF9, HSP70, cyclin B1, poly(A) polymerase (PAP), and 18S and 28S rRNAs. Data were calibrated to GV-stage and analyzed using the ΔΔCt method. The experiment was replicated 9 times. Data were analyzed as a randomized block design using GLIMMIX (SAS; SAS Institute, Inc., Cary, NC, USA). Heat stress for the first 12 h of IVM reduced blastocyst formation after IVF (20.4%v. 30.5%; SEM = 1.9; P < 0.001), but had no effect on total RNA in oocytes (1.9 to 2.2 ng per oocyte; SEM = 0.7; P > 0.7) or in 4- to 8-cell embryos derived from heat-stressed oocytes (2.4 and 2.9 ng per embryo for 38.5 and 41�C, respectively; SEM = 0.7; P > 0.5). Total RNA was higher in blastocysts derived from heat-stressed oocytes (3.7 v. 5.4 ng; SEM = 0.7; P < 0.03). Heat stress during IVM did not alter relative abundance of transcripts examined in oocytes or resulting embryos. However, abundance of 18S, 28S, and GDF9 decreased in oocytes during IVM (P < 0.05). A general trend also existed for abundance to decrease as development after IVF progressed (oocyte >4- to 8-cell > blastocyst). In surrounding cumulus, HSP70 was increased by 41�C at 12 h but reduced by 24 h of IVM compared to controls (P < 0.003). Regardless of IVM temperature, PAP was higher at 12 and 24 h compared to GV stage (P < 0.0001). A stepwise decrease occurred in cyclin B1 from GV stage to 24 h (P < 0.0001). During IVM, no differences were observed in cumulus for 18S or 28S; GDF9 and BMP15 were not detectable. In the context of our study, transcripts examined were not necessarily informative of developmental potential of heat-stressed oocytes. However, increased HSP70 expression in cumulus following exposure to 41�C suggests that consequences of heat stress on oocytes may be mediated, in part, by surrounding cumulus. The results of this study are a first step toward identifying maternal transcripts in oocytes and surrounding cumulus that may be targets for development of therapeutic strategies to improve oocyte quality.

The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress

Zygote ◽  
2011 ◽  
Vol 20 (3) ◽  
pp. 249-259 ◽  
Author(s):  
Hisashi Nabenishi ◽  
Hiroshi Ohta ◽  
Toshihumi Nishimoto ◽  
Tetsuo Morita ◽  
Koji Ashizawa ◽  
...  
Keyword(s):  
Heat Stress ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Formation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Bovine Oocytes

SummaryIn the present study, we investigated the effects of various concentrations of cysteine (0.0, 0.6, 1.2 and 1.8 mM) added to the maturation medium on nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS: set at 39.5 °C for 5 h, 40.0 °C for 5 h, 40.5 °C for 6 h, and 40.0 °C for 4 h versus 38.5 °C for 20 h as the control group). This regime mimicked the circadian rhythm of the vaginal temperature of lactating dairy cows during the summer season in southwestern Japan. Moreover, we also evaluated the oocyte's reactive oxygen species (ROS) and glutathione (GSH) levels and the apoptosis levels of the oocytes and cumulus cells in the presence or absence of 1.2 mM cysteine. As a result, HS in the without-cysteine group significantly suppressed (p < 0.05) both the nuclear maturation rate up to the metaphase (M)II stage and the blastocyst formation rate compared with that of the control group. In addition, this group showed significantly higher (p < 0.05) ROS levels and significantly lower (p < 0.05) GSH levels than those of the control group. Moreover, the level of TdT-mediated dUTP nick end labelling (TUNEL)-positive cumulus cells in the HS without-cysteine group was significantly higher (p < 0.05) than that of the control group. However, the addition of 1.2 mM cysteine to the maturation medium restored not only the nuclear maturation, blastocyst formation rates and GSH contents, but also increased the ROS and TUNEL-positive levels of the cumulus cells, but not oocytes, to that of the control group. These results indicate that the addition of 1.2 mM cysteine during in vitro maturation (IVM) may alleviate the influence of heat stress for oocyte developmental competence by increasing GSH content and inhibiting the production of oocyte ROS followed by apoptosis of cumulus cells.

Expression and localisation of heat shock protein 70 in cultured bovine oocytes and embryos

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 39-50 ◽  
Author(s):  
Sheldon J. Kawarsky ◽  
W. Allan King
Keyword(s):  
Heat Stress ◽  
Elevated Temperature ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Laser Scanning Microscopy ◽  
Meiotic Spindle ◽  
Hsp70 Mrna ◽  
Bovine Oocytes

Effects of elevated in vitro temperature on in vitro produced early bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (hsp70). In vitro matured bovine oocytes, 2-cell and 8-cell embryos, and day 9 hatched blastocysts subjected to control and elevated temperature conditions were analysed by semiquantitative reverse transcription polymerase chain reaction methods for hsp70 mRNA expression. Results revealed the expression of hsp70 mRNA under control conditions and that early embryos can respond to heat stress by transcribing hsp70 mRNA. Confocal laser scanning microscopy used to localise the hsp70 protein in oocytes and embryos revealed that the distribution of hsp70 in the ooplasm of immature and mature oocytes is unaffected by exposure to elevated temperatures and that this protein was closely associated with the meiotic spindle, indicating its possible role in stabilising this structure. In 8-cell embryos derived under control conditions, hsp70 was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed to elevated temperature. In heat-stressed hatched blastocysts, a more even distribution was noted following heat stress relative to corresponding controls, indicating their competence to respond to elevated temperature.

195 HEAT STRESS ALTERS THE TRANSCRIPTOME OF MATURING BOVINE OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 228
Author(s):  
L. A. Rispoli ◽  
R. R. Payton ◽  
C. Gondro ◽  
A. M. Saxton ◽  
J. L. Edwards
Keyword(s):  
Protein Synthesis ◽  
Heat Stress ◽  
Elevated Temperature ◽  
Mitochondrial Function ◽  
De Novo ◽  
Bovine Genome ◽  
Rna Extraction ◽  
Developmental Competence ◽  
The Impact ◽  
Bovine Oocytes

Direct exposure of maturing oocytes to a physiologically relevant elevated temperature reduces embryo development after fertilisation and has been coincident with reduced de novo protein synthesis. Mechanisms responsible for heat-induced reductions in protein synthesis are unknown but may be related to alterations in the transcriptome of the maturing oocyte. To determine the extent to which this may occur, the impact of heat stress on the maternal pool of RNA in bovine oocytes was assessed using microarrays. After maturation for 24 h at 38.5°C (control) or 41°C (first 12 h only, 38.5°C thereafter; heat stress) oocytes were denuded from associated cumulus cells and lysed for RNA extraction or underwent IVF to assess developmental competence. Total RNA from oocytes was amplified by 3′-poly(A) priming or a combination of 3′-poly(A) and internal priming because oocyte transcripts may or may not have a polyadenylated tail. Amplified RNA was hybridised to GeneChip Bovine Genome Arrays (Affymetrix, Santa Clara, CA, USA; 8 oocyte pools per treatment were collected on 7 different occasions and amplified by 2 methods; n = 32 chips). Differential transcript abundance was determined using R and Bioconductor with only probes having a P < 0.01, a fold change of at least 1.3, and called present for at least half the arrays. Functional annotation of selected transcripts was performed using Gene Ontology and KEGG annotations (Bos taurus build 4.0) and DAVID (v 6.7) with significance level set at P < 0.10. Coincident with reduced blastocyst development (28.3 v. 15.2% for control v. heat stress, respectively; SEM = 3.6; P < 0.0003), heat stress altered the abundance of 159 transcripts (22 increased, 137 decreased); 130 of these were annotated. Use of DAVID demonstrated enrichment of genes important for mitochondrial function and RNA processing. Towards validating certain findings, the relative abundance of 3 mitochondrial transcripts (NDUFC2, COQ3, ATP5O) were assessed by quantitative PCR on non-amplified RNA from the oocyte samples used for the microarray study. Gene-specific primers were designed for 5′ and 3′ ends of transcripts when possible. Exposure to elevated temperature during the first 12 h of oocyte maturation reduced transcript levels of NDUFC2 at the 5′ and 3′ ends (P < 0.0001 and P = 0.003), COQ3 at the 3′ end (P = 0.02) and ATP5O at the 5′ end (P = 0.02). In conclusion, exposure of maturing cumulus-oocyte complexes to a physiologically-relevant elevated temperature altered the transcriptome in oocytes, especially certain transcripts important for mitochondrial function. This research was supported in part by USDA National Institute of Food and Agriculture, Hatch Project No. 227701, the state of Tennessee through University of Tennessee AgResearch, Department of Animal Science, and East Tennessee Research and Education Center.

scholarly journals Effect of Heat Stress and Amelioration by Antioxidants on Expression Profile of Pro- and Anti-Apoptotic Genes in in vitro Matured Bovine Oocytes

2019 ◽  
Vol 8 (02) ◽  
pp. 2394-2403
Author(s):  
Jafrin Ara Ahmed ◽  
Nawab Nashiruddullah ◽  
Devojyoti Dutta ◽  
Iftikar Hussain ◽  
Anubha Baruah ◽  
...  
Keyword(s):  
Heat Stress ◽  
Expression Profile ◽  
Apoptotic Genes ◽  
Bovine Oocytes

114 Effect of in vivo heat stress on DNA methylation and DNA hydroxymethylation of bovine oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 183
Author(s):  
F. A. Diaz ◽  
E. J. Gutierrez ◽  
B. A. Foster ◽  
P. T. Hardin ◽  
K. R. Bondioli
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Dna Hydroxymethylation ◽  
Oocyte Collection ◽  
Grade 3 ◽  
Bovine Oocytes

Cattle under the effect of heat stress have reduced fertility, with negative effects on the oocyte observed at the morphological, biochemical, transcriptional and developmental levels. There are no studies evaluating the effect of heat stress on the epigenetic profile of bovine oocytes, which plays a fundamental role in the regulation of gamete development. The objective of this study was to evaluate the effect of in vivo heat stress during the spring to summer transition on DNA methylation and DNA hydroxymethylation of bovine oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. Ten Bos taurus crossbred nonlactating beef cows located at Saint Gabriel, Louisiana, USA (30°16′11.1″ N, 91°06′12.1″ W), were used for oocyte collection once monthly from April to August. Dominant follicle removal was performed 5-7 days before oocyte collection. Cumulus-oocyte complexes were collected through ovum pick-up from follicles &gt;2mm. Germinal vesicle (GV)-stage oocytes (50% of total obtained per cow) were subjected to a standard bovine in vitro maturation protocol to obtain metaphase II (MII) stage oocytes. The DNA methylation and DNA hydroxymethylation of GV and MII oocytes was assessed by fluorescence immunohistochemistry utilising primary antibodies against 5′-methylcytosine and 5′-hydromethylcytosine. Secondary antibodies utilised were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 donkey anti-rabbit IgG. Oocytes were visualised utilising a fluorescence deconvolution microscope and immunofluorescence data were expressed as corrected relative fluorescence per nucleus. The polar body was not included for fluorescence quantification when evaluating MII stage oocytes. Results (least squares means±standard error) were evaluated as cold months (April and May) and hot months (June, July, and August). Results were analysed by the type III test of fixed effects and Tukey media separation utilising Proc Glimmix of SAS 9.4 (P&lt;0.05; SAS Institute Inc., Cary, NC, USA). Maturation rates and percent of grade 1, grade 2, and grade 3 oocytes were square root arcsine transformed for statistical analysis. The number of total oocytes obtained per cow was higher in cold compared to hot months (21.88±2.34 and 14.23±2.17, respectively). Percent of grade-1 oocytes was higher in cold compared to hot months (38.25±3.69 and 27.59±3.09, respectively). There was no difference in percent of grade-2 oocytes between cold and hot months (21.80±2.44 and 22.60±2.20, respectively). There was a lower percent of grade-3 oocytes in cold compared to hot months (39.82±4.54 and 55.87±3.98, respectively). Maturation rate (in vitro maturation) was not different between cold and hot months (81.92±4.04 and 91.11±3.36, respectively). There was no difference between cold and hot months in DNA methylation (417,218.90±71,793.86 and 313,819.88±55,528.01, respectively) and DNA hydroxymethylation (444,931.10±67,920.78 and 352,254.68±56,425.96, respectively) of GV-stage oocytes. There was no difference between cold and hot months in DNA methylation (87,122.36±14,449.47 and 89,807.26±11,303.72 AU, respectively) and DNA hydroxymethylation (102,933.83±15,517.70 and 137,622.45±11,826.86 AU, respectively) of MII-stage oocytes.

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