scholarly journals Evaluation of Seasonal Heat Stress on Transcriptomic Profiles and Global DNA Methylation of Bovine Oocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Fabian A Diaz ◽  
Emilio J Gutierrez-Castillo ◽  
Brittany A Foster ◽  
Paige T Hardin ◽  
Kenneth R Bondioli ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
Molecular Mechanisms ◽  
Developmental Competence ◽  
Beta Catenin ◽  
Hippo Signaling ◽  
Sequencing Analysis ◽  
Dna Hydroxymethylation ◽  
Bovine Oocytes

Heat stress affects oocyte developmental competence and is a major cause of reduced fertility in heat stressed cattle. Negative effects of heat stress on the oocyte have been observed at morphological, biochemical and developmental levels. However, the mechanisms by which heat stress affects the oocyte at the transcriptional and epigenetic levels remain to be further elucidated. Here we aimed to investigate the effect of heat stress on oocyte quality, transcriptomic profiles and DNA methylation of oocytes collected through the transition from spring to summer under Louisiana conditions. Summer season resulted in a lower number of high quality oocytes obtained compared to the spring season. There was no difference in in vitro maturation rates of oocytes collected during spring as compared to summer. RNA sequencing analysis showed that a total of 211 and 92 genes were differentially expressed as a result of heat stress in GV and MII oocytes, respectively. Five common genes (E2F8, GATAD2B, BHLHE41, FBXO44, and RAB39B) were significantly affected by heat in both GV and MII oocytes. A number of pathways were also influenced by heat stress including glucocorticoid biosynthesis, apoptosis signaling, and HIPPO signaling in GV oocytes, and Oct4 pluripotency, Wnt/beta-catenin signaling, and melatonin degradation I in MII oocytes. In addition, fluorescent immunocytochemistry analysis showed no difference in global levels of DNA methylation and DNA hydroxymethylation at either the GV or MII stage between spring and summer oocytes. The results of this study contribute to a better understanding of the effect of heat stress on the molecular mechanisms altered in 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 >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<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.

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 Novel insights into the regulation of chemerin expression: role of acute-phase cytokines and DNA methylation

2019 ◽  
Author(s):  
Kamila Kwiecien ◽  
Piotr Brzoza ◽  
Pawel Majewski ◽  
Izabella Skulimowska ◽  
Kamil Bednarczyk ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Acute Phase ◽  
Molecular Mechanisms ◽  
Retinoic Acid Receptor ◽  
Constitutive Expression ◽  
Sequencing Analysis ◽  
Mrna Levels ◽  
Pleiotropic Actions

AbstractChemerin is a chemoattractant protein with adipokine properties encoded by the retinoic acid receptor responder 2 (RARRES2) gene. It has gained more attention over the past few years due to its multilevel impact on metabolism and immune responses. The pleiotropic actions of chemerin include chemotaxis of dendritic cells, macrophages and natural killers (NK) subsets, bactericidal activity as well as regulation of adipogenesis and glucose metabolism. Therefore, reflecting the pleiotropic actions of chemerin, expression of RARRES2 is regulated by a variety of inflammatory and metabolic mediators. However, for most cell types, the molecular mechanisms controlling constitutive and regulated chemerin expression are poorly characterized. Here we show that RARRES2 mRNA levels in murine adipocytes are upregulated in vitro and in vivo by acute-phase cytokines, IL-1β and OSM. In contrast to adipocytes, these cytokines exerted a weak, if any, response in mouse hepatocytes, suggesting that the effect of IL-1β and OSM on chemerin expression is specific to fat tissue. Moreover, we show that DNA methylation controls the constitutive expression of chemerin. Bisulfite sequencing analysis showed low methylation levels within −735 to +258 bp of the murine RARRES2 gene promoter in unstimulated adipocytes and hepatocytes. In contrast to these cells, the RARRES2 promoter is highly methylated in B lymphocytes, cells that do not produce chemerin. Together, our findings reveal previously uncharacterized mediators and mechanisms controlling chemerin expression in various cells.

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

scholarly journals DNA methylation and mRNA expression profiles in bovine oocytes derived from prepubertal and adult donors

Reproduction ◽  
2012 ◽  
Vol 144 (3) ◽  
pp. 319-330 ◽  
Author(s):  
Mike Diederich ◽  
Tamara Hansmann ◽  
Julia Heinzmann ◽  
Brigitte Barg-Kues ◽  
Doris Herrmann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mrna Expression ◽  
Bos Taurus ◽  
Expression Profiles ◽  
Methylation Status ◽  
Transcript Abundance ◽  
Developmental Competence ◽  
Satellite Sequences ◽  
Bovine Oocytes

The developmental capacity of oocytes from prepubertal cattle is reduced compared with their adult counterparts, and epigenetic mechanisms are thought to be involved herein. Here, we analyzed DNA methylation in three developmentally important, nonimprinted genes (SLC2A1, PRDX1, ZAR1) and two satellite sequences, i.e. ‘bovine testis satellite I’ (BTS) and ‘Bos taurus alpha satellite I’ (BTαS). In parallel, mRNA expression of the genes was determined by quantitative real-time PCR. Oocytes were retrieved from prepubertal calves and adult cows twice per week over a 3-week period by ultrasound-guided follicular aspiration after treatment with FSH and/or IGF1. Both immature and in vitro matured prepubertal and adult oocytes showed a distinct hypomethylation profile of the three genes without differences between the two types of donors. The methylation status of the BTS sequence changed according to the age and treatment while the methylation status of BTαS sequence remained largely unchanged across the different age and treatment groups. Relative transcript abundance of the selected genes was significantly different in immature and in vitro matured oocytes; only minor changes related to origin and treatment were observed. In conclusion, methylation levels of the investigated satellite sequences were high (>50%) in all groups and showed significant variation depending on the age, treatment, or in vitro maturation. To what extent this is involved in the acquisition of developmental competence of bovine oocytes needs further study.

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.

scholarly journals Heat stress and immune response phenotype affect DNA methylation in blood mononuclear cells from Holstein dairy cows

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
A. M. Livernois ◽  
B. A. Mallard ◽  
S. L. Cartwright ◽  
A. Cánovas
Keyword(s):  
Dna Methylation ◽  
Immune Response ◽  
Heat Stress ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Heat Waves ◽  
Promoter Regions ◽  
Blood Mononuclear Cells

AbstractHeat stress negatively affects health and production in cows. Examining the cellular response to heat stress could reveal underlying protective molecular mechanisms associated with superior resilience and ultimately enable selection for more resilient cattle. This type of investigation is increasingly important as future predictions for the patterns of heat waves point to increases in frequency, severity, and duration. Cows identified as high immune responders based on High Immune Response technology (HIR) have lower disease occurrence compared to their average and low immune responder herd-mates. In this study, our goal was to identify epigenetic differences between high and low immune responder cows in response to heat stress. We examined genome-wide DNA methylation of blood mononuclear cells (BMCs) isolated from high and low cows, before and after in vitro heat stress. We identified differential methylation of promoter regions associated with a variety of biological processes including immune function, stress response, apoptosis, and cell signalling. The specific differentially methylated promoter regions differed between samples from high and low cows, and results revealed pathways associated with cellular protection during heat stress.

123 Effect of heat stress on oocyte developmental competence and global gene expression dynamics in Bos taurus crossbred beef cows

2019 ◽  
Vol 31 (1) ◽  
pp. 187 ◽  
Author(s):  
Z. Jiang ◽  
F. A. Diaz ◽  
E. J. Gutierrez ◽  
B. A. Foster ◽  
P. T. Hardin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Ontology ◽  
Heat Stress ◽  
Rna Sequencing ◽  
Molecular Mechanisms ◽  
Global Gene Expression ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Regulation Of Transcription

It is known that animals under the effect of heat stress present reduced fertility. We aimed to investigate the effect of heat stress on the developmental competence and global gene expression profile of oocytes through the transition from spring to summer under Louisiana conditions. Oocytes were collected from 6 crossbred, non-lactating cows once a month from May to July. Temperature and humidity indexes for May and July were 72.48 and 78.06, respectively. An index above 75 indicates that cows are under heat stress. All cows underwent dominant follicle removal, and then 7 days later, ovum pickup was performed to aspirate germinal vesicle (GV) oocytes. Half of the oocytes were processed for RNA-seq as GV, and half were matured in vitro to metaphase II (MII). Smart-seq protocol was followed to prepare RNA sequencing libraries from a pool of 4 oocytes (GV n=6; MII n=6). Sequencing reads were pre-filtered and aligned to the bovine genome, and gene expression values were calculated as transcripts per million. Genes were deemed differentially expressed between different conditions if they showed a false discovery rate P-value&lt;0.05 using DESEqn 2 package. DAVID (https://david.ncifcrf.gov) and IPA (ingenuity pathway analysis) were used to reveal the gene ontology and pathways, respectively. The RNA sequencing showed that a total of 212 genes were differentially expressed as a result of heat stress at the GV stage, with 94 and 118 genes up- and down-regulated, respectively. Gene ontology analysis indicated significant over-representation of elements involved in steroid biosynthetic process, oxidation reduction, and mitophagy in response to mitochondrial depolarization. Several pathways were influenced by heat stress, including glucocorticoid biosynthesis, apoptosis signalling, and HIPPO signalling. At the MII oocyte stage, only 93 genes (19 up-regulated and 74 down-regulated) were significantly differentially expressed in oocytes between July and May groups. Oocytes retrieved on different collection days, from the same cows under the same treatments, showed no difference on maturation rates, suggesting that the in vitro maturation process equalizes the expression of several genes. The primary biological processes significantly affected in MII oocytes were regulation of MAPK cascade, melanosome organisation, and negative regulation of transcription. In addition, we found that UBE2I, a gene involved in phosphorylation-dependent sumoylation of heat shock factor 1 (HSF1), was significantly up-regulated in July compared with May in MII oocytes. Interestingly, only 5 common genes were significantly affected by heat in both GV and MII oocytes: E2F8, GATAD2B, BHLHE41, FBXO44, and RAB39B. Our findings provide new insight into the molecular mechanisms of detrimental conditions (heat stress) on bovine oocytes, which may help to reveal master regulators controlling oocyte competence.

scholarly journals N-acetyl-L-cysteine Improves the Developmental Competence of Bovine Oocytes and Embryos Cultured In Vitro by Attenuating Oxidative Damage and Apoptosis

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 860
Author(s):  
Wu-Sheng Sun ◽  
Hoon Jang ◽  
Mi-Ryung Park ◽  
Keon Bong Oh ◽  
Haesun Lee ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Early Stage ◽  
Developmental Competence ◽  
Beneficial Effects ◽  
Developmental Rates ◽  
A Cell ◽  
Bovine Oocytes ◽  
Dose Dependent

Oxidative stress has been suggested to negatively affect oocyte and embryo quality and developmental competence, resulting in failure to reach full term. In this study, we investigated the effect of N-acetyl-L-cysteine (NAC), a cell-permeating antioxidant, on developmental competence and the quality of oocytes and embryos upon supplementation (0.1–10 mM) in maturation and culture medium in vitro using slaughterhouse-derived oocytes and embryos. The results show that treating oocytes with 1.0 mM NAC for 8 h during in vitro maturation attenuated the intracellular reactive oxygen species (ROS) (p < 0.05) and upregulated intracellular glutathione levels (p < 0.01) in oocytes. Interestingly, we found that NAC affects early embryonic development, not only in a dose-dependent, but also in a stage-specific, manner. Significantly (p < 0.05) decreased cleavage rates (90.25% vs. 81.46%) were observed during the early stage (days 0–2), while significantly (p < 0.05) increased developmental rates (38.20% vs. 44.46%) were observed during the later stage (from day 3) of embryonic development. In particular, NAC supplementation decreased the proportion of apoptotic blastomeres significantly (p < 0.05), resulting in enhanced hatching capability and developmental rates during the in vitro culture of embryos. Taken together, our results suggest that NAC supplementation has beneficial effects on bovine oocytes and embryos through the prevention of apoptosis and the elimination of oxygen free radicals during maturation and culture in vitro.

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