Studies on Gene Expression and Developmental Competence of Bovine Embryos Produced Under Different Conditions of Heat Stress

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

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193 DIFFERENTIAL GENE EXPRESSION IN CUMULUS CELLS OF DEVELOPMENTALLY COMPETENT V. CHALLENGED BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab193 ◽

2009 ◽

Vol 21 (1) ◽

pp. 195 ◽

Cited By ~ 1

Author(s):

R. R. Payton ◽

L. A. Rispoli ◽

J. L. Edwards

Keyword(s):

Gene Expression ◽

Gene Ontology ◽

Heat Stress ◽

Empirical Bayes ◽

Rna Extraction ◽

Cumulus Cells ◽

Developmental Competence ◽

Blastocyst Development ◽

Oocyte Competence ◽

Extracellular Region

It is well established that exposure of cumulus–oocyte complexes (COC) to heat stress during the first 12 h of maturation reduces blastocyst development by 42 to 65%. Previous research supports the notion that some of the effects of heat stress on oocyte competence may be cumulus-mediated. To determine the extent to which this may occur, COC were matured at 38.5°C for 24 h (control) or 41°C for the first 12 h of maturation followed by 38.5°C for remaining 12 h (heat stress). A subset of COC underwent IVF with Percoll-prepared sperm and then was cultured in KSOM containing 0.5% BSA to assess developmental competence. Remaining oocytes were denuded. Cumulus cells, kept separate by treatment, were stored in lysis buffer at –80°C until RNA extraction. Total RNA from cumulus was amplified prior to hybridization to bovine Affymetrix GeneChips (Affymetrix Inc., Santa Clara, CA, USA; n = 8 pools per treatment collected on 8 different occasions; n = 16 chips). Following pre-processing using the MAS5.0 algorithm, microarray data were subjected to linear modeling and empirical Bayes analyses (Bioconductor, Limma package). False discovery rate was controlled using the Benjamini and Hochberg method, and differentially expressed genes were selected by an adjusted P-value (P < 0.05). Functional annotation of selected genes was performed using NetAffx (Affymetrix Inc.) and Database for Annotation, Visualization and Integrated Discovery (DAVID; NIAID, NIH, Bethesda, MD, USA). Heat stress of COC reduced blastocyst development (27.2 v. 16.1% for control v. heat stress, respectively; SEM = 1.6; P < 0.002). Approximately 66 and 65% of 24 000 possible genes were called present (i.e. expressed) in RNA from cumulus of competent (control) v. challenged (heat-stressed) oocytes, respectively. In cumulus from developmentally challenged COC, increased abundance of 42 genes (36 currently annotated) was noted. Use of DAVID demonstrated enrichment of genes important for electron transport and energy generation (NOS2A, MAOB, CYP11A1, HSD11B1L, LTB4DH). Further examination of gene ontology identified genes associated with mitochondrial function (SLC25A10, MAOB, CYP11A1), cell signaling (similar to JAK-3, FSHR, CYP11A1, WNT2B), cytoskeleton (ACTA1), antioxidant activity (GSTA1), and extracellular region (FMOD). In contrast, cumulus from developmentally competent COC had increased expression of 22 genes (20 currently annotated), of which 15% were related to protein binding (CAV1, MMP9, TGFB2) according to DAVID. Further analysis using gene ontology revealed genes associated with extracellular matrix formation (MMP9, MMP19, PCOLCE2) and neural tissue (METRNL). In summary, alterations in cumulus gene expression were associated with differences in developmental competence of oocytes. Additional research is necessary to examine the extent to which identified genes account for functional differences in oocyte competence. This research was supported in part by National Research Initiative Competitive Grant no. 2004-35203-14772 from the USDA Cooperative State Research, Education, and Extension Service.

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Gene expression and developmental competence of bovine embryos produced in vitro under varying embryo density conditions

Theriogenology ◽

10.1016/j.theriogenology.2005.03.019 ◽

2005 ◽

Vol 64 (7) ◽

pp. 1559-1572 ◽

Cited By ~ 26

Author(s):

Alexandre Tavares Duarte de Oliveira ◽

Rui Fernando Felix Lopes ◽

José Luiz Rodrigues

Keyword(s):

Gene Expression ◽

Developmental Competence ◽

Bovine Embryos

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Effects of Astaxanthin-containing Oil on Development and Stress-related Gene Expression of Bovine Embryos Exposed to Heat Stress

Reproduction in Domestic Animals ◽

10.1111/j.1439-0531.2010.01584.x ◽

2010 ◽

Vol 45 (6) ◽

pp. e387-e391 ◽

Cited By ~ 16

Author(s):

T Namekawa ◽

S Ikeda ◽

M Sugimoto ◽

S Kume

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Bovine Embryos ◽

Related Gene

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179 RELATIONSHIP BETWEEN GENE EXPRESSION IN INDIVIDUAL BLASTOMERES OF 2-CELL STAGE BOVINE EMBRYOS AND THE NORMALITY OF FIRST CLEAVAGE

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab179 ◽

2016 ◽

Vol 28 (2) ◽

pp. 220

Author(s):

S. Matoba ◽

M. Kaneda ◽

T. Somfai ◽

T. Nagai ◽

M. Geshi

Keyword(s):

Gene Expression ◽

Target Genes ◽

Calf Serum ◽

Transcript Abundance ◽

Pcr Primers ◽

Developmental Competence ◽

Cleavage Pattern ◽

Bovine Embryos ◽

Culture Dish ◽

Cell Stage

Previously early first and second cleavages after IVF associated with even blastomeres without fragments or protrusions were found to be a potent criterion for the selection of embryos with high development competence (Sugimura et al. 2012 PLOS One 7, e36627). The aim of this study was to examine the relationship between an early normal first cleavage pattern and the transcript abundance in each blastomere in 2-cell stage bovine embryos. IVF-derived bovine embryos were cultured individually in microwells culture dish in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL–1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2. First cleavage and cleavage patterns were categorised as being either normal (the first cleavage within 28 h after IVF with 2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with/without fragment/protrusion and/or later than 28 h after IVF at the first cleavage). Next, cleaved embryos were placed in 0.5% actinase-E in Ca- and Mg-free PBS and blastomeres were separated by pipetting. Individual blastomeres (n = 71, 10 replicates) were analysed for gene expression by quantitative RT-PCR. Primers were designed for 19 target genes related to pluripotency, cell cycle, metabolism, pregnancy reorganization, placentation and fetal growth (NANOG, OCT4, PLAC8, ATP1A1, CCNB1801, CDH1, COX1, CTNNB1, G6PDH, Glut8, MNSOD-3end, SOX2, DYNLL1, IGF1R, IGF2, IGF2R, IGFBP2, IGFBP3, and PMSB1) and a reference gene (PPIA). Transcript abundance of target genes in both of individual blastomeres of cleaved embryos was examined in embryos that cleaved early with a normal cleavage pattern and in those that showed abnormal cleavage pattern. Values were normalised to the average values of the reference genes and means were compared by the student t-test. Transcript abundance of OCT4, ATP1A1, CCNB1801, CDH1, COX1, CTNNB1, MNSOD-3end, IGF2R, and IGFBP2 was significantly higher in blastomeres associated with all categorised abnormal blastomeres compared towith an early normal cleavage (P < 0.05). Furthermore, the expression of PLAC8, IGF1R, and PMSB1 in embryos having 2 uneven blastomeres, Glut8 and SOX2 in 2 uneven blastomeres with fragment/protrusion was higher than that in normal cleavage (P < 0.05). However, the level of G6PDH was lower in embryos having 2 uneven blastomeres than that in those showing normal cleavage (P < 0.05). Our results reveal blastomere gene expression in bovine embryos at the first cleavage may correlated with oocyte developmental competence. This study was supported by JSPS KAKENHI (26450388).

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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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Gene Expression and Developmental Competence of Bovine Embryos Produced In Vitro with Different Serum Concentrations

Reproduction in Domestic Animals ◽

10.1111/j.1439-0531.2006.00653.x ◽

2006 ◽

Vol 41 (2) ◽

pp. 129-136 ◽

Cited By ~ 13

Author(s):

ATD Oliveira ◽

RFF Lopes ◽

JL Rodrigues

Keyword(s):

Gene Expression ◽

Developmental Competence ◽

Serum Concentrations ◽

Bovine Embryos

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175 EFFECT OF HEAT STRESS ON GENE EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS (BOS TAURUS VS. BOS INDICUS)

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab175 ◽

2012 ◽

Vol 24 (1) ◽

pp. 199

Author(s):

C. F. Silva ◽

A. C. S. Castilho ◽

R. A. Satrapa ◽

R. Z. Puelker ◽

E. M. Razza ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Mrna Expression ◽

Reverse Transcription ◽

Target Genes ◽

Bos Taurus ◽

Bos Indicus ◽

Mrna Levels ◽

Bovine Embryos

Heat stress (HS) reduces the production of bovine embryos, especially taurine embryos, which are not adapted to heat. However, little is known about the competence of embryos produced under HS in breeds adapted or not adapted to heat. The aim of this study was to compare the gene expression of PLAC8, HSF1, COX2 and CDX2, related to competence and implantation, in bovine in vitro-produced embryos (Bos taurus vs Bos indicus), submitted or not submitted to HS. Oocytes from Nelore (zebu) and Jersey (taurine) cows were aspirated by ovum pickup, in vitro-matured in TCM-199 medium with bicarbonate containing 10% FCS, 2 μg mL–1 of pyruvate, 75 μg mL–1 of gentamicin, 20 μg mL–1 of FSH and 10 IU mL–1 of LH for 22 h at 38.5°C in 5% CO2 in air. Matured oocytes were fertilized with semen from Nelore (n = 6) and Jersey (n = 6) bulls, respectively, at 38.5°C in 5% CO2 in air. The fertilization medium was TALP-IVF supplemented with 6 mg mL–1 of fatty acid-free BSA, 2 μL mL–1 of pyruvate, 75 μg mL–1 of gentamicin, 11 μg mL–1 of heparin and 44 μL mL–1 of penicillamine, hypotaurine and epinephrine. The day of fertilization was considered Day 0. Twelve hours post-insemination, presumptive zygotes were denuded and randomly divided into 2 groups, nonstressed or stressed and both were in vitro cultured at 38.5°C in 90% N2, 5% CO2 and 5% O2 in SOFaaci medium supplemented with 5% FCS, 5% BSA and 0,2% sodium pyruvate. In the stressed group, 96-h post-insemination embryos were subjected to HS of 41°C for 6 consecutive hours and then returned to 38.5°C. On Day 7, pools with 5 blastocysts [Nelore (n = 9); Nelore HS (n = 7); Jersey (n = 5); Jersey HS (n = 5)] were subjected to RNA extraction (RNeasy, Qiagen Inc., Valencia, CA, USA). The expression of target genes was analysed by real-time reverse transcription PCR with oligo-dT in reverse transcription and bovine specific-primers in PCR. The expression of cyclophilin A was used as an internal control. The mean mRNA levels of target genes among groups were compared by parametric ANOVA, followed by orthogonal contrast. Heat stress reduced (P < 0.05) mRNA expression of CDX2 and PLAC8 in both breeds; additionally, the expression of these genes was higher in the zebu breed when compared with the taurine breed. Messenger RNA expression of COX2 did not differ between groups, under HS or not, in both the Jersey and Nelore breeds. Moreover, HS reduced the mRNA expression of HSF1 (P < 0.05) in Nelore groups, but not in Jersey groups. The highest levels of PLAC8 and CDX2 in nonstressed Nelore embryos indicate better competence and a higher capacity of implantation of these embryos when compared with Jersey and HS embryos in both breeds. Moreover, low HSF1 levels in stressed Nelore embryos indicate the thermotolerance ability of this breed. In conclusion, the data indicate that HS alters the pattern of gene expression in Nelore and Jersey in vitro-produced bovine embryos. This research was supported by FAPESP.

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236 EFFECT OF HEAT STRESS DURING OOCYTE MATURATION ON GENE EXPRESSION OF IN VITRO-FERTILIZED AND PARTHENOGENETIC BOVINE BLASTOCYSTS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab236 ◽

2010 ◽

Vol 22 (1) ◽

pp. 276

Author(s):

F. Q. Costa ◽

M. M. Pereira ◽

S. Wohlres-Viana ◽

R. V. Serapiao ◽

B. C. Carvalho ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

In Vitro Maturation ◽

Rna Extraction ◽

Residual Effect ◽

Blastocyst Stage ◽

Bovine Embryo ◽

Bovine Embryos ◽

Peroxiredoxin 1

Heat stress has been shown to have detrimental effects (41°C) during the first 12 h of in vitro maturation on bovine embryo development (Edwards JL and Hansen PJ 1996 Biol. Reprod. 55, 341-346). However, little is known about the effect on gene expression of in vitro-fertilized andparthenogenetic bovine embryos. This study evaluated the gene expression of in vitro-fertilized andparthenogenetic blastocysts derived fromheat-stressed oocytes. The transcripts evaluated were associated with genes encoding proteins involved in blastocoel formation [aquaporin (Aqp) 3 and Na+/K+-ATPase alpha 1; Watson AJ and Barcroft LC 2001 Frontiers Biosci. 6:d708-730] and cell viability (Bax and Peroxiredoxin 1; Van Delft MF and Huang DCS 2006 Cell Res. 16, 203-213; RaguS etal. 2007 PNAS104, 9747-9752). Oocytes were in vitro matured for 12 h at 41°C followed by 12 h at 38.5°C (heat-stressed oocytes; HS) or for 24 h at 38.5°C (non-heat-stressed oocytes; NHS) under 5% CO2. Heat-stressed and NHS oocytes were in vitro fertilized with Holstein sperm (HS-IVF and NHS-IVF subgroups, respectively) or activated with ionomycin and 6-DMAP (HS-PART and NHS-PART subgroups, respectively). Presumptive zygotes were cultured in CR2aa medium under 5% CO2, 5% O2, and 90% N2 at 38.5°C. Embryos at blastocyst stage with same quality grade for all subgroups were obtained from 3 different replicates and distributed in pools of 10 embryos for relative quantification of the target transcripts. RNA extraction and reverse transcription were performed and cDNA quantified by real-time PCR. Transcripts of H2a gene were used as endogenous control, and statistical analysis was performed by pair-wise, fixed reallocation randomization test. Gene expression comparisons were performed between HS-IVF and NHS-IVF, HS-PART and NHS-PART, NHS-PART and NHS-IVF, and HS-PART and HS-IVF subgroups. Blastocyst rate is shown as mean ± SEM and relative expression as n-fold. The heat stress on oocytes during in vitro maturation decreased (P < 0.05, ANOVA) the development of presumptive zygotes to blastocyst stage at Day 8 for in vitro-fertilized (19.9 ± 2.9% and 10.5 ± 2.0% for NHS-IVF and HS-IVF, respectively) and parthenogenetic (33.0 ± 1.8% and 22.8 ± 2.8% for NHS-PART and HS-PART, respectively) embryos. Embryos from the HS-IVF subgroup showed less (P < 0.01) expression of Na+/K+-ATPase alpha 1 (0.67-fold) than did NHS-IVF embryos, whereas no difference was found for others genes. Embryos from the HS-PART subgroup showed less (P < 0.01) expression of Aqp 3 (0.77-fold) and greater (P < 0.05) expression of Bax (1.40-fold) than did NHS-PART embryos. Expression of Aqp 3 was up-regulated (P < 0.01) in NHS-PART (1.42-fold) embryos when compared with NHS-IVF ones, whereas expression of Na+/K+-ATPase alpha 1 (1.42-fold), Bax (1.67-fold), and Peroxiredoxin 1 (1.40-fold) were up-regulated (P < 0.05) in HS-PART embryos when compared with HS-IVF embryos. In conclusion, heat stress on oocytes during in vitro maturation can affect the amount of transcripts of in vitro-fertilized andparthenogenetic blastocysts, suggesting a residual effect on gene expression of bovine embryos. Financial support was provided by CNPq and FAPEMIG.

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