scholarly journals The Effects of Heat Shock Protein 70 Addition in the Culture Medium on the Development and Quality of In Vitro Produced Heat Shocked Bovine Embryos

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3347
Author(s):  
Konstantina Stamperna ◽  
Themistoklis Giannoulis ◽  
Eleni Dovolou ◽  
Maria Kalemkeridou ◽  
Ioannis Nanas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Culture Medium ◽  
Heat Exposure ◽  
Developmental Competence ◽  
Culture Period ◽  
Negative Effects ◽  
Altered Expression ◽  
Embryo Yield ◽  
Bovine Oocytes

The aims of the present study were to examine the effects of HSP70 addition in the in vitro culture medium of day 3 embryos on their developmental competence and quality. Bovine oocytes (n = 1442) were in vitro matured, inseminated and cultured for the first two days according to standardized methods. The presumptive zygotes were randomly allocated in three experimental groups: Control, C (embryos cultured at 39 °C throughout the culture period), group C41 (temperature was raised to 41 °C from the 48th to 72nd h post insemination (p.i.) and then it returned at 39 °C for the remaining culture period), and group H41 (the temperature modification was the same as in C41 and during heat exposure, HSP70 was added in the culture medium). Cleavage and embryo yield were assessed 48 h p.i. and on days 7, 8, 9, respectively and gene expression in day 7 blastocysts was assessed by RT-PCR. Blastocyst yield was the highest in group C39; and higher in group H41 compared to group C41. From the gene expression analyses, altered expression of 11 genes was detected among groups. The analysis of the orchestrated patterns of gene expression differed between groups. The results of this study confirm the devastating effects of heat stress on embryo development and provide evidence that HSP70 addition at the critical stages can partly counterbalance, without neutralizing, the negative effects of the heat insult on embryos, acting mainly through mechanisms related to energy deployment.

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.

357 MOLECULAR AND SUBCELLULAR CHARACTERIZATION OF BOVINE OOCYTES AND THEIR SURROUNDING FOLLICULAR CELLS IN SUBJECT TO THEIR DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 335
Author(s):  
H. Torner ◽  
D. Janowski ◽  
N. Ghanem ◽  
D. Salilew-Wondim ◽  
H. Alm ◽  
...  
Keyword(s):  
Gene Expression ◽  
Caspase 3 ◽  
Expression Profiles ◽  
Developmental Competence ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Altered Expression ◽  
Developmental Potential ◽  
Expression Of Genes ◽  
Bovine Oocytes ◽  
Oocyte Selection

Though many factors have been shown to affect the oocyte developmental potential, it remains difficult to draw clear and reliable criteria for oocyte selection. With the urgent need for establishing non-invasive means for oocyte selection, the brilliant cresyl blue (BCB) staining test based on glucose- 6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate competent and non-competent bovine oocytes (Alm et al. 2005 Theriogenology 63, 2194-2205). Also it has been hypothesized that there is a correlation between the appearance of light atretic granulosa cells (GC) in the follicle and an increased developmental competence of the oocyte. Here we aim to investigate whether different developmental competent oocytes show differences concerning the degree of apoptosis or in the gene expression pattern of their follicular environment [GC and cumulus cells (CC)]. After follicular aspiration, the immature COCs were separately stained with 26 μM BCB for 90 minutes. Based on their colouration, oocytes were grouped into BCB- (colourless cytoplasm, low developmental competence) and BCB+ (coloured cytoplasm, high developmental competence). The corresponding CC and GC were also grouped according to the colouration of the enclosed oocytes. BCB+ oocytes were found to result in a higher blastocyst rate at Day 8 of in vitro culture (34.1%) compared to BCB- ones (3.9%) (n = 601 COCs). Apoptosis in GC was determined either by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) or by Annexin-V-staining followed by flow cytometric measurement. The degree of apoptosis in GC of BCB+ oocytes was slightly increased in contrast to the BCB- group (17.0 v. 11.0%; P < 0.05). The abundance and activity of protein kinases Akt, MAP kinase, and Caspase-3 were estimated by western blot analysis. CC, GC, and oocytes from the BCB+ group showed a higher ratio of cleaved Caspase-3/Caspase-3 in contrast to all compartments of the BCB- group (P < 0.05). Moreover, a bovine Affymetrix microarray plate form (Affymetrix Inc., Santa Clara, CA, USA) was used to analyze the gene expression profiles in oocytes, CC, and GC. The BCB+ oocytes were found to be enriched with genes regulating the oocyte maturation (EIF3F, PRKCSH) and the transition from maternal to embryonic genome activation (HMG2L1). Also BCB+ derived follicular compartments showed elevated expression of genes related to steroidgenesis, cumulus expansion and gonadotropins. In conclusion, the results demonstrate that the developmental competence of oocytes is associated with the apoptotic level and altered expression of genes in cells of their follicular environment. This work was supported financially by Deutsche Forschungsgemeinschaft (To 138/5-1).

138 THE EFFECT OF FOLLICLE SUPERSTIMULATION ON mRNA LEVELS IN BOVINE OOCYTES COLLECTED BY OVUM PICKUP

2012 ◽  
Vol 24 (1) ◽  
pp. 181
Author(s):  
T. Somfai ◽  
K. Imai ◽  
M. Kaneda ◽  
S. Akagi ◽  
S. Haraguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hormone Treatment ◽  
Developmental Competence ◽  
Mrna Levels ◽  
Dominant Follicle ◽  
Oocyte Collection ◽  
Light Cycler ◽  
Group 3 ◽  
Bovine Oocytes

A previous study revealed that follicle superstimulation significantly improved the developmental competence of immature bovine oocytes collected by ovum pickup (OPU; Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). The aim of the present study was to investigate the effect of follicle superstimulation on the expression of developmentally important genes in bovine oocytes collected by OPU. Follicular oocytes were collected by OPU without (OPU group) or after follicle superstimulation by FSH (FSH/OPU group) by using a 7.5-MHz linear transducer with needle connected to an ultrasound scanner according to Imai et al. (2008). In the FSH/OPU group, after dominant follicle removal from Holstein dry cows by OPU, a CIDR was inserted on Day 5 (dominant follicle removal = Day 0). Cows then received 30 mg of FSH twice a day from Days 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 mg) by IM injection. Cloprostenol (PGF; Clopromate C; Sumitomo Pharmaceuticals Co., Tokyo, Japan; 0.75 mg) was administered in the morning of Day 9 (third day of superstimulation). Oocyte collection by OPU was performed 48 h after PGF administration (Day 11) by the aspiration of follicles larger than 5 mm in diameter. In the OPU group, 3-to-6-mm follicles were aspirated without any previous hormone treatment. In vitro oocyte maturation (IVM) was performed according to Imai et al. (2006 J. Reprod. Dev. 52(Suppl), 19–29). Gene expression was assessed before (0 h IVM) and after IVM (22 h IVM) by RT quantitative PCR. The following genes were investigated: GAPDH, G6PDH, ACTB, H2A, CCNB1, MnSOD, OCT4, SOX2, CX43, HSP70, GLUT8, PAP, GDF9, COX1, ATP1A1, CDH1, CTNNB1, AQP3, DYNLL1, DYNC 1/1 and PMSB1. In brief, mRNA was extracted from 20 oocytes per sample using Qiagen RNeasy Micro kit (Qiagen, Valencia, CA, USA). Gene expression was analysed by a Roche Light Cycler 480 device. The relative expression of each gene was normalized to ACTB. Three replications were performed. Data were analysed by ANOVA. At 0 h IVM, PAP and DYNC 1/1 were found to be down-regulated (P < 0.05) in the FSH/OPU group compared with the OPU group, whereas the rest of the studied genes showed similar expression in the FSH/OPU and OPU groups. At 22 h IVM, PAP and DYNC 1/1 remained down-regulated in FSH/OPU oocytes. However, at this time the expression of GDF9 appeared significantly higher (P < 0.05) in FSH/OPU oocytes than in OPU oocytes. The expression of GDF9 was found to decrease during IVM in both groups; however, this decrease was less drastic in FSH/OPU oocytes. The results suggest that follicle superstimulation caused reduced expression of mRNA levels of PAP and DYNC 1/1 irrespective of maturation status and it also moderated the reduction of mRNA levels of GDF9 during IVM.

263 GENE EXPRESSION PROFILING OF IMMATURE AND IN VITRO-MATURED BOVINE OOCYTES USING AFFYMETRIX GENECHIP TECHNOLOGY

2007 ◽  
Vol 19 (1) ◽  
pp. 248 ◽  
Author(s):  
F. Carter ◽  
T. Fair ◽  
S. Park ◽  
M. Wade ◽  
A. C. O. Evans ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Bovine Genome ◽  
Calf Serum ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Total Rna ◽  
Rna Content ◽  
Bovine Oocytes

Previous studies by our group have demonstrated that oocyte maturation is a crucial event in the determination of subsequent developmental competence. The objective of the current study was to characterize changes in gene expression profiles of bovine oocytes during meiotic maturation. To this end, 5 replicate pools of 200 bovine cumulus–oocyte complexes (COCs)were collected from the ovaries of slaughtered heifers. Upon recovery, 100 COCs from each replicatewere immediately denuded, and the oocytes were snap frozen in liquid nitrogen. The remaining 100 COCs were matured in vitro in TCM-199 supplemented with 10% (v/v) fetal calf serum and 10 ngmL-1 epidermal growth factor for 24 h at 39�C under an atmosphere of 5% CO2 in air with maximum humidity. Following maturation, the remaining COCs were denuded and snap frozen. Total RNA was isolated (mean total RNA content 106.08�38.87 ng per 100 oocytes) and subjected to 2 rounds of amplification incorporating biotin-labeled nucleotides during the second in vitro transcription reaction (mean total RNA content 155.15�51.14 �g per 100 oocytes post-amplification). The resulting labeled antisense RNA was hybridized to a GeneChip Bovine Genome Arrays (Affymetrix, Inc., Santa Clara, CA, USA) (10 chips, 5 replicates each of immature and mature oocytes, n=100 oocytes/chip). Expression data were analysed using Genespring software (Agilent Technologies, Palo Alto, CA, USA), and data were normalized to the median. Overall, 54.9�1.3% and 53.3�3.3% of the 24 178 probe sets representing 23 000 transcripts spotted on the arrays were expressed in immature and in vitro-matured oocytes, respectively. Across the 5 array comparisons, 52 genes were consistently exclusively present in immature oocytes, whereas 16 genes were exclusively present in mature oocytes. A further 821 genes were found to be differentially expressed (≥2-fold) between the 2 groups (P &lt;0.05), of which 209 were up-regulated and 612 were down-regulated in the in vitro-matured oocytes compared with their immature counterparts. The differentially expressed transcripts were classified according to their gene ontology (http://benzer.ubic.ca/ermineJ). The existing Affymetrix annotation was updated by blasting the sequences against bovine, human, and murine databases (≥90% homology; increasing molecular function annotation from 14% to 42%). In terms of olecular function, the majority of these genes were associated with protein or nucleic acid binding (&gt;42%), catalytic activity (24%), signal transduction (7%), transporter activity (5%), and structural molecule activity (5%). In conclusion, we have stablished the molecular transcriptome blueprint of immature and in vitro-matured bovine oocytes. Through comparisons with in vivo-matured oocytes, this resource will be invaluable in determining genes that are involved in controlling the developmental competence of oocytes. This research was funded by the Science Foundation Ireland (02/IN1/B78).

Treatment of buffalo (Bubalus bubalis) donor cells with trichostatin A and 5-aza-2’-deoxycytidine alters their growth characteristics, gene expression and epigenetic status and improves the in vitro developmental competence, quality and epigenetic status of cloned embryos

2016 ◽  
Vol 28 (6) ◽  
pp. 824 ◽  
Author(s):  
M. Saini ◽  
N. L. Selokar ◽  
H. Agrawal ◽  
S. K. Singla ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Trichostatin A ◽  
Developmental Competence ◽  
Simultaneous Treatment ◽  
Altered Expression ◽  
Expression Levels ◽  
Histone 3 ◽  
Donor Cells

We examined the effects of treating buffalo skin fibroblast donor cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a DNA methyltransferase (DNMT) inhibitor, on the cells and embryos produced by hand-made cloning. Treatment of donor cells with TSA or 5azadC resulted in altered expression levels of the HDAC1, DNMT1, DNMT3a, P53, CASPASE3 and CASPASE9 genes and global levels of acetylation of lysine at position 9 or 14 in histone 3 (H3K9/14ac), acetylation of lysine at position 5 in histone 4 (H4K5ac), acetylation of lysine at position 18 in histone 3 (H3K18ac) and tri-methylation of lysine at position 27 in histone 3 (H3K27me3). Moreover, global levels of DNA methylation and activity of DNMT1 and HDAC1 were decreased, while global acetylation of H3 and H3K9 was significantly increased in comparison to untreated cells. Simultaneous treatment of donor cells with TSA (50 nM) and 5azadC (7.5 nM) resulted in higher in vitro development to the blastocyst stage, reduction of the apoptotic index and the global level of H3K27 me3 and altered expression levels of HDAC1, P53, CASPASE3, CASPASE9 and DNMT3a in cloned blastocysts. Transfer of cloned embryos produced with donor cells treated with TSA led to the birth of a calf that survived for 21 days. These results show that treatment of buffalo donor cells with TSA and 5azadC improved developmental competence and quality of cloned embryos and altered their epigenetic status and gene expression, and that these beneficial effects were mediated by a reduction in DNA and histone methylation and an increase in histone acetylation in donor cells.

scholarly journals In vitro maturation alters gene expression in bovine oocytes

Zygote ◽  
2016 ◽  
Vol 24 (4) ◽  
pp. 624-633 ◽  
Author(s):  
Paulo R. Adona ◽  
Cláudia L.V. Leal ◽  
Fernando H. Biase ◽  
Tiago H. De Bem ◽  
Lígia G. Mesquita ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Regulatory Networks ◽  
Developmental Competence ◽  
Protein Protein Interactions ◽  
Developmental Potential ◽  
Oocyte Competence ◽  
Bovine Oocytes

SummaryGene expression profiling of in vivo- and in vitro-matured bovine oocytes can identify transcripts related to the developmental potential of oocytes. Nonetheless, the effects of in vitro culturing oocytes are yet to be fully understood. We tested the effects of in vitro maturation on the transcript profile of oocytes collected from Bos taurus indicus cows. We quantified the expression of 1488 genes in in vivo- and in vitro-matured oocytes. Of these, 51 genes were up-regulated, whereas 56 were down-regulated (≥2-fold) in in vivo-matured oocytes in comparison with in vitro-matured oocytes. Quantitative real-time polymerase chain reaction (PCR) of nine genes confirmed the microarray results of differential expression between in vivo- and in vitro-matured oocytes (EZR, EPN1, PSEN2, FST, IGFBP3, RBBP4, STAT3, FDPS and IRS1). We interrogated the results for enrichment of Gene Ontology categories and overlap with protein–protein interactions. The results revealed that the genes altered by in vitro maturation are mostly related to the regulation of oocyte metabolism. Additionally, analysis of protein–protein interactions uncovered two regulatory networks affected by the in vitro culture system. We propose that the differentially expressed genes are candidates for biomarkers of oocyte competence. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence.

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