Genes In Bovine Embryos Research Articles

Comparison of Gene Expression for Preimplantation Genes in Bovine Embryos Fertilized in vitro

Background: In vitro fertilization (IVF) the technique is useful for understanding early mammalian development and maximizing and speeding up genetic improvement in livestock. Genes that function as genetic markers and are involved in the pre-implantation development of embryos may be used to evaluate the quality of an embryo and the expression of these genes is correlated with the timing of embryonic genomic activation. Methods: Maturing the oocytes of bovine, then fertilizing these matured oocytes and finally culturing the fertilized oocytes to develop into embryos. Determined the gene expression of different certain genes HSP70, OCT4, GLUT1, BAX and DNMT1 in these different cell stages of embryos that were produced. Result: The mRNA expression of these genes was estimated after collecting the embryos listed above that were produced by the in vitro technique. The results showed statistically significant differences in the relative abundance between some of the genes mentioned above in each embryo stage. The timing of the zygotic genome activation process (ZGA) can result in differences in gene expression levels between the early embryonic genes in each developmental stage of in vitro-produced bovine embryos.

The global gene expression outline of the bovine blastocyst: reflector of environmental conditions and predictor of developmental capacity

BackgroundMorphological evaluation of embryos has been used to screen embryos for transfer. However, the repeatability and accuracy of this method remains low. Thus, evaluation of an embryo’s gene expression signature with respect to its developmental capacity could provide new opportunities for embryo selection. Since the gene expression outline of an embryo is considered as an aggregate of its intrinsic characteristics and culture conditions, we have compared transcriptome profiles of in vivo and in vitro derived blastocysts in relation to pregnancy outcome to unravel the discrete effects of developmental competence and environmental conditions on bovine embryo gene expression outlines. To understand whether the gene expression patterns could be associated with blastocyst developmental competency, the global transcriptome profile of in vivo (CVO) and in vitro (CVT) derived competent blastocysts that resulted in pregnancy was investigated relative to that of in vivo (NVO) and in vitro (NVT) derived blastocysts which did not establish initial pregnancy, respectively while to unravel the effects of culture condition on the transcriptome profile of embryos, the transcriptional activity of the CVO group was compared to the CVT group and the NVO group was compared to the NVT ones.ResultsA total of 700 differentially expressed genes (DEGs) were identified between CVO and NVO blastocysts. These gene transcripts represent constitutive regions, indel variants, 3′-UTR sequence variants and novel transcript regions. The majority (82%) of these DEGs, including gene clusters like ATP synthases, eukaryotic translation initiation factors, ribosomal proteins, mitochondrial ribosomal proteins, NADH dehydrogenase and cytochrome c oxidase subunits were enriched in the CVO group. These DEGs were involved in pathways associated with glycolysis/glycogenesis, citrate acid cycle, pyruvate metabolism and oxidative phosphorylation. Similarly, a total of 218 genes were differentially expressed between CVT and NVT groups. Of these, 89%, including TPT1, PDIA6, HSP90AA1 and CALM, were downregulated in the CVT group and those DEGs were overrepresented in pathways related to protein processing, endoplasmic reticulum, spliceasome, ubiquitone mediated proteolysis and steroid biosynthesis. On the other hand, although both the CVT and CVO blastocyst groups resulted in pregnancy, a total of 937 genes were differential expressed between the two groups. Compared to CVO embryos, the CVT ones exhibited downregulation of gene clusters including ribosomal proteins, mitochondrial ribosomal protein, eukaryotic translation initiation factors, ATP synthases, NADH dehydrogenase and cytochrome c oxidases. Nonetheless, downregulation of these genes could be associated with pre and postnatal abnormalities observed after transfer of in vitro embryos.ConclusionThe present study provides a detailed inventory of differentially expressed gene signatures and pathways specifically reflective of the developmental environment and future developmental capacities of bovine embryos suggesting that transcriptome activity observed in blastocysts could be indicative of further pregnancy success but also adaptation to culture environment.

Upregulation of interferon-alpha gene in bovine embryos produced in vitro in response to experimental infection with noncytophatic bovine-viral-diarrhea virus.

In-vitro fertilization is a routine livestock-breeding technique widely used around the world. Several studies have reported the interaction of bovine viral-diarrhea virus (BVDV) with gametes and in-vitro-produced (IVP) bovine embryos. Since, gene expression in BVDV-infected IVP bovine embryos is scarcely addressed. The aim of this work was to evaluate the differential expression of genes involved in immune and inflammatory response. Groups of 20–25 embryos on Day 6 (morula stage) were exposed (infected) or not (control) to an NCP-BVDV strain in SOF medium. After 24 h, embryos that reached expanded blastocyst stage were washed. Total RNA of each embryo group was extracted to determine the transcription levels of 9 specific transcripts related with antiviral and inflammatory response by SYBR Green real time quantitative (RT-qPCR). Culture media and an aliquot of the last embryos wash on Day 7 were analyzed by titration and virus isolation, respectively. A conventional PCR confirmed BVDV presence in IVP embryos. A significantly higher expression of interferon-α was observed in blastocysts exposed to NCP-BVDV compared to the controls (p < 0.05). In this study, the upregulation of INFα and TLR7 genes involved in inflammatory and immune response in BVDV-infected IVP bovine embryos is a new finding in this field. This differential expression suggest that embryonic cells could function in a manner like immune cells by recognizing and responding early to interaction with viral pathogens. These results provide new insights into the action of BVDV on the complex molecular pathways controlling bovine early embryonic development.

75 Expression of lipid metabolism-related genes in bovine embryos cultured invitro with diacylglycerol acyltransferase-1 inhibitor

Intracellular lipids accumulated in embryos produced invitro have been linked to reductions in both quality and post-cryopreservation viability. Diacylglycerol O-acyltransferase 1 (DGAT1) is an enzyme that catalyzes the final step in triglyceride synthesis, which is a major component of the lipid droplets in embryos. Thus, the aim of this study was to evaluate the development and quality, in regard to changes in the expression of candidate genes of lipid metabolism, of bovine blastocysts cultured invitro with DGAT1 inhibitor (A922500 Sigma-Aldrich). Zygotes were cultured in synthetic oviduct fluid supplemented with 5% FCS (TC) or in TC supplemented with 10 or 50 µM DGAT1 inhibitor (T10 and T50, respectively) or TC with 0.1% dimethyl sulfoxide (TDMSO: vehicle for DGAT1 dilution) from 54h post-insemination (hpi; major embryonic genome activation, EGA) until Day 8. Cleavage rate (48 hpi) and blastocyst yield (Day 7-8) were evaluated. Day 7 blastocysts were snap-frozen in LN2 for gene expression analysis. The mRNA abundance of candidate genes related to lipid metabolism (DGAT1, PLIN2, GLUT5, GLUT1, GPX1, PPAR1b, and G6PD) was measured by quantitative PCR. The H2AFZ and ACTB genes were used as housekeeping genes. Statistical analysis was assessed by one-way analysis of variance. No differences were found in cleavage rate, whereas blastocyst yield at Day 8 was higher (P&amp;lt;0.05) for T50 (30.5±0.5%) and TC (29.7±0.5%) compared with T10 (25.6±0.5%) and TDMSO (27.1±0.8%). The expression of genes regulating lipid droplet formation (DGAT1 and PLIN2) was down-regulated in embryos of T10 and T50 groups compared with controls (P&amp;lt;0.05) only for DGAT1, whereas no differences were observed for PLIN2. The expression of GLUT5, a fructose metabolism transporter gene, was only increased in embryos in the T10 group, whereas the relative abundance of GLUT1 (involved in glucose metabolism) was up-regulated in T10 and T50 groups compared with controls (P&amp;lt;0.05). The GPX1 gene was decreased significantly in embryos from both DGAT1-inhibitor groups compared with the controls. The expression profile of PPAR1b and G6PD, lipid metabolism-regulating genes, were not different among embryo groups. In conclusion, supplementation of embryo culture with DGAT1 inhibitor improves development and blastocyst quality in terms of the expression of lipid metabolism-regulating genes, supporting a relationship between lipid metabolism and embryo feature. Funding was provided by MINECO-Spain AGL2015-70140-R &amp;amp; RTI2018-093548-B-I00; J. Giraldo-Giraldo COLCIENCIAS 727/2015-Colombia; Y. N. Cajas, SENESCYT-Ecuador; C. L. V. Leal, FAPESP-Brasil 2017/20339-3.

Effect of exogenous transforming growth factor β1 (TGF-β1) on early bovine embryo development.

SummaryDuring preimplantation development, embryos are exposed and have the capacity to respond to different growth factors present in the maternal environment. Among these factors, transforming growth factor β1 (TGF-β1) is a well known modulator of embryonic growth and development. However, its action during the first stages of development, when the embryo transits through the oviduct, has not been yet elucidated. The objective of the present study was to examine the effect of early exposure to exogenous TGF-β1 on embryo development and expression of pluripotency (OCT4, NANOG) and DNA methylation (DNMT1, DNMT3A, DNMT3B) genes in bovine embryos produced in vitro. First, gene expression analysis of TGF-β receptors confirmed a stage-specific expression pattern, showing greater mRNA abundance of TGFBR1 and TGFBR2 from the 2- to the 8-cell stage, before embryonic genome activation. Second, embryo culture for the first 48 h in serum-free CR1aa medium supplemented with 50 or 100 ng/ml recombinant TGF-β1 did not affect the cleavage and blastocyst rate (days 7 and 8). However, RT-qPCR analysis showed a significant increase in the relative abundance of NANOG and DNMT3A in the 8-cell stage embryos and expanded blastocysts (day 8) derived from TGF-β1 treated embryos. These results suggest an early action of exogenous TGF-β1 on the bovine embryo, highlighting the importance to provide a more comprehensive understanding of the role of TGF-β signalling during early embryogenesis.

Comparison of the developmental competence and quality of bovine embryos obtained by in vitro fertilization with sex-sorted and unsorted semen from seven bulls

In artificial insemination, semen quality is considered to be closely related to the resultant pregnancy rate and the ratio of embryo development into fetus. The aim of this study was to evaluate the quality of sex-sorted versus unsorted semen used for in vitro fertilization (IVF). Semen was collected from seven bulls (A)–(G); each sample was divided in two, sex-sorted or not. Following IVF, the fertilization rate, embryonic cleavage rate, blastocyst formation rate, total cells number (TCN) of blastocysts, inner cell mass (ICM)/TCN ratio and apoptotic cell ratio (ACR) were determined. The results showed that except for bull C, the unsorted semen yielded a significantly higher IVF rate than sex-sorted semen (p<0.05). A significantly higher percentage of embryos reached the two-cell stage using unsorted semen than the matched sex-sorted semen from the seven bulls (both p<0.05), and the blastocyst stage from bulls A, C, D, E and G. However, there was no significant difference in the ICM/TCN ratio between the IVF blastocysts and the sexed blastocysts from bulls A, C, D, E, F and G (p>0.05). Also, there was no significant difference in the expression of IGF1 gene in bovine embryos produced with sorted or unsorted semen (p>0.05). However, the GDF9 gene expression in sexed embryos from bull D was significantly higher than in the others (p<0.05). No significant difference was found in ACR among the seven bulls, while there were differences of Bax and Bcl-2 expression in the sorted sperm of only one bull. The results obtained from the sex-sorted or unsorted semen from the seven different bulls varied, although semen from bull A yielded the highest embryonic cleavage rate, blastocyst rate and ICM/TCN ratio. In conclusion, the quality of unsorted semen was generally better than that of sex-sorted semen in the respect to embryo productivity in vitro. Additionally, individual differences in fertility were observed among different bulls.

123 EFFECTS OF TAURO URSODEOXYCHOLIC ACID ON DEVELOPMENT OF BOVINE EMBRYOS FROM IN VITRO FERTILIZATION

Endoplasmic reticulum stress (ERS) is a novel apoptotic pathway and plays an important role for embryonic development. Tauro ursodeoxycholic acid (TUDCA) is a specific chemical chaperone that can inhibit ERS. In this study, we investigated the effects of TUDCA on the development and mRNA expression of ERS-related genes in bovine embryos from IVF in order to improve the efficiency of embryo in vitro culture. Bovine oocytes collected from ovaries at slaughter were cultured in the maturation medium (TCM-199 + 26.2 mmol L–1 NaHCO3 + 5 mmol L–1 HEPES + 5% fetal bovine serum) for 24 h and fertilized in vitro with bovine sperm. After fertilization, the embryos were respectively placed into the medium (TCM-199 + 3% fetal bovine serum) containing different concentrations of TUDCA (0, 100, 250, 500, 1000 μmol L–1) and cultured in the 5% CO2 at 38.5°C. Blastocyst development was evaluated after 7 days of culture, and then the total cell number and apoptosis index of blastocysts were detected with TUNEL. In addition, X-box binding protein 1 (XBP-1) of embryos at 2-cell, 4-cell, morula, and blastocyst stages was detected with RT-PCR, and the change of the mRNA expression of ERS-related (Grp78, Ire1, Chop) and apoptosis-related (Bax, Bcl-2) genes in blastocyst collected at 7 days of culture were analysed by QRT-PCR. A total of 1336 oocytes were used in this study, and each experimental group comprised 6 replicates. The results revealed that the splicing of XBP-1 was present during the development of bovine embryos, and especially obvious at the 4-cell, morula, and blastocyst stages. When embryos were cultured in medium with different concentrations of TUDCA, compared with the control group (0 μmol L–1), more embryos developed to blastocyst stage with 500 μmol L–1 TUDCA (31.86 ± 7.32% v. 21.11 ± 8.05%; P &lt; 0.05), but the cleavage rate was not significantly different among groups (P &gt; 0.05). The result for TUNEL found that when adding 500 μmol L–1 TUDCA to culture, the bovine embryos significantly improved the total cell number of blastocysts (110. ± 15.21 v. 102.3 ± 8.62; P &lt; 0.05), and the apoptosis index of blastocysts was markedly decreased (3.71 ± 0.91 v. 5.36 ± 1.92; P &lt; 0.05) relative to the control group. Moreover, the result of QRT-PCR analysis showed that treating embryos with 500 μmol L–1 TUDCA significantly reduced the mRNA expression level of Ire1 and Chop genes (P &lt; 0.05) and up-regulated the expression of anti-apoptotic Bcl-2 gene (P &lt; 0.05), while down-regulated the expression of pro-apoptotic Bax gene (P &lt; 0.05). Furthermore, XBP-1 splicing in blastocysts also abated after embryos were treated with 500 μmol L–1 TUDCA. In conclusion, ERS occurs in bovine embryos during in vitro culture, but treating embryos with 500 μmol L–1 TUDCA may reduce ERS to facilitate embryonic development. This work was funded by the China High Technology Development Program (2011AA100607), China Natural Science Foundation (31072033), and Guangxi Science Foundation (2011GXNSFA018084, 2012GXNSFFA060004).

199 IN VITRO-DEVELOPED BOVINE BLASTOCYSTS ARE MARKED WITH ABERRANT HYPER- AND HYPO-METHYLATED GENOMIC REGIONS

Most often, in vitro produced embryos display poor quality and altered gene expression patterns compared to their in vivo counterparts. Aberrant DNA methylation occurring during in vitro embryo development is believed to be one of the multifaceted factors which may cause altered gene expression and poor embryo quality. Here, we investigated the genome-wide DNA methylation patterns of in vitro derived embryos using the recently developed Bovine EmbryoGENE Methylation Platform (BEGMP) array (Shojaei Saadi et al. BMC Genomics 2014 15, 451. doi: 10.1186/1471-2164-15-451) to unravel the aberrantly methylated genomic region in in vitro developed embryos. For this, in vitro and in vivo produced blastocysts were produced and used for genome-wide DNA methylation analysis. In vitro blastocysts were produced from oocytes retrieved from ovaries collected from the local abattoir and matured, fertilized, and cultured in vitro using SOF media. The in vivo blastocysts were produced by superovulation and AI of Simmental heifers followed by uterine flushing. Genomic DNA (gDNA) was then isolated from four replicates (each 10 blastocysts) of in vivo and in vitro derived blastocysts using Allprep DNA/RNA micro kit (Qiagen, Valencia, CA, USA) and the gDNA was then fragmented using the MseI enzyme. Following this, MseLig21 and MseLig were ligated to the MseI-digested genomic fragments in the presence of Ligase enzyme. Methyl-sensitive enzymes, HpaII, AciI, and Hinp1I, were used to cleave unmethlayted genomic regions within the MseI-MseI region of the fragmented DNA. The gDNA was subjected to two rounds of ligation-mediated polymerase chain reaction (LM-PCR) amplification. After removal of the adapters, the amplified gDNA samples from in vivo or in vitro groups were labelled either Cy-3 or Cy-5 dyes in dye-swap design using ULS Fluorescent gDNA labelling kit (Kreatech Biotechnology BV, Amsterdam, The Netherlands). Hybridization was performed for 40 h at 65°C. Slides were scanned using Agilent's High-Resolution C Scanner (Agilent Technologies Inc., Santa Clara, CA, USA) and features were extracted with Agilent's Feature Extraction software (Agilent Technologies Inc.). The results have shown that from a total of 414 566 probes harboured by the BEGMP array, 248 453 and 253 147 probes were detected in in vitro and in vivo derived blastocysts, respectively. Data analysis using the linear modelling for microarray (LIMMA) package and R software (The R Project for Statistical Computing, Vienna, Austria) revealed a total of 3434 differentially methylated regions (DMRs; Fold change ≥1.5, P-value &lt;0.05), of which 42 and 58% were hyper- and hypo-methylated, respectively, in in vitro derived blastocysts compared to their in vivo counterparts. The DMRs were found to be localised in the intronic, exonic, promoter, proximal promoter, and distal promoter, and some of the probes did not have nearby genes. In addition, 10.8% of the DMRs were found to be stretched in short, long, or intermediate CpG islands. Thus, this study demonstrated genome-wide dysregulation in the epigenome landscape of in vitro-derived embryos by the time they reach to the blastocysts stage.

Differential expression of selected candidate genes in bovine embryos produced in vitro and cultured with chemicals modulating lipid metabolism

Lipid accumulated in embryos produced in vitro has been linked to reductions in both quality and postcryopreservation viability. Therefore, the objective of the present study was to investigate the influence of lipid-reducing chemicals on embryo development, quality, and postcryopreservation viability, in addition to expression profiles of selected lipid metabolism-regulating genes. Bovine cumulus-oocyte complexes were matured and fertilized in vitro; eight-cell stage embryos were cultured in IVC medium supplemented with phenazine ethosulfate (PES), l-carnitine (LC), PES + LC, or no supplementation (control). Culturing embryos in medium with LC increased (P < 0.05) blastocyst rate (38.8%) compared with the other groups (control = 28.1%, PES = 27.1%, PES + LC = 26.3%). Embryos cultured with supplements had greater total cell number and fewer apoptotic cells than the control. Cytoplasmic lipid content was reduced, whereas mitochondria density was increased in embryos treated with culture supplements; this was linked to altered expression profiles of selected genes regulating lipid metabolism. For example, transcript abundance of transmembrane lipid gene (SGPP1) was greater in LC- and PES-treated embryos, and they had increased postcryopreservation hatching ability (indicative of embryo cryotolerance). In conclusion, the two lipid metabolism regulators added to the culture media had improved embryo quality and cryotolerance, but embryo development rate and downstream lipid metabolism-regulating genes were more influenced with LC supplementation.

Validation of a reference control for an SYBR-Green fluorescence assay-based real-time PCR for detection of bovine herpesvirus 5 in experimentally exposed bovine embryos

The objective of this study was to optimize an internal control to improve SYBR-Green-based qPCR to amplify/detect the BoHV-5 US9 gene in bovine embryos produced in vitro and experimentally exposed to the virus. We designed an SYBR-Green-based binding assay that is quick to perform, reliable, easily optimized and compares well with the published assay. Herein we demonstrated its general applicability to detect BoHV-5 US9 gene in bovine embryos produced in vitro experimentally exposed to BoHV-5. In order to validate the assay, three different reference genes were tested; and the histone 2a gene was shown to be the most adequate for normalizing the qPCR reaction, by considering melting and standard curves (p < 0.05). On the other hand, no differences were found in the development of bovine embryos in vitro whether they were exposed to BoHV-5 reference and field strains comparing to unexposed embryos. The developed qPCR assay may have important field applications as it provides an accurate BoHV-5 US9 gene detection using a proven reference gene and is considerably less expensive than the TaqMan qPCR currently employed in sanitary programs.

233 ASSOCIATION OF THE BOVINE 2-CELL-STAGE BLASTOMERE TRANSCRIPTOME PROFILE WITH THE INDIVIDUAL DEVELOPMENTAL POTENTIAL OF THE SISTER BLASTOMERE

Transcriptome profiling has been used to identify genes related to developmental competence in bovine embryos and oocytes for several years. However, the direct relationship between the transcriptome profile and developmental potential of the same pre-implantation embryo is missing. Therefore, in the present study, one blastomere of a 2-cell-stage embryo was taken as a biopsy and immediately snap-frozen for transcriptome analysis; the sister blastomere was cultured individually in a well-of-the-well culture system. Frozen individual blastomeres taking the form of 2-cell-stage embryos were pooled together, depending on the developmental destination of the sister blastomeres. Accordingly, three groups were defined: 1) embryos that did not cleave after separation (2CB), 2) embryos arrested before embryonic genome activation (8CB), and 3) embryos that reached the blastocyst stage (BL). On the basis of these developmental phenotypes, blastomeres were pooled and used for transcriptome analysis, using a bovine EmbryoGene microarray platform (Agilent Technologies, Santa Clara, CA, USA). Results revealed 632 genes to be differentially regulated (fold change, P ≥ 1.5, P ≤ 0.05; FDR, P ≤ 0.1) between competent (BL group) and incompetent 2-cell-stage embryos (2CB) as well as 150 genes between the BL group and 8CB. Seventy-seven genes were commonly differentially regulated. Functional annotation analyses of differentially regulated genes indicated those genes to be involved in the protein ubiquitination pathway on the one hand and in the oxidative stress response, including oxidoreductase, peroxidase, and antioxidant activity, as well in oxidative phosphorylation (e.g. NDUFS1, MAPK14, CAT, PRDX1, and PRDX6) on the other hand. Furthermore, selected candidate genes known to function as direct and indirect scavengers of reactive oxygen species (ROS) were analysed for their expression in an independent model for developmental competence; namely, early- and late-cleaved 2-cell-stage embryos. Moreover, ROS detection was performed, showing higher accumulation of ROS in late-cleaved 2-cell-stage embryos, whereas lower ROS levels were detected in early-cleaved 2-cell-stage embryos associated with higher expression of ROS scavengers. The overall findings of the present study indicate the potential of using blastomere biopsies at 2-cell-stage embryos for molecular analysis to understand the molecular mechanisms associated with the further developmental competence of early-stage embryos.

Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro

The aim of this study was to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos and the expression of aquaporin 3 (Aqp3) and Na/K ATPase isoform 1 (ATPAse1) genes in embryos (i) with different ability to undergo rehydration and (ii) following vitrification. In experiment 1, in vitro fertilized presumptive zygotes were co-cultured in SOFaac or modified CR2aa medium and embryos at blastocyst and expanded blastocyst stages at day 7 post-insemination were exposed to NaCl hypertonic medium (900mOsm) for 5min following 120min of culture in isotonic medium in order to evaluate dehydration and rehydration, respectively. No difference (P>0.05) on blastocyst rate was found between CR2aa and SOFaac medium but embryos co-cultured in SOFaac medium underwent greater (P<0.05) dehydration. Embryos at expanded blastocyst stage underwent greater dehydration but slower rehydration than embryos at blastocysts stage (P<0.05). In the experiment 2, the amount of Aqp3 and ATPase1 transcripts were quantified in blastocysts with high or low rehydration after exposure to hypertonic medium. No difference (P>0.05) on relative amount of transcripts was found in either genes. In the experiment 3, expanded blastocysts produced in a co-culture system were vitrified, warmed and then cultured for 72h for analysis of embryo survival and amount of Aqp3 and ATPase1 transcripts. Lower (P<0.05) embryo survival rate was found for vitrified-warmed embryos (57.9%) than for their fresh counterparts (84.6%). There was no difference on expression of ATPase1 gene but lower (P<0.01) amount of Aqp3 transcripts was found in the vitrified-warmed embryos. In conclusion, embryo ability to undergo shrinkage and swelling is influenced by medium used in a co-culture system and by embryo stage. Rehydrating ability of embryos after exposure to NaCl hypertonic medium is not associated with variations on expression of Aqp3 and ATPase1 genes, but the vitrification can alter gene expression of in vitro-fertilized bovine embryos produced in a co-culture system.

Differential expression of heat shock protein genes and their splice variants in bovine preimplantation embryos

Heat shock proteins (HSP) are among the first proteins produced during embryonic development and are crucial to cell function; their activities include folding, unfolding, transport, and localization of proteins and differentiation and regulation of the embryonic cell cycle. Although HSP have been extensively studied in humans and mice, limited information exists on the roles of these genes in bovine embryos. As such, the objectives of this study were to profile the expression of HSP and their splice variants in bovine embryos (degenerates vs. blastocysts) and to carry out association analysis with fertility traits. Quantitative real time PCR analysis revealed differences in expression that ranged from 1.5- to 7.6-fold in degenerate embryos compared with blastocysts. Interestingly, all members of the HSP40 family were found to be upregulated in degenerate embryos. Analysis of current bovine Ensembl data showed that 13 HSP genes have 1 transcript each, 2 genes have 2 transcripts each, and 2 have 3 transcripts each. Some splice variants show differential expression between degenerate embryos and blastocysts, whereas others were not expressed at all in embryos, which implies different functions of these transcripts in embryonic development. The most significant differentially expressed genes were further investigated for association with fertility and development traits. Single nucleotide polymorphisms in DNAJC15 and DNAJC27 were found to be associated with blastocyst rate and fertilization rate, respectively. Collectively, the roles of HSP in fertilization and early development of cattle embryos are important.

Aberrant Expression Patterns of Genes Involved in Segregation of Inner Cell Mass and Trophectoderm Lineages in Bovine Embryos Derived from Somatic Cell Nuclear Transfer

High rates of embryonic, fetal, or placental abnormalities have consistently been observed in bovine cloning. Segregation of inner cell mass (ICM) and trophectoderm (TE) lineages in early embryos is an important process for fetal and placental formation. In mouse embryos, differentiation of ICM and TE is regulated by various transcription factors, such as OCT-4, CDX2, and TEAD4, but molecular mechanisms that regulate differentiation in bovine embryos remain unknown. To clarify gene transcripts involved in segregation of ICM and TE lineages in bovine embryos, we examined the relative abundances of OCT-4, CDX2, TEAD4, GATA3, NANOG, and FGF4 transcripts in blastocyst embryos derived from in vitro fertilization (IVF). Furthermore, transcript levels of such genes in bovine embryos derived from somatic cell nuclear transfer (NT-SC) and in vivo (Vivo) were also compared. OCT-4, NANOG, and FGF4 transcript levels in IVF embryos were significantly higher in ICM than in TE. In contrast, the CDX2 transcript level was lower in ICM than in TE. In NT-SC embryos at the blastocyst stage, transcript levels of all genes except CDX2 were lower than that in Vivo embryos. In the elongated stage, expression levels of the six genes did not differ between NT-SC and Vivo embryos. We observed aberrant expression patterns of various genes involved in segregation of ICM and TE lineages in bovine NT-SC embryos. These results raise the possibility that abnormalities in the cloned fetus and placenta are related to the aberrant expression of genes involved in segregation and differentiation process in the early developmental stage.

In vitroculture and somatic cell nuclear transfer affect imprinting of SNRPN gene in pre- and post-implantation stages of development in cattle

BackgroundEmbryo in vitro manipulations during early development are thought to increase mortality by altering the epigenetic regulation of some imprinted genes. Using a bovine interspecies model with a single nucleotide polymorphism, we assessed the imprinting status of the small nuclear ribonucleoprotein polypeptide N (SNRPN) gene in bovine embryos produced by artificial insemination (AI), in vitro culture (IVF) and somatic cell nuclear transfer (SCNT) and correlated allelic expression with the DNA methylation patterns of a differentially methylated region (DMR) located on the SNRPN promoter.ResultsIn the AI group, SNRPN maternal expression is silenced at day 17 and 40 of development and a third of the alleles analyzed are methylated in the DMR. In the IVF group, maternal transcripts were identified at day 17 but methylation levels were similar to the AI group. However, day-40 fetuses in the IVF group showed significantly less methylation when compared to the AI group and SNRPN expression was mostly paternal in all fetal tissues studied, except in placenta. Finally, the SCNT group presented severe loss of DMR methylation in both day-17 embryos and 40 fetuses and biallelic expression was observed in all stages and tissues analyzed.ConclusionTogether these results suggest that artificial reproductive techniques, such as prolonged in vitro culture and SCNT, lead to abnormal reprogramming of imprinting of SNRPN gene by altering methylation levels at this locus.

Messenger RNA expression patterns of histone‐associated genes in bovine preimplantation embryos derived from different origins

Histone modification genes in bovine embryos: The mRNA expression pattern of histone-related genes was determined in bovine oocytes and embryos. We compared immature and in vitro-matured oocytes, either before or after enucleation and activation, in vitro produced embryos (zygotes, 8-16 cell stages, blastocysts), embryos cloned with female or male donor cells; parthenogenetic embryos, and in vivo-derived blastocysts to detect deviations from the normal expression pattern. A sensitive semi-quantitative endpoint RT-PCR assay was used to reveal differences in histone deacetylation [histone deacetylase 2 (HDAC2)]; histone acetylation [histone acetyltransferase 1 (HAT1)]; histone methylation [histone methyltransferases (SUV39H1, G9A)]; heterochromatin formation [heterochromatin protein 1 (HP1)]; and chromatin-mediated transcription regulation [zygote arrest 1 (ZAR1)]. With the exception of ZAR1, these mRNAs were present throughout preimplantation development. The relative abundance of mRNAs for histone methyltransferases (SUV39H1 and G9A) and for heterochromatin-associated protein (HP1) differed significantly before and after activation of the bovine embryonic genome. The similarity of HAT1 gene expression in 8-16 cell embryos and blastocysts suggests that histone acetylation is primarily affected by in vitro culture only prior to embryonic genome activation. HDAC2 gene mRNA expression was not affected by in vitro culture and/or cloning before and after activation of the embryonic genome. The donor cell line affected mRNA expression patterns of genes involved in reprogramming cloned embryos suggesting epigenetic dysregulation. Results show that both in vitro production and somatic cloning alter the mRNA expression of histone modifying genes in bovine embryos.

Changes in the mRNA Transcripts of Insulin-Like Growth Factor Ligand, Receptors and Binding Proteins in Bovine Blastocysts and Elongated Embryos Derived from Somatic Cell Nuclear Transfer

The objective of this study was to determine changes in the transcription of insulin-like growth factor (IGF)-related genes in blastocyst (BC)- and elongated (EL)-stage embryos produced by nuclear transfer using somatic cells (NT-SC). Bovine BC (day 7)- and EL (day 15)-stage embryos were obtained from NT-SC or in vivo production (Vivo). The relative abundance of mRNA was examined by RT- real-time PCR. The transcript of IGF-II was only detected at the EL stage in both the NT-SC and Vivo embryos. The level of transcription of the IGF-I receptor (r) in the NT-SC embryos was decreased at the EL stage and was significantly (P<0.05) lower than at the BC stage. In contrast, the IGF-IIr levels did not differ significantly between the NT-SC and Vivo embryos, regardless of the developmental stage. IGF-binding protein (IGFBP)-2 levels were markedly decreased in the NT-SC and Vivo embryos at the EL stage (P<0.05). The IGFBP-3 level in Vivo was significantly (P<0.05) increased at the EL stage compared with at the BC stage. However, the IGFBP-3 levels in NT-SC embryos were unchanged and lower (P<0.05) than in the Vivo embryos at the EL stage. These results suggest that there are differences in the levels and changes in the transcription of IGF-related genes in bovine embryos produced by NT-SC compared with those produced by Vivo.

Gene expression in bovine embryos derived from oocytes with different developmental competence collected at the defined follicular developmental stage

The aim of this study was to compare the expression of selected genes in bovine embryos developed from oocytes with different developmental competence. Four oocyte populations were collected, separately either from small (2–5 mm) or medium (6–10 mm) follicles, in the growth/stagnation (G/S) or dominance/regression (D/R) stage of the first follicular wave. They were matured, fertilized and cultured to D7 or D8 blastocysts by a standard protocol. Poly (A)+ mRNA was extracted from pooled blastocysts and the expression of bax-α (Bax), connexin 43 (Cx 43) and connexin 31 (Cx 31) was estimated using real-time RT-PCR. The cleavage rates were significantly higher in oocytes collected from both medium and small follicles, ( p ≤ 0.05 and p ≤ 0.01, respectively) in the G/S than in the D/R stage. There were no significant differences in the D7 blastocyst rates between oocytes from both medium and small follicles in the G/S or D/R stage. But the D8 blastocyst rate was significantly higher in oocytes from small follicles in the G/S stage compared with those in the D/R stage. The relative abundance of Bax and Cx 31 made no significant difference in both D7 and D8 blastocysts developed from oocytes collected from medium or small follicles in the G/S or D/R stages. But the relative abundance of the Cx 43 transcript was significantly higher in D8 blastocysts developed from oocytes collected from both medium and small follicles in the G/S stage compared with those in the D/R stage. We conclude that the relative abundance of Cx 43 can be used as a marker of developmental potential for embryos derived from oocytes with different developmental competence because the level of Cx 43 transcript was greater in embryos derived from oocytes with greater developmental competence compared with those derived from oocytes with lesser developmental competence.

Quantitative expression analysis of blastocyst-derived gene transcripts in preimplantation developmental stages of in vitro-produced bovine embryos using real-time polymerase chain reaction technology

The main objective of the present study was to analyse the quantitative expression pattern of genes from a subtracted blastocyst transcriptome throughout the preimplantation developmental stages of in vitro-produced bovine oocytes and embryos. For this purpose, Day 5 morula (M) cDNAs were subtracted from Day 7 blastocyst (B) cDNAs (B-M) and used to establish a B-M subtracted cDNA library, as reported previously. From the total generated clones, 19 were analysed quantitatively. The mRNA samples isolated from pools of immature oocytes (n = 150), mature oocytes (n = 150) and two-cell (n = 80), four-cell (n = 40), eight-cell (n = 20), morula (n = 6) and blastocyst (n = 3) embryos were reverse transcribed and subjected to real-time polymerase chain reaction (PCR) using sequence-specific primers and SYBR green as the DNA dye. A relative standard curve method was used to analyse the real-time data taking the morula stage as a calibrator. Applying suppression subtractive hybridisation (SSH), a total of 71 clones, which represent 33 different expressed sequence tags, were generated and available for analysis. Most transcripts were analysed for the first time in bovine embryogenesis. The real-time PCR has validated the results of SSH positively for 84% (16/19) of transcripts, whereas 16% (3/19) showed deviation in the expression pattern from the one seen during SSH. Several transcript-specific expression patterns were observed for genes that play decisive roles in bovine embryogenesis. In addition to identification, accurately quantifying the expression profiles of transcripts during development will pave the way towards understanding the molecular mechanisms of embryogenesis and their potential role in early embryo development. Most importantly, the present study has contributed to the enrichment of bovine embryo gene collection by generating new transcripts involved in bovine embryo development.