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Reproduction (2009) 137 271-283
DOI: 10.1530/REP-08-0265
Copyright © 2009 Society for Reproduction and Fertility
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RESEARCH

Analysis of gene transcription alterations at the blastocyst stage related to the long-term consequences of in vitro culture in mice

Raúl Fernández-González, Juan de Dios Hourcade, Irene López-Vidriero1, Alberto Benguría1, Fernando Rodríguez De Fonseca2 and Alfonso Gutiérrez-Adán

Departamento de Reproducción Animal, INIA, Carretera de la Coruña Km 5.9, Madrid 28040, Spain1 Servicio de Genómica, CNB-CSIC, Universidad Autónoma de Madrid, Madrid, Spain2 Fundación IMABIS, Hospital Carlos Haya, Avda Carlos Haya 82, 29010 Málaga, Spain

Correspondence should be addressed to A Gutiérrez-Adán; Email: agutierr{at}inia.es


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Declaration of interest
 References
 
We have reported that in vitro culture (IVC) of preimplantation mouse embryos in the presence of FCS produces long-term effects (LTE) on development, growth and behaviour of the offspring at adult age. To analyse the mechanisms underlying this phenomenon, we have examined development and global alterations in gene expression in the mouse blastocysts produced in the presence of FCS, conditions known to be suboptimal and that generate LTE. Embryos cultured in vitro in KSOM and in KSOM+FCS had a reduced number of cells in the inner cell mass at the blastocyst stage compared with in vivo derived embryos; however, only culture in KSOM+FCS leads to a reduction in the number of trophoblast cells. Gene expression levels were measured by comparison among three groups of blastocysts (in vivo, IVC in KSOM and IVC in KSOM+FCS). Different patterns of gene expression and development were found between embryos cultured in vitro or in vivo. Moreover, when we compared the embryos produced in KSOM versus KSOM+FCS, we observed that the presence of FCS affected the expression of 198 genes. Metabolism, proliferation, apoptosis and morphogenetic pathways were the most common processes affected by IVC. However, the presence of FCS during IVC preferentially affected genes associated with certain molecular and biological functions related to epigenetic mechanisms. These results suggest that culture-induced alterations in transcription at the blastocyst stage related to epigenetic mechanisms provide a foundation for understanding the molecular origin at the time of preimplantation development of the long-term consequences of IVC in mammals.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Declaration of interest
 References
 
The in vitro culture (IVC) of the preimplantation stage mammalian embryo serves as the basis of modern reproductive technologies and embryonic stem cell biology. Such culture of embryos, however, causes a marked reduction in their viability and development. It is well known that the IVC of embryos influences the expression of developmentally important genes in the embryo (Natale et al. 2001, Wrenzycki et al. 2001, Rizos et al. 2002, Lonergan et al. 2003a, 2003b, 2003). After several days in culture, embryos have a typically higher proportion of cells undergoing death and fewer total cells than embryos derived in vivo (O'Neill 1998, Chandrakanthan et al. 2006). Modifications made to culture media can have serious consequences for mRNA expression in the embryo which, in turn, can be associated with alterations in embryo quality. For example, the presence of serum in a standard culture medium alters the pattern of mRNA expression (Wrenzycki et al. 1999, Rizos et al. 2003), affects embryo development (Gutierrez-Adan et al. 2001), affects cryotolerance (Rizos et al. 2003), is associated with a reduction in the pregnancy rate after transfer to recipients (Lazzari et al. 2002), and may produce long-term effects (LTE) on post-natal development and behaviour (Fernandez-Gonzalez et al. 2004). Barker & Osmond (1986) first proposed the ‘foetal origins of adults disease’ hypothesis, which states that aspects of foetal development may predispose offspring to a range of adult diseases. We have found in mice that the alteration of preimplantation development also produces long-term effects; for this reason we named this phenomenon ‘preimplantation origins of adult disease’. Convincing evidence, that indeed the application of IVC can result in altered phenotypes, has been collected from other animal models. For example, a large offspring syndrome has been described in sheep and cattle, which seems to be associated with the IVC of embryos and with the alteration of the genetic imprinting re-established during preimplantation development (Sinclair et al. 2000).

In addition to the effect of IVC on gene expression, the epigenetic reprogramming of the embryo may also be severely affected by IVC, particularly imprinted genes (Doherty et al. 2000, Khosla et al. 2001, Fernandez-Gonzalez et al. 2004). Imprinted genes are particularly implicated in the regulation of foetal growth, placental function, brain development and post-natal behaviour. It has been determined that the formulation of the medium used for embryo culture has a profound effect on the methylation pattern in resultant two-cell embryos (Shi & Haaf 2002), indicating that, in addition to imprinted genes, other epigenetic alterations may intensely alter the gene expression. The subacute nature of some of these aberrant embryo modifications induced by IVC, allows these changes to remain undetected in the short term, and blastocyst production, a hallmark for the efficiency of IVC systems, can often be achieved despite the detrimental environmental effects. In some recent studies (Isles & Wilkinson 2000, Ecker et al. 2004, Fernandez-Gonzalez et al. 2004, 2007), the long-term consequences of IVC produced embryos have been analysed in the mouse. Mice derived from embryos cultured under suboptimal conditions can suffer from obesity, increased anxiety and deficiencies on their implicit memory system. In one of these studies, we observed that only culture in the presence of serum reduces the viability of embryos and it is responsible for alterations in genetic imprinting during preimplantation development (Fernandez-Gonzalez et al. 2004). Similar developmental and behavioural alterations in adult mice derived from in vitro produced embryos not exposed to serum were reported by others (Ecker et al. 2004), suggesting that IVC environments in general, not only those supplemented with serum, are capable of inducing aberrant phenotypes. Taken together, our observations, and the observations of other research teams, suggest that the well-documented developmental alterations seen in mouse, sheep and cattle after embryo IVC and manipulation are probably applicable to most eutherian mammals, including humans. Post-natal survivors of these procedures might have subtle genetic and epigenetic defects that are below the threshold, threaten viability, which are only detected at long term (Fernandez-Gonzalez et al. 2008).

To address the specific effect of FCS on mRNA transcription of blastocyst embryos cultured in vitro, we undertook a detailed analysis of global gene expression in mouse embryos using an Affymetrix (Santa Clara, CA, USA) oligonucleotide array (MOE430 2.0 chip) containing more than 45 000 transcripts and variants, together with a T7-based linear double amplification method. We report here changes in the pattern of gene expression that were evident at the blastocyst stage following IVC in the presence or absence of serum. We report that, when IVC took place in KSOM medium, the presence of serum leads to the altered expression of 198 genes. The functional classification of genes expressed differentially, noting mis-expressed genes related to epigenetic mechanisms which are affected by IVC and which may be responsible for the long-term consequence of suboptimal IVC conditions, is discussed.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Declaration of interest
 References
 
Embryo development and cell number of blastocysts produced in vitro in KSOM or in KSOM supplemented with FCS
Embryos were collected at the zygote stage (day 0.5) and were cultured in vitro 96 h (in KSOM medium either supplemented or not with FCS) or were collected directly from the uterus at the blastocyst stage (day 4). Embryos cultured in KSOM supplemented with FCS were less likely to develop to the blastocyst stage (43%) than zygotes cultured in KSOM (95%; P<0.001). Blastocysts derived from zygotes cultured in the presence of FCS had fewer cells per blastocyst than embryos collected fresh from the reproductive tract or cultured zygotes in the absence of FCS (Fig. 1; P<0.001). No differences were observed between blastocysts cultured in KSOM or produced in vivo. Embryos cultured in KSOM+FCS (that produced long-term deleterious effects) had a reduced number of trophectoderm (TE) cells compared with embryos cultured in KSOM (that did not produce long-term effects). In vivo embryos had more inner cell mass (ICM) cells than embryos produced in vitro with or without FCS, (while having a similar number of TE cells to KSOM but more TE cells than KSOM+FCS (P<0.01)). Blastocyst development to term, after transfer into recipient female, was smaller when IVC was performed in the presence of FCS; and in both groups (IVC with or without FCS) was smaller than embryos produced in vivo (82% in vivo, 44% in KSOM, 23% in KSOM+FCS; the number of recipient mice used was 8, 9 and 13 respectively).


Figure 1
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  		Figure 1 Effect of the preimplantation in vitro culture system (in vitro cultured in KSOM, in vitro cultured in KSOM+FCS and produced in vivo) on blastocysts cell number and on ICM and TE cell number. a,b, significant differences at P<0.05.

 
Differentially expressed genes in blastocysts produced in vitro in KSOM or in KSOM supplemented with FCS
The mouse Affymetrix 430 2.0 microarray chip, which represents the complete mouse genome (45 100 probes), was used to analyse the global pattern of gene expression in the three groups of embryos. Approximately 25% of the transcripts were considered detected in the three groups of embryos; these results are consistent with data reported in the literature (Giritharan et al. 2007). To assess the similarities of the different treatments and to obtain a visual understanding of the relationship between the different experimental groups, Pearson correlations and hierarchical clustering analysis were realised. The Pearson correlation coefficients for the triplicate biological samples related to each of the growth conditions (IVC in KSOM, IVC in KSOM+FCS and in vivo) were respectively 0.92, 0.93 and 0.89, indicating a very high degree of reproducibility between repetitions. The Pearson correlation coefficients of paired comparisons between the three growth conditions were 0.92 for KSOM versus KSOM+FCS, 0.90 for KSOM versus in vivo, and 0.91 for KSOM+FCS versus in vivo. The results of the hierarchical clustering analysis indicate that in vivo embryos, IVC in KSOM and IVC in KSOM+FCS embryos have distinctive transcriptomes, and that all the replicate samples of the three groups self-cluster into one branch (Fig. 2). To evaluate the heterogeneity between individual embryos per treatment (blastocysts produced in vivo or in vitro in KSOM or in KSOM+FCS), we used single embryo qRT-PCR of three housekeeping genes: Gapdh, 18S rRNA and β-actin. The level of embryo-to-embryo variation per group for the three genes did not differ significantly, suggesting low gene expression variability of individual embryos. The lower standard deviation was found in the in vivo group.


Figure 2
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  		Figure 2 Hierarchical clustering analysis results showing cluster image display for genes with colour gradient for standardised gene expression of all samples from different culture system analysed (produced in vivo, in vitro cultured in KSOM and in vitro cultured in KSOM+FCS). Each expression measurement represents the normalised log2 ratio of fluorescence from the hybridised experimental sample to a reference sample. Normalised expression ratios are depicted by a pseudocolour scale with red indicating positive expression above the reference, black indicating equal expression as the reference and green indicating negative expression below the reference. A dendrogram (tree graph) depicts the grouping of the genes based on the similarity between them. Unsupervised clustering in GeneSpring was used to analyse similarities among replicate samples across all treatment groups tested.

 
We then performed pairwise comparison to observe how many genes are statistically different among the three groups of embryos, as is indicated in Supplementary Figure 1, (which can be viewed online at www.reproduction-online.org/supplemental/). At the P<0.01 significance level, microarray analysis revealed that 198 genes were differentially expressed at the blastocyst stage between embryos cultured in vitro in KSOM or in KSOM supplemented with FCS (P<0.01 and FDR <0.1; Table 2, Supplementary Table 2, which can be viewed online at www.reproduction-online.org/supplemental/). This comparison allowed us to analyse the effect of the presence of FCS, since the culture conditions up to the blastocyst stage were the same in the two groups. Thus, a relatively small number of genes were associated with a physiologically altered state of the blastocysts that produced long-term consequences in the offspring (Fernandez-Gonzalez et al. 2004). Out of 198 genes, 100 were more highly expressed in blastocysts cultured without FCS, whereas 98 were more highly expressed in blastocysts cultured with FCS. Because some of the genes were uncharacterised (unknown), we focused our attention on the known genes. Table 1 shows a list of the 30 genes with major variation when we compared transcription of genes in blastocysts produced in KSOM+FCS with transcription of blastocysts produced in vivo or in KSOM without FCS. The list includes genes related to structural constituents, metabolism, growth factors, etc. Table 2 indicates the genes with major variation after culture in KSOM supplemented with FCS vs KSOM. To confirm microarray results obtained in this study, we conducted an additional analysis of six genes (Ghr, Lama1, Sox17, Fgf4, Abcb1b and Rhox5) using real-time RT PCR. Results of this analysis indicated that the six genes tested confirmed the microarray changes (Fig. 3).


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  		Table 2 Principal genes down- and up-regulated after culture in KSOM+FCS versus KSOM.

 

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  		Table 1 Principal genes down- and up-regulated after culture in KSOM+FCS versus KSOM and VIVO.

 

Figure 3
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  		Figure 3 Real-time PCR verification of microarray data. Expression profile of Ghr, Lama1, Sox17, Fgf4, Abcb1b, and Rhox5 genes in murine blastocysts produced by in vitro culture in KSOM supplemented with or without FCS, and in vivo. a,b, significant differences at P<0.05.

 
The gene ontology (FatiGO) was used for categorising embryo expressed sequence tags (ESTs) with respect to gene function, including molecular function and biological process, and cellular component (Al-Shahrour et al. 2007). As indicated in Figs 4 and 5, analysis of 198 genes, differentially regulated in the presence of FCS, provides the following functional categories based on biological processes: molecular metabolic process, primary metabolic process, biosynthetic process, multicellular organism development and characteristics of activated blastocysts (Fig. 4); and based on molecular function: nucleotide binding, transferase activity, hydrolase activity, receptor activity, kinase regulator activity, extracellular matrix structural constituents, etc (Fig. 5).


Figure 4
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  		Figure 4 Fatigo-based comparative analysis of gene ontology of the family of genes statistically different between IVC and in vivo produced mice embryos related to biological processes.

 

Figure 5
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  		Figure 5 Fatigo-based comparative analysis of gene ontology of the family of genes statistically different between IVC and in vivo produced mice embryos related to molecular functions.

 
Differentially expressed genes in blastocysts produced in vitro in KSOM supplemented with or without FCS and in those derived in vivo
When we compared differences between the in vivo control group and the in vitro groups (KSOM supplemented with or without FCS), we observed that the magnitude of change in gene transcription for most of the genes was small (Supplementary Figure 1 and Supplementary Tables 3, 4 and 5, which can be viewed online at www.reproduction-online.org/supplemental/). If we compare the groups of genes that showed more than a one- or four-fold change in KSOM+FCS versus in vivo and in KSOM versus in vivo, a higher number of genes are up-regulated in IVC conditions, indicating that the state of the genome transcription is up-regulated in IVC conditions. Analysis of the common processes (Figs 4 and 5) revealed that the overall differences in biological processes are similar between the FCS groups versus in vivo and KSOM versus in vivo (metabolism, proliferation, apoptosis and morphogenetic pathways); there are differences only in the catabolic process and cell cycle gene groups. Moreover, the principal differences in molecular function are in nucleotide and protein binding, hydrolase activity, oxidoreductase activity, etc. When we compared KSOM with or without FCS, we observed that FCS produces a preferential effect on molecular function (nucleotide binding, transferase binding, hydrolase activity, receptor activity, etc) and biological functions (macromolecule and primary metabolic process, multicellular organism development, cell cycle, response to stress, etc).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Declaration of interest
 References
 
It has been reported that the IVC of preimplantation embryos alters gene transcription when compared with those that develop in vivo; however, it remains to be analysed if these changes are associated with long-term consequences. From some recent studies (Ecker et al. 2004, Fernandez-Gonzalez et al. 2004), we are now gaining insight into the importance of IVC conditions that may influence the developmental programme, not only in the short term for blastocyst morphogenesis, but also in the long term for the growth, viability and physiology of the foetus and offspring. IVC with in KSOM affects the quality of the blastocysts but did not have any long-term consequence in the adult animal (Fernandez-Gonzalez et al. 2007). However, culture in the presence of FCS reduced by half, the capacity of the resulting blastocysts to implant into the uterus following embryo transfer when compared with culture without FCS (Fernandez-Gonzalez et al. 2004), and lead to abnormal foetal growth and development (Khosla et al. 2001, Fernandez-Gonzalez et al. 2004). Using microarray analysis, we have identified a panel of genes that are differentially expressed in in vitro cultured mouse embryos in KSOM, in the presence or absence of FCS, which may explain the biology of blastocysts and the phenotype of the adult derived animals. The differences in gene transcription between blastocysts produced in KSOM and KSOM supplemented with FCS are remarkable, and may be related to the differences in the TE cells number, because the number of cells of their ICMs was similar. The gene expression results paralleled the morphologic results and showed that genes related to cell division, growth factor, apoptosis, development, etc, were misregulated by the presence of FCS. A notable effect of the presence of FCS is the alteration in the expression of genes related to nucleotide binding and imprinting, supporting the view that the differentially expressed genes related to long-term effects are involved in epigenetic modifications that lead to alterations in foetal and placental development that have consequences for adult health (Fernandez-Gonzalez et al. 2007).

In agreement with the quiet embryo hypothesis (Leese 2002) a higher number of genes are up-regulated in IVC conditions, indicating that the condition of the genome is up-regulated in vitro, suggesting a loss of the epigenetic mark. This phenomenon is even more evident when groups of genes, that showed more than one- or fourfold change in in vitro (KSOM w/o FCS) versus in vivo, were analysed. Our results are in agreement with Giritharan et al. (2007), who using the same microarray chip reported that IVC in Whitten's medium produced misregulation of 4660 transcripts, the majority of which were up-regulated by IVC (3693 up-regulated). Also, gene chip technology has shown that out of a total of 22 690 transcripts (Affymetrix array MOE430A), on average 39% were expressed in in vivo developed mouse blastocysts (Rinaudo & Schultz 2004). Blastocysts cultured in Whitten's medium had aberrant expression of 114 genes compared with in vivo developed embryos, and blastocysts cultured in KSOM had only 29 genes misexpressed (Rinaudo & Schultz 2004). Our results identified more misregulated genes in blastocysts produced in KSOM than in a previous report using KSOM in 5% oxygen (Rinaudo & Schultz 2004). These differences may be due to the oxygen tension, the differences in RNA techniques and the gene chip used in this study. Moreover, comparison of IVC using KSOM in 20% oxygen versus in vivo, similar results were reported in a prior publication (Rinaudo et al. 2006; 456 genes in the present comparison of KSOM versus in vivo versus 472 in the past publication). These data have notable differences in the number of genes statistically different with the data of Giritharan et al. (2007) who had used IVC with Whitten's medium versus in vivo. The differences in gene number may be due to the differences in the medium used in the IVC (KSOM versus Whitten's medium), the difference in the number of embryos used, in the amount of cDNA hybridised to the microarray, and/or in the RNA amplification technique (Giritharan et al. protocol is the only that uses linear amplification in contrast to the other studies that use double amplification protocol). However, the classes of genes altered and the biological processes affected are similar.

The effect of culture condition on gene expression has been widely recognised and may be responsible for detrimental effects on the developing embryo and foetus (Kang et al. 2001). Our results show that culture in KSOM did not reduce the number of cells at the blastocyst stage; however, we have identified a number of cellular stressors induced by the culture environment (KSOM with or without FCS), including apoptosis and oxidative stress-related genes (Trp53inp1, Zmat3, Pdrg1, Cdkn1a, Prdx2). In agreement with our results, it has been reported that the culture of mouse zygotes increases Trp53 expression and reduces embryo viability, suggesting that monitoring Trp53 expression in embryos may be a tool for assessing the effects of IVC techniques on embryo viability (Li et al. 2007). The expression of genes related to membrane transport function was also altered by IVC (Prdx2, Slc7a3, Slc15a2) in agreement with previous reports (Rinaudo & Schultz 2004). Also IVC altered the expression of genes related to kinase activity, and kinase regulator activity (Cdkn1a, Pdxk, Pip5k1a, Pdk1, Pftk1, Adk, Etnk1, Cdk2ap1, Nuak1, Ckb, Grk6, etc).

The differences in gene expression between IVC blastocysts with or without FCS should be related to the long-term effect in the offspring (Fernandez-Gonzalez et al. 2004). Differences between KSOM and KSOM+FCS must be due to the presence of inappropriate transcriptional factors in the foetal calf serum, not because of the lack of them, because the only difference between both media is the addition of 10% heat inactivated FCS to the original KSOM. IVC per se affects important players in growth and development like Fgf4, Bmp4, and Cd55. However, IVC with FCS specifically affects important growth factors and cytokines (Pdgfa, Tdgf1, Ghr, Eps8, Mitf, Dusp6, Col4a1, Socs3, Cish), that have been shown to promote blastocyst formation and increases the rate of development in mouse, cow and rat (Hardy & Spanos 2002). Interestingly, three of these genes have been differentially expressed in somatic cell nuclear transfer (Rodriguez-Zas et al. 2008). Moreover, it has been reported that some growth factors may be epigenetic regulators of embryo development (Warburton et al. 1992). Also, FCS specifically affects mitochondrial transporters (Slc25A1, Slc15A2) and the cytochrome P450 family 51 (Cyp51), which modulates serum cholesterol levels in mouse through RNA interference (Xu et al. 2008). In addition, FCS affects the expression of some genes that may be related to epigenetic processes, like nucleotide binding (Idh3g, Pftk1, Abcc4, Hnrpdl, Pdgfra, Rbm3, Myo1b, Tardbp, Amhr2, Kif1b, Pdxk, Abcb1b, Tuba4a, Ddx21, Atp13a3, Ttl, Pabpn1); nucleobase, nucleoside, nucleotide and nucleic acid metabolic processes (Adk, Zic3, Zfp36l1, Tpi1, Elf5, Entpd1, Hibadh, Grhl2, Sox17, Ifnar2, H3f3b, Zfp36l2, Rbm3, Tardbp, 4121402D02Rik, Pom121, Gnpnat1, Prmt2, Phf3, Sod1, Med28, 1110005A23Rik, Pabpn1, Usp39, Ccne1, Polr2a,l, Snrpa1); stress induced laminin glycoproteins of the basement membranes (Lama1 and Lamb1-1); and the protein arginine N-methyltransferase 2 (Prmt2) that interacts with retinoblastoma gene product to recruit a number of proteins, including histone deacetylases (HDACs), chromatin remodelling complex (SWI/SNF), lysine methyl transferase (SUV39H1) and DNA methyltransferase (DNMT1), to regulate the activity of the transcription factor E2F (Yoshimoto et al. 2006). Also embryos cultured in FCS have less expression than in vivo of Terf1, the human telomeric repeat binding factor 1, that it is critical in telomere biology; it has been reported that increase in Terf1 concentration prevents telomere elongation by telomerase (Kuimov 2004). Many of these genes, for which the expression is modified by FCS, can have some epigenetic effect. Epigenetic marks are established early in development, are mitotically, and in some cases meiotically, heritable and can have a profound impact on an organism's phenotype. In addition to DNA methylation, many mediators of epigenetic inheritance involve alterations of chromatin structure and chromatin remodelling. Genes related to nuclear laminin and nucleotide binding, and nucleobase, nucleoside, nucleotide and nucleic acid metabolic processes, are good candidates to be related to chromatin remodelling. Thus, epigenetic modifications provide a mechanism by which the changes associated with embryo programming can occur.

Although it could not be determined if imprinting was affected because the microarray was unable to distinguish maternal from paternal transcripts, the expression of some imprinted genes, like Cd81, that is imprinted in mouse placenta, was expressed at greater levels in in vivo embryos than in in vitro (with or without FCS) embryos. However, the majority of imprinted genes with abnormal expression were present in the embryos cultured in the presence of FCS. For example, FCS affects the expression of Xist, an X-inactive-specific transcript that partly regulates the X-inactivation process. Other imprinted genes that are affected by culture in FCS are Rhox5, Dvl1, E2f7, and Zfp36l2. It has been reported that Rhox5 and Xist were mapped to the X chromosome and were predominantly expressed in female mouse blastocysts (Kobayashi et al. 2006). As with the Xist gene, Rhox5 was predominantly expressed by the paternally derived X chromosome in the blastocyst, indicating sex differences in early epigenetic gene regulation (Kobayashi et al. 2006). This is a member of a homeobox gene cluster that is mainly expressed in reproductive tissues such as those of the testis, ovary and placenta, which may play a role in controlling the development of these organs (Maclean et al. 2005). At present, we have no information on the mechanism that enables preferential expression of these X linked imprinting genes, but it may be related to the differential long-term phenotype that present male and female adult mice, with the female more affected by the culture (Fernandez-Gonzalez et al. 2004, Gutierrez-Adan et al. 2006). Dvl1 encodes a cytoplasmic phosphoprotein that regulates cell proliferation, acting as a transducer molecule for developmental processes, including segmentation and neuroblast specification. Dvl1-deficient mice have abnormal social behaviour (Lijam et al. 1997), something that has also been observed in the long-term effect produced by suboptimal culture conditions (Ecker et al. 2004, Fernandez-Gonzalez et al. 2004). E2f7 is a member of the mammalian E2F transcription factor family that has properties of a transcriptional repressor capable of negatively influencing cellular proliferation (de Bruin et al. 2003), modulates the transcription properties of other E2F proteins, it influences the ability of cells to undergo a DNA damage response, and it is essential for cell survival and embryonic development (Li et al. 2008). Zfp36l2 belong to the tristetraprolin family proteins, it destabilises adenylate uridylate-rich element containing mRNAs encoding cytokines, such as tumour necrosis factor and vascular endothelial growth factor (VEGF; Cao et al. 2008), and it is crucial for female fertility and early embryonic development (Ramos et al. 2004).

It has been reported that while blastocysts exhibited perturbed imprinted gene expression and methylation after culture in vitro in a simple medium, the resultant foetal expression of imprinted genes was correct and the aberrant expression persisted only in the placenta (Mann et al. 2004). Thus, errors arising during preimplantation can result in general epigenetic dysregulation in TE lineages (FCS affects expression at blastocysts of placenta-specific factors Plac8 and Plac1). This could be the link between altered gene expression at blastocyst and long-term effects in the adult (Watkins et al. 2008). IVC in ruminants is also linked with defective oversized foetuses and to overly large placentomes (Bertolini & Anderson 2002). Epigenetic changes in Igf2r are associated with this foetal overgrowth after sheep embryo culture (Young et al. 2001). This syndrome is call large offspring syndrome (resembling the Beckwith–Wiedemann syndrome observed in humans) which, seems to be a consequence of abnormal imprinting alterations resulting from the exposure of in vitro produced embryos to foetal calf serum (Sinclair et al. 2000). In sheep and cattle, in vitro embryo culture with serum has been associated with abnormal physiology, organ and skeletal development (Sinclair et al. 1999, Farin et al. 2001). It will be interesting to analyse if the altered imprinted gene expression observed in mice is also related to the large offspring syndrome in ruminants. In addition, we have identified new genes that are affected by IVC conditions that produce LTE; these are new genetic markers very useful for assaying embryo quality in other species.

Although considerable progress has been made in uncovering genes influencing embryo development, much work still remains to be done to completely characterise the association between gene-expression patterns and embryo, foetal and adult phenotype. Our results show that the differences between blastocysts produced in vitro in culture conditions do not produce long-term effects in adults (KSOM without FCS), and suboptimal culture conditions that produce a long-term effect on the health of the offspring (KSOM with FCS), are related to changes in the expression of nucleotide binding, nucleic acid binding genes, telomeric factors, demethylase and methyltransferase activity, growth factors and imprinted genes, that produce epigenetic modifications that lead to reprogramming errors, some of which will be elicited via direct effects on chromatin structure and remodelling, and some of then via methylation. We conclude that epigenetic modification is best viewed as a component of a broader causal model linking suboptimal IVC with phenotype long-term perturbation through both transcriptional and epigenetic modification of gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Declaration of interest
 References
 
Embryo collection
All mice used in this experiment were B6CBAF1 and were maintained in an animal facility with controlled temperature conditions at 23±1 °C, with a 14h light:10h darkness photoperiod and with free access to water and food. Experimental procedures followed ethical guidelines established by the Federation of European Laboratory Animal Science Associations. One-cell embryos obtained from superovulated female B6CBAF1 mice were cultured for 4 days in KSOM in the presence of 10% foetal calf serum (Group A) or in the presence of 1 g/l BSA (Group B). In vivo blastocysts were isolated 92 h after hCG injection (Group C). All blastocysts were washed twice in M2 and transferred in minimal amount of PBS in sterile Eppendorf microcentrifuge tubes and snap frozen until use for microarray analysis.

Differential ICM and TE cell counts and embryo transfer
In vivo and in vitro produced blastocysts were stained using a modification of a method originally described by Handyside and slightly modified (Biggers et al. 2000). Briefly, blastocysts were washed in KSOM and incubated for 30 min at 37 °C with a rabbit antiserum to mouse (M-5774, Sigma) diluted to 1:10 in KSOM. After 30 min, embryos were transferred through three successive washes with KSOM and then into KSOM with 0.01 mg/ml bisbenzimide (Hoechst 33258, Sigma), 0.005 mg/ml propidium iodide and guinea pig complement (Merck Chemicals) diluted to 1:10. After lysis of trophoectoderm cells, groups of 15–20 blastocysts were transferred to a clean glass slide and compressed under a glass coverslip. Counting was performed at 400x magnification under an epifluorescence microscope (Optiphot-2, Nikon, Tokyo, Japan) using UV-2A prism (Nikon; excitation filter of 330–380 nm, dichromatic mirror 400 nm and barrier filter of 420 nm). The ICM cells fluoresce blue and TE cells stain red/pink.

To analyse blastocyst development to term after embryo transfer, blastocysts produced in vivo or in vitro were transferred into the oviduct of 0.5 days post coitum pseudopregnant CD1 females (Gutierrez et al. 1996). Data were analysed using the SigmaStat (Jandel Scientific, San Rafael, CA, USA) software package. ICM- and TE-cell counting and birth rate were analysed using ANOVA. Significant differences were defined as P<0.05.

mRNA extraction and synthesis of biotinylated cRNA
Total RNA was prepared from three pools of 160 embryos (240 ng total RNA) of each experimental group. RNA was extracted following the manufacturer's instructions using the Strataprep Total RNA microprep kit (Stratagene, La Jolla, CA, USA). Each RNA preparation was tested for degradation using the Agilent 2100 Bioanalyzer (Agilent technologies, Palo Alto, CA, USA). cDNA was synthesised from 75 ng of total RNA using two-cycle target labelling and control reagents (Affymetrix) to produce biotin labelled cRNA. The cRNA preparation (15 µg) was fragmented at 94 °C for 35 min into 35–200 bases in length, and 5 µg were used for the TestChip (Test3, Affymetrix).

Hybridisation, washing and scanning of microarrays
If the quality control was acceptable, then 10 µg of fragmented cRNA were hybridised to the mouse MOE 430 2.0 array (Affymetrix), containing 45 000 transcripts and variants. Each sample was added to a hybridisation solution containing 100 mM 2-(N-morpholino) ethanesulphonic acid, 1 M Na+, and 20 mM of EDTA in the presence of 0.01% of Tween-20 to a final cRNA concentration of 0.05 µg/ml. Hybridisation was performed for 16 h at 45 °C. Each microarray was washed and stained with streptavidin–phycoerythrin in a Fluidics station 450 (Affymetrix) and scanned at 1.56 µm resolution in a GeneChip Scanner 3000 7G System (Affymetrix). Data analyses were performed using GeneChip Operating Software.

Microarray data analysis
Data analysis was performed using Affymetrix microarray suite (MAS) five statistical algorithm. Images from each gene chip were normalised by the global scaling method to a target intensity value of 100. Paired comparisons between different arrays (three replicates of three separate treatment groups for a total of nine independent experiments) were also performed using MAS 5 comparison analysis. In order to evaluate the reproducibility of hybridisation signals between the triplicate biological samples related to each of the growth conditions, and between the paired comparisons of the three growth conditions, Pearson correlation coefficients were computed. Probe sets called ‘Absent’ in all arrays were removed from the analysis. Probe sets were selected for increased expression when all the three possible comparisons were flagged as ‘Increased’ or ‘Marginal Increase’ and when the probe set was detected ‘Present’ or ‘Marginal’ in the experimental replicates. Similar analysis was performed for the decreased probe sets except that the probe sets had to be detected as ‘Present’ or ‘Marginal’ in the Control replicates.

Gene ontology (FatiGO: http://babelomics.bioinfo.cipf.es) was used for categorising embryo ESTs with respect to gene function, including molecular function and biological process (Al-Shahrour et al. 2007).

Quantitative RT PCR (qPCR)
To confirm the ability of this microarray analysis to resolve the differences in expression levels, six genes that showed a significant difference in the embryos IVC were selected. Blastocysts developed in vivo and in vitro (KSOM/BSA and KSOM/FCS) were collected and total RNA was isolated as described above. The quantification of all gene transcripts was carried out by real-time quantitative RT-PCR (Gutierrez-Adan et al. 2004). Three replicate PCR experiments were conducted for the six genes of interest and an internal control. Experiments were conducted to contrast relative levels of each transcript and mouse Gapdh in every sample. PCR was performed by adding 4 l of each sample to the PCR mix containing the specific primers to amplify Gapdh and the selected (Ghr, Lama1, Sox17, Fgf4, Abcb1b, and Rhox5). Primer sequences, annealing temperature, approximate sizes of amplified fragments and GenBank accession number are shown in Supplementary Table 1, which can be viewed online at www.reproduction-online.org/supplemental/. PCR quantification was performed using a Rotorgene 2000 Real Time Cycler (Corbett Research, Sydney, Australia) and SYBR Green (Molecular Probes, Eugene, OR, USA) as a double-stranded DNA specific fluorescent dye. PCR mixture (25 l) contained: 2.5 l 10x buffer, 3 mM MgCl2, 2 U Taq Express (MWGAG Biotech, Ebersberg, Germany), 100 M of each dNTPs and 0.2 M of each primer. In addition, the double-stranded DNA dye, SYBR Green I, (1:3000 of 10 000x stock solution) was included in each reaction. The PCR protocol included an initial step of 94 °C (2 min), followed by 40 cycles of 94 °C (15 s), 56–59 °C (30 s) and 72 °C (30 s). Fluorescent data were acquired at 80–85 °C. The melting protocol consisted of a hold temperature at 40 °C for 60 s and then heating from 50 to 94 °C, holding at each temperature for 5 s while monitoring fluorescence. Product identity was confirmed by ethidium bromide-stained 2% agarose gel electrophoresis. As negative controls, RT-reaction tubes were prepared without RNA or polymerase. In addition, amplified identities were confirmed by the appropriate restriction digestions of PCR products (data not shown). The comparative Ct method was used to quantify expression levels (Bermejo-Alvarez et al. 2008). Quantification was normalised to the endogenous control Gapdh. Fluorescence was acquired in each cycle to determine the threshold cycle or the cycle during the log-linear phase of the reaction at which fluorescence increased above background for each sample. Within this region of the amplification curve, a difference of one cycle is equivalent to doubling of the amplified PCR product. According to the comparative Ct method, the {Delta}Ct value was determined by subtracting the GapdhCt value for each sample from each gene Ct value of the sample. Calculation of {Delta}{Delta}Ct involved using the highest sample {Delta}Ct value (i.e. the sample with the lowest target expression) as an arbitrary constant to subtract from all other {Delta}Ct sample values. Fold changes in the relative gene expression of the target were determined using the formula Formula .

For gene expression analysis of single embryos, we have used the RNA purification and RT methods as described above. Individual cDNA samples were generated from individual blastocysts (10 per treatment: in vivo and in vitro KSOM/BSA and KSOM/FCS). We used 1/10th of a blastocyst per PCR to allow for triplicates for the analysis of three genes per embryo: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA gene (18S rRNA), and β-actin (ACTB). qRT-PCR was performed using a real-time PCR as above described, and the quantification of the mRNA in each sample was realised using a standard curve for each specific gene. To generate the copy number standard curve, cDNAs were amplified by PCR from blastocysts' purified mRNA. PCR products were purified from agarose gel according to the manufacturer's protocol (Elu-quick DNA purification kit, Whatman, Stanford, USA). Following the confirmation of the PCR product by ethidium bromide-stained 2% agarose gel electrophoresis, the expected fragments were eluted, and the concentrations of the PCR fragments were determined spectrophotometrically by measuring absorbance at 260 nm. The standards and cDNA samples were then co-amplified in the same reaction prepared from a master mix. Copy numbers of fragments were empirically determined using the following equation: Copy number of PCR standard=molexAvogadro Constant=grams/fragment molecular weightxAvogadro Constant (Avogadro Constant=6.0221367x1023). The quantified PCR fragments were later serially diluted in five 10-fold serial-dilution increments and were used as known copy number template standards in quantitative PCR. Fluorescence was acquired at each cycle in order to determine the threshold cycle or the cycle during the log-linear phase of the reaction at which fluorescence rises above background for each sample. The Rotor-Gene quantification software generates a best-fit line and determines unknown concentrations by interpolating the noise-band intercept of an unknown sample against the standard curve of known concentrations. Reverse-transcriptase negative controls (samples containing RNA to which reverse transcriptase was not added) were included to exclude PCR amplification of contaminating genomic DNA. One-way repeat-measure ANOVA (followed by multiple pair-wise comparisons using Tukey test) was used for the analysis of the differences in mRNA expression assayed by qRT-PCR.


   Declaration of interest
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Declaration of interest
References
 
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.


   Funding
 
This work was supported by grant AGL2006-04799 from the Spanish Ministry of Education and Science, grants 07/0880 and 07/1226 from Instituto de Salud Carlos III, and Redes Temáticas ISCIII-RETIC RD06 and Proyectos de Excelencia, Consejeria de Innovación, Junta de Andalucia.

Received June 13, 2008
First decision July 17, 2008
Revised manuscript received October 21, 2008
Accepted November 12, 2008

   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Declaration of interest
 References
 

Al-Shahrour F, Minguez P, Tarraga J, Medina I, Alloza E, Montaner D & Dopazo J 2007 FatiGO +: a functional profiling tool for genomic data. Integration of functional annotation, regulatory motifs and interaction data with microarray experiments. Nucleic Acids Research 35 W91–W96.[Abstract/Free Full Text]

Barker DJ & Osmond C 1986 Infant mortality, childhood nutrition, and ischaemic heart disease in England and Wales. Lancet 1 1077–1081.[Web of Science][Medline]

Bermejo-Alvarez P, Rizos D, Rath D, Lonergan P & Gutierrez-Adan A 2008 Epigenetic differences between male and female bovine blastocysts produced in vitro. Physiological Genomics 32 264–272.[Abstract/Free Full Text]

Bertolini M & Anderson GB 2002 The placenta as a contributor to production of large calves. Theriogenology 57 181–187.[CrossRef][Web of Science][Medline]

Biggers JD, McGinnis LK & Raffin M 2000 Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium. Biology of Reproduction 63 281–293.[Abstract/Free Full Text]

de Bruin A, Maiti B, Jakoi L, Timmers C, Buerki R & Leone G 2003 Identification and characterization of E2F7, a novel mammalian E2F family member capable of blocking cellular proliferation. Journal of Biological Chemistry 278 42041–42049.[Abstract/Free Full Text]

Cao H, Urban JF Jr & Anderson RA 2008 Insulin increases tristetraprolin and decreases VEGF gene expression in mouse 3T3-L1 adipocytes. Obesity 16 1208–1218.

Chandrakanthan V, Li A, Chami O & O'Neill C 2006 Effects of in vitro fertilization and embryo culture on TRP53 and Bax expression in B6 mouse embryos. Reproductive Biology and Endocrinology 4 61.[CrossRef]

Doherty AS, Mann MR, Tremblay KD, Bartolomei MS & Schultz RM 2000 Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biology of Reproduction 62 1526–1535.[Abstract/Free Full Text]

Ecker DJ, Stein P, Xu Z, Williams CJ, Kopf GS, Bilker WB, Abel T & Schultz RM 2004 Long-term effects of culture of preimplantation mouse embryos on behavior. PNAS 101 1595–1600.[Abstract/Free Full Text]

Farin PW, Crosier AE & Farin CE 2001 Influence of in vitro systems on embryo survival and fetal development in cattle. Theriogenology 55 151–170.[CrossRef][Web of Science][Medline]

Fernandez-Gonzalez R, Moreira P, Bilbao A, Jimenez A, Perez-Crespo M, Ramirez MA, Rodriguez De Fonseca F, Pintado B & Gutierrez-Adan A 2004 Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior. PNAS 101 5880–5885.[Abstract/Free Full Text]

Fernandez-Gonzalez R, Ramirez MA, Bilbao A, De Fonseca FR & Gutierrez-Adan A 2007 Suboptimal in vitro culture conditions: an epigenetic origin of long-term health effects. Molecular Reproduction and Development 74 1149–1156.[CrossRef][Web of Science][Medline]

Fernandez-Gonzalez R, Moreira PN, Perez-Crespo M, Sanchez-Martin M, Ramirez MA, Pericuesta E, Bilbao A, Bermejo-Alvarez P, de Dios Hourcade J, de Fonseca FR et al. 2008 Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. Biology of Reproduction 78 761–772.[Abstract/Free Full Text]

Giritharan G, Talbi S, Donjacour A, Di Sebastiano F, Dobson AT & Rinaudo PF 2007 Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos. Reproduction 134 63–72.[Abstract/Free Full Text]

Gutierrez A, Meade HM, Ditullio P, Pollock D, Harvey M, Jimenez-Flores R, Anderson GB, Murray JD & Medrano JF 1996 Expression of a bovine kappa-CN cDNA in the mammary gland of transgenic mice utilizing a genomic milk protein gene as an expression cassette. Transgenic Research 5 271–279.[CrossRef][Medline]

Gutierrez-Adan A, Lonergan P, Rizos D, Ward FA, Boland MP, Pintado B & de la Fuente J 2001 Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos. Theriogenology 55 1117–1126.[CrossRef][Web of Science][Medline]

Gutierrez-Adan A, Rizos D, Fair T, Moreira PN, Pintado B, de la Fuente J, Boland MP & Lonergan P 2004 Effect of speed of development on mRNA expression pattern in early bovine embryos cultured in vivo or in vitro. Molecular Reproduction and Development 68 441–448.[CrossRef][Web of Science][Medline]

Gutierrez-Adan A, Perez-Crespo M, Fernandez-Gonzalez R, Ramirez M, Moreira P, Pintado B, Lonergan P & Rizos D 2006 Developmental consequences of sexual dimorphism during pre-implantation embryonic development. Reproduction in Domestic Animals 41 54–62.[CrossRef][Web of Science][Medline]

Hardy K & Spanos S 2002 Growth factor expression and function in the human and mouse preimplantation embryo. Journal of Endocrinology 172 221–236.[Abstract]

Isles AR & Wilkinson LS 2000 Imprinted genes, cognition and behaviour. Trends in Cognitive Sciences 4 309–318.[CrossRef][Web of Science][Medline]

Kang YK, Koo DB, Park JS, Choi YH, Chung AS, Lee KK & Han YM 2001 Aberrant methylation of donor genome in cloned bovine embryos. Nature Genetics 28 173–177.[CrossRef][Web of Science][Medline]

Khosla S, Dean W, Brown D, Reik W & Feil R 2001 Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biology of Reproduction 64 918–926.[Abstract/Free Full Text]

Kobayashi S, Isotani A, Mise N, Yamamoto M, Fujihara Y, Kaseda K, Nakanishi T, Ikawa M, Hamada H, Abe K et al. 2006 Comparison of gene expression in male and female mouse blastocysts revealed imprinting of the X-linked gene, Rhox5/Pem, at preimplantation stages. Current Biology 16 166–172.[CrossRef][Web of Science][Medline]

Kuimov AN 2004 Polypeptide components of telomere nucleoprotein complex. Biochemistry 69 117–129.

Lazzari G, Wrenzycki C, Herrmann D, Duchi R, Kruip T, Niemann H & Galli C 2002 Cellular and molecular deviations in bovine in vitro-produced embryos are related to the large offspring syndrome. Biology of Reproduction 67 767–775.[Abstract/Free Full Text]

Leese HJ 2002 Quiet please, do not disturb: a hypothesis of embryo metabolism and viability. BioEssays 24 845–849.[CrossRef][Web of Science][Medline]

Li A, Chandrakanthan V, Chami O & O'Neill C 2007 Culture of zygotes increases TRP53 [corrected] expression in B6 mouse embryos, which reduces embryo viability. Biology of Reproduction 76 362–367.[Abstract/Free Full Text]

Li J, Ran C, Li E, Gordon F, Comstock G, Siddiqui H, Cleghorn W, Chen HZ, Kornacker K, Liu CG et al. 2008 Synergistic function of E2F7 and E2F8 is essential for cell survival and embryonic development. Developmental Cell 14 62–75.[CrossRef][Web of Science][Medline]

Lijam N, Paylor R, McDonald MP, Crawley JN, Deng CX, Herrup K, Stevens KE, Maccaferri G, McBain CJ, Sussman DJ et al. 1997 Social interaction and sensorimotor gating abnormalities in mice lacking Dvl1. Cell 90 895–905.[CrossRef][Web of Science][Medline]

Lonergan P, Rizos D, Gutierrez-Adan A, Fair T & Boland MP 2003a Effect of culture environment on embryo quality and gene expression – experience from animal studies. Reproductive Biomedicine Online 7 657–663.[Medline]

Lonergan P, Rizos D, Gutierrez-Adan A, Moreira PM, Pintado B, de la Fuente J & Boland MP 2003b Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo. Biology of Reproduction 69 1424–1431.[Abstract/Free Full Text]

Maclean JA II, Chen MA, Wayne CM, Bruce SR, Rao M, Meistrich ML, Macleod C & Wilkinson MF 2005 Rhox: a new homeobox gene cluster. Cell 120 369–382.[CrossRef][Web of Science][Medline]

Mann MR, Lee SS, Doherty AS, Verona RI, Nolen LD, Schultz RM & Bartolomei MS 2004 Selective loss of imprinting in the placenta following preimplantation development in culture. Development 131 3727–3735.[Abstract/Free Full Text]

Natale DR, De Sousa PA, Westhusin ME & Watson AJ 2001 Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR. Reproduction 122 687–693.[Abstract]

O'Neill C 1998 Autocrine mediators are required to act on the embryo by the 2-cell stage to promote normal development and survival of mouse preimplantation embryos in vitro. Biology of Reproduction 58 1303–1309.[Abstract/Free Full Text]

Ramos SB, Stumpo DJ, Kennington EA, Phillips RS, Bock CB, Ribeiro-Neto F & Blackshear PJ 2004 The CCCH tandem zinc-finger protein Zfp36l2 is crucial for female fertility and early embryonic development. Development 131 4883–4893.[Abstract/Free Full Text]

Rinaudo P & Schultz RM 2004 Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos. Reproduction 128 301–311.[Abstract/Free Full Text]

Rinaudo PF, Giritharan G, Talbi S, Dobson AT & Schultz RM 2006 Effects of oxygen tension on gene expression in preimplantation mouse embryos. Fertility and Sterility 86 1252–1265(e1251-1236).[Web of Science][Medline]

Rizos D, Lonergan P, Boland MP, Arroyo-Garcia R, Pintado B, de la Fuente J & Gutierrez-Adan A 2002 Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: implications for blastocyst quality. Biology of Reproduction 66 589–595.[Abstract/Free Full Text]

Rizos D, Gutierrez-Adan A, Perez-Garnelo S, De La Fuente J, Boland MP & Lonergan P 2003 Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression. Biology of Reproduction 68 236–243.[Abstract/Free Full Text]

Rodriguez-Zas SL, Schellander K & Lewin HA 2008 Biological interpretations of transcriptomic profiles in mammalian oocytes and embryos. Reproduction 135 129–139.[Abstract/Free Full Text]

Shi W & Haaf T 2002 Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure. Molecular Reproduction and Development 63 329–334.[CrossRef][Web of Science][Medline]

Sinclair KD, McEvoy TG, Maxfield EK, Maltin CA, Young LE, Wilmut I, Broadbent PJ & Robinson JJ 1999 Aberrant fetal growth and development after in vitro culture of sheep zygotes. Journal of Reproduction and Fertility 116 177–186.[Abstract/Free Full Text]

Sinclair KD, Young LE, Wilmut I & McEvoy TG 2000 In-utero overgrowth in ruminants following embryo culture: lessons from mice and a warning to men. Human Reproduction 15 68–86.[Abstract/Free Full Text]

Warburton D, Seth R, Shum L, Horcher PG, Hall FL, Werb Z & Slavkin HC 1992 Epigenetic role of epidermal growth factor expression and signalling in embryonic mouse lung morphogenesis. Developmental Biology 149 123–133.[CrossRef][Web of Science][Medline]

Watkins AJ, Ursell E, Panton R, Papenbrock T, Hollis L, Cunningham C, Wilkins A, Perry VH, Sheth B, Kwong WY et al. 2008 Adaptive responses by mouse early embryos to maternal diet protect fetal growth but predispose to adult onset disease. Biology of Reproduction 78 299–306.[Abstract/Free Full Text]

Wrenzycki C, Herrmann D, Carnwath JW & Niemann H 1999 Alterations in the relative abundance of gene transcripts in preimplantation bovine embryos cultured in medium supplemented with either serum or PVA. Molecular Reproduction and Development 53 8–18.[CrossRef][Web of Science][Medline]

Wrenzycki C, Herrmann D, Keskintepe L, Martins A Jr, Sirisathien S, Brackett B & Niemann H 2001 Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Human Reproduction 16 893–901.[Abstract/Free Full Text]

Xu B, Wang C, Yang J, Mao G, Zhang C, Liu D, Tai P, Zhou B, Xia G & Zhang M 2008 Silencing of mouse hepatic lanosterol 14-alpha demethylase down-regulated plasma low-density lipoprotein cholesterol levels by short-term treatment of siRNA. Biological & Pharmaceutical Bulletin 31 1182–1191.

Yoshimoto T, Boehm M, Olive M, Crook MF, San H, Langenickel T & Nabel EG 2006 The arginine methyltransferase PRMT2 binds RB and regulates E2F function. Experimental Cell Research 312 2040–2053.[CrossRef][Web of Science][Medline]

Young LE, Fernandes K, McEvoy TG, Butterwith SC, Gutierrez CG, Carolan C, Broadbent PJ, Robinson JJ, Wilmut I & Sinclair KD 2001 Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture. Nature Genetics 27 153–154.[CrossRef][Web of Science][Medline]



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