genomic profiling to improve embryogenesis in the pig

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ARTICLE IN PRESSG ModelNIREP 4969 1–7

Animal Reproduction Science xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Animal Reproduction Science

jou rn al hom epage : w ww.elsev ier .com/ locate /an i r eprosc i

enomic profiling to improve embryogenesis in the pig�

andall S. Prather ∗, Bethany K. Redel, Kristin M. Whitworth, Ming-Tao Zhaoivision of Animal Science, University of Missouri, Columbia, MO, USA

r t i c l e i n f o

rticle history:vailable online xxx

eywords:ranscriptionmbryoNA methylationetabolism

rofiling

a b s t r a c t

Over the past decade the technology to characterize transcription during embryogenesishas progressed from estimating a single transcript to a reliable description of the entiretranscriptome. Northern blots were followed by sequencing ESTs, quantitative real timePCR, cDNA arrays, custom oligo arrays, and more recently, deep sequencing. The amountof information that can be generated is overwhelming. The challenge now is how to gleaninformation from these vast data sets that can be used to understand development and toimprove methods for creating and culturing embryos in vitro, and for reducing reproductiveloss. The use of ESTs permitted the identification of SPP1 as an oviductal component thatcould reduce polyspermy. Microarrays identified LDL and NMDA as components to replaceBSA in embryo culture media. Deep sequencing implicated arginine, glycine, and folateas components that should be adjusted in our current culture system, and identified acharacteristic of embryo metabolism that is similar to cancer and stem cells. Not only willthese characterizations aid in improving in vitro production of embryos, but will also beuseful for identifying, or creating conditions for donor cells that will be more likely to resultin normal development of cloned embryos. The easily found targets have been identified,
and now more sophisticated methods are being employed to advance our understanding ofembryogenesis. Here the technology to study the global transcriptome is reviewed followedby specific examples of how the technology has been used to understand and improveporcine embryogenesis both in vitro and in vivo.
© 2014 Published by Elsevier B.V.

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. Introduction

The basic premise of understanding a cell is that DNAakes RNA makes protein. Thus for a greater understand-

ng of the early mammalian embryo we must understand its

Please cite this article in press as: Prather, R.S., et al., GenomiReprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.201

NA, RNA and protein. The genomes of most species haveow been sequenced and working drafts of their genomesre readily available (Groenen et al., 2012) for a perspective

� This paper is part of a special issue entitled: 4th Mammalian Embryoenomics meeting, Guest Edited by Marc-Andre Sirard, Claude Robert and

ulie Nieminen.∗ Corresponding author at: 920 East Campus Drive, University of Mis-

ouri, Columbia, MO 65211, USA. Tel.: +1 573 882 6414.E-mail address: [email protected] (R.S. Prather).

http://dx.doi.org/10.1016/j.anireprosci.2014.04.017378-4320/© 2014 Published by Elsevier B.V.

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on the pig genome (Prather, 2013). From the basic structureof the genome, predictions can be made about the sequenceof the RNAs, and the control of their transcription. From theRNAs, predictions can be made both about the proteins thatare made and their function. Thus if one were to determinewhich RNAs were present at a specific stage of develop-ment, e.g. the blastocyst stage, then it should be possibleto predict the genes that were transcribed and the proteinsthat are made. Extrapolation of that data should permit thedescription of functional pathways that are present in theblastocyst stage embryo. If this were compared to a blasto-cyst stage embryo that was created in vitro, then it mightbe possible to predict which culture condition to alter to

c profiling to improve embryogenesis in the pig. Anim.4.04.017

make the in vitro produced embryo less different from thein vivo produced embryo.

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dx.doi.org/10.1016/j.anireprosci.2014.04.017


http://www.sciencedirect.com/science/journal/03784320

http://www.elsevier.com/locate/anireprosci

mailto:[email protected]


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2. Ribonucleic acid

Quantification of the different RNAs in a cell can bechallenging. Generally the RNA in focus has been messen-ger RNA (mRNA) because it is easy to isolate due to itspoly(A) tail, and the RNA can be used to predict both thegene and the protein. There are a number of caveats thatthat should be remembered when working with mRNA.First, one should be careful about interpreting the datafrom poly(A) isolated RNA, especially if the samples arecollected during oocyte. There are numerous examples ofpreexisting messages being polyadenylated, translated anddegraded (Dai et al., 2000, 2005). Such polyadenylationcan easily be visualized with an assay that measures thelength of the poly(A) tail, and includes transcripts suchas KPNA7, H1FOO, ID3, and PARL (Dobbs et al., 2010). Thequestion here relates to the isolation of mRNA and doesthe efficiency of mRNA recovery change when the poly(A)tail lengthens. Secondly, it should also be rememberedthat mRNA accounts for only 1–3% of the RNA in a typ-ical cell. Often ignored are ribosomal RNA (>80% of totalRNA), transfer RNA, long coding RNA, signal recognitionparticle RNA, small nuclear RNA, small nucleolar RNA, tel-omerase RNA, micro RNA, Piwi-interacting RNA, and smallinterfering RNA (reviewed by Prather et al., 2013). Evenwhen the focus includes these diverse RNAs, the RNAsthemselves can be edited. Over 100 types of RNA mod-ifications have been identified in all three kingdoms oflife. These include changing adenosine to inosine (result-ing in an A to G conversion in how the ribosome readsthe codon), and methylation of adenosine and cytosine inRNA. For a review of RNA editing (Mallela and Nishikura,2012). RNA editing has obvious ramifications as an RNAsequence may neither predict the DNA sequence fromwhich it was derived, nor the sequence of amino acids thatare translated. Similarly, some of the RNA modificationsmay affect stability, turnover and translation rate. If thisis not complicated enough, proteins can be edited. Inteinsare protein sequences that can be spliced out of polypep-tides (Elleuche and Poggeler, 2010) and even replace otherinteins in cis and trans (Appleby-Tagoe et al., 2011; Arankoet al., 2013). It should be remembered that most of the tech-nologies provide a snapshot of mRNA abundance and donot provide any additional information. Thus the regula-tion of RNA production, post-transcriptional modification,protein production and post-translational modifications allserve to drive a very complex system. For most technolo-gies analysis of RNA abundance is lethal to the cells orembryos. Efforts to develop technologies that do not harmthe embryo include evaluation of the first or second polarbody (Klatsky et al., 2010; Jiao and Woodruff, 2013) as thismay accurately reflect the abundance of message in theoocyte, and thus predict both the abundance of maternalRNAs in the embryo and hence the developmental qualityof the resulting embryo.

3. Profiling technologies


Many technologies are available to quantify RNAs ina sample of cells. Over the past decade the technologyto characterize transcription during embryogenesis has

PRESSn Science xxx (2014) xxx–xxx

progressed from estimating a single transcript to a reli-able description of the entire transcriptome. Northernblots were followed by sequencing ESTs, quantitative realtime PCR, cDNA arrays, custom oligo arrays, and morerecently, deep sequencing (the methods and limitationsof these technologies have been recently reviewed froma pig centric viewpoint (Prather et al., 2013). These studieshave shown a complex metabolic switch at the transitionfrom maternal control of development to zygotic controlof development (MTZ), and these changes are strikinglysimilar across species (Ostrup et al., 2013). Many of thetranscripts that are enriched for prior to the MTZ codefor proteins that have a cytoplasmic function; while thoseenriched for after the MTZ code for proteins that havea nuclear function. Not only is a there a major shift inthe transcriptome and hence metabolism of the embryo,when the embryo reaches the blastocyst stage it shouldbe remembered that the early blastocyst stage embryois composed of at least 3 different cell types that can bedefined by their morphology and expression of Nanog. Thisincludes the trophectoderm, the inner cell mass composedof epiblast (Nanog positive) and the hypoblast (primitiveendoderm that is Nanog negative: those cells separatingthe inner cell mass from the blastocoel cavity) each witha different transcriptional signature (Silva et al., 2009;Lanner and Rossant, 2010). When an intact blastocyst stageembryo is used for RNA isolation all three cell types con-tribute to the final transcript abundance. Thus care shouldbe exercised when interpreting the results as biologicallyimportant differences may be masked (Fig. 1).

To further exacerbate the problem we tend to think ofthe pre-blastocyst stage embryos as being uniform, i.e. allblastomeres are equal. In reality they may not be equal.An example is the maternal Trim28 mutant mouse embryo.Trim28 is a protein that is required for protection of thedifferentially methylated region (DMR) during the globalDNA demethylation observed during the cleavage stages(Messerschmidt et al., 2012) and aberrant expression canresult in 8-cell stage embryos that have a mosaic DNAmethylation pattern (Messerschmidt et al., 2012). Not onlymight there be mosaic patterns of DNA methylation, thepattern of gene expression within cell types, but betweencells has the potential to be quite different. This mosaicexpression may be the result of pulsatile expression ofspecific genes that results in a great deal of variation, or‘noise’ in abundance of a transcript (Levine et al., 2013;Sanchez and Golding, 2013). Thus when data from theseearly embryos is generated it will behoove the reader tobe careful of the interpretation, and remember that theseembryos may have mosaic epigenetic marks and mosaictranscriptional profiles. Unless single cells are measuredthe results will be an average of the cells. Thus the oocyteand preimplantation embryo represent a highly dynamicsystem that on the surface appears to be quite simple, butin reality is quite complex.

This complexity in combination with these technolo-gies now available to describe changes in transcription


and DNA structure provide a wealth of information aboutthe embryo. In fact, the amount of information that cannow be generated is overwhelming. The challenge is nothow to generate the data, but how to glean information

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Fig. 1. Metabolism in the early pig embryo. Glycolysis in somatic cellsgenerally begins with glucose entering the cells via glucose transporters(SLC2Ax) and conversion to glucose 6 phosphate by HK1. For maximumATP generation glucose 6 phosphate is then converted through a seriesof intermediates to phosphoenol pyruvate. Phosphoenol pyruvate is thenconverted to pyruvate by PKM. Pyruvate is converted to acetyl CoA bypyruvate dehydrogenase (PDH) and this serves to charge the TCA cycleand produce ATP. In the early embryo (and other rapidly proliferatingcells) lactic acid may be converted to pyruvate by LDHB. The pyruvatethat is produced cannot enter the TCA cycle due to PDK phosphorylatingPDH. In addition, phosphorylation of the fetal form of PKM results in aninability of phosphoenol pyruvate being converted to pyruvate. However,in the presence of the fetal form of PKM, PGAM1 can perform the reversereaction. These systems serve to charge the glycolysis pathway with inter-mediates, which can now only be metabolized via the pentose phosphatepathway. Low oxygen increases the abundance of TALDO1 and PDK1 thusencouraging metabolism to shift as described by the large red arrows toproduce NADPH and riboses that are necessary for redox balance and DNAsynthesis.A

fdclmatHoa2peaopscoci

cripts, new RNA isoforms and variants. Current technologycan generate 150,000,000 qualified reads per lane. When

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dapted from Redel et al. (2012a).

rom these vast data sets that can be used to understandevelopment and to improve methods for creating andulturing embryos in vitro, and for reducing reproductiveoss. Attempts at profiling from EST sequencing confirmed

any of the genes that were already known to be presentnd differentially regulated, and helped to identify novelranscripts from early embryos (Whitworth et al., 2004;amatani et al., 2006). Similarly, cDNA arrays and customligonucleotide arrays continue to provide informationbout changes in transcript abundance (Whitworth et al.,005; Tsoi et al., 2012) and predict biologically importantathways that change during development. While the gen-ration of this information was a goal, it quickly becamepparent that a description of the changes in abundancef transcripts during embryogenesis was a new startingoint. This description also became a moving target as deepequencing technologies became available. Not only can aomplete description of the all transcripts present in a cellr cells now be generated, but meaningful comparisons


an be made between cells or embryos that are culturedn a slightly different culture environment. In some cases,

PRESSn Science xxx (2014) xxx–xxx 3

altering a single component of the culture media can dra-matically alter the transcriptional profile.

Again, transcriptional profiles represent the startingpoint for asking questions about development, i.e. thetranscriptional profile is not the end. In our opinion, thebiggest challenge of profiling experiments is extractinginformation that can be used to better understand devel-opment, or to improve development of in vitro producedembryos. Some examples will be presented below.

4. Applications to understand and improvereproductive biotechnology

Generation of data that chronicles changes in the abun-dance of individual transcripts and then applying thatinformation to the development of pathways that changeduring embryogenesis may be useful. However, applicationof that information to improve the in vitro development ofembryos is the real test. Many of the applications that weredeveloped here at the University of Missouri have beenrecently reviewed (Prather et al., 2013) and will be brieflyaddressed here again. Our initial profiling experimentswere based on the abundance of ESTs in reproductive tis-sues of the pig (Jiang et al., 2001; Tuggle et al., 2003; Jianget al., 2004). The generation of these ESTs proved usefulas the abundance of a transcript for SPP1 (osteopontin)was found to be higher in the oviduct of a gilt in estrusas compared to other phases of the estrous cycle (Greenet al., 2006). Polyspermy has been, and continues to be, aproblem for in vitro fertilization in the pig (Dang-Nguyenet al., 2011). Since SPP1 was high in the estrous oviduct weobtained purified SPP1 and added it back to the fertilizationsystem. SPP1 resulted in a decrease in polyspermy and anincrease in overall efficiency of fertilization and develop-ment (Hao et al., 2006, 2008). Similarly, an increase in theabundance of transcripts for LDLR and a component of theNMDA Receptor (GRIN3A) indicated to us that the embryomay develop better if the ligands for these receptors wereadded to the culture medium. Additionally, one of our goalswas to remove bovine serum albumin (BSA) from the cul-ture system as different batches (or lots) of BSA promoteddevelopment at different rates. We found that either LDLor NMDA could replace BSA in the culture medium (Spateet al., 2010, 2012). While replacing BSA with either LDL orNMDA could result in normal appearing blastocyst stageembryos, only LDL resulted in piglets after embryo trans-fer. It should also be noted that while LDL can replace BSAin the culture system, LDL is often isolated from biologi-cal samples and may have the same inherent problems asBSA, e.g. batch to batch variation, and possible biologicalcontaminants.

4.1. Deep sequencing: transcripts and genome

Deep sequencing is a powerful tool that has severaladvantages over other technology. First it is not restrictedto a limited probe set. Thus it can reveal new trans-


combined with immunoprecipitation the current tech-nology can be used to describe DNA sequences that are

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into the TCA cycle. Additionally lactate dehydrogenases Aand B (LDHA, LDHB) increase from the germinal vesicle to

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methylated, hydroxymethylated, or are associated withcertain histone modifications for which an antibody isavailable. Deep sequencing can be expensive and thevast amount of data generated can result in challengesfor data storage and data analysis. Deep sequencing hasbeen used to understand differences between embryosthat are created by various techniques and has led toimprovements in the culture systems for creating pigembryos.

Our initial deep sequencing project evaluated blasto-cyst stage embryos. One oviduct of bred gilts was flushedto recover the embryos on day 2. The 2-cell stage embryoswere cultured in vitro for four days. After four days theembryos from the contra-lateral horn of the gilt wererecovered. All embryos were then processed. It was foundthat after four days in vitro the embryos were about onecleavage division behind the in vivo embryos (Bauer et al.,2010a). While there were a number of interesting path-ways that were altered in the embryos cultured in vitro, wefocused on a single transcript for SLC7A1. SLC7A1 is solutecarrier family 7 (cationic amino acid transporter, Y+ sys-tem) which favors the transport of amino acids such asarginine. The abundance of message for this transporteras measured by deep sequencing was 62 times higher inthe in vitro cultured embryos as compared to the in vivocultured embryos. Real time qPCR showed an even greaterdifference. It was concluded that the embryos needed morearginine, and thus the embryo upregulated the expres-sion for a transporter that would carry arginine into thecell. When higher levels of arginine were added to theculture system (three times the level in the base cul-ture system) the abundance of message for SLC7A1 wasreduced to that of the in vivo produced embryo (Baueret al., 2010a). The experiment was repeated with in vitroproduced embryos and this time the oviductal level of argi-nine was added to the culture system (Li et al., 2007). Thehigher levels of arginine in the culture medium resultedin an increase in the percentage of embryos that devel-oped to the blastocyst stage, decreased the abundance ofSLC7A1, but did not change the total cell number (Baueret al., 2010b).

Similarly, the deep sequencing data that first implicatedSCL7A1, also implicated a difference in the one carbon poolby folate KEGG pathway. Down regulation of these genesimplied an impaired folate metabolism. Increasing the con-centration of folate in the culture system increased thenumber of trophectoderm nuclei and total number of nuclei(Redel et al., 2012b).

Also involved with one carbon metabolism is glycine.The glycine transporter (SLC6A9) was 25 times higherin the embryos cultured in vitro as compared to in vivo(Bauer et al., 2010a). Addition of glycine to the culturesystem decreased the abundance of message for SLC6A9(unpublished) and increased the number of nuclei in theresulting blastocyst stage embryos from 47 to 72 nuclei(Redel et al., 2013). The ability of the embryo to dramat-ically alter expression of specific genes in response to asingle component of the culture medium is remarkable.Additional work evaluating message abundance will likely


reveal other components that should be altered to meet theneeds of the early embryo.


4.2. The Warburg Effect

Metabolism of early embryos is not unique. Tumor cellsand stem cells share a number of unique characteristics.They grow better in a low O2 environment, they have amitochondrial morphology that is spherical and withoutcristae, the cells generally do not metabolize glucose well,and pyruvate is converted to lactic acid rather than enteringthe tricarboxcylic acid (TCA) cycle. This type of metabolismhas been termed the Warburg Effect as Otto Warburg is thefirst to characterize this unique metabolic profile (Warburget al., 1927; Warburg, 1956, 2010). Early embryos are verysimilar. For example they grow better at 5% O2 than athigher percentages, they have mitochondria that are spher-ical and without cristae, they extensively use the pentosephosphate pathway, and while glucose use through the TCAcycle can be detected it is very low until the blastocyststage (reviewed in Krisher and Prather, 2012). Inhibitingthe TCA cycle during the morula to blastocyst transitionwith sodium azide or 2,4-dinotrophenol has even beenshown to be beneficial to the development of blastocyststage embryos (Machaty et al., 2001). With this number ofsimilarities a search was made in the transcriptional profilefor a signature similar to what is observed and describedfor the metabolism of tumor cells.

One of the prominent signatures of the Warburg Effectis that of expression of a fetal splice variant of the muscleform of pyruvate kinase (PKM) (Levine and Puzio-Kuter,2010). This fetal form of the PKM the M2 variant is thepredominant form of pyruvate kinase in the early embryo.The unique characteristic of the M2 variant is that it con-tains a tyrosine (not present in the M1 form), that whenphosphorylated has a greatly reduced ability to convertphosphoenol pyruvate to pyruvate (Levine and Puzio-Kuter, 2010). The M2 form of PKM is the predominantform found in early pig embryos (Redel et al., 2012a). Othercharacteristics of the Warburg Effect include expression ofhexokinase 2 (HK2), in contrast to the other hexokinases.Similarly, HK2 is highly expressed in blastocyst stage pigembryos in contrast to low levels of HK1 and HK3 (Redelet al., 2012a). Pyruvate dehydrogenase kinase (PDK) phos-phorylates pyruvate dehydrogenase and thus inactivatesits ability to convert pyruvate to acetyl coA. Searchingthrough the data from (Whitworth et al., 2005) it was foundthat PDK3 levels were highest in germinal vesicle stageoocytes, lower in 4-cell stage embryos, and still lower atthe blastocyst stage. Deep sequencing identified abundantmessage for PKD1, PDK3 and PDK4. Real-time PCR con-firmed that the abundance of PDK1 was higher in embryoscultured to the blastocyst stage in 5% O2 as compared to18% O2 (Redel et al., 2012a). The PDK data is consistent withthe idea that PDK could be preventing the entry of glucosemetabolites into the TCA cycle, and that this inhibition isgreatest in the oocyte as compared to the 4-cell and blas-tocyst stages, and that oxygen tension affects expression ofPDK1 such that at the oxygen tension that supports betterdevelopment there is an increase in the inhibition of entry


blastocyst stages (Whitworth et al., 2005), and that abun-dant LDHB is present in the blastocyst stage embryo. The

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DH monomers form a tetrameric structure and high lev-ls of LDHB favor the conversion of lactic acid to pyruvate.ALDO1 is an enzyme that provides a link between glycol-sis and the pentose phosphate pathway. Similar to PDK1eing increased in blastocyst stage embryos cultured in a

ow O2 environment, TALDO1 expression is enhanced in theow O2 culture thus generating nucleotides for DNA synthe-is as well as NADPH. Another transcript whose abundancencreases from the germinal vesicle stage to the blastocysttage is PGAM1. PGAM1 is generally thought to converthosphoenol pyruvate to pyruvate only in the presencef PKM M2 and does not generate ATP. This transcript isighly abundant in cancer cells (Vander Heiden et al., 2010).owever the reaction does run in reverse and since thebundance of this message increases, as does LDHB, fromhe germinal vesicle stage to the blastocyst stage, we haveoncluded that any lactic acid that is present would be con-erted to pyruvate. The pyruvate could not be converted tocetyl CoA, but would rather be pushed up the glycolysisathway and toward the pentose phosphate pathway. Theet result would be little use of the TCA cycle and signifi-ant use of the pentose phosphate pathway. Thus in manyays the early embryo mimics rapidly proliferating cells

uch as cancer cells and stem cells. The terminology ‘cleav-ge stage embryo’ refers to the observation that the embryoleaves and does not completely replicate itself. The mainellular component that requires replication is the DNA.hunting metabolism toward the pentose phosphate path-ay would achieve that goal. While the argument has beenade that early pig embryos exhibit the Warburg Effect;

n reality only the framework of this argument has beenade. Clearly more studies need to be done to confirm the

resence and function of these different proteins.

. DNA methylation during embryogenesis

An understanding about RNA and protein variety drivesne toward wanting to understand the regulation ofranscription. While DNA methylation clearly regulatesranscription of genomically imprinted genes, similar cor-elations between DNA methylation and transcription atther locations in the genome are not as straightforward.n general DNA methylation appears to heritably maintainhromatin structure in a transcriptional state (reviewedy Reddington et al., 2013). Specifically, methylation ofpG islands in promoters is associated with transcriptionalepression. In contrast, methylation proximal to promotersnd in gene bodies is consistent with active transcription.nd, partial methylation is associated with both transcrip-

ion and the repression of transcription. However it isot clear which is first transcriptional silencing or DNAethylation (Mutskov and Felsenfeld, 2004; Chen et al.,

008). Nevertheless, these correlations of methylation andranscription provide impetus for the study of DNA meth-lation changes during embryogenesis. In pigs, as in mostammals, the first dramatic change in DNA methylation

ccurs after fertilization as the paternal DNA appears to


e demethylated (Kang et al., 2001; Fulka et al., 2006). Inctuality, this apparent demethylation is a result of theen–eleven translocation (TET) family of protein oxidizinghe 5 methylcytosine to 5-hydroxymethylcytosine, and in


pigs this occurs via TET3 (Lee et al., 2014). In contrast thematernally derived DNA is passively demethylated dur-ing the cleavage stages such that most of non-imprintedmethylation marks are removed before the blastocyst stage(Zhao et al., 2013). DNA methyltransferases control sub-sequent cytosine methylation. A number techniques forevaluating DNA methylation have been available for anumber of years and include: sequencing bisulfite con-verted DNA (Morrill et al., 2013), differential micro arrayhybridization of DNA that has been cut with a methyl-ated DNA sensitive restriction enzyme (Bonk et al., 2007,2008), immuno-precipitation of methylated DNA followedby deep sequencing (Fouse et al., 2010) and reduced rep-resentative bisulfite sequencing (Smith et al., 2012). Whilethere are numerous combinations of the above techniques(Prather et al., 2013) the current focus of those working onearly embryos is to develop techniques that would providea global pattern of DNA methylation from a few earlyembryos. By day 12 of gestation in the pig, there is sufficienttissue from which to collect DNA for the highly ineffi-cient method of bisulfite conversion of DNA and still haveenough sample to amplify selected genomic regions andsubmit for deep sequencing (Morrill et al., 2013). Recentlya technique has been reported that has the sensitivity towhere 1 ng of input DNA can be used to provide samplefor immuno-precipitation of methylated DNA followed bydeep sequencing (Zhao et al., 2014). This procedure gener-ated over 80 million 100 bp reads that provided a completecoverage of the genome. Similarly, the input control pro-vided 5X coverage of the genome. Since 1 ng of DNA issufficient, one or two blastocyts stage embryos (100–200nuclei), or a hundred oocytes could provide enough start-ing material. The technology is now available to begin toglobally characterize DNA methylation patterns during theearliest stages of mammalian embryogenesis.

6. Conclusions

The easily identified changes to the culture system havebeen identified, and now more sophisticated methods, suchas single-cell analysis, are being employed to advance ourunderstanding of embryogenesis (Xue et al., 2013). The sen-sitivity of the techniques to detect changes in epigeneticmarks such as DNA methylation, and changes in tran-scription continues to improve. With an increase in thesensitivity comes an increase in the amount of information.Application of these techniques to the early embryo hasrevealed the immense complexity of the seemingly simpleembryo. Improvements in computational tools need to bemade so this information is understandable. Once under-standable, then improvements can be made to the systemof in vitro production of embryos, somatic cell nucleartransfer and other embryo technologies. Finally the reduc-tion of the 30% loss of potential conceptuses that occursduring the first month of development can be addressed.


Conflict of interest statement

The authors have no conflicts of interest to declare.

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Acknowledgements

The authors would like to acknowledge funding fromFood for the 21st Century from the University of Missouri.

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