Reproductive technologies and the porcine embryonic transcriptome.

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ANIREP 4982 1–8

Animal Reproduction Science xxx (2014) xxx–xxx 1

Contents lists available at ScienceDirect

Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Reproductive technologies and the porcine embryonic transcriptome夽

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M.K. Dyck ∗ , C. Zhou, S. Tsoi, J. Grant, W.T. Dixon, G.R. Foxcroft Swine Reproduction and Development Program, Swine Research & Technology Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada

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Article history: Available online xxx

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Keywords: Pig Embryo Transcriptome Assisted reproductive technologies

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1. Introduction

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The domestic pig is not only an economically-important livestock species, but also an increasingly recognized biomedical animal model due to its physiological similarities with humans. As a result, there is a strong interest in the factors that affect the efficient production of viable embryos and offspring in the pig using either in vivo or in vitro production methods. The application of assisted reproductive technologies (ART) has the potential to increase reproductive efficiency in livestock. These technologies include, but are not limited to: artificial insemination (AI), fixed-time AI, embryo transfer, cryopreservation of sperm/oocytes/embryos, in vitro fertilization and somatic cell nuclear transfer (cloning). However, the application of ART is much less efficient in the pig than in many other mammalian species such as cattle. Until recently, the underlying causes of these inefficiencies have been difficult to study, but advances in molecular biology techniques for studying gene expression have resulted in the availability of a variety of options for gene expression profiling such as microarrays, and next generation sequencing technologies. Capitalizing on these technologies the effects of various ARTs on the porcine embryonic transcriptome has been determined and the impact on the related biological pathways and functions been evaluated. The implications of these results on the efficiency of ARTs in swine, as well potential consequences for the developing embryo and resulting offspring, are reviewed. © 2014 Published by Elsevier B.V.

The domestic pig is an economically-important livestock species, with pork constituting 40% of the world’s meat consumption, making it the most important global meat source (Dang-Nguyen et al., 2010). However, pigs are also a well-recognized biomedical animal model (Rogers et al., 2008; Chorro et al., 2009; Vilahur et al., 2011) due

夽 This paper is part of a special issue entitled: 4th Mammalian Embryo Genomics meeting, Guest Edited by Marc-Andre Sirard, Claude Robert and Julie Nieminen. ∗ Corresponding author. Tel.: +1 780 492 0047; fax: +1 780 492 4265. E-mail address: [email protected] (M.K. Dyck).

to its physiological similarities with human (Abeydeera, 2002; Betthauser et al., 2000). As a result, there is a strong interest in the factors that affect the efficient production of viable embryos and offspring in this species using either in vivo or in vitro production methods. As an agricultural species, domestic swine have been raised and selectively bred to produce pigs with desired characteristics for centuries. The application of assisted reproductive technologies (ART) increases reproductive efficiency and genetic gains in this and other livestock species by facilitating the dissemination of genetics from superior sires and dams. From a biomedical and biotechnology point of view, the pig was initially utilized as a model for developing and testing surgical procedures. Later, advances in the area of molecular biology along with the development and

http://dx.doi.org/10.1016/j.anireprosci.2014.05.013 0378-4320/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Dyck, M.K., et al., Reproductive technologies and the porcine embryonic transcriptome. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.05.013

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application of ART formed the basis for genetic manipulation of swine in order to create porcine models that mimic human conditions and disease, among other applications (Whyte and Prather, 2011) or use them as bioreactors (Dyck et al., 2003). Recent research has focused on using the pig as a medical model for renal transplantation (Giraud et al., 2011), cardiovascular-related diseases (Zaragoza et al., 2011), atherosclerosis (Vilahur et al., 2011) and Cystic Fibrosis (Rogers et al., 2008). As well, advances in induced pluripotent stem cell (iPSCs) technologies (Esteban et al., 2009; Roberts et al., 2009; West et al., 2010) make the pig an attractive model for regenerative medicine and stem cell research. Despite the importance of this species for these varied purposes the application of ART in swine is much less efficient in the pig than in other livestock species (Gajda, 2009; Kikuchi et al., 2002). Although a full gambit of ART have been applied in pigs, for research and biotechnology purposes, the regular application of many ART for agricultural purposes is less common in swine production than in species such as cattle. Therefore, an improved understanding of the effects of ART on embryonic gene expression and development in swine will have both agricultural and biomedical benefits.

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1.1. ARTs in swine

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Intellectual and technological advances in the area of reproductive physiology in the past 60 years has resulted in the development of four generations of ART, which are often considered to be either “sperm-based” or “embryobased” technologies (Bertolini and Bertolini, 2009). The first to third generations of ART are general considered to include: (1) Artificial insemination, sperm and embryo freezing. (2) Induced and/or multiple ovulations, ultrasonography as well as embryo transfer. (3) In vitro embryo production and culture, as well as semen and embryo sexing. While the fourth generation of these technologies, which is considered to be more experimental and is still evolving, includes: transgenesis, stem cell biology and “cloning” procedures. The fourth generation of ARTs have the potential to greatly enhance the influence of superior animals on production, as mentioned above, commercial use has been limited to biomedical and biotechnological applications in the pig. 1.2. Gene expression profiling analysis and the porcine embryonic transcriptome The success of ART in swine varies dramatically across these 4 generations. Following artificial insemination, almost 100% of all the fertilized embryos in the pig can develop into blastocysts in vivo (Geisert and Schmitt, 2002). However, porcine embryos derived from in vitro ART manipulation systems, such as in vitro fertilization and culture, cloning, and parthenogenesis, are less competent than their in vivo counterparts, showing slower development, lower cleavage and blastocyst formation rate. Until recently, the underlying causes of these impacts and inefficiencies have been difficult to study. Initial studies in this area relied on morphological evaluations of early embryos, but the lack of “diagnostic” tools to study

embryos limited progress in this area (Alexopoulos and French, 2009; Coy and Romar, 2002; Crosier et al., 2001; McEvoy et al., 2001; Sinclair et al., 1999). Advances in molecular biology techniques for studying gene expression have resulted in high-throughput platforms for gene expression profiling such as next generation sequencing (NGS) technologies and microarrays (Pariset et al., 2009). This, coupled with the ability to amplify the minute amount of genetic material present in the early conceptus, has enabled scientists to better study early embryo development. Next generation sequencing systems, such as RNA-seq, are considered state-of-the-art in this field, as it allows for the precise quantification of transcript levels in tissues or cells of interest and facilitates data mining and identification of transcript isoforms. This technology allows for massive amounts of information to be gathered from minute samples, but the amount of data generated can be problematic and requires sophisticated computational and bioinformatics approaches. Microarrays also represent a powerful method of gene expression profiling, which can analyse expression of thousands of predetermined transcripts at the same time (Hornsh et al., 2009). As a result, microarrays are quite efficient when a large number of different samples are to be studied and work well in a multiuser environment, but they are limited by the gene and sequence knowledge required for the microarray development and data interpretation. As well, although gene expression microarray platforms are available for various species (including the pig), most of these platforms have been designed based on somatic cell gene expression profiles. It has been shown that the embryonic transcriptome differs significantly from that of somatic cells (Vallee et al., 2009) and, therefore, these somatic cell based plat- Q2 forms are not well suited for studies in embryos. This has prompted the development of embryo-specific microarray platforms for both cattle (Robert et al., 2011) and the pig (Tsoi et al., 2012) in order to better study this critical period of development in these important livestock species. There have been efforts to characterize the gene expression profile of in vivo developed porcine embryos and early embryos produced after in vitro manipulations using next generation sequencing (NGS) and microarray platforms. This new ability to study early porcine development has created an environment in which scientists can collaborate with livestock and biotechnology industries to evaluate the effect of various ARTs on the porcine embryonic transcriptome and determine their impact on the related biological pathways and functions. For example, in vitro culture and in vitro production of porcine embryos has proven to be a challenge, however extensive transcriptome profiling of oocyte maturation and early embryonic development has provided insights into the metabolism of the porcine embryo and molecular factors that control its development, which in turn has allowed for dramatic improvements in these techniques (Prather et al., 2013). Despite this, there is still a great deal that is unknown regarding the impact of ARTs on the porcine embryonic transcriptome. Evaluating these impacts, will allow us to better understand the mechanisms involved in early embryonic development in the pig and, in


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turn, improve the efficiency of these techniques with this species. 2. Impact of ARTs on the porcine embryonic transcriptome Across the 4 generations of ART described, there are various degrees of intervention and manipulation involved which impact the embryo and its transcriptome to differing extents. At one extreme, in vitro manipulations such as parthenogenic activation and nuclear transfer involve high degrees of intervention and are well known to impact porcine embryonic development and viability (Isom et al., 2013; Liu et al., 2010; Whitworth et al., 2005, 2011). While at the other extreme, induced ovulation procedures to facilitate fixed-time AI protocols in pigs are considered to have relatively benign effects as a review of the literature would indicate that the induction of ovulation in pigs does not generally affect key fertility parameters such as pregnancy rates and litter sizes (Draincourt, 2013). Although the impact of several technologies on the porcine transcriptome have been evaluated, this paper will concentrate on recent work from our lab on the above mentioned ARTs that represent extremes in the degree of manipulation and expected impact on the embryo. 2.1. Effect of somatic cell nuclear transfer (SCNT), somatic cell chromatin transfer (CT) and parthenogenetic activation (PA) on early porcine embryo transcriptome Embryos generated after in vitro manipulations, such as parthenogenetic activation and nuclear transfer, displayed slower and less effective development, and dysregulation of critical gene networks is assumed to be associated with these deficiencies (Isom et al., 2013; Liu et al., 2010; Whitworth et al., 2005, 2011). Despite the extent of these manipulations, a small percentage of embryos generated by nuclear transfer result in offspring, while porcine parthenotes have developed up to day 29 post-activation (Kharche and Birade, 2013). In a recent study from our group the effects of somatic cell chromatin transfer (CT) and parthenogenetic activation (PA) on the gene expression patterns of hatched blastocyst stage porcine embryos has been characterized (Zhou et al., 2014). Somatic cell nuclear transfer (SCNT) has great potential applications in basic and biomedical research. However, the application of SCNT is limited by a low embryonic survival rate and the high incidence of abnormalities in individuals that develop to term, which are believed to be associated with incorrect or incomplete nuclear reprogramming (Mesquita et al., 2013). Somatic cell chromatin transfer (CT) is a cloning technology that was designed to facilitate the reprogramming process (Rodriguez-Osorio et al., 2009; Sullivan et al., 2004). It involves in vitro remodeling of the donor nuclei prior to transfer into enucleated oocytes to remove nuclear components that may interfere with nuclear remodeling (Sullivan et al., 2004). It is considered that the embryos generated from CT are more competent than embryos produced from traditional SCNT. Although promising results have been reported using chromatin transfer (CT), particularly in

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cattle, the embryos generated still exhibit abnormalities similar to those observed following conventional SCNT (Mesquita et al., 2013; Sullivan et al., 2004). Our studies have shown that significant differential expression of 103 genes was observed in the CT hatched blastocyst (HB) embryos in comparison with in vivo HB stage embryos (Zhou et al., 2014). This is less than the gene expression profile differences between SCNT and in vivo-derived porcine blastocysts reported by (Whitworth et al., 2011), who identified 179 transcripts that were aberrantly reprogrammed. The differences in the number of differentially expressed genes detected between these studies may also be related to the different platforms and bioinformatics approaches used to define the gene expression profiles (i.e. microarray versus RNA-seq). Regardless, these results support the assumption that the gene expression profile of CT embryos are more similar to in vivo embryos and, therefore, are likely to be more competent than conventional SCNT embryos, but still represents a significant deviation in the expected gene expression patterns. Embryos derived from parthenogenetic activation (PA) are valuable for studies on gene imprinting (NaturilAlfonso et al., 2012) and are a potential alternative source of embryonic stem cells (Brevini and Gandolfi, 2008; NaturilAlfonso et al., 2012). However, PA generated embryos are subject to severe development failure (Hao et al., 2004). Several previous studies in different species have reported a large number of differentially expressed genes between PA and in vivo pre-implantation embryos (Isom et al., 2013; Liu et al., 2010; Naturil-Alfonso et al., 2012; Whitworth et al., 2005). In comparison with in vivo HB, comparative microarray analysis in our lab revealed 1492 significantly differentially expressed genes in PA-derived HB (Zhou et al., 2014), which is consistent with previous findings in other species. In addition, approximately half of the differentially expressed genes in CT HB were also differentially expressed in PA HB and displayed the same direction of expression (up- or down-regulation) changes. Given that CT and PA embryos were generated following the same oocyte in vitro maturation processes and embryo in vitro culture conditions, the differential expression of these “commonly differentially expressed genes” in both PA and CT HB embryos probably are associated with these common in vitro manipulation processes. Although the gene expression profile of HB stage embryos derived from CT are more similar to their in vivo counterparts than the conventional SCNT embryos, significant differential expression of critical genes and gene networks were still observed in the CT HB. NOTCH is an important regulator of development in many animal species (Shepherd et al., 2009), and participates in many critical biological processes including cell fate specification, differentiation, proliferation, apoptosis, migration, and angiogenesis (Bolos et al., 2007). Small perturbations in NOTCH activity may lead to numerous developmental defects and diseases (Shepherd et al., 2009). Differential expression of three “notch signalling” pathway-associated genes (HES1, PSEN2, and JAG1) in CT HB was observed in our studies (Zhou et al., 2014). These “notch signalling” pathway-associated genes displayed significant and more dramatic differential expression in PA HB than CT HB. These


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results suggest that the “notch signalling” pathway is dysregulated in both PA and CT HB, and that this dysregulation of “notch signalling” pathway in PA HB is more profound than in CT HB. The altered regulation in Notch signalling probably contributes to the impaired development of both PA and CT-derived embryos. In addition, significant down-regulation of pluripotency regulator (NANOG) and trophectoderm development-associated genes (KRT18, GATA2) were also observed in both CT and PA HB, in comparison with in vivo HB. The altered regulation of these genes and gene networks probably contributes to the impaired development of CT and PA-derived embryos. TP53 (tumour protein p53) is a well-known cell-cycle regulator and apoptosis mediator gene (Molchadsky et al., 2010). Our studies showed that approximately one fourth (23 genes) of all the differentially expressed genes in CT HB were regulation targets of the transcription factor TP53 (Zhou et al., 2014). A total of 136 TP53 regulation target genes were differentially expressed in PA HB, which is more than 5 times of the differentially expressed TP53 targets in CT HB, in comparison with in vivo HB stage embryos. Further results from the IPA upstream regulator analysis suggest that TP53 is significantly activated in both CT HB and PA HB. In addition, ANXA8 (annexin A8) is a member of the annexin (ANXs) family, which is a group of Ca2+ -dependent phospholipid-binding proteins involved in many important biological processes including vesicle trafficking, calcium signalling, cell growth, cell cycle, and apoptosis (Hata et al., 2012). Over expression of ANXA8 has been reported to be associated with cancer and apoptosis (Hammond et al., 2006). In our studies, ANXA8 displayed significantly higher expression in PA HB than CT HB, and no detectable expression of ANXA8 was observed in vivo HB. These results indicate an activated apoptosis process in both PA and CT derived HB, and the activation of this apoptosis process appears to be greater in PA HB than in CT HB. Previous work in our group (Zhou et al., 2013) suggests that “eIF2 signalling”, “mitochondrial dysfunction”, “regulation of eIF4 and p70S6K signalling”, “protein ubiquitination”, and “mTOR signalling” pathways all displayed significant changes from 4-cell to HB stage in vivo, and these pathways are considered to play important roles during early porcine embryonic development. Our studies with CT and PA HB showed that these pathways were the five most significantly changed canonical pathways in PA HB in comparison with in vivo derived HB, and most of the differentially expressed genes associated with these pathways were down-regulated in PA HB. In addition, the transcription factor MYC and MYCN were predicted to be the most significantly activated transcription regulators in all embryonic stages from 8C to HB, in comparison with 4-cell stage. This result suggests that the MYC and MYCN may play important roles during early porcine embryonic development. On the other hand, the transcription factors MYC and MYCN were both predicted to be significantly inhibited in PA HB. In addition, significant down-regulation of pluripotency regulator (NANOG) and trophectodem development-associated genes (KRT18, KRT8, and GATA2) were also observed in PA HB.

Additionally, our studies have showed significant differential expression of 31 genes which occurs during the “normal” blastocyst hatching process in in vivo embryos (Zhou et al., 2012). However, all of these 31 genes were not properly regulated in PA embryos during the blastocyst hatching process including several critical pluripotency, trophoblast development, and implantation-associated genes (NANOG, GATA2, KRT8, LGMN, and DPP4). One must keep in mind that PA embryos lack paternal genetic material, and therefore the related imprinting, which prevents proper development of extra-embryonic tissues and appropriate placentation (Kharche and Birade, 2013). Q3 In addition, altered regulation of “notch signalling”associated genes were also observed during the blastocyst hatching process in PA embryos. Failed regulation in the expression of these critical genes during the hatching process probably contributes to the delayed and less efficient development of PA embryos and their reduced ability to hatch and undergo implantation. Given the extreme perturbances observed in both nuclear transfer embryos and parthonotes it is amazing that they are able to continue development. In the case of nuclear transfer embryos at least a proportion are developmentally competent as demonstrated by the fact that live offspring have resulted following transfer. However epigenetic impacts on gene expression, not only during early development but throughout the lifetime of the resulting animal, need to be considered. 2.2. Effect of hormone (pLH)-induced superovulation on early porcine embryo transcriptome Control and synchronization of ovarian follicular development and induction of ovulation with exogenous hormones can provide practical advantages in livestock management and application of ART such as embryo transfer and related technologies (Draincourt, 2013). Porcine luteinising hormone (pLH) has been effectively used in weaned sows (Cameron et al., 2010; Cassar et al., 2005) and gilts (Degenstein et al., 2008). Referring back to the 4 generations of ART, induction of ovulation for application of fixed-time AI is generally considered to have limited to no impact on embryo quality. However, previous work in our group with gilts has shown that the diameter of the largest follicles prior to ovulation was smaller in pLH-induced animals than control animals, but the resulting embryos appear to be morphologically similar to embryos from noninduced ovulations (Degenstein et al., 2008). Despite this, these embryos may exhibit molecular deviations while displaying similar morphological characteristics to more viable embryos (Nánássy et al., 2008; Rodriguez-Osorio et al., 2009). This raised questions regarding the effects of ovulation induction protocols on factors such as the quality of embryos and the health of offspring resulting from these procedures. Analysis carried out in our group has shown that pLH-induced ovulation in gilts has limited influence on the gene expression profile of blastocyst stage porcine embryos, since only 11 genes showed differential expressions between pLH and control blastocyst (Zhou et al., 2011). This limited number of differentially expressed


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genes only accounts for 0.08% of all the genes detected in control and pLH blastocyst embryos (13,373 genes). However, several of the 11 differentially expressed genes in pLH blastocyst (DPPC, ASNS, SFN, RGN, GNPDA1, MPEG1, SLC41A1, CHAC1, MSMO1, IDI1, B18S) are considered to play important roles during early embryonic development such as ion, lipid, and amino acid homeostasis, trophoblast development, cell cycle, growth, and migration. Dipeptidyl-peptidase 4 (DPP4) is a membrane-bound aminopeptidase, which is a marker for non-invasive trophoblast cells in humans and cattle. DPP4 is associated with placental development and the establishment of proper fetal-maternal interaction (Fujiwara et al., 2005; Mesquita et al., 2013). The down-regulation of DPP4 is positively linked to cell migration and invasion in humans (Sato et al., 2002), and the up-regulation of DPP4 is believed to be associated with the abnormal placental development in cloned cattle (Mesquita et al., 2013). In the present study, up-regulation of DPP4 was observed in pLH blastocysts in comparison with control blastocysts. This result indicates that the embryos produced from the pLH-induced gilts may have altered trophoblast development, which could lead to abnormal placenta development. Asparagine synthetase (glutamine-hydrolyzing) (ASNS) catalyzes the ATP-dependent formation of asparagine and glutamate from aspartate, ATP, and glutamine (Chen et al., 2004; Richards and Schuster, 1998). The transcription of ASNS is highly regulated by nutritional deprivation and other forms of cellular stress (Balasubramanian et al., 2013; Chen et al., 2004; Kilberg and Barbosa-Tessmann, 2002). ASNS can be transcriptionally activated by amino acid and glucose deprivation (Kilberg and Barbosa-Tessmann, 2002), and is involved in maintaining the homeostasis of asparagine, glutamate, aspartate, and glutamine (Balasubramanian et al., 2013). In the present study, significant up-regulation of ASNS was observed in the pLH blastocyst embryos. This suggests a more activated ASNS transcription in pLH blastocyst, indicating that the pLH embryos may experience nutritional limitation or other forms of cellular stress. Stratifin (SFN), also known as 14-3-3 Protein Sigma, is a trophoblast protein associated with cell cycle, growth, and migration (Fu et al., 2000; Laronga et al., 2000). Upregulation of SFN has been reported in less competent porcine embryos during elongation (Blomberg et al., 2008; Blomberg et al., 2005). In this study, SFN showed significant up-regulation in pLH blastocyst, suggesting that the pLH blastocyst maybe affected. Furthermore, significant up-regulation of RGN, GNPDA1, and MPEG1 genes were observed in pLH blastocysts. Regucalcin (RGN), also known as senescence marker protein-30 (SMP-30), is a Ca2+ binding protein which activates (Ca2+ –Mg2+ )-ATPase and is involved in the maintenance of intracellular Ca2+ homeostasis (Takahashi and Yamaguchi, 1997; Wu et al., 2008) and aging (Jung et al., 2004). Studies in rats and chickens suggest that the down-regulation of RGN is associated with increased oxidative stress (Jung et al., 2004). Glucosamine-6-phosphate deaminase 1 (GNPDA1) is an allosteric enzyme that catalyzes the reversible conversion of glucosamine-6-phosphate into fructose-6-phosphate and ammonium (Alvarez-Anorve et al., 2011; Wolosker

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et al., 1998). Glucosamine 6-phosphate deaminase (GNPDA) may have a role in the acrosome reaction (Montag et al., 1999). Macrophage-expressed gene 1 protein-like (MPEG1) is expressed almost exclusively in differentiated myelomonocytic cells in human and murine (Spilsbury et al., 1995). MPEG1 has been used as a macrophage-specific marker gene in mammalian systems (Karlsson et al., 2008) and in in vivo early zebra fish embryos (Zakrzewska et al., 2010). In addition, significant down-regulation of SLC41A1, and significant up-regulation of CHAC1, MSMO1, and IDI1 were observed in pLH embryos. These genes are important in maintaining the ion and lipid homeostasis in porcine embryos. Solute carrier family 41, member 1 (SLC41A1) is a relatively nonselective divalent cation transporter, which transports a variety of divalent metal cations (Goytain and Quamme, 2005a). SLC41A1 proteins are central components of the plasma membrane Mg2+ -uptake system in vertebrate (Mandt et al., 2011), and the transcription of SLC41A1 is up-regulated in response to low magnesium concentration (Goytain and Quamme, 2005b). ChaC, cation transport regulator homolog 1 (CHAC1) is a cation transport regulator that promotes apoptosis (Joo et al., 2012; Mungrue et al., 2009). Methylsterol monooxygenase 1 (MSMO1, also known as sterol-C4-methyl oxidase) and Isopentenyl-diphosphate delta isomerase 1 (IDI1) are both involved in the sterol/cholesterol biosynthetic and metabolic processes (Fukushima et al., 2010). Given the role that these differentially expressed genes are consider to play in embryonic metabolism, and therefore development, further investigation is required to determine if their altered expression is associated with any deficiencies in pLH embryos. In general LH-induced ovulation in gilts appears to have limited influence on the gene expression profile of blastocyst stage porcine embryos, with only 11 genes showing differential expression between pLH and control blastocysts. Despite the concerns that altered expression of certain genes may raise, induction of ovulation with pLH followed by fixed-time insemination has been shown to not impact fertility by our research group (Cameron et al., 2010; Degenstein et al., 2008) and others (Cassar et al., 2005). However this does not eliminate the potential longer term effects this protocol may have on any resulting offspring and further investigation into the impacts of this technology may be needed. 3. Conclusions The application of ARTs has the potential to increase reproductive efficiency in the pig for both agricultural and biotechnological applications, however the efficiency of several ARTs is limited in the pig compared to other livestock species such as cattle (Kikuchi et al., 2002; Gajda, 2009). Our recent ability to evaluate changes in the embryonic transcriptome has put us in a position to determine the effects that ART manipulations are having on the embryo and will provide the information needed to improve their efficiency in this species and others. Here we have highlighted 3 ART technologies that show varying degrees of impact on embryonic viability and survival, which equates


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with varying degrees of alteration or perturbance of the porcine embryonic transcriptome. In the case of parthenogenesis, the embryos are able to complete the earlier stages of development but do not proceed to term and experience arrested development at various stages (Kharche and Birade, 2013). Given the vast number of genes and key developmental pathways that are affected by the parthenogenic process this is not surprising and will be difficult to overcome. The effects on gene expression in nuclear transfer embryos is substantial, but significantly less than that seen in the parthenotes. The fact that nuclear transfer embryos are able to develop to term despite extensive manipulation, is a testament to the embryo’s amazing plasticity and tolerance when it comes to responding to stressors and adapting to in vivo or in vitro environments. This provides hope that transciptomic information will allow us to reduce these impacts and improve on the efficiency of this technology. Perhaps most surprising, is that despite the fact that induced ovulation in pigs is generally not considered to impact fertility, we observed altered expression of genes that could be considered to be important to development. Given what has been observed in nuclear transfer embryos, these differences in gene expression are likely well within the developmental tolerance of these in vivo embryos. However, several studies using animal models have shown that embryos are sensitive to environmental conditions that can affect future pre- and post-natal growth and developmental potential. With this in mind, as progress is made in improving ARTs in this and other species, one must not ignore the potential unintentional epigenetic consequences that may be imposed on the resulting offspring (Kohda, 2013).

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Conflict of interest statement

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The authors declare they have no relationships, either financial or personal, that could inappropriately influence our work.

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References

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Abeydeera, L.R., 2002. In vitro production of embryos in swine. Theriogenology 57, 256–273. Alexopoulos, N.I., French, A.J., 2009. The prevalence of embryonic remnants following the recovery of post-hatching bovine embryos produced in vitro or by somatic cell nuclear transfer. Anim. Reprod. Sci. 114, 43–53. Alvarez-Anorve, L.I., Alonzo, D.A., Mora-Lugo, R., Lara-Gonzalez, S., BustosJaimes, I., Plumbridge, J., Calcagno, M.L., 2011. Allosteric kinetics of the isoform 1 of human glucosamine-6-phosphate deaminase. Biochim. Biophys. Acta 1814, 1846–1853. Balasubramanian, M.N., Butterworth, E.A., Kilberg, M.S., 2013. Asparagine synthetase: regulation by cell stress and involvement in tumor biology. Am. J. Physiol. Endocrinol. Metab. 304, E789–E799. Bertolini, M., Bertolini, L.R., 2009. Advances in reproductive technologies in cattle: from artificial insemination to cloning. Rev. Med. Vet. Zoot. 56, 184–194. Betthauser, J., Forsberg, E., Augenstein, M., Childs, L., Eilertsen, K., Enos, J., Forsythe, T., Golueke, P., Jurgella, G., Koppang, R., Lesmeister, T., Mallon, K., Mell, G., Misica, P., Pace, M., Pfister-Genskow, M., Strelchenko, N., Voelker, G., Watt, S., Thompson, S., Bishop, M., 2000. Production of cloned pigs from in vitro systems. Nat. Biotechnol. 18, 1055–1059. Blomberg, L., Hashizume, K., Viebahn, C., 2008. Blastocyst elongation, trophoblastic differentiation, and embryonic pattern formation. Reproduction (Cambridge, England) 135, 181–195. Blomberg, L.A., Long, E.L., Sonstegard, T.S., Van Tassell, C.P., Dobrinsky, J.R., Zuelke, K.A., 2005. Serial analysis of gene expression during elongation

of the peri-implantation porcine trophectoderm (conceptus). Physiol. Genomics 20, 188–194. Bolos, V., Grego-Bessa, J., de la Pompa, J.L., 2007. Notch signaling in development and cancer. Endocr. Rev. 28, 339–363. Brevini, T.A., Gandolfi, F., 2008. Parthenotes as a source of embryonic stem cells. Cell Prolif. 41 (Suppl 1), 20–30. Cameron, A., Patterson, J., Ambrose, D.J., Foxcroft, G.R., Dyck, M.K., 2010. Synchronizing ovulation for fixed time artificial insemination in cyclic gilts with porcine luteinizing hormone. In: Proceedings of the 21st International Pig Veterinary Society, Vancouver, BC. Cassar, G., Kirkwood, R.N., Poljak, Z., 2005. Effect of single or double insemination on fertility of sows bred at an induced estrus and ovulation. J. Swine Health Prod. 13, 254–258. Chen, H., Pan, Y.X., Dudenhausen, E.E., Kilberg, M.S., 2004. Amino acid deprivation induces the transcription rate of the human asparagine synthetase gene through a timed program of expression and promoter binding of nutrient-responsive basic region/leucine zipper transcription factors as well as localized histone acetylation. J. Biol. Chem. 279, 50829–50839. Chorro, F.J., Such-Belenguer, L., Lopez-Merino, V., 2009. Animal models of cardiovascular disease. Rev. Esp. Cardiol. 62, 69–84. Coy, P., Romar, R., 2002. In vitro production of pig embryos: a point of view. Reprod. Fertil. Dev. 14, 275–286. Crosier, A.E., Farin, P.W., Dykstra, M.J., Alexander, J.E., Farin, C.E., 2001. Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro. Biol. Reprod. 64, 1375–1385. Dang-Nguyen, T.Q., Tich, N.K., Nguyen, B.X., Ozawa, M., Kikuchi, K., Manabe, N., Ratky, J., Kanai, Y., Nagai, T., 2010. Introduction of various Vietnamese indigenous pig breeds and their conservation by using assisted reproductive techniques. J. Reprod. Dev. 56, 31–35. Degenstein, K.L., O’Donoghue, R., Patterson, J.L., Beltranena, E., Ambrose, D.J., Foxcroft, G.R., Dyck, M.K., 2008. Synchronization of ovulation in cyclic gilts with porcine luteinizing hormone (pLH) and its effects on reproductive function. Theriogenology 70, 1075–1085. Draincourt, M.A., 2013. Fixed time artificial insemination in gilts and sows. Tools, schedule and efficacy. In: Rodriguez-Martinez, H., Soede, N.M., Flowers, W.L. (Eds.), Control of Pig Reproduction IX. , Leicestershire, Q4 UK, pp. 89–99. Dyck, M.K., Lacroix, D., Pothier, F., Sirard, M.A., 2003. Making recombinant proteins in animals—different systems, different applications. Trends Biotechnol. 21, 394–399. Esteban, M.A., Xu, J., Yang, J., Peng, M., Qin, D., Li, W., Jiang, Z., Chen, J., Deng, K., Zhong, M., Cai, J., Lai, L., Pei, D., 2009. Generation of induced pluripotent stem cell lines from Tibetan miniature pig. J. Biol. Chem. 284, 17634–17640. Fu, H., Subramanian, R.R., Masters, S.C., 2000. 14-3-3 Proteins: structure, function, and regulation. Annu. Rev. Pharmacol. Toxicol. 40, 617–647. Fujiwara, H., Higuchi, T., Sato, Y., Nishioka, Y., Zeng, B.X., Yoshioka, S., Tatsumi, K., Ueda, M., Maeda, M., 2005. Regulation of human extravillous trophoblast function by membrane-bound peptidases. Biochim. Biophys. Acta 1751, 26–32. Fukushima, R., Kaneto, M., Kitagawa, H., 2010. Microarray analysis of Leflunomide-induced limb malformations in CD-1 mice. Reprod. Toxicol. (Elmsford, NY) 29, 42–48. Gajda, B., 2009. Factors and methods of pig oocyte and embryo quality improvement and their application in reproductive biotechnology. Reprod. Biol. 9, 97–112. Geisert, R.D., Schmitt, R.A.M., 2002. Early embryonic survival in the pig: can it be improved? J. Anim. Sci. 80, E54–E64. Giraud, S., Favreau, F., Chatauret, N., Thuillier, R., Maiga, S., Hauet, T., 2011. Contribution of large pig for renal ischemia-reperfusion and transplantation studies: the preclinical model. J. Biomed. Biotechnol. 2011, 532127. Goytain, A., Quamme, G.A., 2005a. Functional characterization of ACDP2 (ancient conserved domain protein), a divalent metal transporter. Physiol. Genomics 22, 382–389. Goytain, A., Quamme, G.A., 2005b. Functional characterization of human SLC41A1, a Mg2+ transporter with similarity to prokaryotic MgtE Mg2+ transporters. Physiol. Genomics 21, 337–342. Hammond, E.M., Mandell, D.J., Salim, A., Krieg, A.J., Johnson, T.M., Shirazi, H.A., Attardi, L.D., Giaccia, A.J., 2006. Genome-wide analysis of p53 under hypoxic conditions. Mol. Cell. Biol. 26, 3492–3504. Hao, Y., Lai, L., Mao, J., Im, G.-S., Bonk, A., Prather, R.S., 2004. Apoptosis in parthenogenetic preimplantation porcine embryos. Biol. Reprod. 70, 1644–1649. Hata, H., Tatemichi, M., Nakadate, T., 2012. Involvement of Annexin A8 in the properties of pancreatic cancer. Mol. Carcinog. 53, 181–191. Hornsh, H., Bendixen, E., Conley, L.N., Andersen, P.K., Hedegaard, J., Panitz, F., Bendixen, C., 2009. Transcriptomic and proteomic profiling of two


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porcine tissues using high-throughput technologies. BMC Genomics 10, 30. Isom, S.C., Stevens, J.R., Li, R., Spollen, W.G., Cox, L., Spate, L.D., Murphy, C.N., Prather, R.S., 2013. Transcriptional profiling by RNA-Seq of peri-attachment porcine embryos generated using a variety of assisted reproductive technologies (ART). Physiol. Genomics 45, 577–589. Joo, N.E., Ritchie, K., Kamarajan, P., Miao, D., Kapila, Y.L., 2012. Nisin, an apoptogenic bacteriocin and food preservative, attenuates HNSCC tumorigenesis via CHAC1. Cancer Med. 1, 295–305. Jung, K.J., Ishigami, A., Maruyama, N., Takahashi, R., Goto, S., Yu, B.P., Chung, H.Y., 2004. Modulation of gene expression of SMP-30 by LPS and calorie restriction during aging process. Exp. Gerontol. 39, 1169–1177. Karlsson, K.R., Cowley, S., Martinez, F.O., Shaw, M., Minger, S.L., James, W., 2008. Homogeneous monocytes and macrophages from human embryonic stem cells following coculture-free differentiation in MCSF and IL-3. Exp. Hematol. 36, 1167–1175. Kharche, S.D., Birade, H.S., 2013. Parthenogenesis and activation of mammalian oocytes for in vitro embryo production: a review. Adv. Biosci. Biotechnol. 4, 170–182. Kikuchi, K., Onishi, A., Kashiwazaki, N., Iwamoto, M., Noguchi, J., Kaneko, H., Akita, T., Nagai, T., 2002. Successful piglet production after transfer of blastocysts produced by a modified in vitro system. Biol. Reprod. 66, 1033–1041. Kilberg, M.S., Barbosa-Tessmann, I.P., 2002. Genomic sequences necessary for transcriptional activation by amino acid deprivation of mammalian cells. J. Nutr. 132, 1801–1804. Kohda, T., 2013. Effects of embryonic manipulation and epigenetics. J. Hum. Genet. 58, 416–420. Laronga, C., Yang, H.Y., Neal, C., Lee, M.H., 2000. Association of the cyclindependent kinases and 14-3-3 sigma negatively regulates cell cycle progression. J. Biol. Chem. 275, 23106–23112. Liu, N., Enkemann, S.A., Liang, P., Hersmus, R., Zanazzi, C., Huang, J., Wu, C., Chen, Z., Looijenga, L.H.J., Keefe, D.L., Liu, L., 2010. Genome-wide gene expression profiling reveals aberrant MAPK and Wnt signaling pathways associated with early parthenogenesis. J. Mol. Cell Biol. 2, 333–344. Mandt, T., Song, Y., Scharenberg, A.M., Sahni, J., 2011. SLC41A1 Mg(2+) transport is regulated via Mg(2+)-dependent endosomal recycling through its N-terminal cytoplasmic domain. Biochem. J. 439, 129–139. McEvoy, T.G., Robinson, J.J., Sinclair, K.D., 2001. Developmental consequences of embryo and cell manipulation in mice and farm animals. Reproduction (Cambridge, England) 122, 507–518. Mesquita, F.S., Machado, S.A., Drnevich, J., Borowicz, P., Wang, Z., Nowak, R.A., 2013. Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle. Anim. Reprod. Sci. 136, 231–244. Molchadsky, A., Rivlin, N., Brosh, R., Rotter, V., Sarig, R., 2010. P53 is balancing development, differentiation and de-differentiation to assure cancer prevention. Carcinogenesis 31, 1501–1508. Mungrue, I.N., Pagnon, J., Kohannim, O., Gargalovic, P.S., Lusis, A.J., 2009. CHAC1/MGC4504 is a novel proapoptotic component of the unfolded protein response, downstream of the ATF4-ATF3-CHOP cascade. J. Immunol. 182, 466–476. Nánássy, L., Lee, K., Jávor, A., Macháty, Z., 2008. Effects of activation methods and culture conditions on development of parthenogenetic porcine embryos. Anim. Reprod. Sci. 104, 264–274. Naturil-Alfonso, C., Saenz-de-Juano, M.a.d.D., PeÃaranda, D.S., Vicente, J.S., Marco-Jimanez, F., 2012. Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions. PLoS One 7, e51271. Pariset, L., Chillemi, G., Bongiorni, S., Spica, V.R., Valentini, A., 2009. Microarrays and high-throughput transcriptomic analysis in species with incomplete availability of genomic sequences. Biotechnol. Annu. Rev. 25, 272–279 (Special Issue). Prather, R.S., Brown, A., Spate, L.D., Redel, B.K., Whitworth, K., Whyte, J.J., 2013. Transcriptional profiling of oocyte maturation and embryonic development elucudates metabolism and control of development. In: Rodriguez-Martinez, H., Soede, N.M., Flowers, W.L. (Eds.), Control of Pig Reproduction IX. Leicestershire, UK, pp. 71–84. Richards, N.G., Schuster, S.M., 1998. Mechanistic issues in asparagine synthetase catalysis. Adv. Enzymol. Relat. Areas Mol. Biol. 72, 145–198. Robert, C., Nieminen, J., Dufort, I., Gaël, D., Grant, J.R., Cagnone, G., Plourde, D., Nivet, A.-L., Fournier, É., Paquet, É., Blazejczyk, M., Rigault, P., Juge, N., Sirard, M.-A., 2011. Combining resources to obtain a comprehensive survey of the bovine embryo transcriptome

7

through deep sequencing and microarrays. Mol. Reprod. Develop. 78, 651–664. Roberts, R.M., Telugu, B.P., Ezashi, T., 2009. Induced pluripotent stem cells from swine (Sus scrofa): why they may prove to be important. Cell Cycle 8, 3078–3081. Rodriguez-Osorio, N., Wang, Z., Kasinathan, P., Page, G., Robl, J., Memili, E., 2009. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos. BMC Genomics 10, 190. Rogers, C.S., Stoltz, D.A., Meyerholz, D.K., Ostedgaard, L.S., Rokhlina, T., Taft, P.J., Rogan, M.P., Pezzulo, A.A., Karp, P.H., Itani, O.A., Kabel, A.C., Wohlford-Lenane, C.L., Davis, G.J., Hanfland, R.A., Smith, T.L., Samuel, M., Wax, D., Murphy, C.N., Rieke, A., Whitworth, K., Uc, A., Starner, T.D., Brogden, K.A., Shilyansky, J., McCray Jr., P.B., Zabner, J., Prather, R.S., Welsh, M.J., 2008. Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science (New York, NY) 321, 1837–1841. Sato, Y., Fujiwara, H., Higuchi, T., Yoshioka, S., Tatsumi, K., Maeda, M., Fujii, S., 2002. Involvement of dipeptidyl peptidase IV in extravillous trophoblast invasion and differentiation. J. Clin. Endocrinol. Metabol. 87, 4287–4296. Shepherd, A.K., Singh, R., Wesley, C.S., 2009. Notch mRNA expression in drosophila embryos is negatively regulated at the level of mRNA. Processing. PLoS One 4, e8063. Sinclair, K.D., McEvoy, T.G., Maxfield, E.K., Maltin, C.A., Young, L.E., Wilmut, I., Broadbent, P.J., Robinson, J.J., 1999. Aberrant fetal growth and development after in vitro culture of sheep zygotes. J. Reprod. Fertil. 116, 177–186. Spilsbury, K., O’Mara, M.A., Wu, W.M., Rowe, P.B., Symonds, G., Takayama, Y., 1995. Isolation of a novel macrophage-specific gene by differential cDNA analysis. Blood 85, 1620–1629. Sullivan, E.J., Kasinathan, S., Kasinathan, P., Robl, J.M., Collas, P., 2004. Cloned calves from chromatin remodeled in vitro. Biol. Reprod. 70, 146–153. Takahashi, H., Yamaguchi, M., 1997. Stimulatory effect of regucalcin on ATP-dependent calcium transport in rat liver plasma membranes. Mol. Q5 Cell. Biochnol., 149–153. Tsoi, S., Zhou, C., Grant, J., Pasternak, A., Dobrinsky, J., Rigault, P., Nieminen, J., Sirard, M.-A., Robert, C., Foxcroft, G., Dyck, M., 2012. Development of a porcine (Sus scofa) embryo-specific microarray: array annotation and validation. BMC Genomics 13, 370. Vilahur, G., Padro, T., Badimon, L., 2011. Atherosclerosis and thrombosis: insights from large animal models. J. Biomed. Biotechnol. 2011, 907575. West, F.D., Terlouw, S.L., Kwon, D.J., Mumaw, J.L., Dhara, S.K., Hasneen, K., Dobrinsky, J.R., Stice, S.L., 2010. Porcine induced pluripotent stem cells produce chimeric offspring. Stem Cells Dev. 19, 1211–1220. Whitworth, K.M., Agca, C., Kim, J.G., Patel, R.V., Springer, G.K., Bivens, N.J., Forrester, L.J., Mathialagan, N., Green, J.A., Prather, R.S., 2005. Transcriptional profiling of pig embryogenesis by using a 15-K member unigene set specific for pig reproductive tissues and embryos. Biol. Reprod. 72, 1437–1451. Whitworth, K.M., Zhao, J., Spate, L.D., Li, R., Prather, R.S., 2011. Scriptaid corrects gene expression of a few aberrantly reprogrammed transcripts in nuclear transfer pig blastocyst stage embryos. Cell. Reprogram. 13, 191–204. Whyte, J.J., Prather, R.S., 2011. Genetic modifications of pigs for medicine and agriculture. Mol. Reprod. Dev. 78, 879–891. Wolosker, H., Kline, D., Bian, Y., Blackshaw, S., Cameron, A.M., Fralich, T.J., Schnaar, R.L., Snyder, S.H., 1998. Molecularly cloned mammalian glucosamine-6-phosphate deaminase localizes to transporting epithelium and lacks oscillin activity. FASEB J. 12, 91–99. Wu, Y.D., Jiang, L., Zhou, Z., Zheng, M.H., Zhang, J., Liang, Y., 2008. CYP1A/regucalcin gene expression and edema formation in zebrafish embryos exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Bull. Environ. Contam. Toxicol. 80, 482–486. Zakrzewska, A., Cui, C., Stockhammer, O.W., Benard, E.L., Spaink, H.P., Meijer, A.H., 2010. Macrophage-specific gene functions in Spi1-directed innate immunity. Blood 116, e1–e11. Zaragoza, C., Gomez-Guerrero, C., Martin-Ventura, J.L., Blanco-Colio, L., Lavin, B., Mallavia, B., Tarin, C., Mas, S., Ortiz, A., Egido, J., 2011. Animal models of cardiovascular diseases. J. Biomed. Biotechnol. 2011, 497841. Zhou, C., Dobrinsky, J.R., Tsoi, S., Foxcroft, G.R., Dixon, W.T., Stothard, P., Verstegen, J., Dyck, M.D., 2014. Characterization of the altered gene expression profile in early porcine embryos generated from parthenogenesis and somatic cell chromatin transfer. PLoS One 14, e91728. Zhou, C., Tsoi, C., Cameron, A., Dixon, W.T., Foxcroft, G.R., Dyck, M.K., 2011. Transcriptomic effect of porcine luteinizing hormone (pLH) Induced


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ovulation on blastocyst stage porcine embryos. In: Third Embryo Genomics Meeting, Bonn, Germany. Zhou, C., Tsoi, S., Dixon, W.T., Foxcroft, G.R., Dyck, M.K., 2013. Comparative transcriptomic analysis between in vivo derived porcine 4-cell and morula embryos. Soc. Reprod. Fertil. 68 (Suppl.), 87–88.

Zhou, C., Tsoi, S., Dixon, W.T., Foxcroft, G.R., Dyck, M.D., 2012. Stagespecific gene expression profile differences during early porcine embryonic development. In: Applied Reproductive Biology: Making It Relevant Society for the Study of Reproduction 45th Annual Meeting, Philidelphia, PA, USA.


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