Epigenetic Defects Related Reproductive Technologies : Large Offspring Syndrome ( LOS )

Assisted reproductive technologies (ART), such as somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF), have been used to produce genetically superior livestock. Currently, embryos from IVF are commercially available from public or private corporations. However, calves derived by ART techniques frequently suffer with pathological changes in the fetal and placental phenotype, the so-called large offspring syndrome (LOS), and this has significant consequences for development both before and after birth (Behboodi et al., 1995; Constant et al., 2006; Wilmut et al., 2002; Young et al., 1998).


Introduction
Assisted reproductive technologies (ART), such as somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF), have been used to produce genetically superior livestock.Currently, embryos from IVF are commercially available from public or private corporations.However, calves derived by ART techniques frequently suffer with pathological changes in the fetal and placental phenotype, the so-called large offspring syndrome (LOS), and this has significant consequences for development both before and after birth (Behboodi et al., 1995;Constant et al., 2006;Wilmut et al., 2002;Young et al., 1998).
Although the etiology of LOS is not fully understood, these abnormalities may arise from disruptions in expression of developmentally important genes, in particular imprinted genes (Abu-Amero et al., 2006;Amor & Halliday, 2008;Angiolini et al., 2006;Coan et al., 2005;Fowden et al., 2006;Hitchins and Moore, 2002).Genomic imprinting is an important epigenetic mechanism in mammalian development, and is thought to influence the transfer of nutrients to the fetus and the newborn from the mother (Reik & Walter, 2001).Indeed, many imprinted genes are involved in fetal and placental development.Moreover, these imprinting defects cause various developmental disorders in humans, such as Beckwith-Wiedemann syndrome (BWS) (OMIM:130650), Russell-Silver syndrome (OMIM 180860), and Prader-Willi/Angelman syndrome (OMIM 105830) (Enklaar et al., 2006;Horike et al., 2009;Horsthemke & Wagstaff, 2008;Weksberg et al., 2003Weksberg et al., , 2005)).In ruminants, the current study suggests that ART techniques, particularly in vitro culture of preimplantation embryos, have been associated with aberrant imprinted gene expression (Tveden-Nyborg et al., 2008).However, the exact mechanisms that lead to aberrant genomic imprinting after ART remain unknown.
The most likely explanation for the aberrant genomic imprinting in SCNT and IVF cattle may be failures in epigenetic reprogramming and/or maintenance (Bertolini et al., 2002;Beyhan et al., 2007;Blelloch et al., 2006;Everts et al., 2008;Hashizume et al., 2002;Herath et al. 2006;Hochedlinger et al. 2006;Oishi et al. 2006;Pfister-Genskow et al., 2005;Smith et al., 2005;Somers et al., 2006).Genome-wide epigenetic reprogramming in germ cells is essential in order to reset the parental-origin specific marking of imprinted genes.DNA methylation is one of the most important epigenetic marks for the allele-specific silencing of imprinted genes, and its genome-wide profiles undergo drastic changes during gametogenesis (Dupont et al., 2009;Arnaud & Feil, 2005;Bao et al., 2000).Indeed, the genome-wide DNA methylation patterns of the parental genomes are erased and a new methylation pattern is established by de novo methylation during gametogenesis (Arnaud & Feil, 2005;Bao et al., 2000).Therefore, the failures of epigenetic reprogramming could lead to loss of imprinting for many but not all imprinted genes (Reik & Walter, 2001).
A few reports to date have described the aberrant expression of imprinted genes in LOS animals produced by ART techniques.Interestingly, LOS phenotypes are reminiscent of BWS in humans, a loss-of-imprinting pediatric overgrowth syndrome associated with congenital malformations and tumor predisposition (Amor & Halliday, 2008;DeBaun et al., 2003;Maher et al., 2003;Maher, 2005;Manipalviratn et al., 2009;Shiota & Yamada, 2005, 2009).Because the majority of sporadic BWS patients show loss of DNA methylation at KvDMR1, which may function as an imprinting control region (ICR) on the KCNQ1OT1/CDKN1C domain (Mitsuya et al., 1999;Weksberg et al., 2003Weksberg et al., , 2005)), it is possible that LOS is related to the loss of DNA methylation at KvDMR1, leading to diminished expression of Cdkn1c.
In this chapter we highlight some of the epigenetic defects identified in SCNT and IVF cattle and discuss the potential role that imprinted genes may play.

Assisted Reproductive Technologies (ART) and Large Offspring Syndrome (LOS)
LOS calves were first described by Willadsen et al. (1991) following ART technique; the fusion of blastomeres from embryos and enucleated eggs.Since then, oversized neonates and fetuses born after various manipulations of the embryo have been reported not only in calves, but also in sheep (Wilmut et al., 1997(Wilmut et al., , 2002) ) and mouse (Eggan et al., 2001;Fernández-Gonzalez et al., 2004;Wakayama et al., 1998).Up to 40% of SCNT-derived fullterm calves and lambs have LOS, which is characterized by large size at birth, enlarged umbilical cord, enlarged organs, hydrops of the fetus, lethargy, respiratory distress, muscle fiber composition, cerebellar dysplasia and skeletal and facial malformations (Chavatte-Palmer et al., 2002;Constant et al., 2006;Fletcher et al., 2007;Loi P et al., 2006;Maxfield et al., 1997;Schmidt et al., 1996;Walker et al., 1996;Young et al., 1998).Also, it is well known that in high frequency of LOS is also frequently observed in calves that developed from in vitro maturation (IVM) and IVF-derived embryos (Behboodi et al., 1995;Reichenbach et al., 1992;Bertolini et al., 2004).
The most remarkable feature of LOS is large size at birth.Increases in birth weight vary widely; twice the normal birth weight is not uncommon (Young et al., 1998).In our experiments, all calves derived by SCNT (n=7) and IVF (n=2) were shown to be a large size at birth, 1.3 to 2.3 times the normal birth weight.Enlarged umbilical cord was found in almost all of the calves (five of SCNT-derived and two of IVF-derived) (Fig. 1), though abnormality of organs was found only in one SCNT-derived calf in our cases.Placental anomalies, such as a reduced number of placentomes and increased weight of placentomes, lack of placental vascular development, reduced vascularization and poorly developed caruncles were also observed in all LOS cases in SCNT and IVF animals, and are thought to be associated with a high mortality rate and some fetal abnormalities (Bertolini & Anderson, 2002;Chavatte-Palmer et al., 2002;Constant at al., 2006;De Sousa et al., 2001;Hashizume et al., 2002;Hill et al., 2000Hill et al., , 2001)).
While some investigations have previously suggested that reprogramming errors of the donor nucleus following SCNT could affect the fetal and placental development, the etiology of LOS remains unknown (Bertolini et al., 2002;Beyhan et al., 2007;Blelloch et al., 2006;Everts et al., 2008;Hashizume et al., 2002;Herath et al., 2006;Hochedlinger et al., 2006;Oishi et al., 2006;Pfister-Genskow et al., 2005;Smith et al., 2005;Somers et al., 2006).Marques et al. (2004) have previously reported that paternal-allele-specific DNA methylation of the H19 gene was significantly disrupted in spermatozoa from oligozoospermic patients.Although this result strongly suggests that transmission of paternal imprinting errors could affect embryo development, it is not likely that imprinting defects are associated with abnormal spermatogenesis in cattle, since commercially available sperms from healthy bulls are used for IVF.

ART culture may cause epigenetic changes
ART-derived animals can severely influence fetal growth, resulting in LOS, and any disturbance during germ cell development or early embryogenesis has the potential to alter epigenetic reprogramming and/or maintenance (Dupont et al., 2009).The birth of LOS was initially thought to associate with the procedure of ART but it is now recognized that enhanced fetal growth can also result from in vitro culture of oocytes or embryos (Behboodi et al., 1995;Farin et al., 2004;Farin & Farin, 1995;Maxfield et al., 1997;Smith et al., 2009;Walker et al., 1996).
Very limited information is currently available on the effects of in vitro culture; IVM, IVF or SCNT and in vitro development (IVD) on the establishment of imprinting in oocytes or embryos.The influences of in vitro culture on the epigenetic changes are investigated mainly in mouse.The culture medium influences the kinetics of embryo cleavage and embryo morphology up to the blastocyst stage, and can affect the imprinted expression of the H19 gene as well as the DNA methylation status of ICR1, controlling its imprinted manner (Fauque et al., 2007).The presence of serum in culture medium for preimplantation embryos can influence the regulation of multiple growth-related imprinted genes and lead to aberrant fetal growth and development (Khosla et al., 2001).Some researchers reported that ammonium accumulates in culture medium have been linked to aberrant imprinting of H19 and Igf2r (Gardner et al., 2005;Kerjean et al., 2003), however, other researchers have refuted these suggestion that follicle culture system under high ammonia levels showed normal DNA methylation patterns at regulatory sequences of Snprn, Igf2r and H19 (Anckaert et al., 2009a(Anckaert et al., , 2009b)).Mineral oil, which is widely used in in vitro culture, has also been associated with delayed nuclear maturation and reduced development capacity in pig IVM (Shimada et al., 2002).Oil overly extracts steroid hormones in culture medium and reduces steroid hormone level by 55-70% (Anckaert et al., 2009b).Reduced steroid hormones, estrogens or xenobiotic substances with estrogenic effects in culture medium may interfere with normal imprinting establishment (Ho et al., 2006).

LOS in animals is reminiscent of BWS in human
The phenotypes of LOS in animals, such as large size at birth, enlarged umbilical cord and enlarged organs, are reminiscent of BWS in human.Therefore, LOS is speculated to occur primarily as the result of the misregulation of BWS-associated imprinted genes (Fig. 2), while the genomic regions associated with LOS have not yet been determined.BWS is associated with epigenetic alterations at either one of two imprinting control regions on human chromosome 11p15.5,ICR1 and KvDMR1 (Enklaar et al., 2006;Ideraabdullah et al., 2008;Delaval et al., 2006;Mitsuya et al., 1999;Smith et al., 2007;Weksberg et al., 2003Weksberg et al., , 2005;;Owen & Segars, 2009).The domain controlled by ICR1 includes the paternally expressed insulinlike growth factor 2 (IGF2) and the maternally expressed H19 genes (Thorvaldsen et al., 1998;Owen & Segars, 2009).IGF2 is known to be involved in regulation of fetal growth and development (Guo.et al., 2008).H19 is also associated with embryogenesis and fetal growth in mouse (Pachnis et al., 1984), human (Goshen et al., 1993), and sheep (Lee et al., 2002).Several studies have shown that epigenetic alterations in the Igf2/H19 domain are associated with LOS in cattle, sheep, and mice produced by ART techniques (Curchoe et al., 2009;DeChiara et al., 1991;Doherty et al., 2000;Khosla et al., 2001;Li et al., 2005;Moore et al., 2007;Yang et al., 2005;Young et al., 2000Young et al., , 2003;;Zhang et al., 2004).On the other hand, the domain controlled by KvDMR1 contains several maternally expressed genes including CDKN1C, that encodes a cyclin-dependent kinase inhibitor belong to the CIP/KIP family (Yan et al., 1997;Fitzpatrick et al., 2002;Horike et al., 2000).KvDMR1 is a maternally methylated CpG island and includes the promoter of a paternally expressed non-coding RNA (KCNQ1OT1) (Beatty et al., 2006;Mitsuya et al., 1999;).Interestingly, previous studies revealed that KvDMR1 is demethylated in about half of the individuals affected by BWS, and this is associated with the biallelic expression of KCNQ1OT1 and subsequent repression of CDKN1C (Higashimoto et al., 2006;Lee et al., 1999;Mitsuya et al., 1999;Owen & Segars, 2009).Thus, while the Igf2-H19 and Cdkn1c-Kcnq1ot1 gene pairs are good LOS candidates, the phenotypic similarities between LOS and human BWS remain suggestive and deregulation of imprinting remains a plausible candidate mechanism for LOS.

Assessment of the risk of imprinting defects in cattle born following ART
To assess of the risk of imprinting defects in cattle produced by SCNT and IVF, we analyzed DNA methylation status of the Cdkn1c promoter region, KvDMR1 and ICR1, and three promoter regions of other imprinted genes; Peg1/Mest, Klf14 and Gtl2 using CpG methylation sensitive restriction enzymes and bisulfite sequencing (Hori et al., 2010).
Since the use of two restriction enzymes with complementary methylation sensitivities, HpaII and McrBC, is unsurpassed as a simple, rapid method for the analysis of methylation status (Yamada et al., 2004), the HpaII-MspI-McrBC PCR assay is used for screening.HpaII and MspI recognize the CCGG sequence, but HpaII digestion is inhibited by CpG methylation at the internal cytosine while MspI is not.McrBC cleaves DNA containing a methylated cytosine and does not act upon unmethylated DNA (Fiona et al., 2000;Panne et al., 1999).In the case of a fully methylated sequence, amplification would be obtained only  To determine whether hypomethylation at KvDMR1 was linked to the aberrant expression of Kcnq1ot1, Cdkn1c, Igf2, or H19, we performed RT-PCR analysis on samples from two SCNT-and one IVF-derived calves, which showed hypomethylation status at KvDMR1, and compared gene expression patterns with those of a normal calf.In comparison to the normal calf, Kcnq1ot1 transcript levels were increased in three ART-derived calves (two SCNT and one IVF derived calves), whereas the Cdkn1c transcript levels were reduced.No significant differences between three ART-derived calves and the normal calf were detected in H19 or Igf2 expression (Fig. 4

Consideration and prospects
ART-derived embryos, particularly in the cow and sheep, can severely influence fetal growth, resulting in LOS.Disruptions in expression of developmentally important genes, in particular imprinted genes, were found in ART animals, suggesting that any disturbance during germ cell development or early embryogenesis may lead to altering of epigenetic changes.Aberrant gene expression is thought to associate with not only the procedure of ART, asynchronous embryo transfer or progesterone treatment but also in vitro culture of embryos.
The  (2010) suggest that ART techniques might induce an increased risk of epigenetic defects, such as hypomethylation of KvDMR1, because epigenetic changes can be caused by embryo culture itself or the constituents of the culture medium.In humans, a significant deficit in DNA methylation at Kcnq1ot1 in matured oocytes from stimulated cycles matured in in vitro culture (Khoueiry et al., 2008).This paper suggested that hyperstimulation likely recruits young follicles that are unable to acquire imprinting at KvDMR1 during the short in vitro maturation process.In cattle, it is unknown whether hyperstimulation is associated with acquiring imprinting at KvDMR1 of oocytes.A more thorough understanding of the stability of DNA methylation will be important for the continued safeguarding of ART techniques.

Acknowledgements
We thank the members of the laboratory for valuable suggestions.This work was supported by the Program for Improvement of Research Environment for Young Researchers from the Special Coordination Funds for Promoting Science and Technology (SCF) (to SH).

Fig. 1 .
Fig. 1.Phenotype of LOS calf.Left, normal Japanese black calf produced by artificial insemination (body weight at birth: 27kg).Right, LOS Japanese black calf with enlarged umbilical cord produced by SCNT (body weight at birth: 51kg).

Fig. 2 .
Fig. 2. Physical map of imprinting clusters in (a) bovine chromosome 29 and bovine KvDMR1, and (b) human chromosome 11p15.5 and human KvDMR1.Previously identified genes or transcripts (boxes) are drawn approximately to scale.Transcriptional orientation is indicated by arrows and arrowheads.Five and six expressed sequence tags of bovine and human are indicated by filled circle.
from the HpaII-digested template.In contrast, an unmethylated sequence is digested only with HpaII but not with McrBC, and hence amplification would be obtained only from the McrBC-digested DNA.If the target sequence is differentially methylated, such as the imprinting control region, amplification will be obtained from both HpaII-and McrBCdigested DNA.Digestion profiles visualized by PCR amplification from the main organs of seven SCNT-derived and two IVF-derived calves were compared with those of three artificial insemination-derived calves.Lastly, the HpaII-MspI-McrBC PCR assays revealed aberrant KvDMR1 hypomethylation in two of seven SCNT-derived and one of two IVFderived calves.For other imprinting control regions such as ICR1, Peg1/Mest and Gtl2 promoter, PCR amplification was obtained from both HpaII-and McrBC-digested DNA from all samples, indicating that this region is differentially methylated in both normal and SCNT-and IVF-derived calves (Fig.3).For the Cdkn1c and Klf14 promoter, PCR amplification was obtained only from the McrBC-digested DNA, as indicating that both maternal and paternal alleles are unmethylated in all samples.In addition, bisulfite sequencing analyses were demonstrated to confirm the results obtained by HpaII-MspI-McrBC PCR analyses.Bisulfite sequencing is widely recognized to be the gold standard technique to analyze CpG methylation.Finally, these bisulfate sequencing analyses showed strong concordance with the HpaII-MspI-McrBC PCR results.

Fig. 4 .
Fig. 4. (a) Scheme of RT-PCR amplification of Cdkn1c, Kncq1ot1 and H19 from SCNT-No.3 and 5, IVF-No.2 and normal cattle.(b) Putative epigenetic regulation at Kcnq1ot1/Cdkn1c and Igf2/H19 domains of normal and LOS cattle.Transcription is indicated by arrows.Open and filled lollipop indicate unmethylated and methylated CpG site of KvDMR1 and ICR1.
phenotypes of LOS are reminiscent of BWS in humans, an overgrowth syndrome associated with congenital malformations and tumor predisposition.Half of sporadic BWS cases show loss of DNA methylation at KvDMR1, which may function as an ICR on the Kcnq1ot1/Cdkn1c domain.Therefore we examined DNA methylation status of the bovine KvDMR1 in ART cattle.Abnormal hypomethylation status at an imprinting control region of Kcnq1ot1/Cdkn1c domain was observed in two of seven SCNT-derived calves and one of two IVF-derived calves.Moreover, abnormal expression of Kcnq1ot1 and Cdkn1c were observed by RT-PCR analysis.There are very few papers which report KvDRM1 in ARTderived cattle.Coulrey and Lee (2010) reported hypomethylation of KvDMR1 in midgestation bovine fetuses produced by SCNT.Imprinting disruption of KvDMR1 and aberrant expression of Kcnq1ot1 and Cdkn1c identified in SCNT and IVF calves may contribute to LOS in animals conceived using ART techniques.Our findings and those of Couldrey and Lee