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Research Article |
Institute of Molecular Animal Breeding and Biotechnology,3 Gene Center of the Ludwig-Maximilian University, D-81377 Munich, Germany
Department of Analytical Chemistry,4 University of Wuppertal, D-42119 Wuppertal, Germany
Division of Epigenetics,5 Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany
Biotechnology Group,6 Bavarian State Research Center for Agriculture, D-85586 Grub, Germany
Bavarian Research Center for Biology of Reproduction (BFZF),7 D-85764 Oberschleissheim, Germany
Pediatric Endocrinology Section,8 University Hospital, D-72076 Tuebingen, Germany
ABSTRACT
Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable Day-80 bovine fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1 protocol employed 0.01 U/ml of FSH and LH in oocyte maturation medium and 5% estrous cow serum (ECS) in embryo culture medium, whereas the IVF2 protocol employed 0.2 U/ml of FSH and no LH for oocyte maturation and 10% ECS for embryo culture. Comparisons with in vivo–fertilized controls (n = 14) indicated an apparently normal phenotype for IVF1 fetuses (n = 5), but IVF2 fetuses (n = 10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinicochemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2, but not in IVF1, fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1 and IVF2 fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1 fetuses (–16.1% and –9.3%, respectively) and significant hypermethylation in liver of IVF2 fetuses (+11.2%). The 5mC level of cerebral DNA was not affected by IVF protocol. Our data indicate that bovine IVF procedures can affect fetal genomic 5mC levels in a protocol- and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and its correlated endocrine changes.
conceptus, developmental biology, embryo growth factors, in vitro fertilization
INTRODUCTION
Epigenetic modifications of the genome play a significant role in the elaboration of the genetic code and affect early growth and development through their influence on gene expression [1–3]. It is assumed that aberrant epigenotypes of embryos and fetuses cause abnormalities that are frequently observed in ruminants and mice following exposure of oocytes and embryos to chemical and physical agents or manipulations during the use of assisted reproductive technology. One observed abnormality is referred to as large offspring syndrome (LOS), which is found frequently among nuclear transfer (NT) pregnancies but also in a considerable proportion of fetuses and offspring generated by in vitro fertilization (IVF) [4–8]. In the bovine, the phenotypic spectrum of LOS includes altered placental morphology and function, fetal overgrowth, abnormal organ and skeletal development, altered muscle fiber ratio, and endocrinological as well as physiological changes [4, 5, 7–12]. At present, it is not clear whether animals within the presumed normal weight range are truly comparable to in vivo–produced controls [8, 9]. Nuclear transfer calves in the normal range for birth weight showed several physiological alterations that indicate epigenetic perturbations [12].
Epigenetic modifications of the genome include methylation of DNA at cytosine residues; histone protein modifications, such as acetylation and methylation; and the modification and assembly of regulatory protein complexes on DNA. These modifications can be functionally linked and are reversible [1–3]. Methylation of cytosine residues at CpG dinucleotides is the most extensively characterized epigenetic mark in mammals, and it generally is associated with transcriptional silencing and imprinting [1, 3, 13]. During germ cell development, loss of DNA methylation is associated with the erasure of imprinting marks, which subsequently are reset in the developing gametes. After fertilization, during the second phase of large-scale epigenetic reprogramming, genome-wide demethylation is observed in early preimplantation development, followed by remethylation thereafter, but imprinted DNA methylation is maintained [13]. Comparative work demonstrates methylation reprogramming in all mammalian species analyzed, but the extent and the timing vary, consistent with notable differences between species during preimplantation development [13–15]. The presence of DNA methylation in embryos brings about the deacetylation of histone H4 and the methylation of Lys9 of histone H3, and it prevents the methylation of Lys4 of histone H3. This highlights the importance of DNA methylation patterns established during early embryogenesis for setting up the structural profile of the genome [16].
Bovine embryo culture systems and NT can alter the expression of DNA methyltransferases (DNMTs) in bovine preimplantation-stage embryos, and available data suggest that maintenance and de novo DNA methylation can be affected [17]. The analysis of sequence-specific DNA methylation [18, 19] and the investigation of chromosomal or global methylation patterns [20–22] have revealed highly aberrant DNA methylation profiles in bovine preimplantation NT embryos in comparison with IVF embryos. To our knowledge, however, comparisons with ex vivo embryos have not been performed; thus, IVF effects have not been determined. Global DNA hypermethylation in NT embryos was associated with histone H3 Lys9 hypermethylation and poor developmental potential in vitro [22]. Furthermore, significant DNA hypermethylation was detected in liver, but not in placenta, of viable Day-80 bovine NT fetuses with LOS, suggesting that abnormal DNA methylation is maintained in surviving NT embryos and is associated with the LOS phenotype. In that same study, overgrown IVF fetuses that were generated with high concentrations of gonadotropins and serum in the embryo culture medium showed a tendency toward hepatic hypermethylation [23]. A previous study, in contrast, reported significant genomic DNA hypomethylation in skin samples from aborted Day-30 to Day-210 NT fetuses. That same study failed to detect significant differences between IVF fetuses and in vivo controls. However, the specific IVF procedures were not reported, and fetal phenotype was not described [24].
We have analyzed viable Day-80 bovine IVF fetuses in comparison with in vivo–fertilized controls to determine the effects of two bovine IVF protocols, which differed with respect to gonadotropin concentrations during oocyte maturation and serum concentration during embryo culture, on fetal phenotype and genomic cytosine methylation level in tissues derived from the three different germ layers.
MATERIAL AND METHODS
Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co.
Oocyte Recovery and In Vitro Maturation
All cumulus–oocyte complexes (COCs) were obtained from Fleckvieh or Braunvieh ovaries. Ovaries for postmortem collection of oocytes were obtained from commercial abattoirs and transported to the laboratory in PBS at 25–30°C within approximately 2 h. The COCs were collected from batches of 50–100 ovaries as described previously [25]. The COCs for the first IVF protocol (IVF1) were collected in a 50-ml centrifuge tube and washed twice with preincubated (39°C, 5% CO2 in air) Tissue Culture Medium 199 (TCM 199; Invitrogen) supplemented with L-glutamine (100 mg/L), NaHCO3 (3 g/L), Hepes (1400 mg/L), sodium pyruvate (250 mg/L), L-lactic-Ca-salt (600 mg/L), gentamicin (55 mg/L), and 10% (v/v) estrous cow serum (ECS). The maturation medium consisted of supplemented TCM 199 with 0.01 U/ml of bovine FSH and bovine LH (Sioux Biochem). The COCs were washed in maturation medium, transferred to four-well plates (Nunc), and matured in 400 µl of medium for 20–24 h at 39°C in an atmosphere of 5% CO2 in humidified air.
The COCs for IVF protocol 2 (IVF2) were washed twice in TCM 199 (Biochrom) with 10% ECS and washed once in TCM 199 (Biochrom) supplemented with L-glutamine (100 mg/L), NaHCO3 (3 g/L), Hepes (1400 mg/L), sodium pyruvate (250 mg/L), L-lactic-Ca-salt (600 mg/L), gentamicin (55 mg/L; Biochrom), and 10% ECS. The COCs were matured in four-well plates (Nunc) with 400 µl of TCM 199 supplemented with 0.2 U/ml of ovine FSH (Ovagen ICP Bio Ltd.). Oocytes were matured for 20–24 h at 39°C in an atmosphere of 5% CO2 in air and maximum humidity. Additional COC batches for IVF2 were obtained by ultrasound-guided follicle aspiration in vivo as described previously [26]. Follicle aspiration was performed at the Bavarian State Research Center for Agriculture in accordance with the relevant guidelines for the care and use of animals and with approval by the responsible animal welfare authority, the Regierung von Oberbayern. The COCs were aspirated into TCM 199 (Biochrom) with 3% fetal calf serum (Biochrom) and heparin sodium salt (60 µg/ml), filtered with PBS in an Em Con Filter (Immuno Systems, Inc.), and further processed as described above.
IVF and Culture of Embryos
Fleckvieh and Braunvieh semen obtained from commercial artificial insemination (AI) stations (Prüf-und Besamungsstation München-Grub, Poing, Germany and Zweckverband II für künstliche Besamung der Haustiere, Greifenberg, Germany) was used with the corresponding type of oocytes to generate embryos. Matured COCs for IVF1 were washed three times in Tyrode albumin lactate pyruvate (TALP) medium containing 2.2 mg/ml of sodium pyruvate, 10 µg/ml of heparin sodium salt, and 6 mg/ml of BSA. The COCs were transferred to 400 µl of TALP medium. Frozen-thawed spermatozoa were subjected to the swimup procedure for 90 min. Then, the COCs and spermatozoa were coincubated for 18 h in maximum humidity at 39°C and in 5% CO2 in humidified air. The COCs for IVF2 were treated in an identical manner, but TALP medium contained 20 µg/ml of heparin sodium salt.
The presumptive zygotes for IVF1 were mechanically denuded by vortexing and gentle pipetting; washed three times in synthetic oviduct fluid (SOF) culture medium enriched with 5% ECS, 20 µl/ml of 100x basal medium (Invitrogen), and 10 µl/ml of 100x minimum essential medium (Invitrogen); and transferred to 400-µl droplets of medium covered with mineral oil. The culture atmosphere was 5% CO2, 5% O2, and 90% N2 at 39°C and maximum humidity. The presumptive zygotes for IVF2 were treated in an identical manner, but SOF medium contained 10% ECS and 55 µg/ml of gentamicin.
Embryo Transfer, AI, and Recovery of Fetuses
On Day 7 after IVF, embryos were transferred nonsurgically to estrous-synchronized recipient heifers. Control embryos were generated by inseminating Fleckvieh and Braunvieh heifers with frozen-thawed semen. Semen used in IVF1 and IVF2 also was used for in vivo inseminations. Heifers diagnosed as pregnant were slaughtered in a local abattoir on Day 80 after IVF or insemination. The uterus was removed from the recipient and cut open, and fetal blood was collected from the umbilical cord to obtain plasma samples (see below). Fetal weight and length as well as fetal organ weights (heart, kidney, and liver) were recorded. Liver, muscle, and brain tissue samples were collected on ice and stored frozen at –20°C until further processing for DNA isolation. All samples were collected by the same person following standardized procedures. Liver samples were collected from the tip of the lobus hepatis sinister. Muscle samples were obtained from the musculus gluteus maximus. Brain samples were collected from the upper-left hemisphere of the cerebrum. All experiments involving animals were performed in accordance with the relevant guidelines for the care and use of animals and with approval by the responsible animal welfare authority, the Regierung von Oberbayern.
Isolation of DNA and Quantification of 5-Methylcytosine
Total cellular DNA was isolated from tissue samples with the E.Z.N.A. Tissue DNA Kit II (PEQLAB Biotechnologie GmbH). Detailed protocols for the quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis have been published previously [27–29]. Briefly, the cellular DNA was digested to single nucleotides, derivatized with Bodipy FL EDA, and separated by capillary electrophoresis. The DNA samples from liver were analyzed with a Bio-Rad BioFocus 3000TC LIF2 system and the samples from brain and muscle with a Beckman Coulter PACE MDQ LIF system. Electropherograms were analyzed using the software supplied with the respective system, and 5mC levels were determined using derivatization factors established previously [27, 28]. At least three replicates were measured for liver samples, and at least six replicates were measured for brain and muscle samples. We have already demonstrated high reproducibility with both systems [29].
Clinicochemical Parameters in Fetal Plasma
Fetal blood was collected in lithium-Heparin-coated, 1-ml tubes (KABE Labortechnik) and then centrifuged for 10 min at 4500 x g to obtain plasma samples. Isolated plasma aliquots were stored at –20°C until further use. Insulin-like growth factor 1 (IGF1) was determined by IGF-R20 RIA Kit (Mediagnost), with a lower detection limit of 0.15 ng/ml and intra- and interassay coefficients of variation of less than 10%. All other parameters were assayed with an Olympus AU 400 autoanalyzer and adapted reagents from Olympus and Roche. Calibration and quality control were performed according to the manufacturer's protocols and recommendations. The selected parameter list (alanin aminotransferase, alkaline phosphatase, aspartate aminotransferase, calcium, chloride, cholesterol, creatine kinase, creatine kinase muscle-brain, creatinine, 
-glutamyltransferase, glucose, inorganic phosphorus, iron, potassium, sodium, triglycerides, and urea) is part of the clinicochemical screen in the Munich ENU mouse mutagenesis project and is used routinely to uncover clinically relevant phenotypes [30].
Statistics
Both ANOVA and calculation of least-square means were performed with SAS version 8.02 [31] using the general linear model procedure. Treatment group (control, IVF1, and IVF2) means were adjusted for effects of gender (male and female) and genetics (Fleckvieh and Braunvieh). Differences between groups were considered to be significant (two-sided t-test) at P < 0.05. Linear and quadratic regressions were fitted with GraphPad Prism version 3.00 and were considered to be significant at P < 0.05.
RESULTS
Number of Recovered Fetuses
Twenty-nine viable Day-80 fetuses were recovered in total. Fifteen of the fetuses were generated by IVF and 14 by AI. The transfer of 18 IVF1 and 69 IVF2 embryos yielded five (27.8%) and 10 (14.5%) viable Day-80 fetuses, respectively. The collection mode of oocytes used in IVF2, aspiration postmortem or ex vivo, had no effect on the number of transferred embryos that developed into Day-80 fetuses. Thirty-three transferred IVF2 embryos generated with oocytes collected postmortem yielded five (15.2%) fetuses, and 36 IVF2 embryos generated with oocytes collected ex vivo yielded an additional five (13.9%) fetuses. Furthermore, the mean fetal weights (see below) of both IVF2 collectives showed no difference (P = 0.359). Therefore, the data for all IVF2 fetuses (n = 10) were combined for statistical analyses and comparisons with the IVF1 (n = 5) and control (n = 14) groups.
Phenotypic Characteristics of Fetuses
The ANOVA showed that fetal group (control, IVF1, and IVF2) significantly affected fetal weight (P = 0.0025) and length (P = 0.0030) as well as heart (P = 0.0029) and liver (P = 0.0006) weight. The IVF1 fetuses did not differ from in vivo controls, but IVF2 fetuses revealed phenotypic abnormalities. The IVF2 fetuses were heavier (19.9%, P = 0.0008) and longer (4.7%, P = 0.012), with increased heart (25.2%, P = 0.003) and liver (27.9%, P = 0.0002) weights, thus displaying a fetal overgrowth phenotype (Fig. 1). Kidney weight and all relative organ weights, however, were not affected.
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Genomic 5mC Content in Liver, Muscle, and Brain DNA
We analyzed the global cytosine methylation level of DNA samples from fetal liver, muscle, and brain by a highly sensitive capillary electrophoresis method (Fig. 2A). The 5mC content determined for individual samples ranged from 2.29% to 3.61% in liver, from 4.88% to 6.56% in muscle, and from 3.13% to 5.50% in brain. The amounts of 5mC in hepatic DNA (3.036% ± 0.068%; mean ± SEM throughout), muscle DNA (5.971% ± 0.086%), and cerebral DNA (4.777% ± 0.1350%) differed significantly (P < 0.0001). The ANOVA indicated that hepatic 5mC levels were significantly (P = 0.0001) affected by fetal group. Hepatic methylation levels in both groups of IVF fetuses differed significantly from those of in vivo controls. We observed DNA hypomethylation (–16.1%, P = 0.006) in IVF1 fetuses and hypermethylation (+11.2%, P = 0.004) in IVF2 fetuses. A significant but less pronounced DNA hypomethylation (–9.3%, P = 0.043) also was detected in muscle tissue of IVF1 fetuses. Cerebral 5mC level was not affected by fetal group (Fig. 2B).
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Clinicochemical Parameters in Fetal Plasma
A clinicochemical screen of fetal plasma was performed to search for additional potential abnormalities not revealed by fetal phenotype. The ANOVA showed that only four of the 18 parameters were significantly affected by fetal group: creatinine (P = 0.001), glucose (P = 0.037), IGF1 (P = 0.0007), and potassium (P = 0.049). No significant differences were found in these four parameters between IVF1 fetuses and AI controls, but IVF2 fetuses revealed significantly increased levels of creatinine (+37.5%, P = 0.0003), glucose (+70.2%, P = 0.014), IGF1 (+40.8%, P < 0.0007), and potassium (+23.9%, P = 0.019) (Fig. 3).
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Relationships Between Liver 5mC Level, Clinicochemical Parameters, and Phenotype
Regression analyses revealed significant nonlinear relationships between liver weight and liver 5mC and between creatinine and liver 5mC in the data set. Nonlinear regressions became linear when a reduced data set without IVF1 fetuses was analyzed. In contrast, regressions of IGF1 on liver 5mC and of phenotypic parameters on IGF1 were always linear with both data sets (Fig. 4).
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DISCUSSION
In vitro fertilization is practiced routinely in various species, including cattle and sheep, in which its application has been hindered by LOS and associated health problems. The apparently normal phenotype of Day-80 fetuses generated with the IVF1 protocol is consistent with observations that appropriate modifications in IVF protocols can lead to a reduced incidence of LOS [9, 32, 33]. Some features of the overgrown Day-80 fetuses that were generated with the IVF2 protocol have been reported previously, but at much later stages of gestation. This includes a general increase in fetal size and weight and organ-specific organomegaly [4–8]. In sheep and bovine, LOS has been associated with the presence of exogenous protein, particularly serum, during in vitro embryo culture [34, 35]. The major differences between IVF1 and IVF2 protocols used in the present study are the amount and type of gonadotropins in the oocyte maturation medium, the serum batch, and the amount of serum in the embryo culture medium. The IVF1 fetuses were generated with a reduced amount of serum (5% vs. 10% in IVF2) during embryo culture and showed normal fetal and organ weights. We have performed an additional experiment with a single batch of serum and combined either one of the maturation procedures from IVF1 and IVF2 protocols with the embryo culture procedures of the IVF2 protocol. Both combinations yielded overgrown Day-80 fetuses, with virtually identical group means that were indistinguishable from the mean weight of the IVF2 fetuses in the present study (95.3 ± 5.4 and 95.6 ± 5.4 g with n = 8 each vs. 95.1 ± 4.8 g with n = 10 for IVF2). This points to the amount of serum in the embryo culture medium as the cause of the IVF2 protocol–dependent LOS phenotype.
Large-scale DNA methylation reprogramming takes place during preimplantation development and provides a window for epigenetic perturbations that may affect transcription during and after in vitro embryo procedures [13, 35]. In the bovine, the male pronucleus is actively demethylated shortly after fertilization [14, 19–21]. A further passive loss of methylation occurs from the 2- to 8-cell stage [14, 19–21], followed by de novo methylation by the 16-cell stage [21]. Nuclear transfer embryos are characterized by insufficient demethylation and precocious de novo methylation, which appears to be responsible for extensive global, chromosomal, and sequence-specific hypermethylation [18–22]. This observation was recently extended to viable Day-80 NT fetuses with LOS, in which significant hepatic genomic hypermethylation was observed [23]. A previous study, in contrast, reported significant hypomethylation of DNA obtained from skin biopsy samples of viable and aborted NT fetuses. The same study failed to detect significant DNA methylation differences in skin samples of Day-45 to Day-60 IVF-fetuses in comparison to Day-56 to Day-62 in vivo controls [24]. The present data, however, indicate that IVF procedures can significantly affect DNA methylation (i.e., induce DNA hyper- and hypomethylation) in bovine embryos in a tissue-specific manner. A variety of protocols are presently employed to generate bovine IVF and NT embryos, and protocol-specific differences therefore might explain discrepancies in results. Tissue-specific differences, as demonstrated in the present study, also could account for divergent results. We observed no effect of IVF procedures on cerebral DNA methylation level. Both brain and skin are derived from ectoderm, and DNA methylation levels are established around gastrulation and are assumed to be maintained in somatic cells throughout subsequent organogenesis and differentiation [13, 36]. Our results have been derived with a highly accurate method that has been designed to quantify DNA methylation levels only from intact, chemically derivatizable DNA [27–29]. The overall methylation levels detected in the present study ranged from 2.29% to 3.61% in liver, from 3.13% to 5.50% in brain, and from 4.88% to 6.56% in muscle DNA samples. This is in excellent agreement with published data regarding a variety of differentiated mammalian tissues [37, 38].
To our knowledge, all the studies that have investigated bovine preimplantation DNA methylation level and its perturbation by NT used IVF embryos as standard and/or controls [18–22, 39]. The present data indicate that not only NT but also different IVF protocols can significantly affect DNA methylation in bovine embryos. Some of the inconsistencies and variations in DNA methylation data from different studies of bovine NT and IVF embryos [14, 40] therefore might reflect differences in IVF protocols.
The degree of DNA hypermethylation in liver of Day-80 IVF2 fetuses with LOS in the present study (+11.2% vs. AI controls) is very similar to the level of hypermethylation previously detected in Day-80 NT fetuses with LOS (+9.2% vs. AI controls) [23]. Thus, increased hepatic DNA methylation levels clearly are associated with bovine LOS, but whether hypermethylation arises by similar or different mechanisms in NT and IVF embryos is not known. It has been proposed that demethylation of somatic donor nuclei after NT could occur through the same active mechanism by which the paternal genome is demethylated after fertilization [21]. This could provide a link between hypermethylation after NT and IVF procedures. However, our data point toward embryo culture conditions and, therefore, later developmental stages during which hypermethylation in IVF2 fetuses developed. Interestingly, recent studies in mice have provided evidence to suggest that reprogramming after NT occurs progressively during preimplantation development and continues into the early postimplantation period [41]. Embryo culture systems and/or NT can affect the expression of DNMTs in bovine preimplantation-stage embryos. Significant differences in the relative abundance of gene transcripts between in vivo– and in vitro–fertilized or NT-derived blastocysts were reported for DNMT1 and between NT-derived blastocysts and the other experimental groups for DNMT3a, suggesting that maintenance and de novo DNA methylation can be affected by IVF and NT procedures [17]. The reported significant increase in expression of DNMT1 in bovine IVF blastocysts [17] is of particular relevance with respect to the present study, because Dnmt1 overexpression causes genomic hypermethylation and loss of imprinting in the mouse [42].
Global hypermethylation (see above) and sequence-specific DNA hypomethylation [23, 43, 44] have been associated previously with LOS after in vitro embryo culture and/or NT. Our detection of genomic DNA hypomethylation in liver (–16.1% vs. AI controls) and muscle (–9.3% vs. AI controls) tissue of the limited number of IVF1 fetuses is a novel and unexpected finding. Our search for abnormalities in clinicochemical parameters of fetal plasma failed to identify significant differences between IVF1 fetuses and AI controls. Instead, we detected significantly increased IGF1, creatinine, glucose, and potassium levels in hypermethylated overgrown IVF2 fetuses (Fig. 3). All these parameters, particularly IGF1 and creatinine, showed significant relationships with fetal phenotype (Fig. 4). Furthermore, regressions of liver weight, IGF1, and creatinine on liver 5mC levels were significant, linear, and positive when control AI fetuses and IVF2 fetuses were analyzed. This suggests a link between hypermethylation and LOS phenotype of IVF2 fetuses, but a causal relationship has yet to be established. The inclusion of data from hypomethylated IVF1 fetuses yielded nonlinear regressions for liver weight and creatinine (Fig. 4). This might indicate subtle changes in IVF1 fetuses related to hypomethylation.
Altered tissue expression and plasma levels of several IGF system components, particularly IGF2 receptor, have been associated previously with epigenetic change and LOS in sheep, but IGF1 was not investigated in those studies [43, 44]. The essential role of IGF1 for fetal growth was demonstrated by gene-targeting experiments in the mouse [45] and by the phenotype of a human patient with a deletion in the IGF1 gene [46]. Expression of IGF1 is highest in fetal liver and muscle [47], where it regulates eukaryotic initiation factor 4F formation [48]. The IGF1 peptide stimulates myoblast survival, proliferation, and differentiation [49], and it has protein anabolic effects in the sheep fetus [50]. Significant relationships between IGF1 levels in umbilical cord blood and fetal weight and length that are similar to those shown by our data have been reported previously in humans [51]. Fetal and organ weights also were significantly correlated with creatinine (not shown), an end product from creatine and phosphocreatine. Creatine is synthesized in the liver, exported to the circulation, and taken up by muscle, where it is converted to phosphocreatine, an energy source for muscle cells [52]. Thus, more than 90% of the total creatine stores are found in muscle, and serum creatinine is directly correlated with muscle mass [53].
The phenotypic outcome of embryos derived by IVF can be variable. Accumulating evidence suggests that epigenetic mechanisms may be disturbed by manipulation and/or culture conditions in various species [4–8, 17]. We have shown that bovine IVF procedures can affect genomic DNA methylation status in tissue of early fetuses, but the physiological relevance and persistence of these changes have yet to be determined. Tissue-specific transcriptome experiments in combination with genomic and sequence-specific methylation data at different developmental stages could clarify causal relationships and effects of the observed methylation differences.
ACKNOWLEDGMENTS
We thank W. Scholz for excellent technical assistance and P. Rieblinger for animal care.
FOOTNOTES
1 Supported by the Institute for Science and Health (St. Louis, MO) pursuant to Task Order Agreement 03–0900–01RFA02 (to O.J.S.) and a grant from the Deutsche Forschungsgemeinschaft (Priority Program Epigenetics; to F.L.). 
2 Correspondence and current address: Stefan Hiendleder, Department of Animal Science, University of Adelaide, Roseworthy Campus, Roseworthy, SA 5371, Australia. FAX: 61 8 8303 7972; stefan.hiendleder{at}adelaide.edu.au
Received: 18 May 2005.
First decision: 8 June 2005.
Accepted: 21 February 2006.
REFERENCES
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