| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reproductive Technology |
Department of Molecular Animal Breeding and Biotechnology,3 Gene Center of the Ludwig-Maximilian University, D-81377 Munich, Germany
Research Group Epigenetics,4 Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany
Bavarian Research Station for Animal Breeding,5 D-85586 Grub, Germany
Bavarian Research Center for Biology of Reproduction (BFZF),6
D-85764 Oberschleissheim, Germany
Agrobiogen GmbH,7 D-86567 Hilgertshausen, Germany
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
assisted reproductive technology, conceptus, developmental biology, embryo, placenta
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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, which is generally associated with transcriptional silencing and imprinting [1, 3, 6]. During germ cell development, loss of DNA methylation is associated with the erasure of imprinting marks, which are subsequently reset in the developing gametes. After fertilization, during the second phase of large-scale epigenetic reprogramming, a general loss of DNA methylation is observed throughout the preimplantation period of development, but imprinted DNA methylation is maintained [15]. The presence of DNA methylation brings about the deacetylation of histone H4 and methylation of Lys9 of histone H3 and prevents methylation of Lys4 of histone H3. This highlights the importance of DNA methylation patterns established in early embryogenesis for setting up the structural profile of the genome [16].
Analysis of specific sequences by bisulfite sequencing and restriction enzymes [17, 18] and the investigation of chromosomal or global methylation patterns with antibodies against 5-methylcytosine (5mC) [19–21] revealed highly aberrant methylation profiles in bovine preimplantation SCNT embryos. Compared with their normal counterparts, a large fraction of SCNT preimplantation embryos exhibited hypermethylation of DNA on a sequence-specific [17– 19] or global [20, 21] scale, which is presumably caused by inefficient passive demethylation during the initial embryonic cell divisions [19, 20] and/or precocious de novo methylation [20]. Hypermethylation of DNA in SCNT preimplantation embryos is associated with histone H3 Lys9 hypermethylation and is negatively correlated with the developmental potential of embryos [21].
Epigenetic changes have been associated with altered gene expression and fetal overgrowth/LOS after sheep embryo culture [22], but this observation was based on a single locus, and data for the bovine system is lacking. In a more recent study, global hypomethylation was reported for DNA samples from tissues of aborted or viable bovine SCNT fetuses in comparison with in vitro fertilization (IVF) and artificial insemination (AI) controls [23]. However, this investigation described extraordinarily high cytosine methylation levels of up to 36% even in samples from noncloned controls, although it has been demonstrated that less than 6% of cytosine residues are methylated in mammals, including cattle [24, 25]. We have, therefore, designed a standardized experiment to quantify global methylation of cytosine in viable bovine Day 80 fetuses generated by in vitro (SCNT, IVF) or in vivo (AI) procedures with a highly accurate method [26] and explored the relationships between the fetal overgrowth phenotype and 5mC content of liver and cotyledon tissue.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Animals
All germ cells and somatic cells used to generate fetuses were obtained from animals of the Brown Swiss breed. Oocytes for SCNT and IVF were obtained from slaughtered cattle identified by ID number. Nuclear donor granulosa cells were from the cow Hedina (ID 0961238512). Semen for IVF was from the bull Simvitel (ID 276000916363895), a son of the cow Hedina. AI control fetuses were generated from Brown Swiss heifers with semen from the same bull, Simvitel, and were thus half-sibs to IVF fetuses. Simmental heifers were used as recipients for SCNT or IVF embryos throughout. All experiments involving animals were performed according to the relevant guidelines for the care and use of animals under the approval of the responsible animal welfare authority, the Regierung von Oberbayern.
Oocyte Recovery and In Vitro Maturation for Nuclear Transfer or In Vitro Fertilization
Ovaries were transported to the laboratory in phosphate-buffered saline (PBS) at 25–30°C within approximately 2 h and cumulus-oocyte complexes (COCs) were obtained by aspiration.
Class I COCs were washed three times in culture medium TCM 199 (Seromed, Berlin, Germany) supplemented with L-glutamine (100 mg/L), NaHCO3 (3 g/L), Hepes (1400 mg/L), sodium-pyruvate (250 mg/L), L-lactic-calcium-salt (600 mg/L), and gentamicin (55 mg/L; Seromed). Oocytes were transferred to four-well plates (Nunc, Roskilde, Denmark) with 400 µl TCM 199 supplemented with 10% estrous cow serum (ECS) and containing either 0.01 units/ml b-FSH and b-LH (Sioux Biochem, Sioux Center, IA) (oocytes for nuclear transfer) or 0.2 units/ml o-FSH (Ovagen; ICPbio, Auckland, New Zealand) (oocytes for IVF). Oocytes for nuclear transfer were matured for 18 h at 39°C in an atmosphere of 5% CO2 and maximum humidity, exposed for 5 min to modified phosphate-buffered saline (mPBS; PBS plus 4 mg/ml bovine serum albumin) containing 3 mg/ ml hyaluronidase, vortexed for 4 min, and stripped of cumulus cells by gentle pipetting. Oocytes for IVF were matured for 20–22 h at 39°C in an atmosphere of 5% CO2 and maximum humidity.
Karyoplasts
Granulosa cells obtained after ovum pickup were washed twice in saline solution and dispersed by exposure to 0.1% (w/v) trypsin (Gibco, Grand Island, NY). The cell suspension was then transferred into 5-cm culture dishes containing Dulbecco modified Eagle medium (Gibco) supplemented with 10% (v/v) fetal calf serum (FCS; Biochrom, Berlin, Germany), 2 mM L-glutamine, 0.1 mM ß-mercaptoethanol, 2 mM nonessential amino acids, 100 IU/ml penicillin, and 100 µg/ml streptomycin. The cells were cultured until subconfluence at 37°C in a humidified atmosphere of 5% CO2 in air and then frozen in 10% (v/v) dimethylsulfoxide in FCS and stored in liquid nitrogen. For nuclear transfer experiments, the cells were thawed and cultured for three to six passages until confluence just before nuclear transfer.
Nuclear Transfer
The nuclear transfer procedure was carried out at room temperature (20–25°C) using Leitz micromanipulators (Leica Microsystems, Wetzlar, Germany) and a Wilovert stereo microscope. The oocytes with a first polar body were placed in mPBS containing 5 µg/ml cytochalasin B (Sigma) and incubated in this medium for 5–10 min before starting the enucleation. The oocytes were then placed in a small drop of mPBS into a micromanipulation chamber (two cover slips were mounted 1.0 mm apart and the space between cover slips filled with paraffin oil). Enucleation of oocytes was accomplished by aspiration of a small volume of the cytoplasm surrounding the polar body with a micropipette. After manipulation, oocytes were stained with 2 µg/ml Hoechst 33342 dye (Sigma) and observed for a few seconds by epifluorescence microscope (Zeiss, Jena, Germany) to select successfully enucleated oocytes. Single donor cells were transferred into the perivitelline space of enucleated oocytes and the karyoplast-cytoplast complexes (KCCs) were exposed to a double electric pulse of 2.1 kV/cm for 10 µsec using the Zimmermann Cell Fusion Instrument (Bachofer, Reutlingen, Germany). KCCs were placed in the incubator in Ham F-12 medium supplemented with 0.3% bovine serum albumin.
Activation and Culture of SCNT Embryos
Two hours postfusion, the KCCs were activated by a 5-min incubation in 7% ethanol followed by a 5-h culture in 10 µg/ml cycloheximide and 5 µg/ml cytochalasin B (E-Chx). After activation, KCCs were washed three times in culture medium before transfer into 100-µl drops of synthetic oviduct fluid medium (SOF) supplemented with 2% basal medium Eagle (BME) amino acids (Gibco), 1% minimum essential medium (MEM) nonessential amino acids (Gibco), and 10% (v/v) ECS, covered by paraffin oil (Merck, Darmstadt, Germany), and cultured at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. One batch of serum was used for the production of SCNT embryos.
In Vitro Fertilization and Culture of IVF Embryos
Matured COCs were washed three times in fertilization medium (Tyrode albumin lactate pyruvate) supplemented with sodium pyruvate (2.2 mg/ml), heparin sodium salt (2 mg/ml), and BSA (6 mg/ml) and transferred to 400-µl droplets of medium. Frozen-thawed spermatozoa were subjected to the swim-up procedure for 90 min. Then the COCs and spermatozoa (2 x 106 cells/ml) were co-incubated for 18 h in maximum humidity, 39°C, and 5% CO2 in air.
The presumptive zygotes were mechanically denuded by vortexing, washed three times in SOF culture medium enriched with 10% ECS, BME 100x (20 µl/ml; Invitrogen, Karlsruhe, Germany) and MEM (Minimum Essential Medium) 100x (10 µl/ml, Invitrogen), and transferred to 400-µl droplets of medium covered with mineral oil (Sigma-Aldrich, Steinheim, Germany). The culture atmosphere was 5% CO2, 5% O2, 90% N2 and 39°C at maximum humidity. One batch of serum was used for the production of IVF embryos.
Embryo Transfer and Artificial Insemination
On Day 7 after nuclear transfer and IVF, all viable embryos were transferred nonsurgically to synchronous recipients. Two or three embryos were transferred to each recipient. Control fetuses were generated by inseminating heifers with frozen-thawed semen by standard procedures. Pregnancies were confirmed on Day 28 by ultrasonographic examination and on Days 42 and 79 by palpation.
Recovery of Fetuses and Data Collection
Recipient heifers and inseminated controls diagnosed pregnant were killed in a local slaughterhouse on Day 80 after SCNT and IVF and on Day 80.5 after AI. The uterus was removed from the recipient; cut open; and fetus weight, liver weight, placenta weight, number of placentomes, and fetal dimensions (crown-rump length, thorax circumference) were recorded. All uteri were cut open following the horns longitudinally, and photographed to determine the mean length and width of the three largest placentomes. Tissue samples were obtained on ice from liver and cotyledon and stored frozen at –20°C until further processing for DNA isolation.
Isolation of DNA and Microsatellite Typing
Total cellular DNA was isolated from tissue samples with the E.Z.N.A. Tissue DNA Kit II (PEQLAB Biotechnologie GmbH, Erlangen, Germany). The genotype of SCNT fetuses was confirmed by typing genomic DNA samples with a panel of 13 standardized microsatellites recommended for parentage control in cattle by the International Society for Animal Genetics. All SCNT fetuses showed the identical genotype expected from the nuclear donor cells (data not shown).
Quantification of Genomic 5-Methylcytosine Levels
A more detailed protocol has been published previously [26]. Briefly, 5 µg of total cellular DNA were digested to single nucleotides, derivatized with Bodipy FL EDA, and separated by capillary electrophoresis using a BioFocus 3000TC LIF2 system (Bio-Rad Laboratories, Hercules, CA). Electropherograms were analyzed using the software supplied with the system and cytosine methylation levels were determined using derivatization factors established previously [26].
Statistics
Group means were calculated with the program SPSS for Windows version 11.0 (SPSS Inc., Chicago, IL) using the general linear model procedure. Differences between groups (t-test) were considered significant at P < 0.05. Regressions were calculated with the program GraphPad Prism version 3.00 (GraphPad Software, San Diego, CA) and considered significant at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Maturation rate of oocytes for nuclear transfer was 55%, and 203 of 215 matured and enucleated oocytes were successfully fused (94.4%) with nuclear donor cells. By Day 3, 139 KCCs (68.5%) had further developed; this resulted in 63 blastocysts (29.3%) by Day 7. In the IVF group, 120 oocytes subjected to maturation and fertilization procedures yielded 78 Day 3 embryos (65%), of which 32 (26.7%) developed into blastocysts by Day 7.
Phenotypic Characteristics of Fetuses and Fetal Membranes
For a detailed phenotypic analysis of fetuses derived from SCNT, IVF, and AI, fetuses and placentae were recovered 80 days after the initiation of embryonic development. Only SCNT fetuses with a body weight 
109.9 g were selected for further analyses. This yielded a group of overgrown singleton SCNT (n = 6) fetuses. The investigated IVF (n = 5) and AI (n = 5) groups of fetuses were unselected for body weight but had been selected for females and singletons. SCNT and IVF fetuses revealed a distinct overgrowth phenotype (Fig. 1), which was further characterized by quantitative analysis of morphological parameters (see Fig. 3A). In comparison with the AI controls, SCNT and IVF fetuses showed not only a significantly elevated body weight (61%, P < 0.001 and 28%, P < 0.003), liver weight (100%, P < 0.001 and 36%, P < 0.014) and thorax circumference (20%, P < 0.001 and 11%, P < 0.001) but also a higher relative liver weight (25%, P < 0.01 and 7%, not significant). The crown-rump length did not differ among the three groups of fetuses, but a reduced crown-rump length:thorax circumference ratio (1.175 ± 0.017 in SCNT and 1.292 ± 0.018 in IVF vs. 1.390 ± 0.018 in AI, P < 0.001 and P < 0.002) was the external hallmark of the disproportionate overgrowth phenotype observed in both groups of fetuses derived by in vitro techniques.
|
|
The absolute weight of fetal membranes was significantly higher in SCNT fetuses than in IVF or AI fetuses (49%, P < 0.012 and 36%, P < 0.035) but the ratio of fetus weight:placenta weight was not significantly different among the groups. There was no difference in the number of placentomes, but placentomes were significantly longer in SCNT placentae than in IVF or AI placentae (21%, P < 0.034 and 32%, P < 0.007). Placentomes in SCNT fetuses were also wider than in IVF or AI fetuses (36%, P < 0.011 and 21%, P < 0.077) (Figs. 2 and 3B).
|
Global 5-Methylcytosine Content in Liver and Cotyledon DNA and Relationship with Phenotypic Characteristics
In order to analyze the relationship between fetal overgrowth and DNA methylation, we determined the global cytosine methylation level of DNA samples from fetal liver and cotyledon tissue by capillary electrophoresis (Fig. 4A). The SCNT fetuses revealed significant hypermethylation of liver DNA in comparison with AI controls (3.457% ± 0.079% 5mC vs. 3.166% ± 0.087% 5mC, P < 0.03). The cytosine methylation level of liver DNA from IVF fetuses was intermediate (3.342% ± 0.087% 5mC) to but not significantly different (P > 0.18) from the other groups. The average 5mC content of placental cotyledon DNA was 46% lower than in liver DNA (P < 0.0001) but did not differ among fetus groups (Fig. 4B).
|
Regressions of fetal body and liver weight as well as thorax circumference on the cytosine methylation level of liver DNA were positive (P < 0.073–0.079) and showed very similar slopes (Fig. 5, A–C). Furthermore, a significant negative regression (P < 0.021) of the crown-rump length: thorax ratio on liver 5mC was observed (Fig. 5D). This demonstrates a statistically significant association between the level of DNA methylation and an important phenotypic characteristic of the observed fetal overgrowth.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Epigenetic perturbations that could affect the expression of genes involved in embryonic development and fetal growth regulation have been suggested as possible causes of the LOS syndrome [e.g., 9, 27] and epigenetic change at a single locus has been associated with LOS in ovine fetuses [22]. Several groups have observed significant hypermethylation of DNA in bovine preimplantation SCNT embryos on a sequence-specific, chromosomal and global scale [17–21], and the level of methylated cytosine has been correlated with aberrant histone modification and the developmental potential of cloned embryos [21]. The significant hypermethylation (9.2% vs. AI control fetuses) of liver DNA from Day 80 SCNT fetuses investigated in the present study extends these observations to viable cloned fetuses and suggests that aberrant epigenetic modifications due to incomplete reprogramming in the early bovine embryo persist at least until the end of the first trimester of pregnancy. This is consistent with the general stability of established DNA methylation patterns in somatic cells [6], but it is presently unknown if hypermethylation would still be detectable at term. However, DNA methylation data reported for preimplantation embryos [17–21] and our present data obtained from samples of viable fetuses are in striking contrast with the recently reported hypomethylation of DNA obtained from skin biopsies of viable and aborted SCNT fetuses aged 53–62 days and 58–210 days, respectively [23]. A variety of protocols and nuclear donor cells are employed to generate SCNT embryos, and the general difference (hypermethylation vs. hypomethylation) between our data and the data presented by Cezar et al. [23] could therefore be due to differences in SCNT protocols and/or nuclear donor cells. The analysis of overgrown SCNT fetuses in the present study and the different types of tissues used for DNA isolation, liver and placenta in the present study vs. skin in the study of Cezar et al., could also contribute to the observed differences. Methylation of DNA in some samples from the latter study might also have been affected by degradation of DNA in aborted fetuses. The percentage of 5mC reported by Cezar et al. for DNA from individual viable SCNT or IVF fetuses and adult SCNT or AI cattle ranged from 10% to 40% [23], although it is generally established that less than 6% of cytosine residues in mammalian DNA are methylated (e.g., [25]). Similar values have also been reported for various bovine tissues [24]. While we cannot presently explain the absolute differences between our data and the data published by Cezar et al. [23], 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 [26]. In addition, the overall methylation levels detected in our samples ranged from 2.98% to 3.71% 5mC for liver DNA samples and from 1.18% to 2.39% 5mC for cotyledon DNA samples. This is in excellent agreement with published data on a variety of differentiated mammalian tissues [24, 25]. Hypomethylation of placental vs. embryonic DNA has been previously described in the mouse [28, 29].
An additional and interesting result of the present study is the apparent hypermethylation of liver DNA from IVF fetuses. Although the difference in 5mC content between IVF samples and controls was not significant (P < 0.18), it is striking that this value is 
50% of the significant difference between SCNT samples and controls, and thus corresponds to the intermediate phenotypic values observed for IVF fetuses in relation to the phenotypic differences observed between AI controls and SCNT fetuses. Above all, this includes the reduced crown-rump length:thorax ratio that was found to be a highly indicative external hallmark of the overgrowth phenotype observed in both SCNT and IVF fetuses (Figs. 1 and 3A). It has been proposed that demethylation of somatic donor nuclei after nuclear transfer could occur by the same active demethylation mechanism by which the paternal genome is demethylated after fertilization, suggesting that an important trigger for demethylation is remodeling of chromatin by factors present in the oocyte cytoplasm [20]. This could provide a link between fetal overgrowth in bovine SCNT and IVF fetuses, dependent on the in vitro production protocol of IVF embryos. The IVF fetuses investigated in the present study were generated according to a protocol that consistently produces overgrown fetuses, based on differences in oocyte maturation and culture conditions, as compared with a protocol that yields phenotypically normal IVF fetuses (unpublished data). A further indication for such a common mechanism is provided by the positive regressions of phenotypic parameters reflecting the overgrowth phenotype on liver 5mC content, which approached significance at P < 0.073– 0.079, and, most important, the significant negative regression observed for crown-rump length:thorax ratio that was characteristic of the disproportionate overgrowth phenotype observed in SCNT and IVF fetuses, on liver 5mC (Fig. 5D).
The developmental potential and phenotypic outcome of embryos derived by in vitro techniques is so far rather unpredictable. Accumulating evidence suggests that epigenetic mechanisms may be disturbed by manipulation and/or culture conditions [30, 31]. We show for the first time that hypermethylation of fetal DNA is associated with disproportionate overgrowth of SCNT fetuses and is, to a lesser extent, also observed in IVF fetuses. Quantitative analysis of overall DNA methylation further documents the limitations of current embryo technologies and may be a useful parameter for the evaluation and improvement of IVF and SCNT procedures.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Stefan Hiendleder, Department of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University Munich, Hackerstrasse 27, D-85764 Oberschleissheim, Germany. FAX: 49 89 315 2799; S.Hiendleder{at}gen.vetmed.uni-muenchen.de
Received: 29 November 2003.
First decision: 19 December 2003.
Accepted: 24 February 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  H. Niemann, X C. Tian, W A. King, and R. S F Lee Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning Reproduction, February 1, 2008; 135(2): 151 - 163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Wuensch, F. A. Habermann, S. Kurosaka, R. Klose, V. Zakhartchenko, H.-D. Reichenbach, F. Sinowatz, K. J. McLaughlin, and E. Wolf Quantitative Monitoring of Pluripotency Gene Activation after Somatic Cloning in Cattle Biol Reprod, June 1, 2007; 76(6): 983 - 991. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Yang, R. Hao, B. Kessler, G. Brem, E. Wolf, and V. Zakhartchenko Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation Reproduction, January 1, 2007; 133(1): 219 - 230. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Lucifero, J. Suzuki, V. Bordignon, J. Martel, C. Vigneault, J. Therrien, F. Filion, L. C. Smith, and J. M. Trasler Bovine SNRPN Methylation Imprint in Oocytes and Day 17 In Vitro-Produced and Somatic Cell Nuclear Transfer Embryos Biol Reprod, October 1, 2006; 75(4): 531 - 538. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hiendleder, M. Wirtz, C. Mund, M. Klempt, H.-D. Reichenbach, M. Stojkovic, M. Weppert, H. Wenigerkind, M. Elmlinger, F. Lyko, et al. Tissue-Specific Effects of In Vitro Fertilization Procedures on Genomic Cytosine Methylation Levels in Overgrown and Normal Sized Bovine Fetuses Biol Reprod, July 1, 2006; 75(1): 17 - 23. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Mund, B. Hackanson, C. Stresemann, M. Lubbert, and F. Lyko Characterization of DNA Demethylation Effects Induced by 5-Aza-2'-Deoxycytidine in Patients with Myelodysplastic Syndrome Cancer Res., August 15, 2005; 65(16): 7086 - 7090. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
