Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).
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 Ob-erbayern.
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 |xl 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.
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 p-mercaptoethanol, 2 mM nonessential amino acids, 100 IU/ml penicillin, and 100 |xg/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.
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 |xg/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 |xg/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-cy-toplast complexes (KCCs) were exposed to a double electric pulse of 2.1 kV/cm for 10 |xsec using the Zimmermann Cell Fusion Instrument (Bach-ofer, 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 |xg/ml cycloheximide and 5 |xg/ml cytochalasin B (E-Chx). After activation, KCCs were washed three times in culture medium before transfer into 100-|xl 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 (Ty-rode 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-|xl 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 100 X (20 |xl/ml; Invitrogen, Karlsruhe, Germany) and MEM (Minimum Essential Medium) 100X (10 |xl/ml, Invitrogen), and transferred to 400-|xl droplets of medium covered with mineral oil (Sigma-Aldrich, Stein-heim, 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. Briefly, 5 |xg 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 derivati-zation factors established previously.
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 (f-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.