The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from
Nuclear transfer (NT) techniques that use a variety of somatic cells have been used to produce genetically superior animals or transgenic animals that can secrete valuable proteins or pharmaceuticals used in human medicine in their urine or milk [
The mass production of somatic cell nuclear transfer (SCNT) embryos is usually carried out using immature pig oocytes that are obtained from slaughtered ovaries and used as cytoplasts after
The objective of the present study was to examine the feasibility of producing autologous porcine SCNT blastocysts using autologous oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous pig SCNT by examining the effects of electrical fusion and donor cell type on cell fusion and the subsequent
All chemicals were purchased from Sigma-Aldrich (USA), unless otherwise stated. IVM was carried out in TCM-199 media (Invitrogen, USA) supplemented with 10% (v/v) porcine follicular fluid, 0.6 mM cysteine, 0.91 mM pyruvate, 10 ng/ml epidermal growth factor, 75 µg/ml kanamycin, and 1 µg/ml insulin. Porcine follicular fluid (pFF) was collected from follicles of 3-8 mm in diameter, centrifuged at 1900 × g for 15 min, filtered through a 0.2-µm filter, and stored at -30℃ until use. The same batch of pFF was used in all experiments. The
Porcine ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory in saline at approximately 37℃. Follicles of 3-8 mm in diameter were aspirated using an 18 G needle fixed to a 10-ml disposable syringe, and the follicular contents were pooled into 15-ml conical tubes and allowed to settle as sediment. The sediment was suspended in HEPES-buffered Tyrode's medium (TLH) containing 0.05% (w/v) polyvinyl alcohol (PVA) (TLH-PVA) [
Four types of somatic cells were used as donor cells: 1) denuded cumulus cells (DCCs); 2) cultured cumulus cells (CCCs); 3) cultured follicular cells (CFCs); and 4) adult skin fibroblasts. DCCs were collected from IVM oocytes by repeated pipetting in IVM medium that contained 0.1% (w/v) hyaluronidase, washed twice in TLH-PVA by centrifugation, and resuspended in TLH containing 0.4% (w/v) BSA (TLH-BSA) prior to nuclear transfer. CCCs collected from oocytes at 22 h of IVM were cultured for 18 h. Follicular cells collected from the follicular contents at the time of oocyte aspiration were washed twice in TLH-PVA by centrifugation and cultured for 44 h before use. Pig ear skin fibroblasts were cultured until contact inhibited, as described previously [
The base medium for oocyte manipulation was calcium-free TLH-BSA containing 5 µg/ml cytochalasin B. After 40 h of maturation culture, denuded oocytes were incubated for 15 min in a manipulation medium that contained 5 µg/ml Hoechst 33342, washed twice in fresh medium, and then placed into a manipulation medium droplet that was overlaid with mineral oil. Metaphase II (MII) oocytes were enucleated by aspirating the first polar body and MII chromosomes using a 17-µm beveled glass pipette (Humagen, USA), and enucleation was confirmed under an epifluorescent microscope (TE300; Nikon, Japan).
After enucleation, a single cell was inserted into the perivitelline space of each oocyte. Cell-oocyte couplets were placed on a 1-mm fusion chamber that was overlaid with 1 ml of 280 mM mannitol that contained 0.001 mM CaCl2 and 0.05 mM MgCl2. Membrane fusion was induced by applying an alternating current field of 2 V, 1 MHz for 2 sec, followed by two direct current (DC) pulses of 170 V for 50 µsec or 190 V for 30 µsec using a cell fusion generator (LF101; NepaGene, Japan). The oocytes were incubated for 1 h in TLH-BSA and examined for fusion under a stereomicroscope prior to activation.
Reconstructed oocytes were activated by two pulses of 120 V/mm DC for 60 µsec in 280 mM mannitol that contained 0.01 mM CaCl2 and 0.05 mM MgCl2. The oocytes were thoroughly washed in IVC medium, transferred into 30-µl droplets of medium under mineral oil, and cultured for 6 days at 39℃ in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Cleavage and blastocyst formation were evaluated on Days 2 and 6, respectively (the day of SCNT was designated as Day 0). The total cell numbers in the blastocysts were assessed using Hoechst 33342 staining under an epifluorescent microscope.
All of the oocytes used in the respective experiments were randomly allocated to each treatment group, and at least three replications were performed in each experiment. In Experiment 1, SCNT embryos were produced using two types of donor cells (DCC and CCC). The cell fusion rate was examined after applying two electrical fusion stimuli to CCCs and DCCs (2 × 2 factorial design). The
The data were analyzed using the general linear model procedure in the Statistical Analysis System version 9.1 software (SAS Institute, USA), followed by the least significant difference mean separation procedure when treatments differed at
Two electrical field strengths were applied to reconstructed oocytes derived from DCCs or CCCs. As shown in
SCNT embryos derived from DCCs and CCCs were cultured and examined for their developmental capacities
For autologous somatic cell cloning, 1,243 immature oocytes were obtained from 40 pairs of ovaries (31.1 oocytes/pig) and cultured for IVM. After IVM, 74.1% (921/1,243) of the oocytes reached the MII stage. The fusion rate (78 ± 3%) after SCNT was significantly (
A series of experiments was performed to produce cloned blastocysts by autologous SCNT using recipient oocytes and donor cells from the same pig. Our results demonstrated that higher rates of fusion with recipient oocytes could be obtained by culturing cumulus or follicular cells before SCNT. In addition, autologous SCNT blastocysts could be produced via the reconstruction of oocytes with cumulus cells and follicular cells from slaughtered ovaries from the same pig, but their developmental capacities tended to be lower than those of heterologous SCNT embryos derived from adult skin fibroblasts.
Donor cell nuclei are usually introduced into enucleated oocytes by cell fusion [
Even though there was a difference in cell fusion rates, the SCNT embryos derived from DCCs and CCCs did not show any differences in the rates of embryo cleavage or blastocyst formation, which means that the cell culture process itself does not influence embryonic development after SCNT. The donor cell cycle is an important factor in the development of SCNT embryos [
The mean nuclear maturation rate in oocytes collected from individual pigs was 74.1%, which was lower than that (88-98%) observed for heterogeneous oocytes matured in our laboratory [
The fusion rate (52%) of autologous cell-oocyte couplets reconstructed from CCCs (Experiment 3) was low in comparison to that observed in Experiments 1 and 2 (75-77%). It is unclear whether the decreased fusion rate in Experiment 3 can be attributed to the heterologous or autologous origin of the donor cells or to the difference in the batch of oocytes used. Comparative study using heterologous and autologous donor cells with the same batch of oocytes should be performed to clarify the effect of donor cell origin on cell fusion and subsequent SCNT embryo development.
CCC-derived autologous SCNT embryos showed lower developmental capacities to the blastocyst stage than those reconstructed from autologous CFCs and heterologous skin fibroblasts. This result was not consistent with the previous finding in pigs [
In conclusion, the results of the present study show that the culturing of cumulus or follicular cells before nuclear transfer enhances the rate of fusion and that CFCs are superior to CCCs in the production of greater numbers of autologous SCNT blastocysts. The SCNT method established in the present study can be applied to the analysis of the role of mitochondria in the development of autologous or heterologous SCNT embryos. Notwithstanding the successful production of autologous SCNT blastocysts in this study, the low developmental capacity of autologous SCNT embryos remains to be improved. Further studies are needed to establish an effective method for the production of autologous SCNT piglets and to examine the effects of autologous SCNT on economic traits, such as meat quality, milk yield, and fertility.
The authors thank Mr. Bohyun Kwon and Ms. Inyoung Lee for their assistance in the collection and transportation of the ovaries, and Veterinary Services of Gyonggi and Gangwon provinces for their generous donation of porcine ovaries. This work was supported by a Korea Research Foundation Grant (KRF-2004-041-E00342).
Fusion rates of reconstructed oocytes in relation to electrical field strength and donor cell type
abWithin a column, values with different superscripts are significantly different (
abWithin a column, values with different superscripts are significantly different (
*Somatic cell nuclear transfer embryos were produced from donor cells and recipient oocytes that originated from the same pig. abWithin a column, values with different superscripts are significantly different (