S.-Y. Choe

Development and quality of porcine embryos in different culture system and embryo-producing methods.

In this study, the developmental ability and cellular composition of porcine IVF, parthenote and somatic cell nuclear transfer (SCNT) embryos were evaluated following different in vitro culture systems. Group 1, embryos were cultured in NCSU-23 with 5.55 mM D-glucose (NCSU+) until day 6 on 20% O(2) or 5% O(2) (Group 2). Group 3, embryos were cultured in D-glucose-free NCSU-23 (NCSU-) with 0.17 mM Na pyruvate/2.73 mM Na lactate for 58 h and subsequently cultured in NCSU+ until day 6 (NCSU -/+) on 20% O2 or 5% O(2) (Group 4). IVF blastocysts did not differ significantly with O(2) concentrations, but differed significantly with major energy source (glucose and pyruvate/lactate). In Group 3 and 4 IVF blastocysts, the total cell number and apoptosis rates were not significantly different with different O(2) concentrations. Blastocyst rate, total cell number and apoptosis rate in Groups 3 and 4 parthenote embryos also were not significantly different. Parthenote and SCNT, under the same culture treatment, exhibited significant differences in blastocyst and apoptosis rates (47.5 +/- 16.1 vs. 24.0 +/- 4.0 and 4.9 +/- 9.0 vs. 22.8 +/- 23.3). Apoptosis-generating rate increased in the order parthenote, IVF and then SCNT. In conclusion, in vitro development of porcine embryos was not affected by O(2) concentrations but was affected by major energy source. Even so, the concentration of each major energy source and the timing of its inclusion in culture could accomplish relatively high embryonic development, the apoptosis rate stressed that more work still needs to be done in developing a better defined culture system that could support SCNT embryos equivalent to in vivo preimplantation porcine embryos.

Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 microM ionomycin for 5 min (group 3), 5 microM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 microM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.

343 EFFECTS OF EPIDERMAL GROWTH FACTOR SUPPLEMENTATION ON IN VITRO MATURATION AND GENE EXPRESSION OF CANINE OOCYTES

Despite many efforts to improve canine in vitro maturation (IVM), the efficiency is still low compared to that of other mammalian species (Marie et al. 2004). Epidermal growth factor (EGF) has stimulatory effects on the resumption of oocyte maturation and cumulus expansion in vitro and on prei-mplantation embryonic development in mammals by either an autocrine or a paracrine pathway, or a combination of both systems (Paria et al. 2001 PNAS 98, 1047-1052). The present study investigated the effects of EGF supplementation on in vitro maturation and gene expression of canine oocytes. Oocytes were recovered by slicing ovaries recovered from 40 bitches after ovariohysterectomy at random stages of the estrous cycle. Cumulus-oocyte complexes (COCs) were matured in TCM-199 containing 10% FBS, 1 ¼g/mL FSH and LH, and EGF (0, 10, or 30 ng/mL) for 48 or 72 h at 39°C in a humidified atmosphere of 5% CO2 in air. In Experiment I (n = 2520 oocytes), the nuclear maturation status was assessed by fluorescence microscopy after bisbenzimide (Hoechst 33342) staining (10 µg/mL) at 0, 48, and 72 h of incubation. In Experiment II (n = 90 oocytes), expression of transcripts such as EGF receptor (EGFR), luteinizing hormone receptor (LHR), and gap junction protein (GJA5) were determined in 10 intact COCs each at 0, 48, and 72 h, respectively, by reverse transcription-polymerase chain reaction (RT-PCR). At 0 h 10-20% of the oocytes had undergone resumption of meiosis (GVBD<MII). After 48 h of IVM, rate of meiotic resumption for 0, 10, and 30 ng/mL EGF were 28, 35, and 30%, respectively. At 72 h of IVM, oocytes in the 10 ng/mL EGF group had resumed meiosis at a higher frequency (55%; P < 0.05) than in the 30 ng/mL EGF or the control group (39 and 42%, respectively). At 72 h of IVM, the frequency of maturation to the MII stage was significantly higher in the 10 ng/mL EGF group (9.6%) than in the 30 ng/mL EGF or the control group (4.2 and 3.3%, respectively). The expression of EGFR was significantly higher (P < 0.05) in 0 h oocytes than in the 48- or 72-h oocytes. Further EGFR expression levels were decreased in the presence of EGF in a dose dependent manner. Transcripts for LHR were detected at all maturation intervals and its expression patterns were not altered by supplementation with 10 ng/mL EGF. Expression of GJA5 was observed only after 48 h of IVM, and levels of expression were similar in oocytes supplemented with both 10 and 30 ng/mL EGF. In summary, our results indicate that supplementation of canine IVM medium with 10 ng/mL EGF had a positive influence on the progression of maturation to MII at 72 h. The effect may not be related to the alteration of mRNA expression of genes analyzed in the present study, due to the complex patterns regulating meiotic arrest in canine oocytes. This work was supported by Grant no. 204119-03-1-LG000 from ARPC, Republic of Korea.

46 EFFICIENT TRANSFECTION OF PLASMID DNA INTO CELLS FOR USE AS NUCLEAR DONORS

Somatic cell nuclear transfer (SCNT) has the potential to significantly improve the production of valuable livestock that produce recombinant proteins, such as pharmaceutical proteins for human disease or biomaterials for medical use. The success of this potential depends on efficient and optimized protocols for introducing exogenous DNA into cells. In this study, we compared two methods of transfection, Effectene (Qiagen, Inc., Valencia, CA, USA) and electroporation. Plasmid DNA (pEGFF-N1, Clontech, Seoul, Korea) was transfected into fetal fibroblasts (FFB), cumulus cells (CUC), and adult ear skin cells (ESC). Transfection efficiency, chromosome normality, gene expression, and apoptosis were assessed. Cells cultured in α-modified Eagles medium (α-MEM; BioWhittaker, Walkersville, MD, USA) + 10% FBS were transfected with pEGFP-N1. For electroporation, cells (5 × 106 cells/mL) were mixed in 300 μL perrim buffer (75% Cytosalts with 120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4, 5 mM MgCl2, and 25% α-MEM) + 15 μg pEGFP-N1, and subjected to two pulses of 0.38 kV and 400 μF delivered by Gene Pulser (Bio-Rad; BMS, Ltd., Seoul, South Korea). For Effectene transfection, the procedure suggested by the manufacture was followed. Transfected cells were selected with 600 μg/mL G418 (Gibco; KDR Biotech Co., Ltd., Seoul, South Korea) and cultured at 39°C, 5% CO2 in air. Assessments of EGFP transfected cells by green fluorescence was carried out under an inverted epifluorescence microscope (Nicon, Kanagawa, Japan) equipped with a filter for FITC (excitation maximum = 488 nm; emission maximum = 507 nm). Differences among the groups were analyzed using one-way ANOVA after arc-sine transformation of proportional data. Most cells (>80%) in confluence were at G0/G1 phase, and transfection of the gene into all three cell types did not affect the incidence of chromosomal abnormality or change morphology. In addition, the rates of apoptosis assessed by TUNEL did not differ in all three cell types by either method of transfection at different cell passages. However, the efficiency of gene transfection into FFB by Effectene reagent (14.2 ± 1.7%) was significantly (P < 0.05) higher than that by electroporation (5.1 ± 1.0%). Among the three type cells, the efficiency of gene transfection by Effectene and electroporation of FFB (14.2 ± 1.7 and 5.1 ± 1.0%, respectively) was significantly (P < 0.05) higher than those of CUC and ESC (9.4 ± 1.5 and 3.3 ± 0.8; 8.8 ± 0.7 and 2.1 ± 0.4%, respectively). In conclusion, although there were no differences in the alteration of chromosomes, cell morphology, and apoptosis among three cell types transfected with or without plasmid DNA, FFB is the most effective cell type to be transfected. Effectene is superior to other currently available methods for introducing plasmid DNA into a variety of cells. The high level of transfection achieved by Effectene will encourage its use as a tool for producing transgenic embryos and animals by SCNT. This work was supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010.