Hye-Jin Song

Effect of α-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes ☆

Boar semen is extremely vulnerable to cold shock and sensitive to peroxidative damage due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and the relatively low antioxidant capacity of seminal plasma. The present study evaluated the influence of alpha-tocopherol supplementation at various concentrations in the boar semen extender during cryopreservation on post-thawed sperm motility characteristics (total sperm motility, MOT; local motility, LCM; curvilinear velocity, VCL; straight linear velocity, VSL; and average path velocity, VAP), sperm qualities (viability, acrosomal integrity and apoptosis), expression of stress protein (HSP70), and the expression of pro-apoptotic (Bax and Bak) and anti-apoptotic (Bcl-2l and Bcl-xl) genes. Semen collected from 10 Duroc boars was cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of alpha-tocopherol (0, 100, 200, 400, 600 and 800 microM) using the straw-freezing procedure and stored at -196 degrees C for a minimum period of one month. In frozen-thawed groups, sperm motility was significantly (P<0.05) lower than that of fresh sperm. In fresh sperm, HSP70 immunoreactivity expression was observed in the equatorial region, but in frozen-thawed groups, expressions were mostly observed in the sperm head. Higher apoptosis rates were observed in 600 and 800 microM alpha-tocopherol supplemented frozen-thawed groups. In alpha-tocopherol supplemented frozen-thawed groups immediately after thawing, the expression was similar to that of fresh group. But after incubation at 37 degrees C for 3h, the expression in 200 and 800 microM alpha-tocopherol supplemented groups was higher than that of others. Expression of pro-apoptotic genes was significantly higher and anti-apoptotic genes was significantly (P<0.01) lower in alpha-tocopherol supplemented frozen-thawed groups compared to fresh sperm group. In conclusion, alpha-tocopherol, supplemented at 200 microM concentration in boar semen extender during cryopreservation had a positive effect on post-thawed sperm survivability.

Influence of epidermal growth factor supplementation during in vitro maturation on nuclear status and gene expression of canine oocytes.

This study evaluated the effect of epidermal growth factor (EGF) supplementation during in vitro maturation on the meiotic status and the expression of EGF receptor (EGFr), luteinizing hormone receptor (LHr) and gap junction protein α 5 (GJA5) in canine cumulus-oocyte-complexes (COCs). COCs of ≥110 μm diameter, exhibiting dark pigmentation and completely surrounded by three or more layers of cumulus cells collected from anestrus stage ovaries in natural cycle were matured in TCM-199 supplemented with 10% fetal bovine serum, 0.57 mM cysteine, 10 μg/ml LH and FSH, and different concentrations of EGF (0, 10 and 30 ng/ml). Oocytes cultured for 72 h were fixed to assess the nuclear maturation. Expression of EGFr, LHr and GAJ5 was assessed by immunocytochemistry and real-time PCR. Proportion of metaphase II status of oocytes cultured in in vitro maturation (IVM) medium supplemented with 10 ng/ml EGF for 72 h was significantly (P<0.05) higher than 0 and 30 ng/ml EGF supplemented IVM medium (9.8% vs. 6.5% and 5.2%). In both cumulus cells and oocytes, EGFr protein was undetectable, LHr protein level of expression was low and a strong expression of GJA5 protein was observed. The relative abundance (RA) of EGFr transcript revealed low levels and the LHr expression decreased steadily with addition of EGF. However it did not vary among different concentrations of EGF supplementation. The RA of GJA5 transcript exhibited lower level at 10 ng/ml EGF supplementation. In conclusion, the supplementation of 10 ng/ml EGF in IVM media exerted a positive influence on the progression of maturation to MII phase and the expression level of GJA5 at 72 h, but did not demonstrate any stimulatory role on the expression of EGFr and LHr during the maturation of the canine IVM oocytes.

292 Multilineage potential of canine mesenchymal stem cells derived from bone marrow

The purpose of this study is to characterize canine mesenchymal stem cells (MSCs) derived from bone marrow (BM) for use in research on the applications of stem cells in canine models of development, physiology, and disease. BM was harvested antemortem by aspiration from the greater tubercle of the humerus of 30 normal beagle dogs. Canine BM-derived MSCs were isolated according to methods developed for other species and were characterized based on their morphology, growth traits, cell-surface antigen profiles, differentiation repertoire, immunocytochemistry results, and neurotrophic factor expression in vitro. The canine MSCs exhibited a fibroblast-like morphology with a polygonal or spindle-shaped appearance and long processes; further, their cell-surface antigen profiles were similar to those of their counterparts in other species such as rodents and humans. The canine MSCs could differentiate into osteocytes and neurons on incubation with appropriate induction media. RT-PCR analysis revealed that these cells expressed NGF, bFGF, SDF-1, and VEGF. This study demonstrated that isolating canine MSCs from BM, stem-cell technology can be applied to a large variety of organ dysfunctions caused by degenerative diseases and injuries in dogs. Furthermore, our results indicated that canine MSCs constitutively secrete endogenous factors that enhance neurogenesis and angiogenesis. Therefore, these cells are potentially useful for treating dogs affected with various neurodegenerative diseases and spinal-cord injuries.

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.