Poster Presentation

Poster Presentation 1 16-01 DEVELOPMENTAL POTENTIAL OF NUCLEAR-TRANSFERRED EMBRYOS USING A BOVINE MAMMARY EPITHELIAL CELL LINE (BMEC) Satoshi Akagi1, Seiya Takahashi1, Katsuhiro Ohkoshi2, Takato Takenouchi2, Manabu Shimizu3, Masaya Geshi1, Noritaka Adachi1, Daiichiro Fuchimoto2, Yoshiaki Izaike2, and Hisashi Aso4. 1Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Ikenodai 2, Kukizaki, Inashiki-gun, Ibaraki 305-0901, Japan; 2Developmental Biology Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan; 3National Agricultural Research Center for Tohoku Region, Morioka, 020-0198 Japan; 4Department of Animal Production Science, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan In previous report (1), nuclei from a permanent mammary epithelial cell line supported the development of reconstructed embryos only to early cleavage stages but not to the blastocyst stage. We have established a mammary epithelial cell line (BMEC) derived from a mammary alveolus of a Holstein heifer. BMEC cells have the ability to differentiate and proliferate until at least 50 passage (submitted data). In present study, we examined the developmental potential of embryos using BMEC cells for nuclear transfer (NT).

As donors for NT, BMEC cells (passage 15) cultured for 4-5 days after seeding at different cell density of 1.0x105cells/cm2(High-density group) and 0.8x104cells/cm2(Low-density group) were used. These cells became confluent in 2 and 4 days after seeding in High-density and Low-density groups, respectively. Oocytes were enucleated after in vitro maturation for 20 h in TCM 199 supplemented with 10 % fetal bovine serum. BMEC cells were transferred to the perivitelline space of enucleated oocytes. After electric stimulation, the oocytes fused with donors were treated with cytochalasin D (2.5 µg / ml) + cycloheximide (10 µg / ml) for 1 h and cycloheximide alone for further 4 h. NT embryos were cultured in vitro until day 7. In Experiment 1, we examined the effective electric stimulation for oocyte-cell fusion. The oocyte-cell complexes were exposed to 5 different electric stimulation: 1) one DC pulses of 25 V / 150 µm for 10 µsec, 2) two DC pulses of 20 V / 150 µm for 10 µsec , 3) two DC pulses of 25 V / 150 µm for 20 µsec, 4) two DC pulses of 25 V / 150 µm for 30 µsec, 5) two DC pulses of 30 V / 150 µm for 20 µsec. In Experiment 2, we examined the developmental potential of NT embryos fused with two DC pulses of 30 V / 150 µm for 20 µsec in High-density group and with two DC pulses of 20 V / 150 µm for 10 µsec in Low-density group.

The populations of donor cells in the G0 / G1 phase of cell cycle were 83.6 % in High-density group and 60.0 % in Low-density group. In Experiment 1, fusion rates reached maximum with two DC pulses of 30 V / 150 µm for 20 µsec in High-density group and with two DC pulses of 25 V / 150 µm for 10 µsec in Low-density group. The fusion rate (37.5%) in High-density group was significantly (P<0.005) lower than in Low-density group (71.4%). In Experiment 2, there were no significant differences in development rate to blastocyst stage of NT embryo between High-density (17.1%; per fused eggs) and Low-density (25.8%) groups.

The results of this study indicate that the efficiency of oocyte-cell fusion was affected by the culture condition of donor BEMC cells before NT. Reference 1. Zakhartchenko V., Alberio R., Stojkovic M., Prelle K., Schernthaner W., Stojkovic P., Wenigerkind H.,

Wanke R., Duchler M., Steinborn R., Muller M., Brem G., and Wolf E. (1999) Mol Reprod Dev 54: 264-272.

16-02 IN VITRO DEVELOPMENT OF PORCINE OOCYTES ACTIVATED BY ELECTRIC PULSE: EFFECT OF MATURATION CULTURE PERIOD Yinzhong Bing1,2, Limei Che1, Yuji Hirao1, Naoki Takenouchi1, Masashige Kuwayama2, and Takashi Nagai3 1Department of Animal Production and Grasslands Farming, Tohoku National Agricultural Research Center, Morioka, Iwate, 020-0198, Japan; 2Division of Research and Development, Kato Ladies Clinic, Shijuku, Tokyo 160-0023, Japan; 3Developmental Biology Department, National Institute of Agriobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan In vitro development of activated porcine oocytes was strongly affected by the maturation of oocytes before activation (1-2). The present study was designed to evaluate the effect of maturational age of oocytes on their activation and development after stimulation with electric pulse. Porcine oocytes with compact cumulus cells complexes (COCs) were cultured in droplets of modified TCM199 containing FCS, sodium pyruvate, hormones (PMSG, hCG and estradiol-17b) covered with paraffin oil at 38.5OC under an atmosphere of 5% CO2 in air with saturated humidity. In Experiment 1, after maturation culture for 36, 42 and 48 h, oocytes were fixed for examination of the meiotic stages; germinal vesicle break down (GVBD) and metaphase II (MII). In Experiment 2, after maturation culture for 36, 42 and 48 h, oocytes with the first polar body (PB) were subjected to a single DC pulse (1,500 V/cm) of 100 msec in 0.3 M mannitol solution containing MgSO4, CaCl2 and PVA. Activated oocytes were incubated in mNCSU 37 supplemented with 0.4% BSA and 5.0 mg/ml cytochalasin B for 4 h, and then cultured for 8 h in mNCSU 37 supplemented with 0.4% BSA. The nuclear status of activated oocytes was evaluated after fixation. In Experiment 3, activated oocytes were cultured in droplets (1 oocyte/ml) of mNCSU 37 supplemented with 0.4% BSA under a humidified atmosphere of 5% CO2 in air at 38.5OC. After 48 h of culture, cleaved embryos were transferred to new droplets of mNCSU 37 and cultured for 5 days for the development to the blastocsyt stage. The data were tested for significance using ANOVA and Fisher’s PLSD test with P<0.05 as a level of significance. In Experiment 1, the rates of oocytes that underwent GVBD and matured to MII after cultur for 36, 42 and 48 h were not different (GVBD: 95.7, 100 and 100%, respectively; MII: 79.6, 81.6 and 83.6%, respectively). In Experiment 2, the rates of oocytes activated and oocytes with two pronuclei and one PB (2N1PB) did not differ when cultured for 42 h or 48 h. However, the rates were significantly higher than that of oocytes cultured for 36 h (Activation rates: 94, 96% vs 63%; 2N1PB rates: 63, 64% vs 19%; P<0.05, respectively). In Experiment 3, when oocytes were cultured for 42 h and 48 h, the rates of oocytes cleaved were significantly higher than when cultured for 36 h (81, 81% vs 56%, P<0.05, respectively). When oocytes were cultured for 48 h, the rate of blastocyst formation was significantly higher than that of oocytes cultured for 36 h (10% vs 1%, P<0.05). These results indicate that the duration of maturation culture affects subsequent development of porcine oocytes activated by electric pulse and treated with cytochalasin B. The immature cytoplasm, irrespective of nuclear maturation, of oocytes cultured for a shorter period might relate to a lower developmental capability after activation. References 1 Yamauchi, N., Sasada, H., Sugawara, S., and Nagai T. (1996) Reprod Fertil Dev 8, 1153-6. 2 Kikuchi, K., Nagai, T., Ding, J., Yamauchi, N., Noguchi, J., and Izaike Y. (1999) J Reprod Fertil 116,

143-156.

16-03 EFFECTS OF DURATION FOR IN VITRO MATURATION AND 6-DIMETHYLAMINOPURINE ON ACTIVATION OF PORCINE OOCYTES Kohei Fuchinoue1, Takuya Wakai1, Kazumi Saeki1, Manabu Kawahara1, Kohichi Kimura1, Hiroshi Sasada1, and Eimei Sato1 1Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan Since a technique for in vitro maturation of oocytes has been developed in several animals, in vitro matured oocytes have been involved in increasing efficiency of animal cloning, and also several steps before transferring reconstituted embryos should be improved. The present study was conducted to investigate the effects of duration for in vitro maturation of oocytes and treatment of 6-dimethylaminopurine (6-DMAP)on activation in pigs. Porcine oocytes that were collected from ovaries were cultured in vitro in NCSU23 and subjected to the experiments. Oocytes elctro-stimulated at 36 h and 44 h after culture, which resulted in 158/223 (71 %) and 192/251 (76 %) maturation, respectively, showed 18 % and 28 % development at 2-cell stage and 3 % and 3 % at blastocyst, respectively. Manipulation (enucleation of metaphase and injection of the nucleus derived from cumulus cells), which was done at 36 h or 44 h after culture, resulted in a production of 16 % or 30 % cleaved embryos, respectively. The treatment of 6-DMAP to electro-activated oocytes had no effect on their subsequent development to 2-cell. These results showed that the duration for in vitro maturation may affect subsequent development of reconstituted embryos derived from in vitro matured oocytes. 16-04 EFFECT OF ASCORBIC ACID IN CULTURE MEDIUM ON IN VITRO DEVELOPMENT AND POST-CRYOPRESERVATION SURVIVAL OF BOVINE EMBRYOS Ayako Hariyama1, Osamu Dochi1, Shoko Ieda1, Kei Imai2, and Hisaichi Koyama1

1Department of Dairy Science, Rakuno Gakuen University, Ebetsu, Hokkaido 0669-8501, Japan; 2Developmental Biology Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan Previously, we reported that the development rate of blastocysts increased by adding ascorbic acid (AsA) (Thriogenology 2001;55:335). The present study evaluated the effect of AsA concentration in culture medium on in vitro development and post-cryopreservation survival of bovine embryos. Cumulus oocyte complexes (COCs) were aspirated from bovine ovaries collected at a slaughterhouse and were matured in vitro in TCM199 with 5% calf serum (CS) and 0.02 mg/mL of FSH in an atmosphere of 5% CO2 in air at 38.5°C for 20-21 h. After that, the COCs were inseminated with frozen-thawed semen (5×106 sperm/mL) in BO solution (Brackett and Oliphant, 1975, Biol Reprod 12:260-274) containing 10 mM hypotaurine and 4 units/mL heparin. After 18 h of gamete co-culture, the presumptive zygotes were cultured in CR1aa (Rosenkrans et al., 1991, Theriogenology 35:226) + 5% CS supplemented with 0, 0.1, 0.15 or 0.2 mg/L of AsA for 8 days at 38.5°C in an atmosphere of 5% CO2, 5% O2, 90% N2 in air. Embryo development was evaluated for cleavage and blastocyst rate on days 2 and 7 to 8,

respectively, after in vitro fertilization. Blastocysts were frozen in straws with PBS containing 20% CS, 1.5 M ethylene glycol and 0.1 M sucrose. The straws were thawed in a 30°C water bath for 15 sec. The thawed embryos were cultured in TCM199 + 5% CS and 0.1 mM β-mercaptoethanol for 72 h to assess post-thaw embryo survival based on hatching ability. The experiment was replicated six times. Data were analyzed using the chi-square method. No difference in cleavage rate (71.1-76.7%) was seen between treatments. The blastocyst rates with AsA concentrations of 0.15 (42.7%) and 0.2 mg/L (43.0%) were significantly higher than that of the control (31.1%) (P<0.05). There were no differences in the hatching rate after freezing of the blastocysts (6.3-9.7%) between treatments. These results suggest that the addition of AsA to the medium for in vitro culture of IVM-IVF bovine embryos affected their ability to develop to the blastocyst stage, but had no effect on their survival after cryopreservation. Our results suggest that the most suitable concentration of AsA to add is 0.2 mg/L. 16-05 IN VITRO MATURATIONAL AND DEVELOPMENTAL POTENTIAL OF OOCYTES RECOVERED FROM PREPUBERTAL AND ADULT PIGS Koji Ikeda1, and Yoshiyuki Takahashi1 1Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan It has been indicated that oocytes recovered from prepubertal animals have lower developmental competence than those from adult animals. To explore the differences in developmental competence, we examined meiotic progression, p34cdc2 kinase activity, diameter of cytoplasm, and developmental potential of oocytes from prepubertal and adult pigs. Oocyte-cumulus-granulosa cell complexes were recovered from 4 to 8 mm follicles of abattoir-derived ovaries. At the time of recovery, majority (>80%) of oocytes recovered from prepubertal and adult pigs were arrested at GV-I stage, and their subsequent maturation kinetics and maturation rate were not different after culture for 28 to 44 h in the medium containing follicular fluid and gonadotropins. The activity of p34cdc2 kinase was not different between prepubertal and adult pig oocytes immediately after recovery, but was tended to be higher in the oocytes from prepubertal gilts than those from adult pigs after 40 h of maturation culture. The diameters of oocytes recovered from adult pigs before (117.5 µm) and after 40 h of maturation culture (118.4 µm) were larger (P<0.05) than those of prepubertal gilts (115.2 and 116.7 µm, respectively). In vitro development to blastocysts and their cell numbers of parthenotes and nuclear transfer embryos originated from adult pig oocytes were higher than those from prepubertal gilts. In conclusion, oocytes recovered from prepubertal gilts have smaller diameter and lower in vitro developmental potential than those from adult pigs and the differences in developmental potential of oocytes may be independent from their maturation kinetics and p34cdc2 kinase activity. 16-06 SUCCESSFUL CELL CULTURE OF DEAD ANIMAL TISSUES AND INTRODUCTION OF FOREIGN GENE INTO ANIMAL CELLS Kei Katayama1, and Noboru Fujiwara1

1Animal Resource Science Section, Division of Bioresource and Bioenvironmental Science, Graduate School Kyushu University, Fukuoka 812-8581, Japan The present experiments were designed to develop methods for successful cell culture of dead animal tissues and introduction of foreign gene (green fluorescent protein: GFP) into animal cells. Experiment 1: Four kinds of culture media (Opti-MEM, IMDM, DMEM, DMEM/F12) were examined for cell culture to develop better method for tissues from wild and/or domestic animals. Six kinds of tissues (skin, uterus, ovary, gum, ear, muscle) of cow and swine were obtained from a local abattoir and processed for routine cell culture (1). Media were exchanged every 3 days until the cells become confluent. The ovary showed higher cell growth rate than other tissues in both cow and swine for four sorts of media, though the cells from cow failed to show any difference among culture media. Based on these results, we used Opti-MEM for cow and swine tissues, since this medium was considered to be better for any kind of tissues. Experiment 2: For developing effective cell fusion to create some kind of hybrid cells, an electroporation method was employed for animal tissues of cow and swine. First of all, optimal figures of voltage (v: 300-2400v) and capacitances (micro Faraday: µF) were determined using GFP (6 µg/700 µl PBS) as a marker gene together with cultured animal cells (4 × 105 cells). Following electroporation, the treated cells were stained with acridine orange (AO) and ethidium bromide (EB) to observe cell viability under a fluorescent microscope. As the result, around 300v was though to be suitable for better survival rate of cultured cells. On the other hand, the smaller µF , the better cell survivability. The previous reports (2) demonstrated that around 50% of survivability of cultured cells could be optimal for introducing foreign genes into living cells. These results suggest that the introduction of GFP into the cultured cells had no influence on the survivability of cells. References 1 Hager, B., Bickenbach, J.R., and Fleckman, P. (1999) J Invest Dermatol 112,971-976 2 Heiser, W.C. (1994) Analytical Bichem 217, 185-196 16-07 LIPID DROPLET TRANSITION THROUGHOUT SPERM PENETRATION TO PRONUCLEAR FORMATION IN PORCINE OOCYTES Kazuhiro Kikuchi1,3, Hans Ekwall2, Paisan Tienthai1, Yasuhiro Kawai1,4, and Heriberto Rodriguez-Martinez1 Departments of 1Obstetrics and Gynaecology and of 2Anatomy and Histology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden; 3Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan; 4Department of Animal Science, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan Lipid droplet content in mammalian oocytes or embryos differs among species, the bovine and porcine oocytes and embryo showing large amounts. Lipid droplets are considered to be a source for energy supply and to play important roles for oocyte maturation, penetration and development to embryos, which relates to their freezability [Nagashima et al., 1995]. However, little about their function is fully known. In the present study, morphological changes during fertilization in vivo and in vitro were evaluated in the pig. Both in vitro and in vivo matured oocytes, penetrated oocytes and pronuclear oocytes were harvested. In vivo

materials were collected from normally cycling Large white gilts. In vivo matured oocytes were collected just before ovulation, and fertilized oocytes were collected from mated gilts, which were considered to be after 3 h after sperm-oocyte encounter because they were at telophase-II stage. In vivo pronuclear oocytes were also collected after insemination and were considered to be at 6 to 15 h after gamate encounter since they had well-developed pronuclei. In vitro matured oocytes were collected at 48 h in maturation culture [Kikuchi et al., 1999]. In vitro fertilization were carried out in a modified Pig-FM [Suzuki et al., 2000] with ejaculated spermatozoa after preincubation in modified Medium 199 for 2 h. In vitro fertilized oocytes and pronuclear oocytes were collected at 3 h and 10 h, respectively, after coincubation with the spermatozoa. All oocytes were fixed with 2.5% glutaraldehyde solution in cacodylate buffer and processed for transmission electron microscopy (TEM). The analysis of ultrathin sections at TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated to mitochondrial aggregates in the oocytes irrespective of being matured in vivo or in vitro. Immediately sperm penetration, the electron-density of the lipid droplets was lost in either the in vivo and in vitro oocytes. Electron-density resumed in the pronuclear oocytes, fully in the vivo specimens but only partially in the in vitro ones. The amount and size of the droplets seemed, however, to have decreased. The results suggest variations in the morphology and amount of cytoplasmic lipid droplets during pig oocyte activation both in vivo and in vitro. References 1. Nagashima, H., Kashiwazaki, N., Ashman, R.J., Grupen, C.G., and Nottle, M.B. (1995) Nature 374,

416. 2. Kikuchi, K., Kashiwazaki, N., Noguchi, J., Shimada A., Takahashi, R., Hirabayashi, M., Shino, M., Ueda,

M., and Kaneko, H. (1999) Biol Reprod 60:336-340. 3. Suzuki, K., Eriksson, B., Shimizu, H., Nagai, T., and Rodriguez-Martinez H. (2000) Int J Androl 23:

13-21. 16-08 EFFECT OF OOCYTE ACTIVATION TIMING ON NUCLEAR REMODELLING AND DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS Mayuko Kurome1, Naohiro Wako1, Takashi Ochiai1, Takashi Kurihara2, Tatsuya Fujimura3, Yoichi Takahagi3, Hiroshi Murakami3, and Hiroshi Nagashima1 1Meiji University, Kawasaki, Japan; 2Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Japan; 3The Animal Engineering Research Institute Co.,Ltd., Tsukuba, Japan The objective of this study was to investigate the effect of the timing of oocyte activation relative to nuclear transfer on the nuclear remodelling and development of the porcine reconstructed embryos. Oocytes matured in modified NCSU23 were used as recipient cytoplasts. As nuclear donor, cumulus cells were collected from IVM oocytes. Nuclei of the donor cells were transferred into the recipient oocytes by intracytoplasmic injection using a piezo-actuated micromanipulator. Reconstructed embryos were incubated with 7.5 micro-g/ml cytochalasin B for 3-4hr, followed by in vitro culture in NCSU23 + 0.4% BSA. Nuclear transfer was conducted by the following three manners. [I] pre-activation NT : Oocytes were electrically activated 2-2.5hr after nuclear transfer. [II] Immediate NT : Donor nuclei were transferred into recipient oocytes immediately (within 30 min) after activation. [III] post-activation NT : Donor nuclei were injected into activated oocytes at AII/TII stage (peri-AII/TII stage). Transferred nuclei showed premature chromosome condensation (PCC) by 1hr after the nuclear injection in all the three groups. When the reconstructed embryos were examined 3hr after nuclear transfer,

most ( 18/23, 78%) of the embryos in the pre-activation NT group possessed metaphase-like or disarrayed chromosomes (1). In contrast, embryos in the post-activation NT group tended to have early pseudo-nuclei (1) (26/42, 62%). Nuclear status of the reconstructed embryos in the immediate NT group varied from condensed chromosome to early pseudo-nuclei. Examination of the reconstructed embryos at 8hr after activation revealed that 70% (28/40) of the embryos in the pre-activation NT group had multiple pseudo-pronuclei. Proportion of the embryos possessing multiple nuclei were significantly lower ( 13% and 18%, P<0.05) in the other two groups. Blastocyst formation rates of the reconstructed embryos were between 5 (5/107) to 8% (7/90) in the three groups. These developmental rates were significantly lower than that of the control parthenogenetic oocytes (46%, 48/105). These data demostrate that the timing of oocytes activation relative to nuclear transfer influence remodelling patern of the donor nuclei, but has no effect on in vitro development of the reconstructed embryos to the blastocyst stage. Reference 1 Wakayama, T., Perry, A.C., Zuccotti, M., Johnson, K.R., and Yanagimachi, R. (1998) Nature 394,

369-374. 16-09 DEVELOPMENT OF RECONSTRUCTED PORCINE EMBRYOS DERIVED FROM IN VIVO- OR IN VITRO-MATURED OOCYTES AS A SOMATIC CELL RECIPIENT CYTOPLAST Gab-sang Lee1, Sang-hwan Hyun1, Hye-soo Kim1, Dae-young Kim1, So-hyun Lee1, Byeong-chul Oh1, Jong-im Park1, Jeong-mook Lim2, Eun-song Lee3, Sung-keun Kang1, Byeong-chun Lee1, and Woo-suk Hwang1 1College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; 2School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea; 3Departments of Veterinary Medicine, Kangwon National University, Chunchon 200-701, Korea In somatic cell nuclear transfer, cytoplasmic factor of a donor nucleus recipient is important for reprogramming and subsequent development of reconstructed oocytes. This study was conducted to examine whether the use of in vivo-matured porcine oocytes as a recipient cytoplast contribute to enhancing preimplantation development of oocytes reconstructed with fetal fibroblasts. In vivo-matured oocytes were retrieved by flushing the oviducts of prepubertal gilts administrated PGF2α, PMSG and hCG, and, as a control oocytes, oocytes matured in cultured for 44 hours were provided. Only oocytes with normal morphology were selected in both groups. Those were then enucleated and reconstructed with serum-starved fetal fibroblasts by our standard protocols. Reconstructed embryos were then cultured for 168 hours in modified North Carolina State University-23 medium under a humidified atmosphere of 5% CO2, 7% O2 and 88% N2 at 39°C. First cleavage, morula compaction and blastocyst formation were evaluated at 48, 144 and 168 hours of culture, respectively, and total blastomere number in blastocysts were counted by Hoechst 33342 staining at the end of culture. All data were analyzed by a PROC-GLM in SAS program. As shown in Table 1, a total of 335 oocytes (198 in vitro and 137 in vivo) were provided for this study and a significant (P<0.05) model effect was found in the development to the 2-cell (first cleavage), morula and blastocyst stage. Significant increase in the rate of first cleavage was found in IVM oocytes compared with in vivo-matured oocytes. However, more reconstructed oocytes derived from in vivo-matured cytoplasts developed to the morula and blastocyst stages than reconstructed oocytes derived from IVM cytoplasts. There was no significant difference in the number of blastomere between

two groups. Table 1. In vitro development of porcine NT embryos (NT) using in vitro matured oocytes and in vivo matured oocytes.

a-b Values with different superscripts in the same column are statistically different (P<0.05)

These results demonstrated that the source of somatic cell recipient cytoplast is important for the development of porcine reconstructed oocytes and the use of in vivo-matured oocytes as an enucleated oocyte contributes to enhancing preimplantation embryo development. 16-10 SPERM BINDING ACTIVITY OF THE RECOMBINANT PIG ZONA PELLUCIDA GLYCOPROTEINS EXPRESSED IN A BACULOVIRUS/INSECT CELL EXPRESSION SYSTEM Minoru Nakano1, Naoto Yonezawa1, and Toshiyuki Katsumata2 1Department of Chemistry, Faculty of Science, Chiba University, Chiba-shi, Chiba 263-8522, Japan; 2College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Ichikawa-shi, Chiba 272-0827, Japan Zona pellucida surrounding mammalian oocyte is composed of three glycoprotein components called ZPA, ZPB and ZPC. At the initial phase of fertilization, sperm bind to the carbohydrate chains of one of the components in a species-specific manner. We have shown that pig sperm bind to triantennary and tetraantennary neutral complex-type chains localized in the N-terminal region of ZPB (1), while bovine sperm bind to a high-mannose-type chain having five mannose residues (2). In bovine, localization of the sperm ligand chain has not been determined. Here we investigated the sperm binding activity of three recombinant pig zona glycoproteins. We expressed the recombinant proteins in a baculovirus/insect cell expression system and examined their sperm binding activity by a sperm-agarose beads binding assay. As a result, it was shown that pig sperm do not bind to any of the recombinant pig components, but bovine sperm bind to the recombinant pig ZPB. The sperm binding activity of ZPB was enhanced by coexpression of ZPC. The recombinant proteins were recognized by Concanavalin A, suggesting that they have the high-mannose-type chain of bovine sperm ligand. Thus, the carbohydrate moiety of zona glycoprotein was shown to play a major role in species-specific sperm binding. References 1. Nakano, M., and Yonezawa, N. (2001) Cells Tissues Organs 168, 65-75. 2. Amari, S., Yonezawa, N., Mitsui, S., Katsumata, T., Hamano, S., Kuwayama, M., Hashimoto, Y., Suzuki,

A., Takeda, Y., and Nakano, M. (2001) Mol Reprod Dev 59, 221-226. 16-11 EFFECTS OF EXTRACELLULAR AND INTRACELLULAR DIVALENT CATIONS

No. (%) of Oocytes matured Cultured Cleaved (%) Morula Blastocyst

Mean no. of blastomere

In Vitro 198 141 (71.2)a 47 (23.7)a 31 (15.6)a 25.4 In Vivo 137 88 (64.2)b 28 (31.8)b 22 (25.0)b 28.1

ON THE PARTHENOGENETIC ACTIVATION OF PORCINE OOCYTES Konosuke Okada1, Hiroshi Harayama1, and Masashi Miyake1 1Department of Life Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho Nada-ku, Kobe 657-8501, Japan In the previous report porcine oocytes are parthenogenetically activated by the injection of calcium chloride and this activation is accompany with various events of oocyte activation, such as the exocytosis of cortical granule, meiotic resumption, and pronuclear formation (1). While mouse oocytes are parthenogenetically activated by the exposure to culture medium (2) or injection of carrier medium (3) containing divalent cations, it is still unclear the effects of other divalent cations on the induction of parthenogenetic activation in porcine oocytes. The present study was performed to examine effects of treatments with other divalent cations, strontium (Sr2+) and barium (Ba2+), on the parthenogenetic activation of in vitro matured porcine oocytes.

Oocyte cumulus and granulosa cell complexes (OCGCs) were collected from ovaries and the culture for maturation of OCGCs were carried out under an atmosphere of 5% CO2 in humidified air at 38.5℃ for 45-48 h. After the culture for maturation, oocytes were denuded of cumulus cells by hyaluronidase treatment and pipetting, and then used for the following experiments. Some of the oocytes were exposed to calcium-free TL-Hepes medium supplemented with 10 mM BaCl2 ,SrCl2, or CaCl2 for 2 h and then additionally cultured in TL medium for 4 h. The others were injected with 8.2-14.1 pl (0.9-1.6 % of the volume of the porcine oocyte) carrier medium (20 mM HEPES, pH 7.4) supplemented with 100 mM BaCl2, SrCl2, or CaCl2 and then these oocytes were cultured for 6 h in TL medium. All treated oocytes were fixed and stained to assess the rates of the pronuclear formation.

Each exposure of porcine oocytes to extracellular Ba2+, Sr2+, or Ca2+ was not effective for the activation of them at all. On the other hand, the intracellular injection of Ba2+, Sr2+, or Ca2+ effectively induced the pronuclear formation in about a half number of the treated oocytes. These results demonstrate that the intracellular injection of these divalent cations can progress the process of activation in porcine oocytes, also suggesting that the permeability and/or responsiveness of porcine oocytes to these divalent cations are different from those of mouse oocytes. References 1 Machaty Z., Funahashi, H., Mayes, M.A., Day, B.N., and Prather, R.S. (1996) Biol Reprod 54: 316-322. 2 Whittngham, D.G., and Siracusa, G.(1978) Exp Cell Res 113: 311-317. 3 Fulton B.P., and Whittngham, D.G. (1978) Nature 273: 149-151. 16-12 VIABLE OFFSPRING FROM VITRIFIED BLASTOCYST DERIVED FROM NUCLEAR TRANSFER WITH BOVINE CUMULUS CELL

Norio Saito1, Kanako Kaneyama1, Satoko Matoba1, Yutaka Hashiyada2, and Shuji Kobayashi3

1National Livestock Breeding Center (NLBC), Nishigo, Fukushima, 961-8511, Japan; 2Oou Station, NLBC, Shichinohe, Aomori, 039-2567, Japan; 3Niikappu Station, NLBC, Shizunai, Hokkaido, 056-0141, Japan Although there has been increasing number of successful reports on somatic cell cloning in cattle since 1998, little is known about the cryoviability of reconstructed embryos.

Conventional freezing is thought to be less effective for the reconstructed embryos because of disintegration of their zona pellucida and a longer culture period compared with vitrification. The objective of this study is to examine in vivo development of vitrified blastocysts obtained by nuclear transfer. For the nuclear transfer abattoir-derived oocytes and ovum-pickup-derived cumulus cells were used as recipient and donor cells, respectively. After maturation culture in TCM-199 supplemented with 5% calf serum (CS) for 20 h, oocytes with the first polar body were enucleated. Cumulus cells were cultured in Dulbecco’s MEM supplemented with 10% fetal calf serum for 3 to 4 passages. After electrical fusion (a single pulse of 25V for 50µsec) and activating treatment (exposure to 5 µM calcium ionophore for 5 min followed by incubation in TCM-199 supplemented with 5% CS and 10 µg/ml cycloheximide for 5 hours), reconstructed oocytes were cultured in vitro (IVC) in CR1-medium supplemented with 5% CS for 7-8 days, and then blastocysts were vitrified. Vitrification of blastocysts was carried out as reported previously (N. Saito et al. 1988). The vitrification solution (GESX solution) contains 20% glycerol (GL) + 20% ethyleneglycol (EG) + 0.3M sucrose (Suc) + 0.3M xylose (Xyl)+ 3% polyethyleneglycol (PEG). The blastocysts were equilibrated in 3 steps, (1) 10% GL + 0.1M Suc + 0.1M Xyl + 1% PEG for 5 min, (2) 10% GL + 10% EG + 0.2M Suc + 0.2M Xyl + 2% PEG for 5 min and (3) GESX solution. Equilibrated embryos were loaded to 0.25 ml straw and plunged into liquid nitrogen 1 min after transfer to GESX. At the day of transfer, the vitrified blastocysts were warmed and diluted in 0.5M and 0.25M sucrose for 5 min each. One or 2 vitrified (IVC-VIT group) or non-vitrified (IVC group) reconstructed blastocysts per head were non-surgically transferred into non-lactating Holstein cows. The pregnancy rate at 40 days after the last estrus in IVC-VIT and IVC group was 30.8% (8/26) and 35.7% (10/28), respectively, with no significant difference. Among these pregnant cows, 2 and 5 (IVC-VIT and IVC, respectively) were aborted during mid-term of gestation (80-181 days). One and 2 (IVC-VIT and IVC, respectively) completed their gestation terms, and 1 calf from each group is still viable (as of August, 2001). The other calf from IVC group was dead 8 hours after delivered by Caesarian section. Five and 3 (IVC-VIT and IVC, respectively) are pregnant at present. These results indicate that our vitrification method was efficient for cryopreserving the reconstructed blastocysts obtained by somatic cell cloning and that a viable offspring can be obtained after the cryopreservation. Reference 1. Saito, N., and K. Imai (1998) Cryobiol Cryotech, 43:34-39. 16-13 HISTOPATHOLOGICAL OBSERVATIONS OF THE PLACENTAS OF CALVES CLONED FROM SOMATIC CELLS Masumi Sato1, Chikara Kubota2, Shogo Tanaka1, Takeshi Onitsuka3 1Kyushu Research Station, National Institute of Animal Health, 2702 Chuzan Kagoshima, Kagoshima 891-0105 Japan; 2Kagoshima Prefectural Cattle Breeding Development Institute, Kagoshima, Japan; 3Kagoshima Prefectural Kagoshima Central Livestock Hygiene Service Center, Kagoshima, Japan Since 1998, many cloned calves have been produced by nuclear transfer from somatic cells in Japan. In most cases, they showed a heavier birth weight, an increased gestation length and increased rates of abortion/stillbirth. In Kagoshima Prefectural Cattle Breeding Development Institute, 26 calves cloned from somatic cells developed until parturition (1). Thirteen of them, which were derived from abortion/stillbirth or neonatal death, were examined histopathologically. Four calves died of infection with viruses that were causative agents of

abortion/stillbirth. The others died of dystocia caused by a heavy birth weight and severe hemorrhage of the umbilical vessels. Increased gestation length was observed in most cases. Enlargement of the umbilical vessels (2) was also seen in most cases. In this study, to determine the cause of the increased gestation length, we examined the placentas and pituitary of the cloned calves histopathologically. Materials and methods: Procedure of the nuclear transfer was described previously (1). Placentomes of 4 abortion/stillbirth (A/S) and 8 live (including 2 neonatal death) calves were surgically collected at the caesarian sections or collected immediately after the vaginal delivery. Pituitaries of 7 cloned calves derived from either A/S or ND were collected at autopsy. Formalin-fixed, paraffin-embedded tissues from the organs were microscopically examined. In the pituitary anti-adrenocorticotropic hormone (ACTH) antiserum was used for the immunohistochemical detection of ACTH, which is an inducer of parturition, and the number of the ACTH-positive cells were counted using light microscopy. The placentomes were observed with a transmissible electron microscope. Three calves produced by artificial insemination (AI) were sacrificed as controls. Results: In the placentomes of all cloned calves, tight epithelochorial attachments and abnormal structures, characterized by irregular invagination of the epithelochorion and proliferation of the chorial cells, were observed in calves after the full term of gestation, whereas they were not observed in AI calves. Ultrastructurally, irregular length and width of the microvilli were prominently observed between the trophoblasts and the uterine epithelial (u.e.) cells. Tight junctions were sometimes recognized at the microvillous connection. Intact u.e. cells with scarce cytoplasmic organelles connected with trophoblasts in cloned calves, whereas the u.e. cells degenerated before the separation from the trophoblasts in AI calves. In the pituitary anterior lobe, the number of ACTH-positive cells in AI calves was 2-fold increased compared to the cloned calves. Conclusion: Tight epithelochorial attachments in the placentomes and decreased numbers of ACTH-positive cells in the pituitary anterior lobe were observed in this study. The lesions, may have caused the increased gestation length of the calves cloned from somatic cells. References 1 Kubota, C., Yamakuchi, H., Todoroki, J., Mizoshita, K., Tabara, N., Barber, M., and Yang, X (2000)

PNAS 97: 990-995 2 Hill, J.R., Roussel, JB., Edwards, J.F., Hooper, N.L., Miller, M.W., Thompson, J.A., Looney, C.R.,

Westhusin, M.E., Robl, J.M., and Stice,S.L. (1999) Theriogenology 51: 1451-1465 16-14 GROWTH CULTURE OF BOVINE OOCYTES FROM EARLY ANTRAL FOLLICLES IN COLLAGEN GELS -EFFECT OF HYPOXANTHINE IN SERUM-FREE MEDIUM- Shoichiro Senbon1 and Takashi Miyano2 1The Graduate School of Science and Technology and 2Faculty of Agriculture, Kobe University, 1-1 Rokkodai-cho Nada-ku, Kobe City, Hyogo 657-8501, Japan The successful in vitro growth of oocytes has important biotechnological implications through its potential to produce large quantities of oocytes for embryo transfer. Culture systems have been shown to support oocyte growth in mice but there has been little success in applying these methods to other species. In the present study, we compared three culture conditions for bovine growing oocytes from early antral follicles and examined the effect of hypoxanthine on oocyte growth (1). In Experiment 1, early antral follicles (0.5-0.7 mm) were collected, and oocyte-cumulus-granulosa complexes (OCGs) and oocyte-cumulus-complexes (OCs) were dissected from the follicles. Follicles (F), OCGs and OCs were embedded in collagen gels (2)

and cultured in TCM199 containing 10% fetal calf serum, 2.2 mg/ml NaHCO3, 0.08 mg/ml kanamycin and 0.1 mg/ml sodium pyruvate for 16 days. In addition, 4 mM hypoxanthine (Hyp) was supplemented to the medium. After culture, gels and follicles were torn with fine forceps and oocytes were recovered. In F, OCGs and OCs cultured in Hyp-free medium, 21% (17/80), 9% (8/88) and 4% (4/92) of oocytes showed normal morphology, respectively, and Hyp increased the percentages in all groups (F: 37% (19/52), OCGs: 29% (15/51) and OCs: 10% (5/52)). In Experiment 2, early antral follicles were embedded in collagen gels and cultured in serum-supplemented or serum-free medium. Fetal calf serum was substituted by 3 mg/ml BSA in serum-free medium. Oocytes showed normal morphology at 17% (6/35) and 54% (19/35) in serum-supplemented and serum-free medium without Hyp, respectively. Hyp increased morphologically normal oocytes to 24% (16/67) and 91% (61/67), respectively. The mean diameters of oocytes in all groups after culture were significantly larger (103-118µm) than before culture (approximately 95µm). When oocytes were recovered from serum-free medium containing Hyp, 87% (53/61) of the oocytes showing normal morphology was at the germinal vesicle stage. After subsequent maturation culture for 24 hr, 97% (34/35) of oocytes underwent germinal vesicle breakdown and 26% (9/35) reached metaphase II. These results demonstrate that bovine growing oocytes from early antral follicles grow efficiently in serum-free hypoxanthine-supplemented follicle culture system and acquire the meiotic competence. References 1 Harada, M., Miyano, T., Matsumura, K., Osaki, S., Miyake, M., and Kato, S. (1997) Theriogenology 48,

743-755. 2 Torrance, C., Telfer, E. and Gosden, R. G. (1989) J Reprod Fert 87, 367-374. 16-15 MITOCHONDRIAL ACTIVITY IN RESPONSE TO SERUM STARVATION IN BOVINE AND MURINE CELLS Kumiko Takeda1, Satoshi Akagi1, Seiya Takahashi1, Akira Onishi2, Hirofumi Hanada1, and Carl A. Pinkert3 1Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Ikenodai 2, Kukizaki, Inashiki-gun, Ibaraki 305-0901, Japan; 2Developmental Biology Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan; 3Department of Pathology and Laboratory Medicine, Center for Aging and Developmental Biology, University of Rochester, NY, 14642-8645, USA In nuclear transfer procedures, in addition to nuclei, donor cell mitochondria are routinely transferred into recipient oocytes and mitochondrial heteroplasmy has been reported. However, various protocols have resulted in either homoplasmy for recipient mitochondria or varying heteroplasmic levels in cloned animals. Recently, it was demonstrated that microinjection of small numbers of granulosa cell mitochondria into mouse oocytes could prevent oocytes from undergoing apoptosis. Therefore, survival rates of nuclear transferred embryos may reflect mitochondrial viability. In nuclear transfer protocols, donor cells are subjected to serum-starvation prior to electroporation. Therefore, the relationship between culture conditions and mitochondrial activity was explored. Fibroblast cell lines were propagated from murine skeletal muscle (1), or bovine ear epithelium (2), cumulus cells (3) or skeletal muscle (4). Each cell line was cultured in DMEM supplemented with 10% FCS and mitochondria function was assessed during proliferation (A), at confluency (B), and after serum starvation (DMEM + 0.5% FCS) (C) for 6 to 16 days. Cells were stained with 200 nM MitoTracker Red CMXRos

(Molecular Probes) for 20 min, washed twice in PBS and assessed using a fluorescence multi-well plate reader (PE Biosystems) at ex = 590/20 and em = 645/40. We normalized fluorescence intensity to proliferative conditions (A = 100). Comparative fluorescent intensities were: Cell line 1; B = 61, C = 76 (culture for 16 days): cell line 2; B = 37, C = 41 (culture for 16 days): cell line 3; B = 9, C = 11 (culture for 9 days): cell line 4; B = 49, C = 36 (culture for 6 days). The mitochondrial activity per cell was highest under proliferation, significantly lower at confluency (P<0.01), and remained depressed after serum starvation (P>0.10). While serum starvation did not adversely affect mitochondrial activity in these representative cell lines, these results demonstrate that mitochondrial viability is dramatically affected by cell culture conditions. Consequently, specific cell culture parameters provide one explanation for the varying incidence of heteroplasmy identified in cloned animals. 16-16 QUANTITATIVE ANALYSIS OF TELOMERASE AND TELOMERASE REVERSE TRANSCRIPTASE mRNA EXPRESSION IN SINGLE BOVINE EMBRYO DERIVED FROM SOMATIC NUCLEAR TRANSFER AND IN VITRO FERTILIZATION Junko Umakoshi1, Chikara Kubota2, Junichi Todoroki2, and Mitsutoshi Yoshida1 1Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan; 2Kagoshima Prefectural Cattle Breeding Development Institute, Kagoshima 899-8212, Japan Telomerase (Telo) and telomerase reverse transcriptase (TeloRT) identified as the catalytic subunit of telomerase are mainly expressed in germ line stem cells and cancer cells and prevent telomeric shortening. Recently several studies indicated that the telomere length of cloned calves resulting from nuclear transfer of fibroblast-cell was restored. Thus, the reactivation of telomerase component may represent an important step for unlimited proliferation and indefinite survival of cells. However, no detailed quantitative studies on the expression of each telomerase component gene in bovine ova. This study was carried out to examine quantitatively Telo and Telo-RT mRNA expression in bovine ova during in vitro maturation (IVM), and after in vitro fertilization (IVF) and somatic nuclear transfer (NT). Immature oocytes were collected from ovaries of slaughtered cows. The methods for IVM, IVF and NT of bovine ova were largely based on those described previously (Kubota et al., 1998, 2000). Total RNA were isolated from a single oocyte or embryo during IVM (6 hrs interval) and after IVF and NT (3-24 hrs intervals). Quantitative reverse transcription-polymerase chain reaction was carried out with SYBR® Green RT-PCR Kit, specific primers and ABI PRISM® 7700 Sequence Detection System. As an endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was also examined at the same time. Expression of Telo and Telo-RT mRNAs was detectable at all stages of IVM, IVF and NT. During IVM Telo-RT gene expression was increased in oocytes. Following IVF and NT, Telo and Telo-RT mRNA expression drastically increased around the first cleavage. 16-17 GREATER POST-NATAL VIABILITY WITH G0 COMPARED TO G1 DONOR CELLS FOLLOWING SOMATIC CELL NUCLEAR TRANSFER IN CATTLE David Wells1, Andria Miller1, Jan Oliver1, Fleur Tucker1, Jacqui Forsyth1, Martin Berg1, Katie Cockrem1, Björn Oback1, and Robin Tervit1

1AgResearch Ltd., Ruakura Research Centre, PB 3123, Hamilton, New Zealand The importance of cell cycle stage on the ability of differentiated donor cells to be reprogrammed to produce embryos and viable cloned offspring following somatic cell nuclear transfer remains unresolved. Originally, it had been claimed that the use of serum-starved quiescent (G0) cells was a major factor contributing to the first successful births of cloned mammals from cultured cells1. This is questioned by data where randomly picked donor cells from non-serum-starved cultures were also totipotent following nuclear transfer in cattle2 and mice3. However, these studies did not demonstrate which stage or stages of the cell cycle resulted in cloned offspring. Here, we directly compare the developmental potential following nuclear transfer of G0 somatic cells with those selected specifically in G1 of the cell cycle.

In vitro matured bovine MII oocytes were enucleated and reconstructed with adult male skin fibroblasts after 3-4 passages in culture. Two donor cell cycle treatments were investigated. G0 cells were obtained following serum deprivation by culture in medium containing 0.5% FCS for 5 days1. G1 cells were obtained by firstly picking individual mitotic cells with a micro-pipette and allowing them to cleave in medium containing 10% FCS. The resulting cell doublets were then physically separated and single cells fused to cytoplasts within 1-3 hours after mitosis. This timing was prior to S-phase based on negative BrdU incorporation in control cells. Furthermore, the selected G1 population was indeed cycling, as BrdU labeling at 12 h post-mitosis demonstrated progression into S-phase. Following electrical cell fusion, donor nuclei in both cell cycle treatments were exposed to oocyte cytoplasm for 3-6 h before artificial activation involving a combination of ionomycin and 6-dimethylaminopurine. Reconstructed embryos were cultured in vitro and on Day 7, suitable quality blastocysts were transferred singularly to recipient cows and in vivo development monitored regularly.

Compared to G0 cells, the smaller diameter G1 donor cells resulted in lower fusion rates (72.9% vs 58.1%, respectively). There was no effect of G0 or G1 cell cycle stage on subsequent embryonic development to grade 1-3 blastocysts (48.9% vs 50.0%, respectively). Following embryo transfer, the survival of cloned embryos derived from G0 donor cells tended to be greater through the latter part of gestation (figure 1). This difference was particularly evident in relation to live cloned calves following parturition. With G1 cells, 28% of embryos transferred (7/25) resulted in the birth of cloned calves at full term. However, four of these calves died at or within 2 hours of birth, with another two dying within three weeks resulting in an overall efficiency of only 4%. In contrast, G0 donor cells resulted in 39% development to term (7/18), with one calf dying during birth and another at four weeks, culminating in an overall 28% embryonic viability.

In conclusion, a direct comparison between G0 and G1 somatic cells shows no effect in terms of development to blastocyst and Day 35 embryo viability, but suggests longer term reprogramming defects with the G1 cells that are not evident until late gestation and the post-natal period.

Figure 1. Survival of cloned embryos derived from either G0 or G1 adult male skin fibroblasts.

stage of development

D7 D35 D60 D90 D120 D150 D180 D210 D240 Term 2 hrs 1 mnth 8 mnths

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References 1 Campbell, K.H.S., McWhir, J., Ritchie, W.A., and Wilmut, I. (1996) Nature 380:64-66 2 Cibelli, J.B., et al. (1998) Science 280:1256-1258 3 Wakayama, T., and Yanagimachi, R. (1999) Nature Genet. 22:127-128 16-18 ASSAY OF HYALURONIDASE IN FOLLICULAR FLUID Shao-Ping Weng1, Martin Luck2, Cheng-Hsuan Wu1, Ta-Chin Lin1, and Tsung-Cheng Kuo1

1Department of Obstetric and Gynecology, Kuo General Hospital, Tainan, Taiwan; 2Division of Animal Physiology, School of Biosciences, The University of Nottingham, UK Hyaluronidase (Hase) has provoked a great deal of interest because the desire to understand Hyaluronic Acid (HA), which is actively involved in ovulatory processes such as cumulus cell expansion, angiogenesis, and inflammatory reactions. Due to lack of easy examining techniques, Hase remains mysteries to be discovered. The purpose of this project is trying to set up a quantitative assay of Hase. The design of this assay is based on an ELISA-like method developed by Stern and Stern. The first step is biotinylation of Hyaluronic acid, followed by immobilization of bHA to the Covalink plate. Secondly, degrading HA by hyaluronidase. At the end, adding color reagent to the residual avidin-combined bHA and then reading the color density by using a 492 nm filter in an ELISA plate reader. Through many variables crossover reactions, a new developed buffer solution was used to achieve expected reactions. Then, the protocol could work for assaying Hase. This rapid and sensitive assay method seems to be an easier approach to the understanding of Hase. However, early assays for hyaluronidase activity, such as the colorimetric assay of Reissig, still own their positions because of various subtypes of Hase. References 1 Frost, G.I., and Stern, R. (1997) Analy Biochem 251: 263-269. 2 Salustri, A., Camaioni, A., Di Giacomo, M., Fulop, C., and Hascall, V. C. (1999) Human Reprod update

5: 293-301. 16-19 PARTHENOGENETIC DEVELOPMENT IN VIVO AND IN VITRO OF PORCINE OOCYTES MATURED IN NCSU-37 OR M-199 Ryo Yamaguchi1, Asako Wada1, Emina Oka1, Masao Shino1, and Naomi Kashiwazaki1 1Laboratory of Animal Reproduction, Department of Animal Science and Biotechnology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229-8501, Japan The objective of the present study was to compare parthenogenetic development of porcine oocytes in vitro-matured in NCSU-37 [1] or M-199 (Gibco). Porcine cumulus-oocyte complexes were collected from slaughterhouse ovaries and matured in NCSU-37 modified with addition of 10% porcine follicular fluid (pFF) and with hormones (10 iu eCG/ml + 10 iu hCG/ml) during the first 20 h and without hormones during the second 28 h or M-199 supplemented with 0.1 mg/ml sodium pyruvate, pFF and hormones the same as above

NCSU-37. Oocytes with a first polar body were stimulated by 2 DC pulses of 1500 v/cm for 60 µsec. The electro-stimulated oocytes were subsequently cultured in NCSU-37 contained cytochalasin B (5.0 µg/ml: Sigma) for 4 h in order to obtain diploid oocytes. The treated oocytes were cultured in modified NCSU-37 with 1 mg BSA/ml (Fraction V, Sigma) for 144 h. Cultures were performed under humidified atmosphere of 5% CO2 in air at 38.5°C. Some of the oocytes treated with cytochalasin B were surgically transferred to oviducts of synchronized recipient females. Nuclear activation was measured by pronuclear formation at 10 h after electro-stimulation using 1% acetoorcein-staining. Development in vitro was determined by rate of blastocyst formation of the treated oocytes after culture. Parthenogenetic fetuses were surgically collected from uterine horns of the recipients at 28 days post electro-stimulation. As shown Table 1, there was no significantly difference in rates of maturation and nuclear activation between two maturation media. However, proportion of blastocyst formation of oocytes matured in NCSU-37 was significantly higher than that of oocytes matured in M-199 (P<0. 05). Ten and 18 parthenogenetic fetuses were recovered from each one recipient that had been received 200 oocytes matured in NCSU-37 and M-199, respectively. Although the half of the porcine parthenogenetic fetuses reached at the stage of limb-bud formation, some of the fetuses externally showed abnormalities of organ formation, cleft of palate, and ungrowing placenta. Table 1. Parthenogenetic development of porcine oocytes matured in vitro in NCSU-37 or M-199

IVM medium Maturation (%) Nuclear activation (%) Blastocyst formation (%)

NCSU-37 61.3 ± 1.8 67.8 ± 3.0 11.4 ± 1.2* M-199 65.5 ± 2.2 65.9 ± 5.6 7.7 ± 1.1*

*: Significant difference (P<0.05)

The results of the present study indicate that oocytes matured in vitro can develop in vitro to the blastocyst stage and in vivo to day-28 fetuses in the pig. This system for generating maternal parthenogenetic porcine embryos/fetuses will give advantages for study of genome imprinting in mammals. Reference 1. Petters R.M., and Wells K.D. (1993) J Reprod Fertil Suppl 48, 61-73. 16-20 FERTILIZATION AND NORMALITY OF RABBIT OOCYTE INJECTED WITH ROUND SPERM CELL NUCLEOUS Yoshihiko Hosoi1,2, Chikusa Suzuki1, Hiromi Kato2, Kazuya Matsumoto1,2, Kazuhiro Saeki1,2, and Akira Iritani1,2 1Department of Genetic Engineering, College of Biology-Oriented Science and Technology, 2Institution of Advanced Technology, Kinki University, Wakayama 649-6493, Japan Direct sperm injection into mature oocytes (ICSI) could be useful for determining the roles of various sperm components on fertilization and embryonic development. When intact spermatozoa were injected into oocytes, normal fertilization and development were confirmed in many species. In considering application of ICSI for endangered species, even immature spermatozoa are also expected to be used for reproduction in many cases. To determine whether immature spermatozoa can be fertilized normally, we injected rabbit round spermatid cell to

rabbit oocytes (ROSNI) and measured MPF activity in these oocytes. The medium used for culturing oocytes after ICSI or ROSNI was HTF medium supplemented with 10% FBS. New Zealand White (NZW) female rabbits (4-6 months old) were each injected with 75 IU PMSG followed by 75 IU hCG 72 hours later to induce superovulation. Oocytes were collected from oviducts between 14 and 15 hours after hCG injection. They were freed from cumulus cells by treatment with 0.2% hyaluronidase in HTF medium. The spermatozoa from caudae epididymides of NZW male rabbits (6 months old) were used as a control. The immature sperm cell were recovered from fragment of same testis in erythrocyte lysing buffer. Intact sperm or isolated sperm cell nucleus was injected into oocytes. For oocytes activation after ROSNI, 10 µM calcium ionophore A 23187 was used. After injection, oocytes were cultured in HTF under 5% CO2, 95% air, 37°C. For MPF assay, every 10 oocytes were lysed at 1,3,6,9,12 hours after ICSI and stored at -80°C. MPF activity was measured by MESACUP cdc2 kinase assay kit (MBL, Japan) In the first experiment, ICSI and ROSNI were done separately. After ROSNI, fertilized oocytes developed into pronucleus stage (13%) at 5hours and cleaved stage (23%) at 24hours after injection. They were lower than the rate of ICSI (83% and 34%, respectively). MPF activity was decreased 1hour and not detected 3hours after ICSI, however, in case of ROSNI, MPF activity was still maintained over 30% of the level in MII oocyte level at 9 hours after injection. In the second experiment, we examined whether oocyte activation enhances the cleavage rate of ROSNI. The rates of eggs having two pronuclei reached to 36% at 5 hours and 39% of injected oocytes proceeded cleavage stage at 24 hours after injection and some of them reached to blastocyst stage.

These results indicated that the decrease of MPF is important for rabbit normal fertilization and an additional activation of oocytes was effective for subsequent oocyte development after ROSNI. 16-21 FOLLICULAR DEVELOPMENT AND THE OOCYTE MATURATION IN BOVINE FETAL OVARIES BY XENOTRANSPLANTATION Misa Hosoe1, Junko Noguchi2, Kazuhiro Kikuchi2, Hiroyuki Kaneko2, and Tomoyuki Tokunaga1 1Developmental Biology and 2Genetic Diversity Departments, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan Thousands of primordial follicles are present in the ovaries of fetal and neonatal mammals, and also in the ovarian cortex of adult animals. Oocytes from these follicles have been expected as sources for the next generation. However, it is difficult for these oocytes to acquire the complete competence of undergoing maturation, fertilization and embryonic development. The oocytes from fetal or calf ovaries have less developmental competence than those from adult cow ovaries in an in vitro system (1). Recent reports demonstrate that the development of antral follicles was induced in immunodeficient mice after xenograft transplantation of ovaries from sheep, human, etc. (2), though the fertilizability of oocytes from these follicles remained uncertain. In the present study, we investigated whether primordial or secondary follicles from bovine fetal ovaries could produce oocytes with full maturation competence after xenotransplantation to nude mice and in vitro maturation.

Donor ovaries were collected from bovine fetuses (crown-rump length: 46 and 70 cm) in a local slaughterhouse and were transported to the laboratory in saline at 20°C. As recipient

animals, female athymic (nu/nu) mice were used to avoid immunological rejection of the donor tissues. The mice were bilaterally ovariectomized prior to transplantation. Five pieces of donor ovarian cortex (1 mm3) were gently inserted beneath the kidney capsule of the mice. Seven mice were grafted with ovarian tissues from the 46 cm fetus and 4 mice with the 70 cm fetus. Ten to 15 weeks after xenotransplantation, the grafts of the 46 cm fetus were recovered from two mice whose vaginal smears showed the presence of cornified epithelial cells, and were immediately fixed in Bouin solution for histological examination. The other 7 mice in which vaginal cornification occurred (3 of 5 mice with grafts from the 46 cm fetus and 4 of 4 mice with those from the 70 cm fetus) were injected with 10 IU PMSG for stimulating follicular development in the grafts and the grafts were recovered 72 h after PMSG treatment. The oocytes were isolated mechanically from follicles in the grafts and were cultured for 20 h in TCM 199 supplemented with 10% fetal bovine serum. Some oocytes were fixed immediately after maturation culture and stained for assessment of the nuclear stage, and the other oocytes inseminated and fixed at 14 h after insemination.

Histological examination revealed that the formation of antral follicles was noted in the recovered grafts derived from the 46 cm fetus after transplantation although ovarian tissues of the 46 cm fetus contained only primordial follicles before transplantation. Three cumulus-oocyte complexes (COCs) and 7 cumulus-free oocytes were isolated from the grafts of the 46 cm-fetal ovary, 20 COCs and 18 cumulus-free oocytes were isolated from the 70 cm-fetal ovary. When one COC from grafts, which were transplanted from the 46 cm fetus to a mouse, were cultured, it matured to the MII stage. However, when 7 oocytes without cumulus cells from the same grafts were cultured, two oocytes were at the germinal vesicle stage and the others degenerated. Furthermore, when two COCs from 2 mice, which received ovarian tissue from the 46 cm-fetal ovary, were cultured and inseminated, all (2/2) showed degeneration. On the other hand, when 20 COCs from 4 mice with ovarian tissue from the 70 cm fetus were cultured and inseminated, 6 of 20 oocytes (30.0%) were at the germinal vesicle stage, 3 of 20 (15.0%) underwent germinal vesicle breakdown, 7 of 20 oocytes (35.0%) were at the MII stage and 4 of 20 (20.0%) degenerated. No evidence of fertilization was observed in any of the inseminated oocytes.

The present results suggest that xenotransplantation is effective in promoting the growth of primordial follicles from bovine fetal ovaries, and provides maturational competence to the oocytes in the follicles. References 1. Betteridge, K.J., Smith, C., Stubbings, R.B., Xu, K.P., and King, W.A. (1989) J Reprod Fertil Suppl 38:

87-98. 2. Weissman, A., Gotlieb, L., Colgan, T., Jurisicova, A., Greenblatt, E.M., and Casper, R.F. (1999) Biol

Reprod 60:1462-1467. Poster Presentation 2 17-01 PRODUCTION OF CHIMERIC CHICKEN USING SOMATIC CELLS DERIVED FROM HENS Shizuka Aritomi1, and Noboru Fujihara1 1Animal Resource Science Section, Division of Bioresource and Bioenvironmental Sciences, Graduate School Kyushu University, Fukuoka 812-8581, Japan

In the previous studies, we succeeded in the production of chimeric chickens by transferring blastodermal cells (1). In this experiment, we tried to develop techniques for producing somatic- and germline chimeras by the transfer of cultured cells which were produced by electrofusion of the somatic cells from adult hens with blastodermal cells. The cultured somatic cells obtained from White Leghorn hens were used as donors and unincubated fertile Rhode Island Red eggs (stage X embryos) were used as recipients. The cultured cells and Rhode Island Red’s blastodermal cells were fused with electroporation in the two-phase polymer systems (2). Immediately after the electrofusion, the cell clusters and debris were washed with medium. Unincubated fertile Rhode Island Red eggs (stage X) were employed as recipient eggs after kept at 4°C for 24-48 hours. A window was opened on the blunt edge of the egg just above the blastoderm. A capillary filled with 3-5 µl of fusing cell suspension was injected into the subgerminal cavity of recipient egg. After the injection, the window was closed with adhesivetape and the eggs were set in an incubator maintained at 37°C with 50% relative humidity, turning through 90°/h for 19 days, and transferred to a hatcher at 37°C and 85% relative humidity at 20 days of incubation until hatched. From 21-day old embryos (dead chick), some tissues (brain, heart, liver, gonad, skin, muscle, intestin) were removed to extract DNA and analyzed by PCR method for confirming the existence of donor cell specific DNA. In the embryos treated with electroporation, only one embryo showed donor derived feather pigmentation. By PCR analysis, donor-chromosome-specific DNA was detected in the skin and muscle. This finding suggests the possibility of producing somatic-chimera by transferring cultured cells fused with electroporation. References 1. Aritomi, S., and Fujihara, N. (2000) Asian J Androl 2 (4): 271-275 2. Hui, S. W., Stoicheva, N., and Zhao, Y. L. (1996) Biophysical J 71: 1123-1130 17-02 EFFECT OF ACTIVATION TREATMENTS ON BLASTOCYST DEVELOPMENT IN BOVINE NUCLEAR TRANSFER BY INTRACYTOPLASMIC INJECTION (ICI) Masatsugu Asada1, Sachiko Ikumi1, and Yutaka Fukui1

1Laboratory of Animal Genetics and Reproduction, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan

It has already been obtained normal fetuses in mice and pigs that a donor nucleus from somatic cells was directly injected into the enucleated oocytes by piezo-micro manipulator. Chemical agents or electrical pulse has been attempted as activating treatments. In the bovine somatic cell cloning, a generic way of electrically stimulation has been attempted mostly for the activation of the nuclear injected oocytes. However, the electrically stimulation was required an expensive equipment. In this study, we investigated the effect to the developmental capacity of nuclear injected oocytes by chemically treatment as a simple activation method after ICI. In Experiment 1, for the reprogramming of donor nuclear must be exposed to constant time in the cytoplast. Therefore, bovine oocytes after ICI with the bovine cumulus cell nuclear were examined the time to the post-activation (0, 1, 3 and 6 h) with 7% ethanol. Experiment 2, it is well known that the calcium oscillation pattern is different with the various chemically activation methods. Ethanol treatment for donor nuclear injected oocytes may be the easiest way among the chemically activation treatments. Therefore, the efficiently to the developmental capacity of nuclear injected oocytes was investigated with 7% ethanol or 5 µM Ca ionophore A23187. The

blastocyst rate was not affected by 0, 1, 3 and 6 h of post-activation times. The present results showed that the chemically activations did not result in significant difference between ethanol and Ca ionophore activations. It is conluded that the time to the post-activation of donor nuclear tendeds to be a high blastocyst rate in the groups of 0 and 1 h, and ethanol treatment is a efficient method for activation following ICI. Reference 1 Wakayama, T., Perry, A.C.F., Zuccotti, M., Johnson, K.R., and Yanagimachi, R. (1998) Nature 394:

369-374. 17-03 FETAL AND PLACENTAL LESIONS AND CLINICAL OBSERVATIONS ON SOMATIC CLONES Pascale Chavatte-Palmer1,2, Christophe Richard2, Yvan Heyman2, Farah Kesri2, Michel Guillomot2, Nathalie Cordonnier3, Le Bourhis Daniel4 and Jean-Paul Renard2 1INRA, Biologie du Développement et Biotechnologie, Domaine de Vilvert, 78352 Jouy en Josas cedex, France; 2INA P-G, Dept. des Sciences Animales, GER Génétique, Elevage et Reproduction, 16 rue Claude Bernard, 75231 Paris cedex 05, France; 3Laboratoire d'Anatomo-Pathologie, Unité Biologie de la Reproduction, Ecole Nationale Vétérinaire d'Alfort, 7 avenue du Général-de-Gaulle, 94704 Maisons-Alfort cedex, France; 4UNCEIA, Technical services , 94 703 Maisons-Alfort, France Although healthy animals may be born after nuclear transfer procedures using somatic cells nuclei, the success of this procedure is generally poor (2 to 10%) due to gestational and neonatal losses. In our laboratory, late gestation losses account for about 50% of the gestations and around 20% of the newborn calves do not survive.

The gross pathology and histological examination of placenta and organs in 5 abnormal fetuses show that kidneys and liver are heavier than liver from normal controls, in respect to bodyweight. Steatosis and a decrease in hematopoïetic centers in the liver have also been noted in some individuals. Despite the important hydrops in all pathological pregnancies, little inflammatory tissue could be demonstrated although edema was present in the placenta.

In 11 surviving cloned calves, no abnormal white blood cell profiles could be demonstrated, in contrast to one animal that died from thymic aplasia (1). Recently, hypotrophic thymuses were found in two neonatal calves at post-mortem, awaiting histological analyses. Hematological analyses at birth show that Mean Corpuscular Volume is significantly larger in clones compared to controls (P<0.05), suggesting that the clones hematological system may not be as mature as normal calves. These data suggest that lesions unrelated to genetic imprinting perturbations may be present in clones. Further hormonal assays are currently being completed on these individuals and will be presented in the poster. Reference 1 Renard, J. P., Chastant, S., Chesné, P., Richard, C., Marchal, J., Cordonnier, N., Chavatte, P., and Vignon,

X. (1999). The Lancet 353, 1489-1491. 17-04

THE EFFECTS OF 6-DIMETHYLAMINOPURINE, CYTOCHALASIN B AND CYCLOHEXIMIDE ON THE PARTHENOGENETIC DEVELOPMENT OF ELECTRICALLY ACTIVATED IVM PORCINE OOCYTES

Christopher G. Grupen1, Stephen M. McIlfatrick1, Simon Maddocks2, and Mark B. Nottle1 1Reproductive Biotechnology Division, BresaGen Limited, Adelaide, South Australia, Australia; 2Department of Animal Sciences, Adelaide University, Adelaide, South Australia, Australia A number of treatments have been reported to increase the developmental potential of artificially activated oocytes (1, 2). The aim of the present study was to determine the effects of 6-dimethylaminopurine (6-DMAP), cytochalasin B (CB) and cycloheximide (CHX) on the parthenogenetic development of electrically activated in vitro matured (IVM) porcine oocytes. Following IVM, oocytes were denuded and those with a polar body were electrically activated. Pulsed oocytes were incubated for 3 h in culture medium containing: 1) no supplement (untreated control); 2) 2 mM 6-DMAP; 3) 7.5 µg/ml CB; 4) 10 µg/ml CHX; or 5) 2 mM 6-DMAP and 7.5 µg/ml CB. The treated oocytes were then cultured in vitro (IVC). Some oocytes from each group were stained with orcein after 16 h IVC to examine nuclear structures. Parthenogenetic cleavage and development to the blastocyst stage were assessed for the remaining oocytes after 48 h, 6 d and 7 d IVC. Blastocysts that had formed after 7 d IVC were differentially labelled and the numbers of trophectoderm and inner cell mass cells were counted. Oocytes that received electrical pulses alone displayed high rates of meiotic resumption (98%) and pronuclear formation (97%). Subsequent incubation with 6-DMAP, CB, CHX, or 6-DMAP and CB combined did not affect the rates of meiotic resumption and pronuclear formation. Exposure to 6-DMAP and CB, either alone or in combination, increased the proportion of oocytes that had a single polar body compared with untreated and CHX-treated oocytes (92-98% vs 67%; P < 0.05). The incidence of cleavage to the 2-cell stage did not differ significantly between the treatments. The incidence of blastocyst formation in oocytes exposed to 6-DMAP and CB combined was higher after 6 and 7 d IVC compared with the other groups (39% vs 10-25% and 47% vs 17-32%, respectively; P < 0.05). Oocytes incubated with 6-DMAP alone displayed a higher rate of blastocyst formation after 6 d IVC compared with untreated oocytes (25% vs 11%; P < 0.05) and oocytes incubated with either CB (25% vs 13%; P < 0.05) or CHX (25% vs 10%; P < 0.05) alone. Oocytes incubated with CB alone also formed blastocysts at a lower rate after 7 d IVC compared with those exposed to 6-DMAP alone (17% vs 32%; P < 0.05). Incubation with 6-DMAP alone generated blastocysts that had a greater number of trophectoderm and total cells compared with the combined 6-DMAP and CB treatment (48.8 ± 4.6 vs 38.1 ± 3.0 and 55.5 ± 5.2 vs 43.2 ± 3.4, respectively; P < 0.05). The number of inner cell mass cells and the proportion of total cells allocated to the inner cell mass did not differ significantly between the treatments. Our results demonstrate that incubation with 6-DMAP, either alone or in combination with CB, increased the developmental potential of electrically activated IVM porcine oocytes. This effect was correlated with an increased incidence of diploid parthenote development. Blastocyst cell number data suggest that exposure to CB was detrimental to parthenote development. References 1 Cha, S.K., Kim, N.-H., Lee, S.M., Baik, C.S., Lee, H.T., and Chung, K.S. (1997) Reprod. Fertil. Dev. 9,

441-446. 2 Liu, L., Ju, J.-C., and Yang, X. (1998) Mol. Reprod. Dev. 49, 298-307. 17-05

BOVINE IMMATURE OOCYTES ACQUIRED DEVELOPMENTAL COMPETENCE DURING MEIOTIC ARREST IN VITRO Shu Hashimoto1, Naojiro Minami2, Ryo Takakura1, and Hiroshi Imai2 1Embryo Transplantation Laboratory, Snow Brand Milk Products Co. Ltd., 119 Uenae, Tomakomai, Hokkaido 059-1365; 2Laboratory of Reproductive Physiology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan To improve the developmental competence of bovine immature oocytes, various approaches have been attempted. Especially, holding oocytes at germinal vesicle stage before in vitro maturation has been examined because oocytes should be required the time to acquire greater developmental competence during meiotic arrest. This idea is based on the facts; mammalian oocytes are arrested at diplotene stage until Gn-RH surge occurs for the resumption of meiotic maturation. On the other hand, oocytes aspirated from follicles resume meiotic maturation spontaneously. Furthermore, the developmental competence of oocytes recovered from large follicles was higher than that of oocytes recovered from small follicles. From these observations, the developmental competence of oocytes is suggested to be acquired progressively during follicular growth.

In the present study, effects of addition of bovine serum albumin (BSA) or fetal bovine serum (FBS) to the media and of oxygen tension (5% vs. 20%) during meiotic arrest by 100-µM butyrolactone I (BL I) on the developmental competence of bovine oocytes were investigated. The proportion of oocytes developed to the blastocyst stage arrested by BL I in FBS supplemented medium under 5% O2 (37%) was higher (P<0.05) than that of oocytes arrested under other conditions (5-24%) or non-arrested oocytes (23%). Furthermore, time course of nuclear maturation of BL I treated oocytes was examined. The results demonstrated that oocytes treated with BL I commence the resumption of meiotic maturation and reach MII stage 5.5 h earlier than non-arrested oocytes in average. From the results, the developmental competence to the blastocyst stage of GV-arrested oocytes matured for 15.5 or 21 h was compared with that of non-arrested oocytes matured for 21 h or 26.5 h. The developmental rate to the blastocyst stage of BL I treated oocytes matured for 15.5 h or 21 h was higher (P<0.05) than that of non-treated oocytes matured for 21 h or 26.5 h.

Results of the present study demonstrated that bovine immature oocytes arrested by BL I in vitro acquire the developmental competence to the blastocyst stage during the meiotic arrest. 17-06 NUCLEAR TRANSPLANTATION USING RECIPIENT OOCYTES AND DONOR CELLS OBTAINED FROM THE SAME INDIVIDUAL COW Kanako Kaneyama1, Syuji Kobayashi2, Satoko Matoba1,Yutaka Hashiyada3, Miharu Yonai1, and Norio Saito1

1National Livestock Breeding Center (NLBC), Nishigo, Fukushima, 961-8511, Japan; 2Niikappu Station, NLBC, Shizunai, Hokkaido, 056-0141, Japan; 3Oou Station, NLBC, Shicinohe, Aomori, 039-2567, Japan A lot of cloned animals have been produced by somatic cell nuclear transplantation since 1998. In those cases, however, recipient oocytes and donor cells were not obtained from the same animals. Therefore each reconstructed oocyte has a different source of cytoplasm with different mitochondria DNA, so that this difference in mitochondria DNA in cytoplasm may affect the performance of cloned animals obtained from the same donor nucleus but with oocytes from

different animals. The objective of this study was to examine effects of using the same cow as a source of recipient oocytes and donor cells to produce cloned cattle in terms of cloning as a strict definition of a copy of a self. Forty-two cumulus oocyte complexes were aspirated from a Holstein cow using ovum pick-up and incubated in TCM199 supplemented with 5% calf serum (CS) for 20 h for in vitro-maturation. Thirty-one maturated oocytes were stripped of cumulus cells, and enucleated. The cumulus cells that were retrieved from the same cow were incubated in Dulbecco's MEM supplemented with 10% fetal calf serum for 3 to 4 passages. Twenty-nine oocytes were successfully enucleated, and a cultured single cumulus cell was introduced into their perivitelline space, and then they were electrically fused using a single pulse of 25V for 50µsec in Zimmerman medium. After the electrical stimulation, reconstructed oocytes were exposed to 5µM calcium ionophore for 5min for a further activation, and incubated in TCM199 + 5% CS + 10µg/ml cycloheximide for 5 h. Then, 20 fused oocytes were cultured in CR1aa medium supplemented with 5% CS. Out of these 18 cleaved, and 12 developed to the blastocyst stage at day 7 or 8 (day 0 = day of nuclear transfer). The fusion rate, cleavage rate, and blastocyst rate of nuclear transplanted oocytes were 69.0% (20/29), 90.0% (18/20) and 60% (12/20), respectively. The morphologically normal blastocysts were transferred to 9 Holstein cows on day 8. Pregnancies were diagnosed 40 and 80 days after the last estrus with ultrasonography. The pregnancy rate was 33.3% (3/9) at 40 and 80 days after the estrus, and 2 pregnant cows were aborted at 144 and 176 days of gestation. One pregnant cow delivered a calf of 70 kg body weight by Caesarian section, and the calf was dead 15 h after the delivery. These results indicated that recipient oocytes and donor cells from the same cows can be used for nuclear transplantation resulting in the birth of a cloned calf, but the cause of the large offspring syndrome observed in the calf was not identified. Reference 1 Goto, Y., Kaneyama, K., Imai, K., Shin-noh, M., Tsujino, T., Nakano, T., Matsuda, S., Nakane, M., and

Kojima, T. (1999) Anim Sci J 70:243-245. 17-07 EVALUATION OF SOME CHEMICAL STIMULI FOR INDUCTION OF ACTIVATION IN PORCINE OOCYTES MATURED IN VITRO Manabu Kawahara1, Kohei Fuchinoue1, Takuya Wakai1, Kohichi Kimura1, Hiroshi Sasada1, and Eimei Sato1 1Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan During the process of production of cloned embryos, activation of reconstituted embryos is crucial for their subsequent development. The present study was conducted to demonstrate the effect of some chemical stimuli on activation of in vitro matured oocytes in pigs by examining the rate of pronuclear fomation, cleavage and cell number of blastocysts obtained. Porcine oocytes that were collected from ovaries were cultured in vitro in NCSU23 and subjected to the experiments. In Experiment 1, the treatment of either ionomycin or electric stimulus induced pronuclear formation at a rate of 22/32(68.8%) or 18/28(64.3%) of matured oocytes, respectively, whereas no pronuclear formation was observed in those treated with cycloheximide. In Experiment 2, the combination of two or three treatments, in which ionomycin was followed by electric stimulation and/or cycloheximide, showed 78.9 and 81.8 % cleavage, and 31.1 and 53.0 % blastocyst formation, respectively, compared to the corresponding rates of 45.3 and 62.9 %, and 20.8 and 13.9 % in single treatment with

ionomycin and with electric stimulation, respectively. The mean cell number of blastocysts obtained in groups after the combined treatments was higher than that in groups after a single treatment. In conclusion, our results showed that the combined treatment of ionomycin with electrical stimulation and cycloheximide may be an effective tool for the activation of reconstituted embryos in pigs. 17-08 DEVELOPMENT OF PORCINE EMBRYOS RECONSTRUCTED WITH FETAL FIBROBLASTS TRANSFECTED WITH GREEN FLUORESCENT PROTEIN GENE Dae-young Kim1, So-hyun Lee1, Byeong-chul Oh1, Hye-soo Kim1, Gab-sang Lee1, Sang-hwan Hyun1, Jong-im Park1, Eun-song Lee2, Jeong-mook Lim3, Sung-keun Kang1, Byeong-chun Lee1, and Woo-suk Hwang1

1College of Veterinary Medicine, Seoul National University, Seoul, 151-742, Korea; 2Department of Veterinary Medicine, Kangwon National University, Chunchon, 200-701, Korea; 3School of Agricultural Biotechnology, Seoul National University, Suwon, 441-744, Korea Production of transgenic pig using somatic cell nuclear transfer technique has a tremendous value in the field of biotechnology and medicine (1). The objective of this study was to evaluate whether transfection of somatic cells with foreign gene affected the development of reconstructed embryos to the blastocyst stage or not. Green fluorescent protein (GFP) gene was used as a foreign gene and non-transfected fetal fibroblast was utilized as a positive control of donor cell. Monolayers of porcine fetal fibroblasts were established by our standard procedure and subsequently transfected with pEGFP-N1TM (Clontech, USA) by LipofectAmine PlusTM (Life Technologies, USA). Oocytes matured in culture for 44-46 h were provided as recipient oocytes. Reconstructions of oocytes with either transfected or non-transfected fetal fibroblasts were achieved by electric fusion and activation using a single DC pulse of 1.8 kV/cm for 30 µs in Ca2+ and Mg2+-containing 0.26 M mannitol solution. Reconstructed oocytes were subsequently cultured in NCSU-23 medium for 144 h for the evaluation of development. The rates of cleavage, blastocyst formation and number of cells in blastocysts did not differ significantly (P>0.05) between oocytes reconstructed with transfected and non-transfected cells (Table 1). Table 1. evelopment of porcine nuclear transfer embryos

These results indicate that no significant decrease in embryo development was found in

porcine oocytes reconstructed with GFP-transfected fetal fibroblast and that GFP gene could be safely used as a marker of foreign gene in porcine transgenesis. We are currently undertaking a series of experiments to examine fetal development of the reconstructed embryos by transfer to the uterine horn of surrogated mother. Reference 1. Betthauser, J., Forsberg, E., Augenstein, M., Childs, L., Eilertsen, K., Enos, J., Forsythe, T., Golueke, P.,

Jurgella, G., Koppang, R., Lesmeister, T., Mallon. K., Mell, G., Misica, P., Pace, M., Pfister-Genskow, M., Strelchenko, N., Voelker, G., Watt, S., Thompson, S., and Bishop, M. (2000) Nat. Biotechnol, 18,

Type of fetal fibroblasts

No. reconstructed

No. cleaved (%)

No. of blastocysts (%)

No. of cells in blastocysts

GFP transfected 218 122 (56.0) 23 (18.9) 44.3 ± 15.1 (n = 4) Non-transfected 229 103 (45.0) 21 (20.4) 46.3 ± 6.4 (n = 4)

1055-1059. 17-09 IMPROVED DEVELOPMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED FROM G1/S DONOR CELLS AND ACTIVATED RECIPIENT CYTOPLASTS Satoshi Kurosaka1, Yasumitsu Nagao1, Naojiro Minami1, Masayasu Yamada1, and Hiroshi Imai1 1Laboratory of Reproductive Physiology, Graduate School of Agriculture, Kyoto University, Kyoto, Kyoto, 606-8502, Japan We previously reported that recipient cytoplasts prepared by simultaneously activation and fusion or activation at 2 hours before fusion have a similar potential to support in vitro development of bovine nuclear transfer embryos using unsynchronized adult somatic cells (1, 2). In this study, developmental potential in vitro of reconstructed embryos using these two types of recipient cytoplasts and adult somatic cells synchronized in G0 or G1/S phase was examined. The donor cells were synchronized in G0 phase by serum starvation or in G1/S phase by treatment with aphidicolin. Two activation/fusion protocols were used; 1) unactivated cytoplasts were fused with donor cells at 24 hours post maturation (hpm) followed by treatment with cycloheximide (CH, 10µg/ml) for 6 hours (F24 protocol), and 2) recipient cytoplasts were activated at 22 hpm by ethanol (7%, 7 minutes) followed by treatment with CH for 2 hours, and fused with donor cells at 24 hpm followed by treatment with CH for additional 4-5 hours (A22F24 protocol). In F24 protocol, developmental rates to the blastocyst stage of nuclear transfer embryos reconstructed from G0 and G1/S donor cells were 32.7% and 37.7%, respectively. In A22F24 protocol, developmental rates of the embryos reconstructed from G0 and G1/S donor cells were 29.2% and 50.9%, respectively. These results suggest that synchronization of donor cells in G1/S phase by aphidicolin is effective to obtain diploid donor cells having high developmental potential after nuclear transfer, and that G1/S cells are particularly suitable for donor to the recipient cytoplasts activated at 2 hours before fusion. References 1. Kurosaka, S., Ohashi, A., Nagao, Y., Minami, N., Yamada, M., and Imai, H. (2000) Biol Reprod 62 (suppl

1) 127. 2. Kurosaka, S., Nagao, Y., Minami, N., Yamada, M., and Imai, H. (2001) Theriogenology 55, 239. 17-10 THE REGULATORY ROLE OF THE FIRST ROUND OF DNA REPLICATION IN THE REPROGRAMMING OF GENE EXPRESSION IN MICE Serguei Medvedev1, Tomoyuki Tokunaga1, Richard M. Schultz2, Takashi Nagai1, Natalia Bossak1, and Yoshiaki Izaike1. 1Developmental Biology Department, National Institute of Animal Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan; 2Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA The first round of DNA replication has been suggested to play a key role in the activation of zygotic transcription in mice by facilitating the chromatin remodeling (1). We report here that

initial phase of the first round of DNA replication is essential for the initiation of zygotic transcription in mice by providing opportunities for the assembling of transcription complexes. Using a paternally-inherited chicken β-actin promoter-driven green fluorescent protein gene (cβA-GFP), whose expression is readily detected in preimplantation mouse embryos, we determined the effect of inhibiting DNA replication at different times during S phase on the initiation of zygotic transcription. We observed that treatment of 1-cell embryos with the inhibitor of DNA polymerases, aphidicolin just before mid S phase did not prevent expression of the transgene, since the fluorescent signal monotonically increased in the cleavage-arrested embryos over the course of 100 hours. When aphidicolin was added prior to the initiation of S phase, however, no expression of the reporter transgene was detected. Similar results were obtained with 4 independent transgenic lines. In addition, inducing histone hyperacetylation by butyrate treatment stimulated transgene expression in embryos arrested at mid S phase, but it showed no effect on the initiation of transcription in embryos arrested at the beginning of S phase. Results of these studies suggest that changes in chromatin structure as a consequence of either DNA replication or histone acetylation are mechanistically linked to the initiation of transcription in the embryo, and as such may also be involved in the process of nuclear reprogramming that occurs during cloning. Reference 1. Wolffe, A.P. (1994). Curr Opinion Gen Dev 4:245-254. 17-11 CAPRINE SOMATIC CELL NUCLEAR TRANSFER USING IN VIVO MATURED OOCYTES COLLECTED BY LAPAROSCOPIC FOLLICULAR ASPIRATION Katsuhiro Ohkoshi1, Seiya Takahashi2, Shin-ichiro Koyama1, Satoshi Akagi2, Noritaka Adachi2, Tadashi Furusawa1, Jun-ichiro Fujimoto3, and Tomoyuki Tokunaga1 1Developmental Biology Department, National Institute of Animal Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan; 2Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Ikenodai 2, Kukizaki, Inashiki-gun, Ibaraki 305-0901; 3National Children’s Medical Research Center, Taishido 3-35-31, Setagaya-ku, Tokyo, 154-8509, Japan In our recent study, we have examined the developmental potential of the somatic cell nuclear transferred (SCNT) goat eggs produced using in vitro maturation (IVM) and culture (IVC) systems (1), and obtained a SCNT goat offspring. However, the numbers of SCNT goat eggs that developed to the blastocyst stage was low and as the main cause, it was considered that the maturation of oocytes was incomplete. It seemed to improve the developmental ability of SCNT goat eggs by using the in vivo matured oocyte as a recipient. In this study, we established the collection method of in vivo matured oocytes by the laparoscopic follicular aspiration. And the development of SCNT goat eggs that produced with in vivo-matured oocytes as the recipient was examined.

Japanese native (Shiba) goats were used. To induce synchronization of estrus, a sponge containing 1g of progesterone was inserted into the vagina of each goat for 14 days. Those animals were treated with ovine FSH (Ovagen, ICP. Ltd., total 14mg each animal) in a series of 8 injections over 4 days. The first FSH was administered on the morning of Day 9 of sponge insertion. And on the morning of Day 13, 50 µg of GnRH (Conceral, Takeda Yakuhin) was injected into each animal. After 29 hrs of GnRH injection, laparoscopic follicular aspiration was performed. After removal of cumulus cells, collected oocytes with the first polar body were selected and enucleated for nuclear transfer. Anterior pituitary cells isolated from an adult male

Shiba goat were transfected with GFP gene, puromycine resistant gene and hammer head ribozyme gene against mRNA of caprine growth hormone. The single donor cell was inserted into the perivitelline space of each enucleated oocyte and fused by one electric pulse of 20V for 10 µsec. SCNT goat eggs were cultured in chemically defined medium (IVD-101, Research Institute for the Functional Peptides Co.Ltd.,) at 38.5°C in 5% CO2, 5% O2, 90% N2 for 9 days.

By laparoscopic follicular aspiration, 150 oocytes were collected from 12 females. After removal of cumulus cells, 61% of them excluded the first polar body. The percentages of SCNT goat eggs produced with in vivo-matured oocytes developing the blastocyst stage (20%) were significantly higher (p<0.05) than that with IVM oocytes (3%)(unpublished data). At present, several recipient females become pregnant after transfer of SCNT blastocysts. This work was supported by the Organized Research Combination System, Ministry of Education, Culture, Sports, Science and Technology, Japan. Reference 1. Ohkoshi, K., Takahashi, S., Akagi, S., Watanabe, S., Yamaguchi, M., Fujimoto, J., Izaike, Y., and

Tokunaga, T., (2000) 14 th I.C.A.R. 17-12 AN ATTEMPT FOR THE ISOLATION OF ICM-DERIVED CELL LINES FROM BOVINE CLONED EMBRYOS Jong-Im Park1, Kyeong-Hee Jang2, Sung-Keun. Kang1, and Woo Suk Hwang1

1Department of Theriogenology, College of Veterinary Medicine, 2School of Agricultural Biotechnology, Seoul National University, Shillim-Dong, Kwanak-Ku, Seoul, 151-742, Korea

The present study was carried out to isolate an embryonic cell line derived from ICM (inner cell mass) of bovine cloned embryos reconstructed with somatic cells. Cell lines used as donor for nuclear transfer (NT) were isolated from bovine ear skin and cultured in DMEM supplemented with 10% fetal calf serum (FCS)(1). After cultured for less than 8 passages, donor cell cycles was arrested at quiescent stage by culturing in DMEM supplemented with 0.5% FCS for 3-7 days. For preparing recipient cytoplasts, oocytes collected from slaughtered cows were matured in TCM-199 supplemented with 10% FBS for 18-20 hrs, and were enucleated. Single donor cell was injected into perivitelline space of each enucleated oocyte. After fusion and activation, reconstructed embryos were cultured in mSOF medium for 7-10 days. Bovine fetal fibroblasts (BFF), bovine ear fibroblasts (BEF), bovine oviduct cells (BOC), and mouse embryonic fibroblasts (MEF) were used as feeder cell lines. Expanded and hatched blastocysts derived from NT and in vitro fertilization (IVF) were placed into 4-well dishes and cultured on the various feeder layers in DMEM supplemented with 15% FCS, and 105 unit of leukemia inhibitory factor (LIF). After 4-6 days of culture, ICM of blastocysts attached to feeder layer were picked up and subsequently cultured for another 6-10 days. Within 4 days, the efficiency of attachment to 4 types of feeder layers was higher in NT-derived blastocysts than that in IVF-derived counterpart (49/133 vs. 85/293, respectively). MEF showed higher efficiency of attachment than other feeder cell types in both NT- and IVF-derived blastocysts. Five colonies (3, NT; 2, IVF) were isolated from 45 blastocysts (20, NT: 25, IVF) attached to MEF feeder layer. These colonies were disaggregated, continued to proliferate for several weeks, and stained for expression of stage-specific embryonic antigen (SSEA)-1 and alkaline phosphatase (AP) activity. The SSEA-1 antibody was detected in 2 lines from NT-derived blastocysts, however, AP activity was not detected in both lines. These findings indicate that undifferentiated embryonic cell lines can be derived from nuclei of terminally differentiated

adult somatic cells Reference 1. Shin, S. J., Lee, B. C., Park, J. I., Lim, J. M., and Hwang, W. S. (2001) Theriogenology 55, 1697-1704. 17-13 EFFECTIVE CRYOPROTECTANTS FOR THE CRYOPRESERVATION OF ANIMAL TISSUES Keishi Saruwatari1, and Noboru Fujihara1 1Animal Resource Science Section, Division of Bioresource and Bioenvironmental Science, Graduate School Kyushu University, Fukuoka 812-8581 Japan In these decades, many kinds of animals are on the verge of extinction in the world. A lot of techniques for restoring these endangered animals have been developed, including nuclear transfer, creation of chimeric animals and animal cloning. To make this kind of tools much more successful, basical researches are needed to be done just now. One of these fundamental researches may be the usage of some tissues from dead animals, e.g. cropreservation, tissue culture, cell fusion and nuclear transfer.In this experiment, based on these things, we tried to preserve several tissues from dead animals (cow and pig) and reuse them for cell culture after cryopreservation of animal tissues using three kinds of cryoprotectants, dimethylsulfoxide (0.15 - 3.5 M), dimethylformamide (0.25 - 3.5 M) and methylacetamide (0.15 - 2.0 M). Several tissues of the ear, gum, muscle, ovary, skin and uterus were obtained from the cattle of a local slaughterhouse, wiped once with 70 % alcohol cotton and washed three times in PBS added with 5 % Antibiotic-Antimycotic. These tissues were cut into small block (ca. 1cm3) and put into 4.5 ml cryotube containing 200 µl cryomedium (PB1) with different concentration of cryoprotectants described above. The tubes were kept on the ice for 5 min and then added 4 ml cryomedium on the ice, followed by freezing at -80°C in a deep freezer for two weeks. Following cryogenic storage, the tissues were thawed at 37°C in a water bath, washed three times in PBS to remove the cryoprotectants. The thawed tissues were treated with 0.25 % trypsin for 24 hr at 4°C, washed twice with 10 % FCS D-MEM / Ham-F12 by repeated centrifugation at 1500 rpm for 5 min. The collected cells were cultured in collagen-coated microplate 96 wells for three days until the determination of cellular survivability. Cellular viability was examined by the method of cell staining with acridine orange and ethidium bromide solution under a flourenscent microscope (1). Each tissue showed most effective cryoprotectant, respectively the tissues of the ear, uterus and skin have been effectively stored in the solution containing 3.0 M DMSO. For preserving gum tissues, 0.5 M MA was effective. The ovarian tissues showed 0.5 M DMSO to be most effective cryoprotectant. These results suggest that some of animal tissues may need suitable cryoprotectant and its effective concentration for each organ of different animal species. Reference 1. Gill, C.W., Fischer, C.L., and Lovrekovich, H. (1979) Medical Instrumentation 13 (1) 64-65. 17-14 GENE EXPRESSION IN BOVINE EMBRYOS RECONSTRUCTED WITH TRANSGENIC CELLS

Ken Sawai1, Satoru Moriyasu1, Hiroki Hirayama1, Soichi Kageyama1, Akira Minamihashi1, Masaru Okabe2, Masahito Ikawa2, and Sadao Onoe1 1Department of Animal Biotechnology, Hokkaido Animal Research Center, Shintoku, Hokkaido, 081-0038, Japan; 2Genome Information Research Center, Osaka University, Yamadaoka, Suita, Osaka, 565-0871, Japan It is probable that the reprogramming and gene expression of donor nuclei after fusion with recipient oocyte are important factors in development of reconstructed embryo. In a previous study (1), we demonstrated that the developmental competence of bovine embryos reconstructed with enhanced green fluorescent protein (EGFP) transfected cells was the same as when non-transgenic cells were fused, and we reported that EGFP was observed in almost all blastocyst stage embryos. The present study was conceived to examine the onset of gene expression in bovine embryos reconstructed with EGFP transfected cells. Linearized DNA fragment containing EGFP and neomycin resistance gene isolated from plasmid pCXNeo-EGFP [pCX-EGFP (2) containing neomycin resistance gene] was used for transfection. Bovine fetal fibroblast cells derived from a day 100 fetus were transfected with the gene by liposome mediation. Transformed cells were selected with medium containing G418 for two weeks. Donor cells featuring observed EGFP expression were injected into enucleated oocytes. After cell fusion, the reconstructed embryos were treated with 10μg/ml cycloheximide for activation and were transferred to mTALP medium with 0.1% BSA. From 16 h of culture, the embryos were cultured individually (1 embryo/drop) in microdrops (50μl) of mTALP supplemented 3% calf serum. The developmental stages and EGFP expression of embryos were examined every 12 h from Day (D) 1 to D7 (D0: the day of nuclear transfer). The EGFP expression in embryos was evaluated as belonging to one of three levels (+: weak; ++: middle; +++: strong) by the intensity of cellular fluorescence using direct observation with fluorescence microscope. The developmental rates to the 4-cell≦ (D2), 8-cell≦ (D3) and 16-cell≦ (D4) stages of reconstructed embryos were 69, 49 and 43%, respectively. The percentages of embryos with observed EGFP at each stage was 52, 86 and 100%, respectively. 31% (57/185) of the reconstructed embryos developed to the blastocyst stage, and EGFP expression (level ++≦) was detected in all of the 57 embryos. In these embryos developed to the blastocyst, levels + and ++ of the EGFP expression were first detected at 1-cell to 32-cell and 4-cell to blastocyst stage, respectively. The maximum rates (level +: 53%; level ++: 53%) of embryos were obtained at the 8-cell stage (P<0.05). The onset stage of level +++ expression was 8-cell to blastocyst stage, and no difference was detected in each population (6-27%). The EGFP expression (level +≦) in embryos developed to blastocyst at the final stage (BC embryos) as compared to embryos which could not reach blastocyst stage (arrested embryos), the expression rate at 8-cell stage was significantly (P<0.05) higher in BC embryos (74%) than arrested embryos (39%). These results suggested that the gene expression in bovine embryos reconstructed with somatic cells begins from the early developmental stage and the degrees of expression may increase from the 8-cell stage. References 1. Sawai, K., Moriyasu, M., Hirayama, H., Kageyama, S., Minamihashi, A., Okabe, M., Ikawa, M. and

Onoe, S. (2001) J. Reprod. Dev. Suppl. 2. Ikawa, M., Kominami, K., Yoshimura, Y., Tanaka, K., Nishimune, Y., and Okabe, M. (1995) Dev Growth

Differ 37, 455-459. 17-15 STUDIES ON THE NUCLEAR REQUIREMENT FOR NORMAL KINETICS OF CELL CYCLE REGULATORS IN PORCINE OVA: MPF AND MAP KINASE ACTIVITIES

DURING MEIOSIS Koji Sugiura1, Kunihiko Naito1, Huruna Naruoka1, and Hideaki Tojo1

1Laboratory of Applied Genetics, Department of Animal Resource Sciences, Graduate School of Agricultural Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan The cloned embryo seems to cleave normally, although its nucleus was exchanged for a somatic cell nucleus. We examined, therefore, the requirement of nucleus for the normal kinetics of cell cycle regulators, MPF and MAP kinase, in porcine ova. Porcine follicular oocytes were used for the experiment after enucleation (1), and their MPF and MAP kinase activities were assayed as histone H1 (2) and MBP kinase activity (3), respectively. The phosphorylation states of ERK1/2, major MAP kinases in maturating porcine oocytes, were detected by Western blotting analyses. The activation of MPF at the start of oocyte maturation was detected in enucleated porcine oocytes and the level was comparable to the control denuded oocytes. The phosphorylation and the activation of MAP kinase was induced and maintained through maturation, even in the absence of a nucleus and spindle. These results indicate that nuclear material is dispensable for the normal kinetics of cell cycle regulators at the start of oocyte maturation. As the start of maturation was the transition from G2-phase to M-phase, these results might indicate the dispensability of nucleus also the G2/M transition during the early embryonic development. In contrast, the reactivation in the second meiosis was not observed in the enucleated oocytes. Furthermore, the reactivation of MPF was induced in enucleated oocytes by injection of germinal vesicle materials from fleshly isolated oocytes. This result suggests that germinal vesicle is necessary for the M1/M2 transition, a meiotic unique kinetics. References 1. Sun, F.Z., and Moor, R.M. (1991) Development 111, 171-180. 2. Naito, K., and Toyoda, Y. (1991) J. Reprod. Fertil. 93, 467-473. 3. Sugiura, K., Naito, K., Iwamori, N., Kagii, H., Goto, S., Ohashi, S., Yamanouchi, K., and Tojo, H. (2001)

Mol Reprod Dev 59, 215-220. 17-16 EFFECTS OF AMINO ACIDS ON THE DEVELOPMENT OF PARTHENOGENETIC DIPLOIDS IN THE PIG Nguyen Van Thuan1, and Masashi Miyake1 1Department of Life Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho Nada-ku, Kobe City, Hyogo 657-8501, Japan The aim of this study was to investigate the effects of amino acids on the in-vitro development of parthenogenetic diploids to the blastocyst stage in the pig. Oocyte cumulus and granulosa cell complexes (OCGCs) were cultured in-vitro for 48 h. Matured oocytes were subjected to a single electro-stimulation (El-St; 100 µsec, 1,500 V/cm) and then treated with 5.0 µg/ml cytochalasin B for 4 h. Culture media were based on Whitten's medium including 0.5 mg/ml hyaluronic acid (mWM). In Exp. 1, effects of replacing bovine serum albumin (BSA) with 0.01, 0.05, 0.1, 0.5, 1.0, and 5.0 mg/ml polyvinyl alcohol (PVA) on the development of diploids to the blastocyst stage were examined. In Exp. 2, diploids were cultured for 0, 48 or 72 h in mWM containing 0.5 mg/ml of PVA (WMPVA), and then cultured under the presence of Eagle’s Basal Medium essential amino acids without glutamine (E-AA) and Minimum Essential Medium

non-essential amino acids (NE-AA) until 168 h after El-St. In Exp. 3, diploids were cultured in WMPVA including E-AA or NE-AA for the first 48 h and subsequently cultured in WMPVA including E-AA + NE-AA (AA) up to 168 h after El-St. In Exp. 4, diploids were cultured in WMPVA for the first 48 h under the presence of basic E-AA, polar E-AA, or non-polar E-AA and then cultured in WMPVA including AA up to 168 h after El-St. All experimental groups were compared with a group cultured in mWM including 4 mg/ml of BSA (control).

Exp. 1 showed that diploids could develop up to the blastocyst stage in a medium supplemented with 0.01 to 5 mg/ml PVA (15 to 53%), and the addition of more than 0.5 mg/ml PVA supported the same rate of development to the blastocyst stage as BSA (49 to 53%, vs 63%) did. However, the rates of expanded blastocyst at 168 h after El-St were lower in all media with PVA (11-20%) than in the medium with BSA (39%, P<0.05). In Exp. 2, the rates of diploids developed to the blastocyst and expanded blastocyst stages were lower in the addition of AA at 0 h group (22 and 7%, respectively) than in the addition at 48, 72 h groups and control group (57-61 and 35-39%, respectively). The results of Exp. 3 and 4 showed that the presence of E-AA, especially non-polar E-AA, during the first 48 h after El-St resulted in the inhibition and delay of the first division of diploids and most of them ceased their development at the 4-cell stage.

These results indicate that porcine parthenogenetic diploids can develop up to the blastocyst stage in a protein-free and amino acids free medium containing PVA. Only PVA in a protein-free medium, however, can not support the expansion of the blastocyst. The addition of AA later than the 4-cell stage (48 h after El-St) stimulates the expansion of blastocyst, and increases the number of cells in the blastocyst. The presence of non-polar E-AA during the first 48 h after El-St causes 4-cell block in the early development of porcine parthenogenetic diploids. References 1. Kurebayashi, K., Miyake, M., Katayama, M., Miyano, T., and Kato, S. (1996) Theriogenology 46,

1027-1036. 2. Petters, R. M., and Wells, K. D. (1993) J Reprod Fertil 48, 61-73. 3. McKiernan, S. H., Clayton, M K., and Bavister B D. (1995) Mol Reprod Dev 42, 188-199. 4. Lane, M., and Gardner D. K. (1997) J Reprod Fertil 109, 153-164. 5. Steeves T. E., and Gardner D.K. (1999) Biol Reprod 61, 731-740 17-17 BIRTH SITUATION AND TELOMERE LENGTH OF JAPANESE BLACK CALVES PRODUCED BY NUCLEAR TRANSFER Manami Urakawa1, Katsuyoshi Uruno1, Atsushi Ideta1, Yutaka Sendai2, Hiroyoshi Hoshi2, Tokihiko Sawada3 and Yoshito Aoyagi1

1ET Center, ZEN-NOH, Kamishihoro, Katougun, Hokkaido, 080-1407, Japan; 2Research Institute for the Functional Peptides, Shimojo, Yamagata, Yamagata, 990-0823, Japan; 3Department of Surgery, Kidney Center, Tokyo Women’s Medical University, Kawata, Shinzyuku, Tokyo, 162-0054, Japan Whereas there have been few reports on the birth situation and telomere length of cloned calves, it is still unclear to completely understand the interrelation between them. This study carried out to examine the birth situation, relevance between histological inspection and biochemical examination of blood, telomerase activity and telomere length of live calves derived from nuclear transfer. The NT group consists of live calves from nuclear transfer ( a still birth was removed) using bovine fetal fibroblast cells and embryonic stem - like cells for donor cells

(n=10). The control group consists of calves from artificial insemination and embryo transfer (n=5). The parturition was either spontaneous (NT group; n=4, control group; n=5) or induced by PGF2α (NT group; n=6). The birth situation regarding pregnancy period, birth weight, success in standing and suckling was examined. The relevance to histological inspection of the main organs and biochemical examination of blood of 42 items was also examined. The telomerase activity of the total proteins extracted from lung cells was measured using TeloChaser (TOYOBO, TLK-101 ). The telomere length was determined by terminal restriction fragment using heart and lung or ear hypodermic cells. The birth weights of the NT group were 33.1 ± 6.8kg (spontaneous) and 36.6 ± 4.9kg (induced). The pregnancy periods were 289.0 ± 3.6 days (spontaneous) and 297.7 ± 10.1 days (induced). The birth weight of calves in the control group was 29.6 ± 5.7kg (spontaneous), and the pregnancy period was 287.0 ± 5.4 days (spontaneous). There was a significant difference in pregnancy period and birth weight of calves in the NT group where the parturition was induced for the control group (P<0.05, Student-t test). Standing and suckling were confirmed in all calves of the NT group and the control group. There was relevance between the histological necrobiosis image at part of the heart muscle and the creatine phosphokinase value in the blood of calf in the NT group, which had induced parturition. The relative telomerase activities of the NT group varied between 23 and 190%, when the control group was adjusted to 100%. The mean telomere length of the NT group was 18-20Kb, and in comparison with the mean telomere length (14-20kb) of the control group calves, shortening or elongation of the telomere length could not be identified. These results indicate that there was no difference in birth situation or telomere length in calves of the NT group by spontaneous parturition and the control group. 17-18 CHARACTERISTICS OF CLONED CATTLE Yutaka Yamada1, Minoru Sakaguchi1, Hiroya Kadokawa1, Masao Kishi2, and Ryo Takakura2 1Department of Animal Production and Grassland, National Agricultural Research Center for Hokkaido Region, Sapporo, Hokkaido, 062-8555, Japan; 2Embryo Transplantation Laboratory, Snow Brand Milk Products Co. Ltd., Tomakomai, Hokkaido, 059-1365, Japan The objectives of this study were to clarify the normality and similarity of cloned cattle derived from blastomeres (32 to 64-cell stage). Six cloned calves of Japanese Black breed derived from 3 different embryos (female single calf, female twin calves and male triplet calves) were used. The growth performances were examined under the conventional management. The weight at birth and at 18 months of female twin calves, female single calf and male triplet calves were 37.5 ± 5.0 kg, 373.5 ± 53.0 kg; 32.0 kg, 389.0 kg and 23.0 ± 2.7 kg, 346.3 ± 11.2 kg; respectively. The meat production performances were examined in castrated triplet cattle. They were fattened from 19 months until 26 months of age. Mean body weight at the start of fattening and the end of fattening were 349.3 ± 9.1 kg, 535.3 ± 17.0kg, respectively. The changes of body weight, withers height, body length, chest girth and shank circumference were very similar within three cattle. The carcass grades of all cattle were judged as the same class and the dressed weights were 304.3 ± 9.0 kg. The carcass traits such as rid eye area, thickness of subcutaneous fat, Beef Marbling Standard Number, Beef Color Standard Number, Beef Fat Standard Number showed the similar figures. The reproductive performances were examined in 3 female heifers. Single female showed a normal estrus behavior and she calved normally at 27 months of age. Twin female heifers showed no estrus behavior until 30 months of age. They were conceived by artificial insemination after the treatment of PG and GnRH. They calved at 40 and 42 months of age, respectively. One of them had a stillbirth and the fetus had a longer

body length (BW: 33.5 kg, Body length; 105cm). From the above results, it is concluded that the cloned cattle showed normality and similarity in growth performances and meat productivity, while further research is necessary to clarify the characteristics of the reproductive performance in female. 17-19 AN ABILITY OF CULTURED CHIKEN BLASTODERMAL CELLS TO RECONSTITUTE THE GERMLINE OF BLASTODERMAL CHIMERA Yuko Matsubara1, Azusa Hirota2, Hideo Sobajima3, Akira Onishi1, Hiroshi kagami4, TakahiroTagami5, Takashi Harumi1, Michiharu Sakurai6, Akiko Sano1, and Mitsuru Naito1 1Natinal Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan; 2Saitamaken Prefectural Livestock Center, Sugahiro 784, Konan, Osato, Saitama, Japan 360-0100; 3Gifu Prefectural Poultry Experimental Station,Sakoma 2672-1, Seki, Gifu 501-3924, Japan; 4Shinshu University, Minamiminowa 8340, kamiina, Nagano 399-4598, Japan; 5National Institute of Livestock and Grassland Science, Ikenodai 2, Kukizaki, Inashiki, Ibaraki 305-0901, Japan; 6National Institute of Animal Health, Kannondai 3-1-5, Tsukuba, Ibaraki 305-0856, Japan The use of transgenic animals is gaining considerable attention in the study of developmental biology and development of biomedical products. So far many efforts to develop the methods of producing transgenic birds have been made. Only the method that could produce transgenic birds was DNA maicroinjection into the germinal disc of chick zygotes. In this case, the frequency of production of transgenic birds is low, with approximately 0.4% of injected zygotes giving rise to germline trasngenic birds. The transgenic birds generated to date have all been mosaic, transmitting the introduced gene construct to between 1% and 5% of their offspring (1). Therefore, presently there were no efficient methods to produce transgenic birds yet. Chicken blastodermal cells of freshly laid eggs are known to have an ability of reconstituting somatic cells and germ cells when injected into recipient embryos to form chimeras (2). Avian germline chimeras are one of the most useful tools for the production of transgenic birds. In this study, chicken blastodermal cells were cultured in order to select only cells integrated an exogenous gene and injected these cells into recipient embryos. The central disc of the area pellucida (stage X) of chicken blastodermal cells is considered to contain primordial germ cells or their precursor cells (3). Cells derived from the central disc of the area pellucida proliferated by about 7 times in culture for 3 days without a feeder layer. These cultured cells showed alkaline phosphatase activity for 2 to 5 days in culture. It was also found that these cultured chicken blastodermal cells contained the anti SSEA-I and/or EMA-I antibody positive cells. We obtained three chimeric chickens judged by the feather color by transfer of two–day or five-day cultured Barred Plymouth Rock donor cells to White Leghorn recipients. It was proved that one male of chickens was a germline chimera. References 1. Love, J., Gribbin, C., Mather, C., and Sang, H. (1994) Biotechnology 12,60-63. 2. Pettite, J. N., Clark, M.E., Verrinder Gibbins, A. M., and Etches, R. J. (1990) Development 108,185-189. 3. Kagami, H., Tagami, T., Matsubara, Y., Harumi, T., Hanada, H., Maruyama, K., Sakurai, M., Kuwana, T.,

and Naito, M. (1997) Mol Reprod Develop 48,501-510.