Current Status and Perspectives in Reproductive Biology and Biotechnology

Transcription

1 [Satellite Symposium of the JSAR Annual Meeting 2004] Current Status and Perspectives in Reproductive Biology and Biotechnology Abstracts for Section 3: The 9th Asian Symposium on Animal Biotechnology (ASAB)

2 Development of a New Vitrification Container for Freezing of Bovine Embryo 1 Hoon Teak Lee, 1, 2 Young Mi Kim, 1 Sang Jun Uhm and 2 Dae Hwan Ko 1 ARRC, Konkuk University, Seoul; Department of Life Science, 2 Sangji Youngseo College, Wonju, Korea This study was to investigate the development of new container for the effective cryopreservation of bovine blastocysts. Vitrification has been used to eliminate ice crystal formation during the cryopreservation of mammalian embryos. However, this method may introduce some problems such as loss of eggs during cryopreservation (EM grid) and damage to the zona pellucida. Thus our study examined an alternative container (paper) for the vitrification of in vitro produced bovine blastocysts. Oocytes were aspirated from slaughterhouse ovaries and cultured in TCM-199 supplemented with 25 mm NaHCO3, 10% (v/v) FBS, 0.22 mm sodium pyruvate, 25 mm gentamycin sulfate, 10 ァカ /ml FSH (Follitropin V; Vetrepharm, Canada) and 1 ァカ /ml estradiol-17_ for 24 hr. Matured oocytes were cocultured with sperm (1 _ 10 6 /ml) for hr. Cleaved embryos were cultured in 50 ァ of CR1aa medium containing 0.4% BSA for 5 days. For the vitrification, in vitro produced embryos at blastocyst stage were exposed to 5.5 M ethylene glycol freezing solution (E.G 5.5) for 20 sec, loaded on containers; grid, straw and paper and then immediately plunged into -196_ LN 2. The blastocysts were thawed serially every one minute with sucrose solution 0.5, 0.25 and M, and transferred to the culture solution with 10% FBS. As results, there were no significant differences in recovery rates among EM-grid, straw and paper as 78.1, 74, and 95.4%, respectively. However, survival rates of embryos frozen-thawed with EM-grid (68%) and paper (71.9%) showed significantly higher than those with straw (51.8%). In addition, after transferring those embryos, we found that pregnancy rates were not different between EMgrid (21.6%) and paper (29.4%) but significantly lower than those of unfrozen embryos (61.3%). Therefore, our results suggest that it is possible to use papers as a vitrification container with taking advantages such as simple, cheap and efficient.

3 Diversification of Cyclooxygenase-2-derived Prostaglandins in Ovulation and Implantation Hiromichi Matsumoto Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan Prostaglandins (PGs) have long been known to participate in female reproductive functions. Mice with null mutation for the gene encoding cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG biosynthesis, were shown to be infertile with abnormalities in ovulation, fertilization, implantation and decidualization. However, the specific PG and its mode of action were unknown. Further studies revealed that mice deficient in EP 2, a PGE 2 -receptor subtype, have reduced litter size, apparently resulting from poor ovulation but more dramatically from impaired fertilization. Superovulation regimen and subsequent in vitro culture system demonstrated that the ovulatory process, not follicular growth, oocyte maturation, or fertilization, was primarily affected in adult COX-2- or EP 2 -deficient mice. Furthermore, when oocytes obtained from the deficient mice were matured and fertilized in vitro, they were found to be capable of subsequent preimplantation development. However, severely compromised ovulation in adult COX-2- or EP 2 -deficient mice was not manifested in immature (3-wk-old) COX-2- or EP 2 -deficient mice, suggesting that the process of ovulation was more dependent on PGs in adult mice. Although the processes of implantation and decidualization were defective in COX-2-deficient mice, these events were found to be normal in EP 2 -deficient mice, as determined by embryo transfer and experimentally induced decidualization. During the peri-implantation period, increased vascular permeability and angiogenesis at the site of blastocyst apposition in the uterus are two hallmarks. The present study shows that although the proangiogenic vascular endothelial growth factor (VEGF) and its receptor, Flk-1, are primarily important for uterine vascular permeability and angiogenesis prior to and during the attachment phase of the implantation process, VEGF in complementation with the angiopoietins and their receptor, Tie-2, directs angiogenesis during decidualization following implantation. The reporter and mutant mice show that COX-2-derived PGs are important for uterine vascular permeability and angiogenesis during implantation and decidualization, suggesting that one cause of the failure of these latter processes in COX-2-deficient mice is the deregulated vascular events in the absence of COX-2. The attenuation of uterine angiogenesis in these mice is primarily due to defective VEGF signaling but not due to the defective angiopoietin system. Collectively, present study suggests that whereas COX-2-derived PGE 2 is essential for ovulation via activation of EP 2, COX-2-derived prostacyclin is involved in implantation and decidualization via activation of peroxisome proliferator-activated receptor δ). This COX-2-derived prostacyclin signaling also influences uterine angiogenesis primarily via affecting the VEGF system during implantation.

4 ICSI in Laboratory Animals Atsuo Ogura RIKEN Bioresource Center, Tsukuba, Ibaraki, Japan ICSI (Intracytoplasmic sperm injection) has been widely used in the field of basic reproductive researches and human infertility clinics. Historically, ICSI in laboratory animals started with the golden hamster and since then it has provided invaluable information on the mechanisms of mammalian fertilization including sperm nucleus decondensation, oocyte activation, pronuclear formation etc. Due to the recent advances in animal genetic engineering and germ cell technologies, the ICSI techniques, especially those of mice, are extensively used to identify the biological significance of the genes of interest or to confirm the genetic normality of gametes produced by experimental manipulations in vitro. In some of these experiments, immature sperm cells (spermatids) or abnormal sperm are the only male gametes available. Fortunately, in mice, practically high rates of embryo development into offspring can be obtained as long as we use postmeiotic spermatogenic cells as male gametes; i.e., round spermatids, elongated spermatids, and spermatozoa. This high fecundity enables us to apply the mouse ICSI technique to transgenesis, gene therapy, germline stem cell and gene targeting studies. By contrast, for other laboratory animals including rabbits and monkeys, immaturity of spermatogenic cells significantly decrease the efficiency of microinsemination. No or very few offspring have been born following microinsemination using spermatids in these species. This is due to inadequate potential of the immature sperm cells to complete the fertilization process, but not due to immaturity of their genome. Physically unstable chromatin structure and low oocyte-activating capacity are the major cause of the fertilization failure. In this lecture, the recent advances of ICSI in laboratory animals and its application to genetics and biomedical researches are presented.

5 Current Advance in Nuclear Transfer Technology in Korea Choong-Saeng Park and Seong-Keun Cho Laboratory of Animal Reproduction, College of Agriculture and Life Sciences, Gyeongsang National University, Jinju, Korea The genetic improvement of farm animals has been increased greatly by artificial insemination and multiple ovulation-embryo transfer. The nuclear transfer (NT) technology has been developed to increase the frequency of genes of genetically outstanding animals in a population. The introduction of new genetic materials into a population and the production of knock-in and knock-out animals for xenotransplantation have also been attempted by transgenic and somatic cell nuclear transfer (SCNT) technologies. Furthermore the human somatic cell nuclear transfer-embryonic stem (SCNT-ES) cell lines will be contribute to develop new technologies in cell therapy. During the period of 15 years since 1988, the research works in the field of embryo cloning and SCNT have also been performed with successful results in Korea. Some of the interesting results related to embryo cloning, SCNT, transgenic SCNT, and human SCNT-ES cell lines from Korean laboratories are discussed by animal species in my talk.

6 Nuclear Transfer Technology in Cattle and Swine Yukio Tsunoda and Yoko Kato Laboratory of Animal Reproduction, College of Agriculture, Kinki University, Nara, Japan Since the first successful nuclear transfer of somatic cells in mammals in 1997, animal cloning have been in the spotlight, not only in the field of animal science, but also in the medical field dealing with xenotransplantation for humans. Cloned calves and piglets with normal fertility have been produced by nuclear transfer of somatic cells from various organs, including bone marrow mecenchymal stem cells (1). Somatic cells are either fused with or directly injected into enucleated oocytes (2) whose chromosomes are previously removed by aspiration from oocyte cytoplasm or by chemical assisted method (3, 4). Oocyte cytoplasm has the ability to convert differentiated somatic cell nuclei into a state that resembles the conditions that occur at fertilization (5). Although the molecular nature of nuclear preprogramming factors is not known, it has been reported that the process of nuclear reprogramming is not directly regulated by maturation promoting factor or mitogen activated protein kinase (6). In this symposium, we will show recent data on cloning in our lab. Recent publications 1. Kato, Y. et al. (2004) Biol. Reprod., 70, Kawano, K., Kato, Y. and Tsunoda, Y. (2004) Cloning and Stem Cells, 6, Yin, X.J. et al. (2002) Biol. Reprod., 67, Kawakami, M. et al. (2003) Cloning and Stem Cells, 5, Tani, T., Kato, Y. and Tsunoda, Y. (2001) Biol. Reprod., 64, Tani, T., Kato, Y. and Tsunoda, Y. (2003) Biol. Reprod., 69,

7 Sperm Vector Method as a New Technology for Transgenic Pig Production Hiroshi Nagashima Laboratory of Developmental Engineering, Department of Life Science, Meiji University, Kawasaki, Japan In recent years, transgenic pigs have become indispensable to biomedical research, and are employed in xenotransplantation and as models of human diseases. However, conventional pronuclear microinjection, i.e., the injection of DNA constructs into the pronuclei of fertilized eggs, does not efficiently yield transgenic pigs, and has proven to be a bottleneck in producing the transgenic pigs needed for their various practical applications. Recently, somatic cell nuclear transfer has been developed as a new technology for the production of genetically modified pigs. Because of its technological complexity in practice, however, the production of cloned pigs is still far from adequate. Perry et al. (1999) have reported a highly efficient method for the production of transgenic mice, the so-called sperm vector method, in which sperm are co-incubated with DNA and microinjected into the ooplasm (intracytoplasmic sperm injection, ICSI). This method allows insertion of foreign genes into the embryonic genome by introducing DNA molecules bound to the sperm head into the ooplasm. In the course of establishing the sperm-vector method as a viable option for producing transgenic pigs we examined various factors with the potential to impact the efficiency of ICSI to porcine IVM oocytes. We succeeded in producing live transgenic pigs after transfer of embryos produced by the spermvector method. Furthermore nuclear donor cell were established to clone the transgenic pig obtained. We have been also pursuing cryopreservation of the in vitro produced porcine embryos, aiming at establishing a banking system of the transgenic embryos. Our study demonstrates that the sperm-vector method using IVM oocytes can be a feasible, alternative way for production of transgenic pigs.

8 Future Directions for Mammalian Cloning M.B. Nottle, L.F.S. Beebe, S.J. Harrison, R. Faast, M Moretta and S.M. Mcilfatrick Cell Biology Group, Department of Obstetrics and Gynaecology, University of Adelaide,Adelaide, South Australia, Australia Current cloning efficiencies are relatively low compared with in vitro embryo production. It is generally believed that this is due to inadequate nuclear reprogramming which in turn is the result of a number of factors including the method of activation and type of donor cell used. We have produced cloned piglets using fetal and adult fibroblasts, as well as Gal knockout pigs using gene targeting of fetal fibroblasts. Currently our efficiency is around 3% of embryos transferred resulting in liveborn. To increase this efficiency further, work is focussed on improving methods for activation and using a less differentiated cell type for cloning. Following fertilisation, the oocyte is subjected to multiple changes in intracellular calcium which last for several hours, depending on the species. In contrast, cloned embryos are activated using one or two electrical pulses or chemical treatments which result in one (or two) calcium oscillations. We have previously shown that the while one electrical pulse may be sufficient to reduce MPF levels to levels seen immediately following fertilisation, MPF activity is abnormally high around the time of the first cleavage. As MPF can cause irreversible damage to the chromatin and aneuploidy, abnormally high MPF levels around this time may be a major contributing factor to the failure of cloned embryos to develop. Hence further examination of the relationship between MPF activity, method of activation and the development of cloned embryos is warranted as ultimately the method used should duplicate the changes in intracellular calcium levels seen following fertilisation. This will require a better understanding of the events surrounding fertilisation. Recent work in mice has suggested that the use of embryonic stem cells can increase cloning efficiencies compared with somatic cells. It follows therefore, that the use of less differentiated cells such as adult stem cells which have been shown recently to exist in some (if not all) tissues may improve cloning efficiencies compared with adult fibroblasts for example. We have recently isolated mesencyhmal stem cells from bone marrow and other tissues and are currently examining their suitability for cloning. However for applications such as xenotransplantation where multiple genetic modifications are required, embryonic stem cells which have an infinite capacity to divide will need to be isolated. These and other studies, would be greatly facilitated by a marker of early gene expression. As such, we are examining the suitability of Oct 4 as a measure of developmental competence.