CURRENT STATUS AND PERSPECTIVES IN ANIMALREPRODUCTION AND SAFETY ASSESSMENT OF
CLONING ANIMALSThe 3rd Korea-Japan Joint Symposium of Animal Reproductive
Biology and Biotechnology(09:50-17:30, 18th October, Thursday, 2007)

Yayoi Auditorium-Ichijo Hall
(http://www.a.u-tokyo.ac.jp/yayoi/)
College of Agricultural and Life Sciences, The University of Tokyo
Yayoi 1-1-1, Tokyo 113-8657, JapanCURRENT STATUS AND PERSPECTIVES IN ANIMAL REPRODUCTION AND
SAFETY ASSESSMENT OF CLONING ANIMALSThe 3rd Korea-Japan Joint Symposium of Animal Reproductive Biology and Biotechnology(18th October, Thursday, 2007)

Yayoi Auditorium-Ichijo Hall
(http://www.a.u-tokyo.ac.jp/yayoi/)
College of Agricultural and Life Sciences, The University of Tokyo
Yayoi 1-1-1, Tokyo 113-8657, JapanPROGRAMM
18th October, Thursday, 2007
09:50 – 10:00 Opening Remarks
Dr. Masugi NISHIHARA
President of Japanese Society of Animal Reproduction (JSAR)The University of Tokyo, Japan
10:00 – 10:30 Recent Advances in Animal Biotechnology in Korea
Dr. Hoon-Taek LEE
President of Korean Society of Animal Reproduction (KSAR)
Konkuk University, Korea
10:30 – 11:00 Recent Research Trends in Animal Reproduction and Biotechnology in Japan
Dr. Eimei SATO
Former President of Japanese Society of Animal Reproduction (JSAR)
Tohoku University, Japan
11:00 – 11:30 Present status of somatic cell cloned cattle produced in Japan
Dr. Shinya WATANABE
National Institute of Livestock and Grassland Science, Japan
11:30 – 12:00 MicroRNA system in fertilized and cloned mouse embryos
Dr. Nam-Hyung KIM
National Research Laboratory of Molecular Embryology
Chungbuk National University, Korea
12:00 – 13:30 Lunch
13:30 – 14:00 Production of cloned cats and dogs by somatic cell nuclear transfer
Dr. Il-Keun KONG
Sunchon National University, Korea
14:00 – 14:30 From somatic cell to stem cell through nuclear transfer
Dr. Teruhiko WAKAYAMA
Center for Developmental Biology, RIKEN-Kobe, Japan
14:30 – 15:00 Characterization of somatic nuclear remodeling and reprogrammingin mouse cloned embryo
Dr. Nguyen Van THUAN
Konkuk University, Korea
15:00 – 15:30 Transgenesis and assisted reproductive technologies in rat
Dr. Naomi KASHIWAZAKI
Azabu University, Japan2

15:30 –16:00 Improvement of culture media for in vitro embryo production
Dr. Hiroyoshi HOSHI
Functional Peptide Research Institute, Japan
16:00 – 16:20 Coffee break
16:20 – 17:20 Cellular and molecular mechanisms regulating function of the corpus luteum
Dr. Joy L. PATE
Former President of the Society for the Study of Reproduction (SSR)
Ohio State University, USA
17:20 – 17:30: Closing Remarks
Dr. Noboru MANABEThe University of Tokyo, Japan

Correspondence: Dr. Noboru MANABE
Animal Resource Science Center, The University of Tokyo
Ago 3145, Kasama 319-0206, Japan
Tel: +81-299-45-2606, Fax: +81-299-45-5950
E-mail: amanabe@mail.ecc.u-tokyo.ac.jp3

ABSTRACTS

4

PREFACE: CURRENT STATUS AND PERSPECTIVES IN REPRODUCTIVE
BIOTECHNOLOGY AND SAFETY ASSESSMENT OF CLONING ANIMALS
Noboru MANABE
Animal Resource Science Center, The University of Tokyo
Ago 3145, Kasama 319-0206, Japan(amanabe@mail.ecc.u-tokyo.ac.jp)
Since “Dolly”, the first somatic cell cloned-sheep, was born in 1996, studies on the somatic cellcloning technique have been of interest around the world. The studies will bring fast and efficient
improvement in farm animals, with precise sex determination and multiplication of elite animals including
genetically modified ones. When combined with transgenic technologies, it will open new fields inmedical applications such as the establishment of genetically defined models for xeno-transplantation anda source for human therapeutics. In Japan, the first somatic cell cloned calves was born in 1998. To date,more than 500 cloned calves have been born. The somatic cell cloning technique will be considered as astandard technique for beef cattle breeding. Recently, Japanese people have interesting in the food safetyincluding the beef of somatic cell cloned cattle. To assess the safety of the cloning animals as food, it isessential to understand the current status and perspectives in reproductive biotechnology. The 3rd
Korea-Japan joint symposium of animal reproductive biology and biotechnology will be held tounderstand the current status and perspectives in reproductive biotechnology in Korea and Japan.
Professor Hoon-Taek LEE (President of Korean Society of Animal Reproduction: KSAR) will give alecture on the recent advances in animal biotechnology in Korea. Professor Eimei SATO (Former Presidentof Japanese Society of Animal Reproduction: JSAR, and President of Japanese Society of Animal
Sciences) will give a lecture on the recent research trends in animal reproduction and biotechnology inJapan. In Korea and Japan, the importance of animal biotechnology is highly appreciated. Animal
biotechnology researchers in government, university, institutes and industries have tried its vast application
not only in production of superior quality animals but also in medicine, genetic engineering, transgenesis,
cloning and stem cell. Professor LEE will focus on animal transgenesis, cloning and stem cell researches in
Korea. Professor SATO will present the history and importance of research activities of animal
reproduction in Japan. Dr. Shinya WATANABE will give a lecture on the present status of somatic cellcloned cattle produced in Japan. As mentioned above, from 1998 to September 2006, 511 somatic cellcloned cattle have been produced in Japan. He is a major member of government committee to assess thesafety of somatic cell cloned cattle-beef as food in Japan, and will report the studies on genetic similarities
and muzzle prints, hematology and clinical chemistry findings, pathology, growth performance,
reproductive performance, meat productive performance and milk productive performance. Moreover, hewill present that the somatic cell cloning technology does suffer from problems such as the large calf
syndrome at birth, extremely low survival rates before weaning in cattle and abnormal gene expression ofembryos. Professor Nam-Hyung KIM will present the study on micro RNA, small non-coding RNA
molecules of 22 nucleotides in length that can post-transcriptionally regulate gene expression bybase-pairing to mRNA, in fertilized and cloned embryos. Professor Il-Keun KONG will show us thesomatic cell nuclear transfer (SCNT) for production of cloned-cats and dogs. Dr. Teruhiko WAKAYAMA,
who obtained the first somatic cell cloned mouse, will give a lecture titled “From somatic cell to stem cellthrough nuclear transfer”. Mice can be cloned from cultured and non-cultured, adult-, fetus-, male- or
female-derived cells. Recently, he was able to improve the success rate of cloning in mice up to six times
using dimethyl sulfoxide, trichostatin A or by serial cloning using nuclear transfer- embryonic stem
(NT-ES) cell technique. However, the overall efficiency of mouse cloning is less than 10% and theoffspring that survive to term have shown many abnormalities, which are probably caused by incomplete
genomic reprogramming after nuclear transfer into the oocyte cytoplasm. Moreover, he could get themouse from nuclear transferred-embryonic stem (ntES) cell lines. He will talk about the similarity between
ntES and embryonic stem (ES) cells, and the possibility to establish ES/ntES cell lines without ethicalproblem. Professor Nguyen Van THUAN, who recently moved from Wakayama’s laboratory to Lee’slaboratory in Konkuk University, will talk about the histone deacetylase, scriptaid, rescues full-term
development in cloned inbred mice by enhancing zygotic nascent mRNA production. Professor Naomi
KASHIWAZAKI will show us the transgenesis in rat, very important experimental animal. He will explain
the detailed techniques on advancements in assisted reproductive technologies (ARTs) and the applicationof cryopreserved sperm, ovaris and intracytoplasmic sperm injection (ICSI). Dr. Hiroyoshi HOSHI, who is5

a leader of the researches for improvement the follicle, oocyte and embryoculture system, will give a
focused lecture on in vitro growth, maturation and development of oocytes derived from bovine early
antral follicles. Finally, Professor Joy L. PATE (Former President of the Society for the Study of
Reproduction: SSR) will give a special lecture on cellular and molecular mechanisms regulating functionof the corpus luteum in cow. The corpus luteum (CL) dominates the estrous cycle, and so the regulation of
CL function is very important for management the transplanted embryos with SCNT. Her lecture will be
fruitful for all members of KSAR and JSAR.

6

RECENT ADVANCES IN ANIMAL BIOTECHNOLOGY IN KOREA
Sang-Jun UHM, Mukesh Kumar GUPTA, Ji-Hoon YANG, Young-Tae HEO, Hoon-Taek LEE
Departments of Biotechnology and Bioscience, and Animal Biotechnology
Bio-Organ Research Center, Konkuk University
1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Korea(htl3675@konkuk.ac.kr)
The importance of animal biotechnology is highly appreciated in Korea. Recently, animal
biotechnology researchers in government, university, institutes, and industries have tried its vast
application in bio-medicine, genetic engineering, transgenesis, cloning, stem cell, and production ofsuperior quality animals. This review focuses on animal transgenesis’ cloning, and stem cell research in
Korea. Through transgenesis, the mouse models for human diseases and animal bioreactors such as
chicken (enhanced green fluorescent protein gene), goat (bovine growth hormone gene), pig (humanerythropoietin and thrombopoietin genes), and cow (human lactoferrin gene) have been produced for
production of proteins of pharmaceutical importance. These animals were produced by utilization oftechniques such as pronucleus microinjection, gene recombination and transfer in somatic cells, andsomatic cell nuclear transfer (SCNT) techniques. The SCNT technique has also been used for conservation
as well as production of superior and rare animals. This technique has also been applied for the productionof bio-organs for xenotransplantation in human. In the arena of stem cell research, embryonic and adultstem cells have been applied for therapy of human incurable disease using mice and pig as model systems.
The Korean government promotes advancement of these researches for the next generation under networkwith industry, university and institute. In the present and future, the successful achievements of theseanimal biotechnology researches would contribute to bio-medicine, genetic engineering as well as
agricultural industries.

7

RECENT RESEARCH TRENDS IN ANIMAL REPRODUCTION AND BIOTECHNOLOGY IN
JAPANEimei SATO
Laboratory of Animal Reproduction, Tohoku University
Sendai 981-8555, Japan(eimei@mail.bios.tohoku.ac.jp)
Scientific activities regarding animal reproduction and technology in CJK regions are strong.
Japanese scientists in this field have joined several domestic societies including the Japanese Society ofAnimal science, Japanese Society of Veterinary Science, Japanese Society of Animal Reproduction (JSAR),
Japan Embryo Transfer Society, Japan Society of Fertilization and Implantation, Japanese Society of
Mammalian Ova Research and others.
JSAR has a role as a branch of the International Congress of Animal Reproduction and has
joined a working group to establish a new international society, the World Congress of Reproductive
Biology (WCRB) with the Australian Society of Reproductive Biology, Society for the Study of
Reproduction(SSR) in USA, Society for the Study of Fertility (SSF) in the UK and the first meeting isscheduled to be held in Hawaii in 2008.
History and activities of JSAR
JSAR was established in 1948 to encourage the study of reproductive biology in wild, domestic,laboratory, and companion animals. This was two years before the establishment of the British Society for
Reproduction and Fertility, SSF and 19 years before the US Society for the Study of Reproduction, SSR.
Members of JSAR have contributed to the cutting edge of research in reproductive biology in this country,
which promotes the use and development of high level biotechnology. Previously, members of JSAR haveplayed a key role in developing techniques for artificial insemination, fertilized egg transfer, and in vitro
fertilization to increase the productivity of animals. Currently, members are actively involved indeveloping new technologies, such as transgenic techniques and cloned animals. They are also involved inresearch on endocrine disruptors.
The society has about 850 members from academic, governmental, and business institutions aswell as veterinary clinicians. The interests of the members are necessarily diverse including sexual
maturation, oogenesis, spermatogenesis, ovulation, gestation, and lactation. There is also research onpractical themes such as the reproductive failure of dairy cows and in vitro fertilization. Several cloned
domestic animals, including cows, pigs and goats have been produced by members of this Society.
The Society holds an annual fall meeting at locations throughout the country at which members
and guests make presentations and hold symposia. Special lectures by foreign researchers are a highlight ofthe meetings. The Society has been publishing an official journal, the Journal of Reproduction andDevelopment (JRD) since 1955. This bimonthly journal is currently up to volume number 53. Both theabstracts and full-text articles are accessible online at: http://jrd.jstage.jst.go.jp/en/. Impact factor of JRD
was announced to be 1.300 and 1.149 on May, 2006 and 2007, respectively.
The JSAR Outstanding Research Award is given for outstanding basic and applied research inanimal reproduction. The JSAR Innovative Technology Award is given for research which contributed tothe development of innovative technology in animal reproduction. The JSAR Young Investigator Award isgiven for outstanding research in animal reproduction performed by an exceptionally promising young
investigator who has not reached the age of 35 before April 1st of the year when the award is presented.
JSAR made an academic agreement with the Korean Society of Animal Reproduction (KSAR) in 2005 to
hold a joint symposium of JSAR and KSAR every year. In 2007, the third Korean-Japanese Seminar on
Reproductive Biology is scheduled to be held at the University of Tokyo, on October 18 under the title of“Current Status and Perspectives in Reproductive Biotechnology and Safety Assessment of Cloning
Animals”.
Papers in the last 99th annual meeting of JSAR in 2006
The animal species in the papers presented at the 99th annual meeting1) are shown in Table 1.
Some 51.9% of the papers refer to work with laboratory animals. Domestic animals accounted for 43.0%.
The ratio of cattle and pigs is high in domestic animals. As for subjects, 29.1% of the papers were8

classified as technology in animal reproduction, including freezing and drying of gametes and transgenic,
animal cloning (Table2). A total of 70.9% of the papers were classified as reproductive biology. The fields
of endocrinology, gametogenesis and oocyte maturation, follicular development and luteinization werenumerous in the paper on reproductive biology. Our papers published in the international journal were
presented originally at the annual meeting of JSAR 2-5).
Recent progress in biotechnology research has been remarkable in Japan, as well as Korea. Since
the report of a cloned sheep by nuclear transfer using adult mammary gland cells, the birth of cloned
calves, pigs, goats and other domestic animals has been reported first or in succession in Japan. The ratioof papers related to animal cloning is also dramatically increasing for domestic and laboratory animals at
the annual meeting of JSAR.
Cloned animals have the greatest impact in animal biotechnology
The cloned sheep was produced from the viewpoint of animal husbandry and more efficient
breeding of good domestic animals. In the field of animal husbandry, animal biotechnology is often
considered to consist of two categories. One is techniques such as animal reproduction, animal breeding,animal feeding, animal hygiene, and animal management, which have developed on the basis of"traditional technology" and have already yielded a number of practical techniques that contribute to theindustry. The other is the technology that has emerged with the development of modern biology andconsists primarily of cell manipulation such as gene recombination and cell fusion. Techniques represented
by "cloned sheep" have been developed on the basis of both of these categories, and techniques such as
artificial insemination, microscopic insemination, selective production of males or females, in vitro
fertilization and embryonic transplantation, and storage of sperm and embryos by freezing have advanced
until they now play important roles in medicine, as well as animal industry.
The birth of the cloned sheep is influencing the frontier of research in animal biotechnology inJapan, as well as worldwide. It has effects on research of animal biotechnology itself by requiring new
research subjects, modifications to the strategy of ongoing research projects, and challenges to schemes
formerly considered impossible 6-7).
I object to restrictions on cloning research
Discussion over restrictions on cloning research has become heated since the birth of the "cloned
sheep". I agree that application of the cloning technique to humans must be made carefully, because it may
shake the foundation of humans as higher animals: Individuals die and they produce the next generation bysexual reproduction. However, I object to the contention raised by some people that cloning research ofeven domestic animals should be restricted. Cloning research has a major impact on various fields ofanimal biotechnology, and restricting research would cause great loss to society. Also, if the restrictions areapplied formally, it may lead to restrictions on animal biotechnology research itself, which would havemassive negative effects on society. I personally think that on cloning research using domestic animals
should go no further than associations formulating independent guidelines that investigators respect.
External report of the Science Council of Japan in 2000
Public interest in cloned animals is extremely high. However, lack of information and the
difficulty of the available information result in anxiety and skepticism over cloned animals. In thesecircumstances, we thought that it is necessary to establish a “Guideline Concerning Research on Clone
Individuals of Industrial Animals” prepared by scientists directly and indirectly involved in research intoclones of industrial animals, thus, promoting research on biotechnology, including research on cloned
animals. Thus, the Japan National Committee of Animal Science of the 17th Science Council of Japan
organized the “Subcommittee For the Manual of Research on Cloning of Industrial Animals” and has
intensively evaluated the issue. Then, in March 27, 2000, we announced our basic attitude to research oncloned individual industrial animals and the desirable state of research in the future. We made proposals
concerning the importance of public information and educational activities concerning clone research onindustrial animals and the need to establish a pioneer biotechnology research organization under the nameof the Chairman of Japan National Committee of Animal Science, Science Council of Japan, Dr. Seiki
Watanabe.
Guide for Studies on Industrial Animal Clones
The guide was released under the name of the Japan National Committee of Animal Science,
Japan National Committee of Veterinary Medicine, and Japan National Committee of Breeding of the 17th

9

Science Council of Japan. In this guide, several points were emphasized. In studies related to industrial
cloned animals, all investigators should promote studies beneficial for life science and strive to promote
both animal and human welfare, aim to move research forward with the ultimate goal of improving humanwelfare. To achieve these objectives, investigators must adhere to the basic principles listed below:(1) Investigators must adhere to national laws, regulations, policies and/or guidelines.
(2) In regard to foreign laws, regulations, policies and/or guidelines, basic common provisions must be
adhered to with due consideration of Japanese national laws, regulations, policies and/or guidelines
unless the content is based on specific religious beliefs and/or cultures.
(3) Studies that may conflict with (1) and (2) and those that may provoke social or ethical arguments must
be deliberated upon by consultation with related academic associations and society in general. For thispurpose, an ethical committee must be established in each research institution before the execution ofa study, and the scientific necessity, significance, influence on society and/or the ethical aspects of thestudy must be fully investigated. Discussion at the ethical committee must be recorded and archived,
and must be immediately disclosed upon request. In areas of controversy, the opinions of competentauthorities must be collected via the head of the institute.
Future State of Research stated by the Science Council of Japan in 2000
The development of animal biotechnology research represented by animal cloning studying will
have a very significant impact on the future of the Japanese animal industry in the 21st century. Applied life
science research is rapidly progressing worldwide, and to keep up with this, Japan must develop long-termplans and redress the balance. A keen desire to learn in future generations, inspiring the spirit of inquiry,
the training of researchers with a strong desire to advance science, and the execution of creative studies arevery important not only for the Japanese animal industry, but also for the general progression of applied
life science. In regard to these points, the promotion of the following items should be particularly focusedon:(1) Educational activity for industrial animal cloning research: To obtain sound public understanding ofindustrial animal cloning research and animal biotechnology, it is necessary to promote educational
activities to correct social prejudice and excess expectations. To establish the position of industrial
animal cloning research in the life science field, insemination of knowledge ranging from the basic
research level to general life-supporting mechanisms must be promoted without excessive bias ofapplication, and the educational efforts intended for the sound intelligent development of junior, highschool and university students aspiring to careers in applied life science will be supported. For
students in non-scientific fields, balanced and accurate knowledge will be made accessible to increase
and maintain high standard of public knowledge of animal biotechnology.
(2) Establishment of research organizations for advanced animal biotechnology including clone research:
For the development of advanced animal biotechnology in Japan, it is essential to establish a strong
research system including basic life science such as embryology, reproductive biology, cell biology,
molecular biology, genetics, animal behavior, and ecology, in addition to related fields such as
traditional animal production, veterinary medicine, and breeding. Success in the production of cloned
animals is based on the long history of basic research that started with the production of Xenopus
laevis by somatic cell nuclear transfer achieved by Dr. J.B. Gordon in the 1960’s in England. The
production of cloned sheep would not have been possible without the tradition and tremendous
accumulation of the results of basic studies in England on the molecular and cell biology related to thecell cycle and regulatory mechanisms underlying expression of genetic information stored inchromatin. When the reprogramming mechanisms in the nucleus of differentiated cells are elucidated
in the near future following the rapid progress in genome research in domestic animals, epoch-making
biotechnology may be developed. The same will be true life sciences fields other than animal
biotechnology.
To develop new biotechnology for the animal industry keeping pace with Western countries, it isessential to establish a comprehensive research organization for advanced applied animal science with astrong research system. Thus, the Japan National Committee of Animal Science strongly and actively
promote the establishment of the research organization described above.
References
1) Journal of Reproduction and Development, 52 (suppl.): J1-146, 2006 (in Japanese)
2) Yokoo,M., Sato,E. Cumulus-oocyte complex interactions during oocyte maturation. International
Review of Cytology, 235: 251-291, 2004.

10

3) Abe,Y., Hara,K., Matsumoto,H., Kobayashi,J., Sasada,H., Ekwall,H., Rodriguez-Martinez,H., Sato,E.
Feasibility of a nylon mesh holder for vitrification of bovine germinal vesicle oocytes in subsequent
production of viable blastocysts. Biology of Reproduction, 72:1416-1420, 2005.
4) Tanemura,K., Ogura,A., Cheong,C., Gotoh,H., Matsumoto,K., Sato,E., Hayashi,Y., Lee,H.W.,
Kondo,T. Dynamic rearrangement of telomeres during spermatogenesis in mice. Developmental
Biology, 281:196-207, 2005.
5) Miyake,Y., Matsumoto,H., Yokoo,M., Miyazawa,K., Kimura,N., Tunjung,W.A.S., Shimizu,T.,
Sasada,H., Aso,H., Yamaguchi,T., Sato,H.: Expression and glycosylation with polylactosamine of
CD44 antigen on macrophages during follicular atresia in pig ovaries. Biology of Reproduction, 74:
501-510, 2006.
6) Sato,E. A new frontier of animal biotechnology research with the birth of the cloned sheep. Journal of
Mammalian Ova Research, 16: 83-85, 1999.
7) Sato,E., Yoshida,N., Kimura,N., Yokoo,M. New frontiers of animal biotechnology in the field ofanimal reproduction. Asian-Australasian Journal of Animal Sciences, 14 (suppl.): 19-27, 2001.
8) Japan National Committee of Animal Science: External report of Science Council of JapanEstablishment of the “Guideline concerning research on clone individuals of industrial animals”.
Journal of Mammalian Ova Research, 18: 130-132, 2001 (the original was published in 2000 in
Japanese).
Table 1. Animal species in the papers at the annual meeting of JSAR and SSR

Domestic animals
cattle
horse
pigs
sheep
goat
mink
dog
cat
chickenquail
Human
Laboratory animals
mouse
rat
hamsterguinea pig
rabbitmonkeyMastomys natalensis
Suncus murinus
Wild animals
fish
Other(cultured cells)

JSAR

SSR

77 (43.0%)
39
1
24
0
5
0
3
2
3
0
1 (0.6%)92 (51.9%)
50
32
2
0
1
3
1
1
2 (1.1%)2 (1.1%)1 (0.6%)195 (36.8%)
76
8
44
49
1
1
1
3
11
1
40 (7.6%)
235 (44.3%)
151
59
5
2
2
16
0
0
15 (2.8%)
15 (2.8%)30 (5.7%)

JSAR: The 99th annual meeting of JSAR at Nagoya, SSR: The 39th Annual meeting of SSR at Omaha.
Note: There are papers using two or more animal species.

11

Table 2. Subjects in the papers at the annual meeting of JSAR and SSR

Reproductive technology
Freezing and freeze-dry of gametes
X and Y sperm
Transgenic
IVF and ICSI
ES cells and stem cells
Clone and parthnogenesis
Transplantation of gonads
Reproductive biology
Sex differentiation
endocrine
gametogenesis and maturation
gonadal function 8 18
follicular development and luteinization
fertilizationembryogenesis
implantation and placenta
Fallopian tube and uterus
pregnancy and delivery
sexual maturation
Reproductive immunology
Comparative animals
Stress, chemicals and irradiation
DNA imprinting
cancersexual behavior
lactationothers
Total

JSAR

SSR

51(29.1%)
13
1
9
5
7
13
3
124(70.9%)
2
25
30
8
17
0
10
6
4
5
0
0
1
5
5
0
4
2
0
175

54(10.4%)
11
1
18
1
5
17
1
464(89.6%)
12
75
76
18
77
4
37
37
30
17
4
4
4
22
8
22
1
2
12
516

JSAR: The 99th annual meeting of JSAR at Nogoya, SSR: The 39th Annual meeting of SSR at Omaha
Note: There ere papers working in 2 or 3 categories. ICSI: intracytoplasmic sperm injection. ES cells:
embryonic stem cells

12

PRESENT STATUS OF SOMATIC CELL CLONED CATTLE PRODUCED IN JAPANShinya WATANABE
National Institute of Livestock and Grassland Sciences
Tsukuba 305-0901, Japan(shw@affrc.go.jp)

Since the first somatic cell cloned calves were born in Japan (Kato et al., 1998), 511 somatic cellcloned cattle have been produced in this country [as of September, 2006; press release from Ministry ofAgriculture, Forestry and Fisheries, Japan (http://www.s.affrc.go.jp). According to the press release data,
which is accumulated from 1998 to 2006 (September), 68% of newborns were surviving the perinatal
period. However, improvement in efficiency of cloned cattle production is still on the way. Althoughvoluntary moratorium on cloned cattle products was made by the Japanese government on November,
1999, and has remained in effect even now, studies on cloned cattle and their offspring have been
conducted by many institutes in Japan. Namely, it have been reported at least 64 papers, which employed
171 clones and 32 offspring of clones, since 2000. These studies covered following seven research fields:(1) Genetic similarities and muzzle prints, (2) Hematology and clinical chemistry findings, (3) Pathology,(4) Growth performance, (5) Reproductive performance, (6) Meat productive performance and (7) Milk
productive performance. No remarkable differences in health status and reproductive performance were
found among conventionally bred cattle, somatic cell cloned cattle surviving to adult-hood and offspring ofsomatic cell cloned cattle. Similarities in growth performance and meat quality were observed between
nuclear donor cattle and their clones. Also, growth curves of the offspring and their full siblings wereresembled. As for the milk and meat produced from cloned cattle, studies on composition analysis andtoxicology have been carried out in our country (Japan Livestock Technology Association, 2002). This
study concluded that there were no remarkable differences between products from clones and comparators,
and no harmful effects attributable to these products. Another data accumulation concerning milk and meatproduced from offspring of clones is progressing since 2004 in a “Research Project for Utilizing Advanced
Technologies in Agriculture, Forestry and Fisheries” from the Agriculture, Forestry and Fisheries ResearchCouncil, Ministry of Agriculture, Forestry and Fisheries, Japan. Conclusions of this study might appear inthis autumn.

13

MICRORNA SYSTEM IN FERTILIZED AND CLONED MOUSE EMBRYOS
Nam-Hyung KIM
National Research Laboratory of Molecular Embryology, Department of Animal Science, Chungbuk
National University
Cheongju, 12 Gaeshin-dong, Heungduk-gu, Vheongju, Chungbuk 361-763, Korea(nhkim@chungbuk.ac.kr)
MicroRNAs (miRNAs) are small non-coding RNA molecules of 22 nucleotides in length thatcan post-transcriptionally regulate gene expression by base-pairing to mRNAs. To elucidate the role ofmicroRNA pathway in early embryo development, we initially evaluated gene expression of dicer, GW182
and Ago2 in mouse oocytes and embryos in vitro. The transcript levels of these genes in GV stage oocytes
steadily decreased up to the 2-cell embryo stage, and expression remained stable during morulae andblastocyst formation. Their protein syntheses were additionally observed in mouse oocytes and early
embryos. Silencing of their mRNA expression by RNA interference (siRNA) did not inhibit development
up to the blastocyst stage. However, significantly, enhanced apoptosis and lower cell numbers wereobserved in siRNA-treated blastocysts. Real time RT-PCR experiments confirmed the decreased
expression of selected transcription factors, including POU domain, class 5, transcription factor 1 (Pou5f1),
SRY-box containing gene 2 (Sox2), and Nanog homeobox (Nanog). Moreover, POU5F1 protein
expression was suppressed by Dicer1 or QW182 siRNA. Our data showed that microRNA pathway isimplicated in gene transcription regulation at the blastocyst stage.
In addition, in order to get insights into the role of miRNAs in early embryogenesis, mouse
fertilized and cloned embryos were chosen as model to investigate various miRNAs, such as miR-15a, -16,
-21 and -302. TaqMan® MicroRNA Assays were employed to quantify the precise expressions of thesemiRNAs. The results revealed that expression of miR-15a, miR-16 and miR-21 expression were
down-regulated from zygote to 2-cell and then gradually increasing from 4-cell to blastocyst in
vitro. Interestingly, the significantly higher expressions in in vivo-and cloned derived blastocysts were
found for three miRNAs compared with that in in vitro-derived blastocysts. In general miR-15a wasexpressed lowestly in preimplantation embryos among the three miRNAs, while miR-16 was the highest
one. As the low levels of miR-15a, we carried out the microinjection of mature miR-15a and -302 into
fertilized and cloned zygotes. Data showed that miR-15a and 302 expression were significantly increased
in microinjected blastocysts. Although their microinjection did not affect the embryo development up to
blastocyst stage, significantly higher apoptosis in microinjected blastocysts was observed (p<0.05). Taken
together, the changes in miR-15a, 16, 21 and 302 expressions in fertilized and cloned embryos strongly
indicated their possible role in early embryogenesis.

14

PRODUCTION OF CLONED CATS AND DOGS BY SOMATIC CELL NUCLEAR TRANSFER
Il-Keun KONG
Division of Applied Life Science, Gyeongsang National University
Jinju 660-701, Gyeong-Nam Province, Korea(ikong@sunchon.ac.kr)
The somatic cell nuclear transfer (SCNT) technique has been used to produce cloned animals invarious species, including cattle, sheep, goat, pig, cat and dog. Domestic cat and dog is a useful researchmodel to develop assisted reproductive technologies for the conservation of endangered felids and canis.
The current applications for cat and dog cloning include targeted genotype replication of pets and the
production of models for the study of human and animal diseases. A second point of application is the
production of transgenic cats and dogs, genetically engineered “designer pets” with favorable attributes,
such as an allergen-free cat and a mini-cat. To date several groups have successfully cloned kittens andpups (Gomez et al., 2004; Shin et al., 2002; Yin et al., 2005; Lee et al., 2005). The cloned male cats havenormal reproductive success and lie within the normal range of gonadal hormone production. All F1
kittens were produced by natural breeding and delivery, and are still alive and display normal healthy
growth. Serial cloning is an excellent way to study the effect of progressively accumulating somatic
mutations on development, health and reproductive performance. Moreover, recloning existing cloned
fetuses or animals has the distinct advantage of reproducing unique genetic characteristics. We were also
the first to successfully produce a cloned transgenic cat expressing an exogenous red fluorescence protein.
Whole body red fluorescence was detected in the liveborn transgenic cats, but not in the non-TG cat at
more than 2 months after birth. Red fluorescence was detected in tissue samples, including hair, muscle,
brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue and even excrement from thetransgenic cat. Our nuclear transfer procedure using genetically modified somatic cells will be useful forthe efficient production of transgenic cats. The cloned dog, Snuppy, was produced from in vivo ovulated
and matured oocytes instead of in vitro matured oocytes, because nobody has been able to establish an invitro maturation system yet. A cloned toy poodle was also cloned from somatic cells derived from an aging(14 year-old) female dog, and a wolf. The application of SCNT procedure to produce genetically modified
cat and dog will be valuable for the biomedical modeling of human disease, as well as for the productionof designer pets.

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FROM SOMATIC CELL TO STEM CELL THROUGH NUCLEAR TRANSFER
Teruhiko WAKAYAMALaboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN
Kobe 650-0047, Japan(teru@cdb.riken.go.jp)

Mice can now be cloned from cultured and non-cultured, adult-, fetus-, male-, or female-derived
cells. Recently, we were able to improve the success rate of cloning in mice up to six times using dimethyl
sulfoxide (DMSO), trichostatin A (TSA) or by serial cloning using NT-ES cell technique. However, theoverall efficiency of mouse cloning is less than 10% and the offspring that survive to term have shown
many abnormalities, which are probably caused by incomplete genomic reprogramming after nuclear
transfer into the oocyte cytoplasm. Even the technique itself remains imperfect, embryonic stem (ES) celllines can be generated from adult somatic cells via nuclear transfer. These cells, called ntES cells, exhibit
full pluripotency in which they can be made to differentiate along prescribed pathways in vitro, andcontribute to the germ line following injection into blastocysts. We have demonstrated that those ntES cells
are indistinguishable from ES cell derived from fertilized embryos. For safety application of thesetechniques to regenerative medicine, there are several problems which have to be solved. For example, it
has been widely assumed that fresh oocytes are required for nuclear transfer and the establishment of ntES
lines, or derivation of ES or ntES cells arises from the need to destroy the embryos. Several approaches
that circumvent this problem have recently been reported, such as ES cell derivation from single
blastomere without destroy the embryo, or use aged fertilization-failure (AFF) oocytes as recipient, whichusually discarded in infertility clinics. In this symposium, I will talk about the similarity between ntES and
ES cells, and the possibility to establish ES/ntES cell lines without ethical problem.

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THE HISTONE DEACETYLASE, SCRIPTAID, RESCUES FULL-TERM DEVELOPMENT IN
CLONED INBRED MICE BY ENHANCING ZYGOTIC NASCENT MRNA PRODUCTION
Nguyen Van THUAN
Department of Animal Biotechnology, Konkuk University
1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Korea(Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, Kobe, Japan)(vanthuan@konkuk.ac.kr)
Inbred mouse models serve as a biological test ground and genetic background is an importantcriterion when selecting a strain for a particular experiment. Moreover, inbred mouse strains such as
C57BL/6 are used for transgenesis research to identify functional genes or as genetic models for diseasesusceptibility via somatic cell nuclear transfer (SCNT). Since Cumulina, the first B6D2F1 mouse clone
produced by somatic cell SCNT (Wakayama, 1998), the success rate of cloning in mice has been extremely
low compared with other species. Most of the important inbred mouse strains, such as C57BL6 and C3H,
have never been cloned, except for the DBA2 strain (Wakayama, 2001) and mouse strain 129 (Inoue et al.,2003) but this was achieved with extremely low efficiency. A variety of approaches have been used withthe aim of improving the efficiency of SCNT cloning: changing the timing and conditions of activation andfusion, treating donor cells or SCNT embryos with dimethyl sulfoxide, cyclin-dependent kinase inhibitor(roscovitin), or 5-aza-2′-deoxycytidine; altering the timing of removal of the oocyte chromosomes; serial
nuclear transfer; removal of donor cell cytoplasm; and clone–clone aggregation. Recently, our laboratory
found that treatment of SCNT mouse embryos with 50–100 nM Trichostatin A (TSA) for 10 h after nuclear
transfer can enhance the generation of cloned B6D2F1 mice (Kishigami, 2006). However, application ofthese methods to inbred mouse strains such as B6 and C3H has never been successful.
Here, we show for the first time that treatment of SCNT embryos with scriptaid (SCR), a histone
deacetylase inhibitor with low toxicity that enhances transcriptional activity and protein expression, all theimportant inbred mouse strains can be cloned, such as C57BL6, C3H, DBA2 and 129. Moreover, using
SCR treatment, we obtained healthy GFP-expressing male ICR mice with donor cells collected from thetail of a 2.5 year-old GFP-expressing ICR mouse. Most importantly, the placentae of cloned mice weresignificantly smaller than those of untreated control clones, although they were still heavier than theplacentas of ICSI-fertilized fetuses. In addition, our results clearly show that the full-term developmental
ability of cloned mouse is donor cell strain-dependent. Moreover, the success of somatic reprogrammingand cloning efficiency via NT technique is clearly linked to the competent synthesis of nascent mRNA atthe 2-cell embryo stage. We also found that increases of histone acetylation in the pronuclei of cloned
1-cell embryos is essential for somatic cell chromosome reprogramming, but is not sufficient for full
reprogramming of somatic cells to allow full-term development. In this symposium, I will discuss thecharacterization of somatic nuclear remodeling and reprogramming and the correlation between histone
acetylation and transcriptional activities during preimplantation and full-term development of SCNT
embryos in mice.

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TRANSGENESIS IN RATS
Naomi KASHIWAZAKI
Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University
Sagamihara 229-8501, Japan.
(nkashi@azabu-u.ac.jp)
The rat is one of the most important experimental animals for biomedical andphysiological research. During the last two decades, there have been advancements in assisted
reproductive technologies (ARTs), genetic engineering and molecular biology. The synergy has
allowed genetic modifications including transgenesis. Transgenic rats are extremely valuable
for studying, and can be produced by exogenous DNA microinjection into the pronucleus aswell as intracytoplasmic sperm injection (ICSI) with exogenous DNA. Transgenesis throughDNA microinjection accounts for most of the rat transgenic lines, which were over one hundred.
Cryopreservation of rat gametes and embryos has also been developed to bank the genetic
resources efficiently. However, transgenesis in rats is still hampered by the lack of embryonicstem cell lines in which generation of knockout (KO) mice by homologous recombination isroutinely used. Although French research group reported generation of cloned rats to control
oocyte activation through the targeting of proteasome and kinase inhibitors in 2003, this cloning
approach that enables to produce KO rats, is not available yet. The first KO rat models, on theother hand, were generated in 2003 by the combination of N-ethyl-N –nitrosourea (ENU)
mutagenesis and a screening assay. The application of cryopreserved sperm, ovaris and ICSI
will likely increase the generation of KO rats from ENU-mutagenized males. In this symposium,
I will talk about ARTs related to transgenesis in rats.

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IN VITRO GROWTH, MATURATION AND DEVELOPMENT OF OOCYTES DERIVED FROMBOVINE EARLY ANTRAL FOLLICLES
Hiroyoshi HOSHI, Joon-Ho CHO, Takehiro ITOH and Yutaka SENDAI
Research Institute for the Functional Peptides
Yamagata 990-0823, Japan(hoshih@func-p.co.jp)
Mammalian ovary contains a large number of resting follicles. Limited numbers of these follicles
are activated to enter the growth phase characterized by proliferation of granulosa cells and by increase insize of the oocytes. However, most of these follicles gradually become atretic during in vivo growth. The
large store of these small follicles creates a potential source of oocytes for animal reproduction,conservation of endangered animals and human infertility therapy. So far, it has been more difficult toestablish a complete in vitro culture system for growing oocytes of early antral follicles from cattle.
Essential factors that could achieve a full growth of oocytes from the small follicles have not been fully
understood yet. We have recently established a novel culture system “cumulus-oocyte complexes with
granulosa cells (COCGs) cultured on collagen-coated flat substratum” which was beneficial for along-term survival of growing oocytes from bovine early antral follicles. This improved culture system
was applied for identifying factors which could attain the complete growth of oocytes from bovine early
antral follicles (follicle diameter; 400-700 µm, oocyte diameter; 90-99 µm).
In the present study, KGF, also known as FGF 7, was found to be important factor for in vitro
growth of oocytes. After a subsequent in vitro maturation, the in vitro grown oocytes exposed to KGF
increased the rate of meiotic competence (MII phase) compared to the control. Furthermore, the in vitro
grown oocytes cultured in KGF-containing medium resulted in high rate of developmental competence
after in vitro maturation, fertilization and embryo culture. Preliminary results indicated that KGF
stimulated the oocyte growth directly via KIT ligand (KITLG), KITLG receptor (KIT) signaling pathway
in bovine COCGs from the early antral follicles. These results may support the in vitro production of large
numbers of fully grown oocytes for embryo transfer and nuclear cloning.

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CELLULAR AND MOLECULAR MECHANISMS REGULATING FUNCTION OF THE CORPUS
LUTEUM
Joy L. PATE
Department of Animal Sciences, Ohio Agricultural Research and Development Center,
Ohio State University,
2029 Fyffe Road, Columbus, OH, USA(pate.1@osu.edu)
In cows, the corpus luteum (CL) dominates the estrous cycle, because it persists for the majorityof the cycle, and as long as progesterone is elevated, ovulation of another follicle is prevented, due tonegative feedback on GnRH/LH release. Near the end of the estrous cycle, the uterus releases
prostaglandin F2α, which causes regression of the CL, allowing a new follicle to ovulate. If an embryo ispresent, it is critical that the CL be rescued from luteolysis and continue progesterone production, an event
known as maternal recognition of pregnancy. Despite the central importance of the CL in regulation of the
estrous cycle and maintenance of pregnancy, the exact mechanisms that regulate whether the CL regresses
or is maintained are poorly understood. For some time, a focus of our research has been to understand themechanism of action of PGF2α on luteal cells that would initiate luteolysis. During the studies of functional
effects of the prostaglandin on luteal cells, it was observed that, although PGF2α effectively inhibited
LH-stimulated progesterone, it did not kill luteal cells in culture. This was very surprising, because it iswell accepted that PGF2α is the luteolytic agent in the cow. The latter observation provided the first cluethat the action of PGF2α in the CL might require participation of cell types in addition to the steroidogenic
cells represented in the culture system. The CL is a very heterogeneous tissue. Abundant endothelial cells,
pericytes, extracellular matrix fibroblasts, and immune cells all contribute to support of the steroidogenic
cells. Much of the work on the role of endothelial cells has been done here in Japan. In the past few years,
considerable data have been collected in this laboratory and others to support the hypothesis that theimmune system may be involved in regression of the corpus luteum. The proinflammatory cytokines,
IFN-γ and TNF-α are cytotoxic to luteal cells. Class II MHC molecules are expressed in the CL,
suggesting that antigen presentation may serve to activate the resident lymphocytes to produce theproinflammatory cytokines. In fact, cultured luteal cells are potent stimulators of T cell proliferation invitro. However, we have recently determined that the majority of MHC molecule expression within the CL
is localized to the capillary endothelial cells at midcycle, and may serve a completely different role thanactivation of immune cells to a proinflammatory state. Further, analysis of luteal cell-stimulated T
lymphocytes by flow cytometry revealed that the class of T cells that proliferated to the greatest extent wasthe γδ set of T cells. Since γδ cells can be either pro- or anti-inflammatory and typically respond to self
antigens rather than being MHC restricted, entirely new possibilities for immune cell regulation of luteal
function will be proposed.

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