The 2nd Joint Symposium of KSAR & JSAR

“Novel Approaches to Mammalian Oocytes by Young Scientists from East Asia”
Busan Exhibition & Convention Center (BEXCO),Busan, Korea September 19, 2006

Abstracts from Japanese Speakers

General introduction “Novel Approaches to Mammalian Oocytes by Young
Scientists from East Asia”
Takashi MIYANOFirst successful in vitro fertilization of bovine and porcine oocytes were achieved in Japan in 1970s.
Since then, the Japanese researchers have been leading the study of Animal Reproduction,
especially in vitro maturation and fertilization of oocytes and in vitro culture of embryos. Theprogress has been accelerated by the introduction of the oocyte micromanipulation technology, suchas ICSI and somatic cell nuclear transfer for producing cloned animals. Besides Japan,
oocyte-manipulating technology in Korea has also been accelerated recently, and now the Korean
Reproductive Biology has become the front-runner in this field. These technologies have beenapplied to animal reproduction as well as human infertility treatment. In the Reproductive Biology
in Korea and Japan, some basic studies approaching mammalian oocytes have started, such as basicmolecular and cellular mechanisms underling the oocyte maturation-fertilization and embryo
development in mammals. The purposes of the seminar are to organize the new knowledge ofOocyte Science from both Korean and Japanese sides and to establish the cooperation between
young scientists.

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Shugoshin protects centromeric cohesion of sister chromatids
Tomoya KITAJIMA
Meiosis consists of two rounds of chromosome segregation following a single replication of theDNA to produce haploid gametes. Cohesion between sister chromatids is established during S phase,and homologous chromosomes are also physically linked by chiasmata, which are formed byrecombination. At the anaphase onset of meiosis I, cohesion is destroyed along the chromosomearms, leading to resolving chiasmata, and thereby homologous chromosomes segregate to the
opposite sides. However, at centromeres cohesion is protected to hold sister chromatids together
until meiosis II. We identified the novel protein Sgo1 (shugoshin), which protects centromericcohesion of sister chromatids in fission yeast meiosis. We also show that shugoshin homologues inhuman protect centromeric cohesion during mitotic pro- and prometaphase.

3Mechanism of chromosome separation in mammalian oocyte maturation
Jibak LEE
Progression of anaphase (sister chromatid separation) in somatic cells requires anaphase-promoting
complex/cyclosome (APC/C)-mediated degradation of Securin and Cyclin B. After degradation ofSecurin, Separase is activated and can cleave cohesin protein which connects sister chromatids until
the onset of anaphase. In meiosis, however, homolog separation does not require theAPC/C-mediated degradation in Xenopus laevis. Here I will introduce recent our study
investigating the function of the essential APC/C activators, Cdc20 and Cdh1 in mouse oocytes.
Further, I will also introduce our study on meiosis-specific cohesin Rec8 in mammalian oocytes.

4Mechanism for establishment of sex-specific imprinting in mouse gametogenesis
Yayoi OBATA
Oocyte- and sperm-specific imprinting / DNA methylation, generated in mammalian gametogenesis
is one of the requirements of a functional gamete. By now none of the specific mechanisms for
sex-specific DNA methylation have been fully understood. To clarify this issue, bipotential germ
cells were transferred into fully grown oocytes, and DNA methylation of imprinted genes wasanalysed in reconstituted oocytes after 5 days culture. The results showed that both Igf2r and H19
were methylated in oocytes, reconstituted by non-growing oocytes with absence of oocyte-specificimprinting. This suggests that non-growing oocytes have no blocking mechanism for H19
methylation.

5The immunoglobulin superfamily protein “Izumo” is required for sperm to fusewith eggs
Naokazu INOUE
Representing the 60 trillion cells that build a human body, a sperm and an egg meet, recognize eachother, and fuse to form a new generation of life. The factors involved in this important membranefusion event, fertilization, have been sought for a long time. Recently, CD9 on the egg membrane
was found to be essential for fusion, but sperm-related fusion factors remain unknown. Here, byusing a fusion-inhibiting monoclonal antibody and gene cloning, we identify a mouse sperm fusion
related antigen and show that the antigen is a novel immunoglobulin superfamily protein. We havetermed the gene Izumo and produced a gene-disrupted mouse line. Izumo -/- mice were healthy but
males were sterile. They produced normal-looking sperm that bound to and penetrated the zona
pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of
the antibody against human Izumo left the sperm unable to fuse with zona-free hamster eggs.

6Maternal contribution of the nucleolus to the zygote
Sugako OGUSHI
Oocytes and spermatozoa are equipped with complementary organelles and molecules necessary forthe fertilization and further development. We found that the nucleolus in the oocyte is one of suchorganelle. Fully grown mammalian oocyte have large compact nucleoli, in contrast to the absenceof nucleoli in spermatozoa. Since the oocyte nucleolus is transcriptionally inactive and disappears
during maturation, the role of the nucleolus in maturation, subsequent fertilization and early
development has not been elucidated. In the present study, pig oocytes at the germinal vesicle(GV)-stage were enucleolated (removal of the nucleolus by micromanipulation), and subjected to in
vitro maturation followed by intracytoplasmic sperm injection (ICSI) or electro-stimulation toenable the pronucleus formation and early development. In summary, the prominent nucleoli in pig
GV oocytes are not required for oocyte maturation and fertilization, however, they are indispensable
for nucleolus formation in male and female pronuclei in the zygote. This confirms that thenucleolus in fertilized oocytes is of maternal origin.

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Generation of Progeny from Embryonic Stem Cells by Microinsemination ofMale Germ Cells from Chimeric Mice
Eiji Mizutani
Mice chimeric for embryonic stem (ES) cells have not always successfully produced ES-derivedoffspring. Here we show that the male gametes from ES cells could be selected in male chimericmice testes by labeling donor ES cells or host blastocystes with GFP. Male GFP-expressing
ES-derived germ cells occurred as colonies in the chimeric testes, where the seminiferous tubules
were separated into green and non-green regions. When mature spermatozoa from green tubules
were used for microinsemination, GFP-expressing offspring were efficiently obtained. Using areverse study, we also obtained ES-derived progeny from GFP-negative ES cells in GFP-labeled
host chimeras. Furthermore, we showed this approach could be accelerated by using roundspermatids from the testes of 20-day-old chimeric mice. Thus, this technique allowed us to generate
the ES cell-derived progeny even from the low contributed chimeric mice which can not produce
ES-origin offspring by natural mating.

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