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Institute for Biogenesis Research
Research Description

Ryuzo Yanagimachi, D.Sc.
Professor Emeritus

 

Profile:
Founder and First Director of the
Institute for Biogenesis Research

 

 

Phone:  (808) 956-8746
Fax:  (808) 956-7316
Email: yana@hawaii.edu
Biomedical Tower, IBR Building Manoa Campus

Yana1

D.Sc. Embryology, Hokkaido University, Japan 1960
B.Sc. Biology, Hokkaido University, Japan 1953

Reproductive Biology

For nearly four decades, Dr. Ryuzo Yanagimachi (fondly nicknamed “Yana”) has worked on pioneering research on gametes and fertilization biology, assisted reproduction, and biotechnology, including the therapeutic use of cloning technology.  His discoveries are of such fundamental importance in reproductive and developmental biology, that they have now been taught in textbooks for many years.

Yana is tireless in pushing the leading edge of his field.  To accomplish this, one of his visions was to establish a center of excellence for the study of reproductive and developmental biology.  In 2000, he founded and became the first Director of the Institute for Biogenesis Research (IBR), which is housed in the Department of Anatomy, Biochemistry and Physiology.  The IBR faculty, which began only with Yana, has grown to include graduate faculty members, ranging from junior to senior level and whose research programs are supported by grants from the National Institutes of Health and private foundations.  Today, research in the IBR has expanded to include stem cell biology, genome structure and its role in embryogenesis, transgenesis technology, and the elucidation of molecular mechanisms during normal and abnormal embryo development.

  Grad
 

Yana dons full regalia to honor Kellie L. K. Tamashiro, his last M.S. student (2000) who graduated at University of Hawaii before he retired. Kellie was one of over 100 graduate students and postdoctoral fellows mentored by Yana over the past four decades.

Although Yana has retired recently, he shows no signs of slowing down and continues to do research.  He has over 300 publications and still counting.  He has mentored over 100 young people in their graduate and postdoctoral research.  While he no longer officially mentors graduate students, his door is always open to young people seeking his scientific advice.

The following is Yana’s own words, putting into perspective his discoveries and numerous accomplishments.  May they inspire the next generation of scientists…

Summary of the Work and Publications by Ryuzo Yanagimachi
(Listed more or less chronologically)

(1) Pioneering study of in vitro fertilization (IVF)

I considered that IVF was the only way to precisely analyze the process and mechanism of mammalian fertilization.  We succeeded in fertilizing golden hamster eggs in vitro without any contributions from the female genital tract.  This was considered the first successful in vitro capacitation of mammalian spermatozoa.  Later, I succeeded in capacitating guinea pig spermatozoa and fertilizing the eggs in vitro.  Dr. Lopata and I were among the first to observe sperm penetration into human eggs in vitro.

Yanagimachi, R. and Chang, M.C. 1963. Fertilization of hamster eggs in vitro. Nature 200:281-282.

Yanagimachi, R. 1972. Fertilization of guinea pig eggs in vitro. Anatomical Record.174:9-20.

 
IVF
 

The first demonstration of mammalian IVF. A hamster sperm penetrated the egg coat called zona pellucida and is about to fertilize the egg in a culture dish.

The first demonstration of mammalian IVF. A hamster sperm penetrated the egg coat called zona pellucida and is about to fertilize the egg in a culture dish.

(2) Pioneering study of sperm capacitation, acrosome reaction and hyperactivation

When I started to work on mammalian fertilization, the phenomenon of sperm acrosome reaction was already known, but no one but Dr. C.R. Austin had paid attention to this important biological process.  I was the first to start analyzing the mechanism of the acrosome reaction in mammals.  Later, I discovered a phenomenon of sperm hyperactivation, which, like the acrosome reaction, is essential for normal fertilization.

Yanagimachi, R. 1969. In vitro acrosome reaction and capacitation of golden hamster spermatozoa by bovine follicular fluid and its fractions. Journal of Experimental Zoology 170:269-280.

Yanagimachi, R. 1970. The movement of golden hamster spermatozoa before and after capacitation. Journal of Reproduction and Fertility 23:193-196.

Katz, D.F. and Yanagimachi, R. 1981. Movement characteristics of hamster and guinea pig spermatozoa upon attachment to the zona pellucida. Biology of Reproduction 25:785-791. .pdf

(3) Discovery that sperm acrosome reaction is essential not only for sperm penetration through the zona pellucida, but also for sperm fusion with egg

We discovered that sperm acrosome reaction is essential not only for sperm passage through the zona pellucida, but also for membrane fusion between sperm and egg.  Calcium-dependence of sperm-egg fusion was first reported by us.

Yanagimachi, R. and Noda, Y.D. 1970. Physiological changes in the post-nuclear cap region of mammalian spermatozoa: A necessary preliminary to the membrane fusion between sperm and egg cells. Journal of Ultrastructure Research 31:486-493.

Yanagimachi, R. 1988. Sperm-egg fusion. In: Duzgunes, N. and Bronner, F, editors. Current Topics in Membranes and Transport, Vol. 32. Florida: Academic Press. p. 3-43.

Takano, H., Yanagimachi, R. and Urch, U. 1993. Evidence that acrosin activity is important for the development of fusibility of mammalian spermatozoa with oolemma: Inhibitor studies using the golden hamster. Zygote 1:79-91.

(4) Examination of fertilization processes in living state and by electron microscopy

I was the first to examine the entire process of sperm penetration through the zona pellucida (of the golden hamster egg) using the phase-contrast microscopy.  Later the processes of zona penetration and of sperm fusion with the egg were examined in detail by electron microscopy.

Yanagimachi, R. 1966. Time and process of sperm penetration into hamster ova in vivo and in vitro. Journal of Reproduction and Fertility 11:359-370.

Yanagimachi, R. and Noda, Y.D. 1970. Electron microscope studies of sperm incorporation into the hamster egg. American Journal of Anatomy 138:429-462.

Yanagimachi, R. and Phillips, D. 1984. The status of acrosomal caps of hamster spermatozoa immediately before fertilization in vivo. Gamete Research 9:1-19

 
Fig4

Transmission electron micrographs of hamster sperm fertilizing egg. Left: A view of the sperm head 3 minutes after insemination. Egg microvilli, the cytoplasmic extensions, are contacting sperm plasma membrane. Right: Microvilli fused with sperm plasma membrane. Egg cytoplasm is in direct contact with sperm nucleus (black, triangular structure).

(5) Use of lectins for the studies of gamete membranes and fertilization

We used plant lectins to demonstrate “mobility” of sperm plasma membrane components and the importance of saccharide moiety of zona pellucida glycoproteins for sperm-zona interaction.

Oikawa, T., Yanagimachi, R. and Nicolson, G.L. 1973. Wheat germ agglutinin blocks mammalian fertilization. Nature 241:256-256.

Nicolson, G.L. and Yanagimachi, R. 1974. Moility and the restriction of mobility of plasma membrane lectin-binding components. Science 184:1294-1296.

Yanagimachi, R. and Nicolson, G.L. 1976. Lectin-binding properties of hamster egg zona pellucida and plasma membrane during maturation and preimplantation development. Experimental Cell Research 100: 249-257.

(6) Use of zona pellucida-free hamster eggs for assessment human sperm fertility and examination of human sperm chromosomes

After finding that zona-free hamster eggs can fuse with acrosome-reacted spermatozoa of a wide variety of other species, inducing humans, we proposed that zona-free hamster eggs can be used to assess fertilizing potential of human spermatozoa.  We found that we can analyze chromosome constitution of individual spermatozoon after its entry in zona-free hamster egg.  This was the first system allowing us to examine chromosome constitutions of human spermatozoa.

Yanagimachi, R., Yanagimachi, H. and Rogers, B.J. 1976. The use of zona-free animal ova as test-system for the assessment of fertilizing capacity of human spermatozoa. Biology of Reproduction 15:471-476. .pdf

Rudak, E., Jacobs, P.A. and Yanagimachi, R. 1978. Direct analysis of the chromosome constitution of human spermatozoa. Nature 274:911-912.

Lee, J.D., Kamiguchiu, Y. and Yanagimachi, R. 1996. Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes. Human Reproduction 11:1942-1946. .pdf

(7) Demonstration of the importance of Ca ions for sperm acrosome reaction, sperm-egg fusion, and egg activation

We were the first to demonstrate that extracellular Ca ions are essential for sperm acrosome reaction and hyperactivation as well as for sperm-egg fusion.  The evidence that liberation of intracellular Ca ions within egg cytoplasm triggers egg activation was first presented by us using calcium ionophore.

Yanagimachi, R. and Usui, N. 1974. Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa. Experimental Cell Research 89:161-174.

Yanagimachi, R. 1978. Calcium requirement for sperm-egg fusion in mammals. Biology of Reproduction 19:949-958. .pdf

Steinhardt, R.A., Epel, D., Carrol, E.J. and Yanagimachi, R. 1994. Is calcium ionophore a universal activator for unfertilized eggs. Nature 252:41-43.

(8) Demonstration that cortical granule materials and sperm plasma membrane contribute to the establishment of polyspermy block of the oocyte

Brros,C. and Yanagimachi, R. 1971. Induction of zona reaction in golden hamster eggs by cortical granule material. Nature 233:268-269.

Barros, C. and Yanagimachi, R. 1972. Polyspermy-preventing mechanisms in the the golden hamster egg. Journal of Experimental Zoology 180:251-266.

Maleszewski, M., Kimura, Y. and Yanagimachi, R. 1996. Sperm membrane incorporation into oolemma contributes to the oolemma block to sperm penetration. Molecular Reproduction and Development 44:256-259.

(9) Studies of spermatozoa in vivo

We reported that uncapacitated (hamster) spermatozoa contact firmly to the epithelium of lower segment of the oviduct, where they are capacitated then migrate towards the ampullary region of the oviduct to meet eggs.  Adovarian movement of the oviduct which begins about the time of ovulation directs capacitated spermatozoa to the ampullary region of the oviduct.

Yanagimachi, R. and Chang, M.C. 1963. Sperm ascent through the oviduct of the hamster and rabbit in relation to the time of ovulation. Journal of Reproduction and Fertilization 6:413-420.

Battalia, D.E. and Yanagimachi, R. 1979. Enhanced and coordinated movement of hamster oviduct during periovulatory period. Journal of Reproduction and Fertilization 56:515-520.

Smith, T.T. and Yanagimachi, R. 1991. Attachment and release of spermatozoa from the caudal isthmus of the hamster oviduct. Journal of Reproduction and Fertilization 91:567-573.

(10) Comprehensive reviews of normal fertilization in mammals

Yanagimachi, R. 1981. Mechanisms of fertilization in mammals. In: Mastroianni, L. and Biggers, J.D., editors. Fertilization and Embryonic Development. New York: Plenum Press. p. 81-182.

Yanagimachi, R. 1994. Mammalian fertilization. In: Knobil, E. and Neil, J.D., editors. The Physiology of Reproduction, 2nd edition. New York: Raven Press. p. 189-317.

Yanagimachi, R. 2003. Fertilization and development initiation in orthodox and unorthodox ways: From normal fertilization to cloning.  In: Ebashi, S., editor. Advances in Biophysics 37. Japan Sci. Soc. Press/Elsevier. p. 49-89.

(11) Pioneering intracytoplasmic sperm injection (ICSI)

We were the first to perform mammalian ICSI.  We demonstrated using the golden hamster that sperm nuclei injected into oocytes transform into normal-looking pronuclei.  Even nuclei of testicular (hamster) spermatozoa and freeze-dried (human) spermatozoa transformed into pronuclei after injection into (hamster) oocytes.  Later, we perfected mouse ICSI technique which is used widely today.

Uehara, T. and Yanagimachi, R. 1976. Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biology of Reproduction 15:467-470. .pdf

Kimura, Y. and Yanagimachi, R. 1995. Intracytoplasmic sperm injection in the mouse. Biology of Reproduction 52:709-720. .pdf

Yanagimachi, R. 2005. Intracytoplasmic injection of spermatozoa and spermatogenic cells: Its biology and applications in humans and animals. Reproductive Biomedicine Online 10:247-288. .pdf

yana2
   
Kimura

Dr. Y. Kimura, Yana’s postdoc, manipulates mouse gametes during ICSI with the aid of a joystick. As seen in the video monitor, a holding pipet (left) positions the egg as an injection needle (right) penetrates the egg plasma membrane and deposits a sperm head into the cytoplasm.

Yana and a forerunner of the microscopes for micromanipulating gametes. With the aid of this very basic microscope, Yana and his team were the first to successfully perform mammalian ICSI. By contrast, today’s microscopes are augmented by state-of-the-art technology (compare with the next picture).

 

12) ICSI-mediated transgenesis

We produced transgenic animals (mice) by co-injecting a transgene and a sperm head (nucleus) into an unfertilized egg.  We are in the process of increasing its efficiency.

Perry, A.C.F., Wakayama, T., Kishikawa, H., Kasai, T., Okabe, M., Toyota, Y. and Yanagimachi, R. 1999. Mammalian transgenesis by intracytoplasmic sperm injection. Science 284:1180-1183. .pdf

Keneko, T., Moisyadi, S., Suganuma, R., Horn, B., Yanagimachi, R. and Pelczar, P. 2005. Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection. Theriogenology 64:1704-1715. .pdf

Suganuma, R., Pelczar, P. Spetz, J.F., Hohn, B., Yanagimachi, R. and Moisyadi, S. 2005. Tn5 transposase-mediated mouse transgenesis. Biology of Reproduction 73:1157-1163. .pdf

 
Fig5

The first demonstration of ICSI-mediated transgenesis (a.k.a. Honolulu technique) in mouse. Transgenic mouse gave birth to green pups, passing on her ability to produce the jellyfish green fluorescent protein (GFP) to her young. She was created, by injecting an egg with a sperm head and GFP transgene that integrated into the genome. By GFP-tagging genes of interest, the fluorescence can be used as a reporter of gene expression in transgenic mice.

Ampoules of freeze-dried mouse sperm. Sperm with valuable genetic material can be shipped to researchers in any part of the world. Appropriate medium is simply added to freeze-dried sperm prior to use in ICSI.

(13) Freeze-drying spermatozoa

We demonstrated that freeze-dried mouse spermatozoa are not alive in the conventional sense, but they are able to produce normal live offspring by ICSI.  Freeze-dried spermatozoa can be shipped by ordinary air mail to any parts of the world at ambient temperature.

Wakayama, T. and Yanagimachi, R. 1998. Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nature Biotechnology 16:639-641. .pdf

Kusakabe, H., Szczygiel, M.A., Whittingham, D.G. and Yanagimachi, R. 2001. Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa. Proceedings of the National Academy of Sciences USA 98:13501-13506. .pdf

Ward, M.A., Kenoko, T., Kusakabe, H., Biggers, J.D., Whittingham, D.G. and Yanagimachi, R. 2003. Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection. Biology of Reproduction 69:2100-2108. .pdf

(14) Use of pre-spermatozoal cells for the production of normal offspring

We reported birth of normal mouse offspring after injection of the nuclei of round spermatids into oocytes (ROSI).  Later we obtained live offspring using nuclei of spermatocytes even though success rates are not as high as those after ICSI or ROSI.

Ogura, A., Matsuda, J. and Yanagimachi, R. 1994. Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proceedings of the National Academy of Sciences USA 91:7460-7462. .pdf

Kimura, Y. and Yanagimachi, R. 1995. Development of normal mice from oocytes injected with secondary spermatocytes nuclei. Biology of Reproduction 53:855-862. .pdf

Kimura, Y., Tateno, H., Handel, M.A. and Yanagimachi, R. 1998. Factors affecting meiotic and developmental competence of primary spermatocytes nuclei injected into mouse oocytes. Biology of Reproduction 59:871-877. .pdf

Yanagimachi, R. 2005. Intracytoplasmic injection of spermatozoa and spermatogenic cells: Its biology and applications in humans and animals. Reproductive Biomedicine Online 10: 247-288. .pdf

(15) Cloning

We were the second in producing live cloned mammals after the Dolly (sheep).  Unlike Dolly, we obtained many cloned animals (mice), even clone of clone of clone.  We produced the first cloned males, first clones from ES cells, and first clones from cerebral neurons, etc.  We reported genome-wide aberrations of gene expressions in cloned offspring.

B-day
Fibro

Cumulina, the world’s first cloned mouse, on her second birthday. She provided the genetic material to generate the first series of clone of clone of clone.

Fibro, the world’s first cloned male mouse. He hangs from a bar, showing off his male prowess

Wakayama, T., Perry, A.F., Zuccotti, M., Johnson, K. and Yanagimachi, R. 1998. Full term development of mice from enucleated oocytes injected cumulus cell nuclei. Nature 394:369-374. .pdf

Humphrey, D., Eggan, K., Akutsu, H., Binszkiewicz, D., Yanagimachi, R. and Jaenisch, R. 2001. Epigenetic instability in ES cells and cloned mice. Science 293:95-95. .pdf

Yanagimachi, R. 2002. Cloning: Experience from mouse and other animals. Molecular and Cellular Endocrinology 187:241-248. .pdf

Yamazaki, Y., Low, E.W., Marikawa, Y., Bartolomei, M., McCarrey, J. and Yanagimachi, R. 2005. Adult mice cloned from migrating primordial germ cells. Proceedings of the National Academy of Sciences USA 102:11361-11366. .pdf

International Awards and Recognition for Ryuzo Yanagimachi’s Scientific Work

Zoological Society Prize (Japan), 1977
Research Award, Society for the Study of Reproduction (USA), 1982
The University of Hawaii Board of Regents Medal for Excellence in Research (USA), 1988
Marshall Medal, Society for the Study of Fertility (UK), 1994
International Prize for Biology (Japan), 1996
Honorable Ph.D. degree from University of Rome (Italy), 1997
Distinguished Andrologist Award, American Society of Andrology (USA), 1998
Induction to the Polish Academy of Science (Poland), 1998
R.E. Smith Lecture, University of Texas M.D. Anderson Cancer Center (USA), 1998
Seidel Lecture, Society for the Study of Fertility and Deutche Geselischaft zum Studien der Fertilitat und Sterilitat (Germany), 1998
Carl G. Hartman Award, Society for the Study of Reproduction (USA), 1999
Honorable Ph.D. degree from University of Pavia (Italy), 1999
Pioneer Award, International Embryo Transfer Society, 2000
Induction to the National Academy of Sciences (USA), 2001
Honorable Ph.D. degree from Hokkaido University (Japan), 2002
Induction into the Hall of Honor of the National Institute of Child Health & Human Development (USA), 2003