Reproductive Bioscience and Biotechnology

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Reproductive Bioscience and Biotechnology -Basics,
Application and Venture Business
Bioscience and biotechnology in the field of
animal and human reproduction are talked by 15
Japanese professors. Lectures series include
basics, application and venture business in
these fields. Recent topics of the research will
be also introduced. Lectures will be carried out
in February -March, 2006
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Introduction Reproductive bioscience and
Biotechnology by Prof. Eimei Sato
During the past decade, new bioscience
andbiotechnology in reproduction based on
moleculargenetics and embryology has been
rapidly developedIVMFC, freezing of embryos,
sexing of embryo andsperm, knock-out by
homologous recombination oftargeted genes,
transgenic animals by means of ES cells,and
animal cloning by nuclear transfer of somatic
cells.These bioscience and biotechnology enable
us to make anew field in the research of
laboratory animals,domestic animals, endangered
species and humanbeings. Recent advance in
reproductive bioscienceand biotechnology,
especially in Japan will besummarized in this
lecture.
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The present era is a wonderful period in which
to be involved in reproductive research.
(Robert H.Foot, Jacob Gould Schurman Professor
Emeritus of Animal Science, Cornell University,
USA)
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(No Transcript)
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French edition 1991 English revised edition
1993 Edition Marketing. Paris ISBN 2-7298-9354-7
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Advances in understanding the underlying
processes have continued at an ever increasing
pace throughout the 20th century. Progress has
been from the purely descriptive to the
experimental level and from simple biology to
advanced biochemistry and so to molecular
biology. At the endocrine level, between 1940
and 1970, the chemical structures of
gonadotropins and prolactin as well as their
hypothalamic neuroendocrine regulators
were characterized. Pathways of synthesis and
metabolism of sex steroids and neuroamines were
thoroughly investigated, leading to definitive
descriptions and substantial understanding of
endocrine control of puberty, spermatogenesis,
oogenesis, fertilization, implantation,
pregnancy, parturition, lactation and
menopause.
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At the cellular level, all main steps, puberty,
spermatogenesis, oogenesis, fertilization,
implantation, pregnancy, parturition, lactation
and menopause were almost perfectly described
during the first decades of 21th century,
offering a solid basis for electron microscopic
and immunocytological studies which have
developed since the 1950s and have supported
the physiological understanding of hormone
secretion and action. Since the 1970s, it has
become clear that growth factors and
proto-oncogenes play a major role in the
translation of endocrine, paracrine and autocrine
message. Monoclonal antibodies and molecular
probes (immunocytochemical and in situ
hybridization) have brought sensitive and
specific tools revealing information at the
level of cellular and molecular structures.
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Brain Hypothalamus
External environmental factor Sexual behavior
GnRH
Anterior pituitary
GTH (FSH, LH)
Gonad
FolliclogenesisOvulation Spermatogenesis
Accessory gland Luteinization
Sexual steroid
Inhibin
Endocrine regulation in the gonad
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Granulosa cells
Follicular growth
Cumulus cells
FSH/LH
Primary oocytes
Antral follicles
Preantral follicles
Oogonium
Primordial follicles
Primary follicles
Non-growing follicle pool
Growing follicle pool
Ovuratory follicles
Oocyte stage of meiosis
Meiotic maturation
Arrest at prophase I (GV stage)
Initiation of meiosis
Resumption of meiosis
Arrest at metaphase II
Mitosis
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What is a ligand ? Gonadotrophin ?
Activate
Inactivate
Activate
G protein receptor?
GPR3
GPR3
?
?
In oocyte
cAMP?
cAMP?
PI3-K ?
MOS ?
PKA?
PKA?
Akt / PKB ?
MAPK ?
Inactive MPF
Active MPF
?
T161
T14
Y15
T161
Spindle formation Chromosome aliment Maintenance
of the MII arrest
GV
GVBD
M I
M II
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1
2
3
Gonadotrophin surge
Cumulus expansion
Pre-surge
HA-CD44interaction? Phosphorylation of CX43 Gap
junctions of Cx43 close
HAS2 ?, CD44 ?, CX43 ? Gap junctions of CX43 open
Lower level of CX43 expression Gap junctions of
CX43 open
cAMP, Ca2
(Cumulus cells)
cAMP, Ca2
A temporary Increase in cAMP
cAMP
Under a threshold in cAMP
(Oocyte)
Active MPF
Switch on activation of MPF
pre-MPF
Meiotic resumption
Arrest at GV stage
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Based on these development in the research in
reproductive bioscience, techniques manipulating
reproductive phenomena have been developed.
Superovulation Synchonization of estrus
Diagnosis of pregnancy Contraception
Gene targeting in embryo stem cells (ES cells)
allows specific mutation to be introduced into
germ line in virtually any genes so that the
gene function can be studied by mutational
analysis in vivo. In the mouse, by using this
technology, functions of various genes have been
shown.
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Molecular basis of meiotic arrest at metaphase
II - c-mos knockout mouse -
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Background
Meiosis I
Meiosis II
S phase
Pro. I
Meta. I
Meta. II
FSH/LH
- Overview of oocyte maturation -
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Endogenous c-mos
BI
H
B
BI
X
X
X
X
B
H
H
B
B
X
MOS105
MOS102
Targeting vector
NXH
B
B
X
BI
BI
H
AT/pau
N
XH
BSK
DT
neor
Targeted c-mos
Production of c-mos knockout mice by homologous
recombination in ES cells.
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- Effects of Mos in mouse oocyte maturation -
First polar body emission
GVBD
Mos is not essential for GVBD and 1st PB
emission.
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- Effects of Mos in mouse oocyte maturation -
GV stage
Metaphase I
Metaphase II
/
ch
GV
Metaphase II
GV stage
Metaphase I
pb
pb
-/-
ch
ch
ch
ch
GV
Morphology of oocytes during in vitro maturation
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C-mos knockout
?-tubulin
Mos participates in chromosome condensation and
the reorganization of microtuble.
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- Effects of Mos in mouse oocyte maturation -
GV stage
Metaphase I
Metaphase II
pb
pb
ch
-/-
ch
ch
ch
GV
ch chromosome pb polar body pn pronucleus
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- Effects of Mos in mouse oocyte maturation -
MPF activity
12
10
8
histone H1 kinase activity (fold increase)
6
4
-/- (Activated)
2
0
Culture period (h)
Mos plays crucial role in reactivation of MPF,
which result in preventing spontaneous
activation.
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- Effects of Mos in mouse oocyte maturation -
Morphology of fertilized oocytes at 6-7 h after
insemination.
Number of oocytes ()
Total oocyte examined
Genotype
Sperm Tail
Sperm Tail
CH
PN
/
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35 (100)
0 (0)
- / -
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9 (47)
10 (53)
PN pronuclei CH metaphase-like chromosome
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Metaphase III
Mos participates the pronuclei formation and is
important to progress into interphase after
fertilization.
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Summary

• Mos was not essential for GVBD and first polar
  body emission, but was essential for maintaining
  condensation of chromosome.
• Mos played a crucial role in the reactivation of
  MPF after meiosis I to enable oocytes to progress
  from meiosis I to meiosis II.
• In mos knockout oocytes, some oocytes were
  transformed into metaphase III instead of
  interphase after fertilization.

Mos is a key coordinator that controls the
meiotic/mitotic conversion before and after
entry into meiosis II.
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Reproductive Biotechnology the past, present
and future-
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The story begins when artificial insemination
(AI) was in its infancy in this country and
continue cloning. In Artificial Insemination to
cloning, tracing 50 years of research Cornell
University, 1998.
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The clone sheep produced at Losline Institute, UK
has a great impact in biology, animal production
and medicine in the world and then clone animals
were born in a number of mammalian species. The
production of a somatic cell clone by fusing a
cell from the fetus and adults with an oocyte was
the event with the greatest impact in the recent
development of animal biotechnology. The year
1997 will be long remembered as a special year in
the research of reproductive biotechnology.
This clone sheep was produced from the motive
of animal husbandry of more efficient production
of good domestic animals.
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Nuclear transfer technique
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The clone cattle and miniature pig born in Tohoku
University
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In the field of animal husbandry, animal
biotechnology is often considered to consist of
2 categories. One is the technique 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
are contributing to the industry. The other is
the technology that has emerged with the
development of modern biology and consists
primarily of cell manipulation such as gene
recombination and cell fusion. Techniques
represented by clone sheep have been developed
on the basis of both of these categories.
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Biotechnology in animal reproduction and
reproductive medicine
Artificial insemination Freezing of Semen Embryo
transfer Splitting of embryo Sexing of embryo In
vitro fertilization IVMFC Microscopic
insemination Ovum pick up (OPU) Nuclear
transfer Gene incorporation Gene-knock-out
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Licenses involved in Animal reproduction and
human medicine

• ? Artificial insemination in domestic animals
• Embryo transfer in domestic animals
• (License approved by the law )

• Embryologists in Assisted Reproductive
• Medicine
• (Japanese Society of Mammalian Ova Research)

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The birth of the clone sheep is influencing the
frontier of research of biology, animal
production and medicine. It has effects on
research of animal biotechnology itself by
necessitating setting of new research subjects,
modifications of the strategy of ongoing
research projects, and challenges to schemes
formerly considered impossible.
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1) Studies of ova make clones more genuine
clones 2) Strategy to produce gene-knock-out pigs
modified due to the birth of the clone
sheep 3) The somatic cell cloning technique saves
endangered species 4) The "serial
culturing" technique of germ sell series may
circumvent the disadvantages of somatic cell
clones 5) The clone technology also changes
genome science In Sato,E. A new frontier of
animal biotechnology research with the birth of
the cloned sheep, J.Mamm.Ova.Res., 1683- 85, 1999
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In the cow, 100 - 200 thousand ova are
produced in the ovary and are potentially
available. Many of them die in the course of
oogenesis, and usually only 1 of them is
ovulated in each estrus cycle, and 100 - 200 of
them are ovulated even in the lifetime. Thus,
there is a mechanism in the mammalian ovary to
selectively ovulate a small number of ova and
induce death in most of them. I have started
experimenting on the assumption that selective
oogenesis can be avoided, and oocyte death can
be prevented, by releasing ova from the ovary.
Collection of more oocytes from one animal is
expected to become possible as a result of such
investigation of the oogenesis, and the
availability of more oocytes from the same will
make animal clones produced by nuclear
transplantation more like clones.
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The research of oocytes may lead to the
establishment of new biology or medical
technology. Cells constituting the body are
conditioned to perform specific functions. Genes
of these cells have been considered to be
irreversibly modified to perform only specific
functions. However, the birth of the "clone
sheep" demonstrated that the nucleus of a cell
that performs a specific function reclaims
"omnipotential of differentiation" as it is
transplanted to an ovum. Can such a function of
the ovum be explained from the knowledge
accumulated to date?
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oocyte
granulosa cell
fertilization reprograming Hyaluronic acid
,CD44,MAP kinase
Flt-1 Flk-1/KDR bFGF-R
cumulus cell
capillary vessel
granulosa cell
follicular atresia oocyte apoptosis Hyaluronic
acid
VEGF 120 bFGF
? Differentiation and degeneration of oocyte ?
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The somatic cell cloning technique saves
endangered

species The number of existing species has
decreased markedly in 20th century. Mammals that
have ever appeared on the earth are classified
into 35 orders by phylogenetic taxonomy, but 17
of them have been extinguished to date. On the
species level, there were 4,226 mammalian species
in the year 1600, when taxonomy was established,
but 36 of them have been extirpated, and 120 are
on the verge of extinction. The situations of
the birds, amphibians, and reptiles are similar.
Whether such a state is a result of prosperity
of the mankind or is an outcome of the natural
life spans of species is an open question, but
everyone would agree that aggressive measures to
protect endangered species must be
considered today.
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Concerning endangered species, not only
conservation of their habitat but also technical
intervention to avoid extirpation has become
necessary. Furthermore, in species extinction
of which has not been avoided (or has been
judged to be unavoidable), consideration of
preservation of genes and analysis of the causes
of extinction as well as measures for future
restoration may be needed.
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In such a trend, gene preservation projects have
been initiated for part of the species (including
the Japanese crested ibis). Discussion as to
how such genes should be utilized and whether
individuals of the species should be restored by
the nuclear transplantation technique or not has
begun, and it has gained greater reality with the
birth of the clone animals.
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(No Transcript)
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Studies on contraception as well as sterility in
humans and fertlity control in domestic mammals,
companion animals, endangered species and
laboratory animals are so important for the
wellbeing of mankind.
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The subject for a paper Role of reproductive
biotechnology for the wellbeing of mankind