Manufacture Gametes by the
Technique of Nuclear Transfer
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Assisted Reproductive Technology (ART)
Artificial Insemination (AI)
In vitro fertilization (IVF)
Gametes Intra-Fallopian Transfer (GIFT)
Tubal Embryo Transfer (TET)
Intra-Cytoplasmic Sperm Injection (ICSI)
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Cow AI
Swine AI
Mare AI
Ewe AI
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The oocytes are retrieved
from ovary.
The eggs are fertilized in vitro.
(The Infertility Center of St. Louis Website)
http://www.in-vitro-fertilization.com/index.html
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Fallopian tube
Eggs and sperm
The eggs and sperm are released into the
fallopian tube.
(The Infertility Center of St. Louis Website)
http://www.in-vitro-fertilization.com/index.html
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A
B
C
Sperm aspiration for ICSI.
(The Infertility Center of St. Louis Website)
http://www.in-vitro-fertilization.com/index.html
6
A
B
C
The procedure of intra-cytoplamic sperm injection (ICSI).
A. Immobilizing the sperm's tail before picking it up.
B. B.Injection of sperm into the egg.
C. C. Fertilized egg demonstrating the two nuclei--one from the father,
one from the mother.
(The Infertility Center of St. Louis Website)
http://www.in-vitro-fertilization.com/index.html
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Age-related aneuploidy
Cytoplasmic transfer
Nuclear transplantation
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Ooplasmic transplantation by electrofusion with donated ooplast.
A-C. A large gap is made over the zona pellucida of an egg hold by the holding pipette
and the polar body is removed.
D-F. A gap is made opposite of the polar body in the donor egg and ooplasm are
removed.
G-H. The ooplasm is placed underneath the zona of the recipient egg and electrofusion
is performed. A spermatozoon is injected into the fused hybrid cell.
(Cohen, J. et al., 1998)
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Ooplasmic transplantation by injection.
A.
B.
C.
D.
E.
F.
A spermatozoon is aspirated into the ICSI needle.
The membrane of a donor egg is broken by the ICSI needle containing a spermatozoon.
Ooplasm is aspirated from the areas opposite the polar body.
The needle is moved to a recipient egg.
The membrane is broken, and cytoplasm and spermatozoon are deposited into the egg.
The egg is cultured.
(Cohen, J. et al., 1998)
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Table. Cytoplasmic transfer case performed.
(Barritt, J. W. et al., 2001)
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Nuclear transplantation.
• Enucleation of immature oocytes.
• Grafting, electrofusion, and reconstitution.
(Tsai, M. C., et al., 2000)
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A
B
C
A. Removal of the nucleus surrounded by a small
amount of ooplasm from a human germinal vesicle
stage oocyte.
B. An isolated human germinal vesicle karyoplast.
D
C. Transfer of an isolated human germinal vesicle
karyoplast into an enucleated oocyte.
D. A grafted human oocyte (couplet of a karyoplast
and a cytoplast) aligned between two
microelectrodes.
(Palermo, G. D., et al., 2002)
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What are the treatment options for
patients without gametes leading
to their own genetic children?
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Sperm or oocyte donation
what else?
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Methods of producing diploid embryos by using primary spermatocytes as
male gametes.
The cell-cycle stages of primary spermatocytes and oocytes were synchronized at the GV
(Method A), Met I (Method B), or Met II (Method C) stages.
(Ogura, A., et al., 1998) 16
Table Development after oviductal transfer of oocytes fertilized
with primary spermatocytes (Method B)
(Ogura, A., et al., 1998) 17
Pups born after microfertilization with primary spermatocytes.
a. Just after birth. The smallest pup (female, Center) died soon
after birth, although no gross abnormality was found.
b. The male pup
grew normally and developed into a fertile adult (arrow).
(Ogura, A., et al., 1998) 18
The sequence of events after injection
of a primary spermatocyte nucleus
into mature oocytes.
A. The nucleus of the primary spermatocyte
was injected into an oocyte.
B. After the spermatocyte nucleus transformed
into metaphase I chromosomes, the oocyte
was electro-activated.
C. The oocyte extruded two polar bodies.
D. The nucleus of the first polar body (PbI) of
spermatocyte origin was transferred into
another mature oocyte.
E. When spermatocyte chromosomes were
arranged in the metaphase II plate, the
oocyte was electro-activated.
F-G. The activated oocyte extruded two polar
bodies to form two pronuclei. Such eggs
could develop into fertile offspring.
(Yanagimachi, R., 2001)
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Manufacturing oocytes.
• Manufacturing oocytes.
• Enucleation of an immature oocyte.
• Grafting, electrofusion, reconstitution
and haploidization.
(Tsai, M. C., et al., 2000)
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Production of artificial gametes.
(a) fertile woman/sterile man.
(Trounson, 2001)
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Production of artificial gametes.
(b) sterile woman/fertile man.
(Trounson, 2001)
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Production of artificial gametes.
(c) sterile woman/sterile man.
(Trounson, 2001)
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Strategies potentially achieving somatic haploidization
G0/G1 somatic cells
(2N and 2C)
M II oocytes
extrusion of one
polar body
somatic cell-oocyte
complex
(1N and 1C)
G0/G1 somatic cells
G2/M somatic cells
(2N and 2C)
(2N and 4C)
GV oocytes
(G2/M stage)
extrusion of one
polar body
somatic cell
(1N and 1C)
GV oocytes
somatic cell-oocyte
complex
(1N and 2C)
somatic cell-oocyte
complex
(1N and 1C)
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Strategy I
2N/2C somatic cell divided
by the second meiotic
division;
Strategy II
2N/2C somatic cell divided
by the first meiotic division;
Strategy III
2N/4C somatic cell divided
by the first and second
meotic divisions.
(Theriogenology, 2007)
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Metaphase II chromosomes
Donor oocyte
Enucleation
Ooplast
Nucleus injection
1h
Somatic cell chromosomes
Metaphase entry
13 h
Sperm injection
Oocyte activation
Anaphase entry
5h
Pseudopolar body
Haploid set of chromatids
5h
Female pronucleus
Male pronucleus
30 h
2-cell embryo
(Modified from Tesarik, J., et al., 2001)
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Removal of a germinal vesicle from a mouse oocyte.
Grafted oocytes with mouse cumulus cells.
Grafted oocyte with a cumulus cell aligned between
two microelectrodes.
(Palermo, G. D., et al., 2002)
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Mouse reconstituted oocyte with
human endometrial cell showing
segregated chromosomes in both the
ootid and the polar body.
A normal haploid set of chromosomes
stained with Giemsa in a reconstituted
oocyte after extrusion of the first polar
body.
(Palermo, G. D., et al., 2002)
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Table Maturation of intact and enucleated mouse oocytes fused to
somatic or embryonic cells
(Fulka Jr, J., et al., 2002)
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1
3
5
1.
Somatic cell nucleus introduced into an
enulceated GV stage mouse oocyte. Fixed 1 h
post-introduction of fusion.
2.
Somatic cell nucleus introduced into an
enucleated GV stage mouse oocytes. Fixed 20 h
post-introduction of fusion. Note the nucleus
enlargement and a well visible nucleolus.
3.
Mouse oocyte metaphase I spindle with an
equatorial arrangement of meiotic chromosomes
while the mitotic chromosomes are located on
spindle poles.
4.
Mouse anaphase-telophase I oocyte with somatic
cell chromosomes located outside the spindle
(arrow).
5.
Enucleated mouse oocyte fused to somatic cell
and matured for 16 h. Note that in this case the
first polar body was extruded but the metaphase II
plate is evidently abnormal (arrow).
6.
Enculeated mosue oocytes fused to somatic cells
and thereafter matured in vitro did not extrude the
first polar body and are arrested in metaphase Ilike stage with chromosomes dispersed
chaotically on the spindle.
2
4
6
(Fulka Jr, J., et al., 2002)
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Variation in number of mouse cumulus chromosomes remaining in
the oocyte after the emission of a pseudo polar body.
N is the total number of oocytes examined.
(Tateno, H. et al., 2003)
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Fluorescence microscopy of an oocyte incubated for 2–3 hours
after enucleation, followed by injection of a mouse cumulus
cell nucleus.
a. Immunocytochemical staining of spindle.
b. 4,6-diamidino-2-phenylindole [DAPI] staining of chromosomes. Spindle
microtubules are aberrantly assembled, and chromosomes are
distributed in a disorderly fashion.
(Tateno, H. et al., 2003)
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Table Chromosome number analysis of artificial mouse zygotes and oocytes
created by injection of a somatic mouse cumulus cell nucleus into nonenucleated and enucleated metaphase II oocytes.
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(Heindryckx, B., et al., 2004)
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Table Chromosome number analysis of artificial mouse zygotes and
oocytes created by injection of a somatic mouse cumulus cell
nucleus into non-enucleated and enucleated metaphase II
oocytes after different time intervals between nucleus injection
and artificial activation
(Heindryckx, B., et al., 2004)
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Table Developmental potential of non-enucleated metaphase II
oocytes injected with cumulus cell nucleus (artifical zygotes)
(Heindryckx, B., et al., 2004)
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A. A somatic cell synchronized at G2/M phase was transferred into the
perivitelline space of an enucleated GV oocyte.
B. The somatic cell is shown fused into an enucleated GV oocyte 30 min
after fusion. (Bar = 30 m).
(Chang, C. C., et al., 2004)
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Schematic summary of the timing of oocyte maturation and activation
in control and reconstructed oocytes.
The gray bars represent the duration of in vitro maturation. The black bars represent the duration
of activation (6 h). The timing of polar body extrusion was approximately 9–10 h postmaturation
and 5–7 h postfusion in control and reconstructed oocytes, respectively. The dotted boxes
represent the durations of PB extrusion (PBE).
Arrows represent the expected MI, MII, and PN stage for control and manipulated oocytes.
(Chang, C. C., et al., 2004)
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Table The nuclear morphology of control and reconstructed ocytes
during in vitro maturation.
(Chang, C. C., et al., 2004)
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Table The nuclear morphology of control and reconstructed oocytes
at the expected pronuclear stage (6 h after activation treatment).
(Chang, C. C., et al., 2004)
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Nuclear morphology of control (A and B) and manipulated oocytes (C–F) 6 h
after activation treatment.
A. A control oocyte had two wellformed pronuclei (arrows).
B. The pronuclei had interphase-like chromatin under epifluorescence microscopy.
C. Treconstructed oocytes do not have pronuclear membrane visible under light microscopy.
D-F. Their nuclear morphology was one (D) and two (E) pseudo-PN with interphase-like
chromatin or uneven condensed chromosomes (F). (Bar = 30 mm).
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(Chang, C. C., et al., 2004)
Conclusions
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Nuclear transplantation itself does not appear to interfere
with chromosomes segregation.
Nuclear transplantation can possibly rescue oocytes with
damaged mitochondria.
Immature ooplasm might support separation of somatic
chromosomes to expected numbers, but its efficiency was
low.
A high proportion of G2/M somatic nuclei appear to undergo
meiosis-like division, suggesting geminal vesicle cytoplast
retained capacity for meiotic division.
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