Transcript ART - Texas A&M University

ART
Assisted
Reproductive
Techniques
Hunter O’Reilly, “Madonna con Clon” 2001
ART
Methods or procedures designed to
Remedy dysfunctional reproduction
Increase reproductive success
Expand knowledge in fields of genetics and
reproductive physiology
ART
Techniques include
IVF - in vitro fertilization
ICSI - intracytoplasmic sperm injections
PGD - preimplantation genetic diagnosis
GIFT - gamete intrafallopian transfer
ZIFT - zygote intrafallopian transfer
Sex determination
NT - nuclear transfer
In Vitro Fertilization
 in vitro = in glass
Involving the insemination of an embryo
that was previously fertilized in culture into
a female recipient.
In Vitro Fertilization
 Methodology
1. Superovulation & harvest of oocytes
* Kept at 37°C in balanced salt solution
* Cyropreserved
2. Insemination of oocyte with sperm cell
* Sperm is allowed to “naturally swim” to
oocyte and join for ferilization
Courtesy of http://www.gfmer.ch/Livres
/FIV_atlas/FIV_Images.htm
In Vitro Fertilization
Methodology
3. Culture of embryos
* Micromanipulation for biopsy occurs here
* Grown to the blastocyst stage
Courtesy of http://www.reproductivegenetics.com
4. Embryo transfer
* Transfer of embryo from culture to
implantation into female recipients
Courtesy of http://www.shadygrovefertility.com
In Vitro Fertilization
 First successful attempt to conceive a human
child in 1978.
 Louise Brown
Courtesy of http://www.babycenter.com/general/9811.html
• British baby girl, born July 25, 1978
• Today is 26 and lives in Bristol, England
• Is a postal worker and recently married
 Since then, 1 million babies born via IVF
 Spawning 115,000 births in U.S. alone
 Cost $7-10,000
• Vary in donor age and reproductive complication
Courtesy of http://news.bbc.co.uk/1/hi/health/3091241.stm
In Vitro Fertilization
Advantages
Correct oocyte fertilized to correct sperm
Embryo biopsy to assess genetic status
Remedies the issue of infertility affecting 6.1 million
people (male and female)
Increase reproductive potential of genetically superior
animals
Intracytoplasmic sperm injections
 aka. Microinsemination
 Process by which a single sperm cell is
injected into an oocyte
 Regardless of quality or level of maturity
 Non-motile sperm cells can be used
 In case of male infertility due to
 Low motility, viability, and quantity
 Abnormally shaped
Courtesy of http://www.ivf.com/insem.html
Intracytoplasmic Sperm Injections

Procedure
1. Gamete cells harvested and prepared as in
IVF
2. Sperm cells cultured in medium and oocytes
exposed to hyaluronidase
* Enzyme destroys cumulus layer to expose
oocyte for ICSI
3. Micropipette equipped with gentle vacuum
holds egg in place
4. Hollow needle containing sharp edge is
used
* To draw up a single sperm cell
* Penetrate the zona pellucida of the oocyte
* Deposit sperm cell into cytoplasm of oocyte
Courtesy of http://www.ivf.com/ivf_icsi.html
Intracytoplasmic Sperm Injections
 Procedure (cont’d)
6. Embryo culture observation
* Formation of 2 pronuclei
* Signaling successful fertilization
7. Embryo culture and maintenance
Courtesy of http://www.ivfmississippi.com/
* Only healthiest embryos selected
* Embryo biopsy or screening can occur at this time
8. Embryo transfer
* Stage of embryo development dependent upon species
* Livestock 5-6 day cultures
Preimplantation Genetic
Diagnosis
 Purpose
 Screening of embryos for genetic
mutations, chromosomal errors, sex
selection and determination for sexlinked chromosomal abnormalities.
 Procedure
1. Removal of 1st polar body from
oocyte
2. Removal of one of the cells in
developing
embryo in the 6-12 cell stage
3. Removal of cells from trophectoderm
Courtesy of http://www.ivf.com/insem.html
Preimplantation Genetic Diagnosis
 Analysis for genetic disorders
 FISH (Fluorescence in situ hybridization)
* Fluorescent probes for specific chromosomes
* Binding will cause illumination and indicate presence of
chromosome of interest
 PCR (Polymerase Chain Reaction)
* Amplification of specific DNA fragment or sequence of genetic
material within the cell
* Results obtained within a day
 Risk of harm during procedure exists
Alternative ET Methods
GIFT
Gamete intrafallopian transfer
Both sperm and oocyte are placed inside female fallopian
tubes
Natural fertilization
 ZIFT
Zygote intrafallopian transfer
IVF embryo inserted into female’s fallopian tubes
Differs from standard IVF, not implanted in uterus
Sex Determination (sperm & embryo)
 In mammalian species, the male determine sex or gender
of offspring via sperm cells (X or Y).
 Selection between X and Y sperm cells
 PGD: development of probes or localization of sex
chromosome
 Microsort sperm separation: separation of X and Y
chromosome bearing sperm
* X chromosome = negatively charged, higher mass than Y bearing
sperm
* Y chromosome = positively charged, lower mass, cell surface antigen
Embryo or fetus prenatal testing
* Ultrasound scanning (non-invasive)
* Amniocentesis & CVS (invasive)
Sperm Sex Sorting
Various procedures
Electrophoresis
Sample in buffered media exposed to anode/cathode
Sperm cells move according to cell surface charge
Sedimentation
Sample allowed to settle in density gradient media
Equilibrium point = sperm’s specific gravity
Immunological
Y-bearing sperm cell surface antigen, highly conserved
Developed antibody either binds to or destroys Y-sperm
Sperm Sex Sorting
Combo Procedure
 Convection counter
streaming-galavanization
Electrophoresis +
Sedimentation
DNA content = X > Y
Dr. Larry Johnson, USDA
Beltsville
Courtesy of XY Inc., http://www.xyinc.com/sexselect/moflo.php
Sex Determination
 Screening of sex-linked chromosome abnormalities
 ie. Duchenne’s muscular dystrophy or hemophilia
 Afflicting only males, found on Y chromosome
 Only female embryos selected for implantation
 The skinny on the old wives’ tale…
 Intercourse timing and ovulation induction has not shown to
modulate sex ratio.
Nuclear Transfer
 Definition
 Involves the insertion of a nucleus from a differentiated somatic
cell into an un-nucleated oocyte.
 Nucleus of the somatic (adult) cell is “reprogrammed” into a
pleuropotent cell by the cytoplasm of the oocyte.
 Functions on the principle that the cytoplasm from the oocyte
contains factors that can reactivate all genes in any somatic or
differentiated nucleus converting it back into a stem cell.
 Can also be accomplished with fusing and entire diploid cell into
an oocyte and switched on with an electrical impulse
* Reconstructed embryo
Nuclear Transfer
 History
 Techniques began in 1950 for the study of frog
development.
 Spread into livestock cloning in 1980s.
 Successful attempts
 Megan and Morag created in 1995
* Lambs created from week-old embryo cultures
* Marked 1st time an animal was derived from
cultured cells
Courtesy of http://www.islamset.com/healnews/cloning/wilmut.html
 Roslin Inst. & PPL Therapeutics
* Created 1st animal from a somatic (adult) cell in
1996
* Birth of Dolly in 1997
* Dolly has a baby lamb named Bonnie
Courtesy of http://www.roslin.ac.uk/public/cloning.html
Nuclear Transfer
 Cloning of Dolly
 Nucleus of mammary gland cell of 6-yr
old sheep was “transferred” into an
oocyte.
 Normal development of a 5-6 day
culture was placed inside a female
recipient.
 Proved that somatic cells are not “fixed
in their roles.”
 Received the Science Breakthrough of
the Year
Courtesy of http://www.roslin.ac.uk/public/cloning.html
Jury is still out on: Dolly’s Problems
 Premature aging
 Dolly was confirmed to be “chromosomally older” than her birth
date according to her telomere length.
 One of the obstacles in cloning.
 Age of cell harboring nucleus to be transferred remains and
continues to age from this point despite new cytoplasm.
 As cells divide and differentiate, they age and have a limited
lifespan.
* DNA stretches and chromosome ends (telomeres) fray and shorten.
* DNA is rendered susceptible to errors and mutations.
Cloning via Nuclear Transfer
 In 1963, Chinese embryologist Tong Dizhou, cloned a carp.
Obscured in a Chinese science journal never translated to
English.
 Success in mice, cattle, sheep, goats, and pigs, rhesus monkey,
garu, cat, rabbit, mule, deer, horse, rat, and fruitfly.
 Success rates leading to live births are very low in all species.
 Success rate of 1%.
 Differences exist in early embryo development.
 Death occurring in late pregnancy: improper placental development.
 Death occurring right after birth: neonate much larger than normal.
 High incidence of genetic abnormality.
Cloning Cats
Cells from Rainbow were cloned to
get Cc.
Surprisingly the cats don’t look or
act alike.
Rainbow is a typical calico with
splotches of brown, tan and gold on
white.
Cc has a striped gray coat over
white.
Rainbow is reserved.
Cc is curious and playful.
Rainbow is chunky.
Cc is sleek.
Copycat or Cc for short was
born Dec 22, 2001 at Texas
A&M University, she is the
first cat successfully cloned.
Cc is a clone of Rainbow
What else has A&M cloned?
Second Addition, a Boer goat
Second Chance, first
First piglets cloned
was born in 2001
cloned bull
born first animal
werecloned
born August
12, resistance, was born
86was
Squared,
for disease
Dewey, the world's first deer
in 1999.
2001.
in November 2000.
clone, was born May 23, 2003.
And still more clones coming
Cloning Abnormalities
 Chromatin structure
 Somatic cells vary compared to gamete cells.
 Transferred nucleus must be “reprogrammed” immediately
during activation of reconstructed embryo.
 Abnormalities are due to
 Improper methylation of DNA and inappropriate reconfiguration
of chromatin following fertilization.
• Causing deregulation of expression of genes.
• Normal embryo development failure.
Limitations of Nuclear Transfer
 Success requires an intact nucleus with functional
chromosomes.
 Most preserved species have lost their DNA integrity
* Fragmented DNA
* Complete genome is destructed
 As of yet, no hope for the rebirth of the Tasmanian tiger or
Siberian mammoth.
 Farm animal production and cloning
 Integration into breeding program to profit.
 Care taken on genetic diversity preservation.
?? Questions??
Hunter O’Reilly, “Unique Clones” 2001