ART

1. ART Assisted Reproductive Techniques Hunter O’Reilly, “Madonna con Clon” 2001

2. ART • Methods or procedures designed to • Remedy dysfunctional reproduction • Increase reproductive success • Expand knowledge in fields of genetics and reproductive physiology

3. 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

4. In Vitro Fertilization • in vitro = in glass • Involving the insemination of an embryo that was previously fertilized in culture into a female recipient.

5. 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

6. In Vitro Fertilization • Methodology 3. Culture of embryos * Micromanipulation for biopsy occurs here * Grown to the blastocyst stage 4. Embryo transfer * Transfer of embryo from culture to implantation into female recipients Courtesy of http://www.reproductivegenetics.com Courtesy ofhttp://www.shadygrovefertility.com

7. In Vitro Fertilization • First successful attempt to conceive a human child in 1978. • Louise Brown • 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://www.babycenter.com/general/9811.html Courtesy of http://news.bbc.co.uk/1/hi/health/3091241.stm

8. 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

9. 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

10. 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

11. Intracytoplasmic Sperm Injections • Procedure (cont’d) 6. Embryo culture observation * Formation of 2 pronuclei * Signaling successful fertilization 7. Embryo culture and maintenance * 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 Courtesy of http://www.ivfmississippi.com/

12. Preimplantation Genetic Diagnosis • Purpose • Screening of embryos for genetic mutations, chromosomal errors, sex selection and determination for sex-linked 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

13. 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

14. 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

15. 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)

16. SpermSexSorting • 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

17. 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

18. 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.

19. 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

20. 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 • 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.islamset.com/healnews/cloning/wilmut.html Courtesy of http://www.roslin.ac.uk/public/cloning.html

21. 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

22. 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.

23. 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.

24. 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 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.

25. What else has A&M cloned? Second Addition, a Boer goat was born in 2001 Dewey, the world's first deer clone, was born May 23, 2003. Second Chance, first cloned bull was born in 1999. First piglets cloned were born August 12, 2001. 86 Squared, first animal cloned for disease resistance, was born in November 2000.

26. And still more clones coming

27. 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.

28. 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.

29. ?? Questions?? Hunter O’Reilly, “Unique Clones” 2001