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Scientific References
Germ Line Cells Are Totipotent:
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Ronan O'rahilly And Fabiola Muller, Human Embryology & Teratology (New York: Wiley-Liss, 1994); also, O'Rahilly and Muller, ibid., (3rd ed., 2001). [Note: O'Rahilly is one of the originators of The Carnegie Stages of Early Human Embryological Development, and has sat on the international Nomina Embryologica Committee for decades -- DNI).
... Primordial germ cells (PGC), or gonocytes, are generally believed to be both extragonadal and extraembryonic in origin. They are difficult to recognize in very young human embryos. Claims for them have been made as early as in the blastocyst, and they are believed to be segregated at latest by 2 1/2 weeks and possibly much earlier. At 4 weeks they can be identified in the umbilical vesicle (yolk sac) and hindgut. A week or so later, they have migrated to the gonads. When the primordial germ cells have settled in the gonad, they become more spherical, stain less intensely with alkaline phosphatase, undergo mitosis, and are then referred to as oogonia or spermatogonia, depending on whether they are situated in an ovary or a testis.. ... The unifying feature in the formation of primordial germ cells would seem to be the exemption of those cells from the processes of regional, somatic differentiation. . (pp., 23-24)
... Cells differentiate by the switching off of large portions of their genome. Future somatic cells thereby lose their totipotency and are liable to senescence, whereas germ cells regain their totipotency after meiosis and fertilization. (p. 39)
... Stem cells comprise a small subpopulation of multipotent or pluripotent, ultrastructurally unspecialized, slow-cycling cells that possess the ability of self-renewal and can produce cells that are destined to differentiate. (In contrast, primordial germ cells and those of a morula are totipotent; i.e., they can develop into any type of embryonic tissue and can even form an entirely new embryo) ... In addition to embryonic stem cells from preimplantation blastocysts, stem cells can be obtained from an adult. (p. 136) ... Ethical concerns are intensified by the experimental finding in primates (in contrast to the mouse) that embryonic stem cells are totipotent and can develop into a complete embryo with a primitive streak. ... If the source of the stem cells is a human embryo or fetus, however, ethical and legal issues have to be considered. (pp. 136-137)
Germ Line Cells Are Diploid:
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RONAN O'RAHILLY AND FABIOLA MULLER, Human Embryology & Teratology (3rd ed.)(New York: Wiley-Liss, 2001): Gametogenesis is the production of germ cells (gametes), i.e., spermatozoa and oocytes. ... The gametes are believed to arise by successive divisions from a distinct line of cells (the germ plasm), and the cells that are not directly concerned with gametogenesis are termed somatic. ... The 46 human chromosomes consist of 44 autosomes and two sex chromosomes: X and Y. In the male the sex chromosomes are XY; in the female they are XX. Phenotypic sex is normally determined by the presence or absence of a Y chromosome. ... During the differentiation of gametes, diploid cells are termed primary, and haploid cells are called secondary, e.g., secondary oocyte. Diploid refers to the presence of two sets of homologous chromosomes: 23 pairs, making a total of 46. This is characteristic of somatic and primordial germ cells alike. Haploid is used for a single set of 23 chromosomes, as in gametes. (p. 19)
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TOM STRACHAN and ANDREW READ, Human Molecular Genetics: Second Edition (New York: Wiley-Liss, 1999): A subset of the diploid body cells constitute the germ line. These give rise to specialized diploid cells in the ovary and testis that can divide by meiosis to produce haploid gametes (sperm and egg). ... The other cells of the body, apart from the germ line, are known as somatic cells ... most somatic cells are diploid ... (p. 28)
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KEITH MOORE AND T.V.N. PERSAUD, The Developing Human: Clinically Oriented Embryology (6th ed. only) (Philadelphia: W.B. Saunders Company, 1998): Meiosis is a special type of cell division that involves two meiotic cell divisions; it takes place in germ cells only. Diploid germ cells give rise to haploid gametes (sperms and oocytes). (p. 18)
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BRUCE M. CARLSON, Human Embryology & Developmental Biology (St. Louis, MO: Mosby, 1999): "In a mitotic division, each germ cell produces two diploid progeny that are genetically equal." (p. 2)
Inner Cell Mass (ICM) Cells of Blasotcyst Are Totipotent:
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RONAN O'RAHILLY AND FABIOLA MULLER, , Human Embryology & Teratology (3rd ed.)(New York: Wiley-Liss, 2001): Future somatic cells thereby lose their totipotency and are liable to senscence, whereas germ cells regain their totipotency after meiosis and fertilization. ... As soon as a cavity can be detected (by light microscopy) in the cellular mass of the morula, the organism is termed a blastocyst. This occurs when about 16-32 cells are present. The embryo is about 4 days in age and is not yet attached to the uterine mucosa. ... The appearance of the blastocyst demonstrates the differentiation into (1) trophoblast (or trophectoderm), the peripherally situated cells and (under the influence of E-cadherin) in first epithelium formed, and (2) embryonic cells proper. The latter, at first few in number, form the inner cell mass (ICM). The trophoblast at the future site of attachment is sometimes termed polar, the remainder being called mural. The cells of the ICM (inner cell mass) are considered to be totipotent initially. They give rise directly to various lines of embryonic stem cells. (p. 39)
...Cells of early free blastocyst (stage 3) are totipotent: The embryo enters the uterine cavity after about half a week, when probably at least 8-12 cells are present and the endometrium is early in its secretory phase. Each cell (blastomere) is considered to be still totipotent (capable. on isolation, of forming a complete embryo), and separation of these early cells is believed to account for one-third of cases of monozygotic twinning. (p. 37)
Cells of Early Human Embryo Are Totipotent; Twinning (cloning by blastomere separation and blastocyst splitting):
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RONAN O'RAHILLY AND FABIOLA MULLER, , Human Embryology & Teratology (3rd ed.)(New York: Wiley-Liss, 2001): Biopsy of an embryo can be performed by removing one cell from a 4-cell, or two cells from an 8-cell, embryo. This does not seem to decrease the developmental capacity of the remaining cells. ... The embryo enters the uterine cavity after about half a week, when probably at least 8-12 cells are present and the endometrium is early in its secretory phase. Each cell (blastomere) is considered to be still totipotent (capable, on isolation, of forming a complete embryo), and separation of these early cells is believed to account for one-third of cases of monozygotic twinning. (p. 37) ... Cells differentiate by the switching off of large portions of their genome.
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RONAN O'RAHILLY and FABIOLA MULLER, Human Embryology & Teratology (New York: Wiley-Liss, 1994): ... The embryo enters the uterine cavity after half a week, when probably at least 8-12 cells are present and when the endometrium is early in its secretory phase (which corresponds to the luteal phase of the ovarian cycle). Each cell (blastomere) is considered to be still totipotent (capable, on isolation, of forming a complete embryo), and separations of these early cells is believed to account for one-third of cases of monozygotic twinning. (p. 23)
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BRUCE CARLSON, Human Embryology & Developmental Biology (St. Louis, MO: Mosby, 1999) (2nd ed.):
... Mammalian embryogenesis is considered to be a highly regulative process. Regulation is the ability of an embryo or an organ primordium to produce a normal structure if parts have been removed or added. At the cellular level, it means that the fates of cells in a regulative system are not irretrievably fixed and that the cells can still respond to environmental cues. Because the assignment of blastomeres into different cell lineages is one of the principal features of mammalian development, identifying the environmental factors that are involved is important. (pp. 44-49); ... Of the experimental techniques used to demonstrate regulative properties of early embryos, the simplest is to separate the blastomeres of early cleavage-stage embryos and determine whether each one can give rise to an entire embryo. This method has been used to demonstrate that single blastomeres, from two- and sometimes four-cell embryos can form normal embryos, ... (p. 44); ... Fate mapping experiments are important in embryology because they allow one to follow the pathways along which a particular cell can differentiate. Fate mapping experiments, which involve different isozymes of the enzyme glucose phosphate isomerase, have shown that all blastomeres of an eight-cell mouse embryo remain totipotent; that is, they retain the ability to form any cell type in the body. Even at the 16-cell stage of cleavage, some blastomeres are capable of producing progeny that are found in both the inner cell mass and the trophoblastic lineage. (p. 45); ... Another means of demonstrating the regulative properties of early mammalian embryos is to dissociate mouse embryos into separate blastomeres and then to combine the blastomeres of two or three embryos. The combined blastomeres soon aggregate and reorganize to become a single large embryo, which then goes on to become a normal-appearing tetraparental or hexaparental mouse. By various techniques of making chimeric embryos, it is even possible to combine blastomeres to produce interspecies chimeras (e.g., a sheep-goat). (p. 45); ... Blastomere removal and addition experiments have convincingly demonstrated the regulative nature (i.e., the strong tendency for the system to be restored to wholeness) of early mammalian embryos. Such knowledge is important in understanding the reason exposure of early human embryos to unfavorable environmental influences typically results in either death or a normal embryo. (p. 46).
... Some types of twinning represent a natural experiment that demonstrates the highly regulative nature of early human embryos, ... (p. 48); ... Monozygotic twins and some triplets, on the other hand, are the product of one fertilized egg. They arise by the subdivision and splitting of a single embryo. Although monozygotic twins could ... arise by the splitting of a two-cell embryo, it is commonly accepted that most arise by the subdivision of the inner cell mass in a blastocyst. Because the majority of monozygotic twins are perfectly normal, the early human embryo can obviously be subdivided and each component regulated to form a normal embryo. (p. 49)
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WILLIAM J. LARSEN, Essentials of Human Embryology (New York: Churchill Livingstone, 1998): [Monozygotic twinning in humans] "If the splitting occurred during cleavage -- for example, if the two blastomeres produced by the first cleavage division become separated -- the monozygotic twin blastomeres will implant separately, like dizygotic twin blastomeres, and will not share fetal membranes. Alternatively, if the twins are formed by splitting of the inner cell mass within the blastocyst, they will occupy the same chorion but will be enclosed by separate amnions and will use separate placentae, each placenta developing around the connecting stalk of its respective embryo. Finally, if the twins are formed by splitting of a bilaminar germ disc, they will occupy the same amnion." (p. 325)
"Twinning" Most Genetically Accurate Form of Cloning:
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TOM STRACHAN and ANDREW P. READ, Human Molecular Genetics 2 (New York: John Wiley & Sons, Inc, 1999): The term 'clones' indicates genetic identity and so can describe genetically identical molecules (DNA clones), genetically identical cells or genetically identical organisms. Animal clones occur naturally as a result of sexual reproduction. For example, genetically identical twins are clones who happened to have received exactly the same set of genetic instructions from two donor individuals, a mother and a father. A form of animal cloning can also occur as a result of artificial manipulation to bring about a type of asexual reproduction. The genetic manipulation in this case uses nuclear transfer technology: a nucleus is removed from a donor cell then transplanted into an oocyte whose own nucleus has previously been removed. The resulting 'renucleated' oocyte can give rise to an individual who will carry the nuclear genome of only one donor individual, unlike genetically identical twins. The individual providing the donor nucleus and the individual that develops from the 'renucleated' oocyte are usually described as "clones", but it should be noted that they share only the same nuclear DNA; they do not share the same mitochondrial DNA, unlike genetically identical twins. ... Nuclear transfer technology was first employed in embryo cloning, in which the donor cell is derived from an early embryo, and has been long established in the case of amphibia. ... Wilmut et al (1997) reported successful cloning of an adult sheep. For the first time, an adult nucleus had been reprogrammed to become totipotent once more, just like the genetic material in the fertilized oocyte from which the donor cell had ultimately developed. ... Successful cloning of adult animals has forced us to accept that genome modifications once considered irreversible can be reversed and that the genomes of adult cells can be reprogrammed by factors in the oocyte to make them totipotent once again. (pp. 508-509)
References From IVF Textbooks and IVF Clinic Web Sites:
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GEOFFREY SHER, VIRGINIA DAVIS, and JEAN STOESS, In Vitro Fertilization: The A.R.T. of Making Babies (copyright 1998 by authors; information by contacting Facts On File, Inc., 11 Penn Plaza, New York, NY 10001): (2) the fertilized egg, which has not yet divided, is now known as a zygote; (3) the egg begins to divide and is now known as an embryo; at this point each blastomere, or cell, within the embryo is capable of developing into an identical embryo." (p. 20)
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PETER BRINSDEN (ed.), A Textbook of In Vitro Fertilization and Assisted Reproduction, 2nd edition (New York: The Parthenon Publishing Group, 1999); Kay T. Elder, "Laboratory techniques: Oocyte collection and embryo culture", p. 197: "Surprisingly, fragmented embryos, repaired or not, do implant and often come to term. This demonstrates the highly robust nature of the human embryo, as it can apparently lose over half of its cellular mass and still recover."
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PROFESSOR DR. MITHHAT ERENUS, "Embryo Multiplication": "In such cases, patients may benefit from embryo multiplication, as discussed in the study by Massey and co-workers. ... Since each early embryonic cell is totipotent (i.e., has the ability to develop and produce a normal adult), embryo multiplication is technically possible. Experiments in this area began as early as 1894, when the totipotency of echinoderm embryonic cells was reported ... In humans, removal of less than half of the cells from an embryo have been documented. No adverse effects were reported when an eighth to a quarter of the blastomeres were removed from an embryo on day 3 after insemination. ... Further evidence supporting the viability and growth of partial human embryos is provided by cryopreservation. After thawing four-cell embryos, some cells may not survive, leaving one-, two-, or three-cell embryos. These partial embryos survive and go to term, but at a lower rate than whole embryos. ... Based on the results observed in lower order mammals, the critical period of development to ensure success in separating human blastomeres should be at the time of embryonic gene expression, which is reported in humans to be between the four- and eight-cell stages. .... The second potential method of embryo multiplication is blastocyst splitting. ... Embryo multiplication by nuclear transfer has been used in experimental cattle breeding programs. ... IVF clinics routinely replace multiple (three to four) embryos into the uterus to increase the chances of a successful pregnancy. For couples who have less than three quality embryos for transfer, blastomere separation could be of benefit." [http://www.hekim.net/~erenus/20002001/asistedreproduction/micromanipulation/embryo_multiplication.htm]
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THE TWINS FOUNDATION (http://twinsfoundation.com/ru-v9n1-1994.htm) New Ways to Produce Identical Twins -- A Continuing Controversy
Identical twins occur naturally approximately 3.5 times out of every 1000 human births. And, to date, scientists still don't know why and can't predict that they will, in any given birth, occur. However, in the last half of this century, and indeed, in the past ten to fifteen years, scientific advances have impacted on twins and other multiples and their families in numerous ways.
Now, a new method of actually producing identical twins looms near. Called "blastomere separation" (the separation of a two- to eight-cell blastomere into two identical demi-embryos), it is potentially one method of helping infertile couples have children through in vitro fertilization (IVF).
The following is excerpted from the medical journal Assisted Reproduction Reviews, May 1994. Dr. Joe B. Massey, who heads an in vitro clinic in Atlanta. Dr. Massey reviews the advances in blastomere separation and discusses the potential indications, benefits, limitations, and ethics of using this method to produce monozygotic twin embryos for IVF patients. The Twins Foundation, by presenting Dr. Massey's material for your information neither advocates nor rejects any such procedures.
Embryo Multiplication by Blastomere Separation-One Doctor's Proposal [Massey] In spite of many advances in human vitro fertilization (IVF), there are still many problems. While leading clinics now have success rates of about 30%, many other clinics lag behind. Still, the number of couples undergoing IVF continues to increase despite high costs.
According to Dr. Massey, "Observations on the potential impact of removing less than half of the cells from the human embryo have been well documented in pre-clinical embryo biopsy studies. (For more on this story see Research Update Vol. 9, No. 1, 1994)
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ETHICS COMMITTEE OF THE AMERICAN SOCIETY FOR REPRODUCTIVE MEDICINE, "Ethical Considerations of Assisted Reproductive Technologies": Originally published as a supplement to the ASRM medical journal (Fertility and Sterility 1994;62:Suppl 1), Ethical Considerations for Assisted Reproductive Technologies covers the American Society for Reproductive Medicine's position on several aspects of reproductive medicine, including: ... the moral and legal status of the preembryo, ... the use of donor sperm, donor oocytes and donor preembryos, ... the cryopreservation of oocytes and preembryos, micro techniques such as: zona drilling, microinjection, blastomere separation (cloning), and assisted hatching ... [http://www.asrm.com/Media/Ethics/ethics94.html]
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AMERICAN SOCIETY OF REPRODUCTIVE MEDICINE (http://www.asrm.com/Media/Ethics/embsplit.html) Because early embryonic cells are totipotent, the possibility of splitting or separating the blastomeres of early preimplantation embryos to increase the number of embryos that are available for IVF treatment of infertility is being discussed. Because embryo splitting could lead to two or more embryos with the same genome, the term "cloning" has been used to describe this practice. ... Splitting one embryo into two or more embryos could serve the needs of infertile couples in several ways. For couples who can produce only one or two embryos, splitting embryos could increase the number of embryos available for transfer in a single IVF cycle. Because the IVF pregnancy rate increases with the number of embryos transferred, it is thought that embryo splitting when only one or two embryos are produced may result in a pregnancy that would not otherwise have occurred. For couples who produce more than enough embryos for one cycle of transfer, splitting one or more embryos may provide sufficient embryos for subsequent transfers without having to go through another retrieval cycle, thus lessening the physical burdens and costs.
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