Scientific References: Human Genetic Engineering
(including Cloning): Artificial Embryos, Oocytes, Sperms, Chromosomes and Genes

Dianne N. Irving
Compiled by Dianne N. Irving, M.A., Ph.D.
Copyright May 25, 2004
Reproduced with Permission

Genetic engineering is the artificial construction, deconstruction, or reconstruction of the genetic composition of organisms and their components or precursors. Like all technologies, it can be used for the good or for the hindrance of individual human beings or of society. Advances in human genetic engineering have been rapid since the mapping of the human genome, and already great medical benefits have been achieved. However, it has also become clear that without serious public input into public policy making decisions on these issues, a great deal of unethical research and abuse of human subjects will continue to go forward, with no public scrutiny or professional or legal accountability. Yet such public input must be well-informed, based on objective, accurate and current science, and understood in relation to the broader ethical, social and political issues.

Accessing those scientific facts, however, is sometimes difficult, especially for those with little background in the various sciences that are involved in human genetic engineering. This effectively renders the public incapable of entering into meaningful discussions in "the public square", or able to draft effective legislation without gapping loopholes. To that end, this selected bibliography for human genetic engineering has been compiled, with specific reference to the use of artificial embryos, germ-line cells, genes and chromosomes.

It is hoped that these scientific references might help and encourage more serious and meaningful public input into public policy discussions involving the rapidly expanding field of human genetic engineering.

The references listed below are literally the "tip of the iceberg", and were selected almost entirely from searches on Entrez PubMed, a service of the National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health, including citations from MEDLINE and additional life science journals (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed). Selected abstracts and full articles have been quite extensively reduced for brevity so as to simply indicate the type of research performed. The full URLs for each scientific study are provided if more information is desired. It is not necessary to understand all of the details in the scientific studies listed below. Simply paging through them can enable one to see roughly that such research has been on-going for some time now, and that it urgently needs to be seriously addressed.

Although there is necessarily some overlap, the articles listed are generally ordered in terms of the following categories of research involving the use of:

I. Artificial embryos
II. Artificial germ-line cells
..... A. Artificial primitive, primary and secondary oocytes
..... B. Artificial spermatogonia, spermatocytes, and sperms
III. Artificial genes and chromosomes

Within each of these categories, the use of human materials is listed first (sometimes using human patients, especially those involved in IVF "therapies"), followed by human/non-human chimeras (e.g., transgenic animals), and finally non-human animal studies (where most of the genetic engineering techniques to be used with humans are first introduced and refined).

Finally, a few words about correctly identifying and defining these techniques. First, genetic engineering is the overall category. Under that heading we find many different kinds of techniques that may be used to genetically engineer - what is referred to in scientific and policy making circles as the use of "converging technologies". That is, the techniques that are usually special to different fields of science are now being combined in order to genetically engineer. When searching for "genetic engineering", for example, "cloning" is only one of many techniques that are identified.

Second, in understanding how to genetically engineer an entire organism, e.g., a human being, scientists often keep the biological steps used by nature in mind. First there are elements (like carbon or oxygen) which make up molecules -- including DNA molecules, the smallest genetic component. DNA molecules make up genes, which make up chromosomes, found inside and outside the nucleus of cells. Cells make up tissues, which make up organs, which finally compose a whole organism. One can engineer at any of these stages of development, using a variety of genetic engineering techniques. Investigations into each of these steps are represented in the studies below.

Third, it is possible to genetically engineer a human being starting with various components. For example, one can use DNA-recombinant gene transfer to insert a "foreign" gene into sperm or oocytes that will then be used to reproduce a human being (using either sexual or asexual reproductive processes, or a combination of both). One can insert a "foreign" gene into an artificial chromosome, into pronuclei, or even into a very early human embryo (including the single-cell zygote). This "foreign" gene will then be copied and passed down through the generations, because it will have become "part" of the genetic makeup of every cell in the new human organism - including his/her germ-line cells.

One can also "reconstruct" -- or, genetically engineer -- each of these various cells/organisms using several techniques, e.g., by adding artificial genes and chromosomes, by transferring the pronuclei, nuclei, or mitochondria of one cell into another cell, etc. Donor and recipient cells used can be any human cell type (somatic or germ-line), including those from early embryos, fetuses, young children or adult human beings. Note the common use of certain "euphemisms" in many of these articles in order to deflect attention away from what is really going on, e.g., the products of these techniques are often referred to as "stem cells", "reconstructed oocytes" after nuclear transfer and activation, or as "fertilized oocytes". But many of these products are actually new genetically engineered human embryos, human beings - and hence the real ethical concern.

It is hoped that these scientific references might help and encourage more serious and meaningful public input into public policy discussions involving the rapidly expanding field of human genetic engineering.


Selected Bibliography:

I. Artificial Embryos

Note: All embryos reproduced by cloning or other genetic engineering techniques are "artificial embryos" - asexually reproduced human beings. This includes the use of all types of cloning techniques, as well as other genetic engineering techniques, e.g., pronuclei transfer, mitochondrial transfer, DNA-recombinant gene transfer (somatic or germ line), transfer of all other kinds of cell constituents. Such techniques may produce living embryos for pure research or for implantation; cells derived from these embryos can be used for "therapies", test materials for biological/chemical agents, vaccines, pharmaceuticals, and the production of transgenic animals, etc.

-- 22 Biotechnology Law Report 376, No. 4 (August 2003)

Social and Ethical Issues in Nanotechnology: Lessons from Biotechnology and Other High Technologies, Joel Rothstein Wolfson

Nanotechnology can be used to clone machines as well as living creatures. ... Proponents of nanotechnology postulate a world where DNA strands can be custom built by repairing or replacing sequences in existing strands of DNA or even by building the entire strand, from scratch, one sequence at a time. With enough nanorobots working quickly enough, one could build a DNA strand that will produce a perfect clone. The same issues will arise, or re-arise, if nanotechnology is successful in promoting cloning of DNA segments, cells, organs, or entire organisms. ... ... It is likely that nanotechnologyÕs efforts will lead to twists in the assumptions that lead to the resolution of cloning issues in terms of genetic bioengineering. Policy makers should anticipate, now, that in setting the boundaries for bioengineered cloning, the need to foresee issues that will arise from cloning by nanotechnology and be ready to reevaluate cloning regulation before nanotechnology perfects its own methods of cloning. http://www.blankrome.com/publications/Articles/WolfsonNanotechnology.pdf

-- J Reprod Immunol. 2002 May-Jun;55(1-2):149-61

New techniques on embryo manipulation. Escriba MJ, Valbuena D, Remohi J, Pellicer A, Simon C. (Spain)

For many years, experience has been accumulated on embryo and gamete manipulation in livestock animals. The present work is a review of these techniques and their possible application in human embryology in specific cases. It is possible to manipulate gametes at different levels, producing paternal or maternal haploid embryos (hemicloning), using different techniques including nuclear transfer. At the embryonic stage, considering practical, ethical and legal issues, techniques will be reviewed that include cloning and embryo splitting at the cleavage stage, morula, or blastocyst stage. [PMID: 12062830] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12062830

-- News Release, University of Mass., Amherst, Sept. 3, 1999

"UMass is Issued Patent For the Biotechnology Behind Cloned, Transgenic Cattle"

The technology enables scientists to create identical cells and animals either or without genetic modifications. The technique was first announced by UMass and ACT with the birth of George and Charlie, the first cloned transgenic cattle produced from genetically altered bovine somatic (body) cells. Details of the technique were published in the May 22, 1998 issue of the journal, Science. Unlike other cloning methods, which rely on nuclear transfer using germ line embryonic cells or on using somatic cells in a quiescent, or inactive state, this method covers transfer of somatic cells during any phase of the cell cycle except quiescence. The technology involves an improved method of nuclear transfer involving the transplantation of differentiated somatic cells into an oocyte, or egg cell, from which the nucleus has been removed. Transfers are performed between same-species, non-human mammalian cell and egg donors. This technology applies to embryonic stem cell technology because nuclear transfer is a way of genetically modifying embryos that can then produce embryonic stem cells. http://www.umass.edu/newsoffice/archive/1999/090399patent.html

-- Hum Mol Genet. 2004 May 1;13(9):935-44. Epub 2004 Mar 11

Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Ekstrand MI, Falkenberg M, Rantanen A, Park CB, Gaspari M, Hultenby K, Rustin P, Gustafsson CM, Larsson NG. (Sweden)

Mitochondrial DNA (mtDNA) copy number regulation is altered in several human mtDNA-mutation diseases and it is also important in a variety of normal physiological processes. Mitochondrial transcription factor A (TFAM) is essential for human mtDNA transcription and we demonstrate here that it is also a key regulator of mtDNA copy number. [W]e generated P1 artificial chromosome (PAC) transgenic mice ubiquitously expressing human TFAM. Interestingly, the expression of human TFAM in the mouse results in up-regulation of mtDNA copy number without increasing respiratory chain capacity or mitochondrial mass. It is thus possible to experimentally dissociate mtDNA copy number regulation from mtDNA expression and mitochondrial biogenesis in mammals in vivo. In conclusion, our results provide genetic evidence for a novel role for TFAM in direct regulation of mtDNA copy number in mammals. [PMID: 15016765] [PubMed - in process] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15016765

-- Biol Reprod. 2001 Jul;65(1):253-9

Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos. Mitalipov SM, Nusser KD, Wolf DP. (USA)

This study determines ..., in inducing artificial activation and development of rhesus macaque parthenotes or nuclear transfer embryos. Exposure of oocytes arrested at metaphase II (MII) to ionomycin followed by 6-dimethylaminopurine or to electroporation followed by cycloheximide and cytochalasin B induced pronuclear formation and development to the blastocyst stage at a rate similar to control embryos produced by intracytoplasmic sperm injection. Parthenotes did not complete meiosis or extrude a second polar body, consistent with their presumed diploid status. In contrast, oocytes treated sequentially with ionomycin and roscovitine extruded the second polar body and formed a pronucleus at a rate higher than that observed in controls. Following reconstruction by nuclear transfer, activation with ionomycin/6-dimethylaminopurine resulted in embryos that contained a single pronucleus and no polar bodies. All nuclear transfer embryos activated with ionomycin/roscovitine contained one large pronucleus. However, a third of these embryos emitted one or two polar bodies, clearly containing chromatin material. In summary, we have identified simple yet effective methods of oocyte or cytoplast activation in the monkey, ionomycin/6-dimethylaminopurine, electroporation/cycloheximide/cytochalasin B, and ionomycin/roscovitine, which are applicable to parthenote or nuclear transfer embryo production [PMID: 11420247] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11420247

-- Hum Reprod. 2000 Sep;15(9):1997-2002

In-vitro development of mouse zygotes following reconstruction by sequential transfer of germinal vesicles and haploid pronuclei. Liu H, Zhang J, Krey LC, Grifo JA. (USA)

We evaluated whether mouse oocytes reconstructed by germinal vesicle (GV) transfer can develop to blastocyst stage. The oocytes were artificially activated with sequential treatment of A23187 and anisomycin; fertilization was then established by transfer or exchange of pronuclei with those of zygotes fertilized in vivo. Type 1 zygotes were constructed by placing the male haploid pronucleus from a zygote into the cytoplasm of an oocyte that underwent GV transfer, in-vitro maturation and activation; for type 2 zygotes, the female pronucleus was removed from a zygote and replaced with the female pronucleus of an oocyte subjected to GV transfer, in-vitro maturation and activation. Karyotypes of activated oocytes and type 2 zygotes were also subjected to analysis. When cultured in human tubal fluid (HTF) medium, reconstructed oocytes matured and, following artificial activation, consistently developed a pronucleus with a haploid karyotype; the activation rate for this medium was two- to three-fold higher than that of oocytes cultured in M199 (87% versus 30% respectively). Following transfer of a male pronucleus, only 47% of the type 1 zygotes developed to morula or blastocyst stage and embryo morphology was poor. In contrast, 73% of the type 2 zygotes developed to morula or blastocyst stage, many even hatching, with few morphological anomalies. Normal karyotypes were observed in 88% of the type 2 zygotes analyzed. These observations demonstrate that the nucleus of a mouse oocyte subjected to sequential nuclear transfer at GV and pronucleus stages is, nonetheless, capable of maturing meiotically, activating normally and supporting embryonic development to hatching blastocyst stage. In contrast, the developmental potential of the cytoplasm of such oocytes appears to be compromised by these procedures. [PMID: 10967003] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10967003

-- Methods Mol Biol. 2004;254:149-64

A method for producing cloned pigs by using somatic cells as donors. Lai L, Prather RS.

Based on the source of donor cells, NT can be classified into embryonic cell NT and somatic cell NT. Somatic cell NT was first reported in 1996 and includes more practical applications. Most importantly, it provides a promising method for producing transgenic animals. This concept is exemplified by the generation of transgenic sheep, pigs and calves, along with gene-targeted sheep and pigs, derived from NT approaches by using transfected somatic cells. For pigs, somatic cell NT has another specific significance, as it allows the use of genetic modification procedures to produce tissues and organs from cloned pigs with reduced immunogenicity for use in xenotransplantation. However, when measured as development to term as a proportion of oocytes used, the efficiency of somatic cell NT, has been very low (1-2%). Several variables influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to NT, the method of artificial oocyte activation, embryo culture, possible loss of somatic imprinting in the nuclei of reconstructed embryos, failure of adequate reprogramming of the transplanted nucleus, and the techniques employed for NT. In some species (e.g., pigs) there is an additional difficulty in that at least four good embryos are required to induce and maintain pregnancy. Procedures for NT include the following steps: acquisition of recipient oocytes and donor cells, enucleation (removal of the chromosome from recipient oocytes), insertion of donor nuclei into enucleated oocytes, artificial activation of reconstructed oocytes, and embryo transfer (transfer of the reconstructed embryos into a surrogate). [PMID: 15041761] [PubMed - in process] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15041761

II. Artificial Germ-Line Cells

Note: Keep in mind that the term "germ-line cells" covers both male and female cells at many different stages of maturity: immature primitive germ-line cells (which are both diploid and totipotent, and found in the early human embryo as early as 21/2 weeks post fertilization or cloning), immature and mature cell stages. The most mature stages are the sperm and the secondary oocyte (the sex gametes). The sperm is haploid. The secondary oocyte is diploid until and unless it is fertilized by a sperm. Once fertilization/cloning has taken place, there is no longer just an "oocyte", a "fertilized oocyte", or a "reconstructed oocyte" - but rather a new living organism - a single-cell human being called a zygote.

A. Artificial Primitive, Primary and Secondary Oocytes

-- Politics and the Life Sciences 17, 1 (March, 1998):3-10

"Transplanting Nuclei between Human Eggs: Implications for Germ-Line Genetics." Bonnicksen, Andrea L.

A recent theoretical proposal suggests that diseases linked to mutations in mitochondrial DNA (mtDNA) might be circumvented by transferring the nucleus from the egg of a woman with a mitochondrial disease to a donor egg from which the nucleus has been removed and discarded. Egg cell nuclear transfer would be a straightforward technique for preventing serious diseases. However, its impact on all subsequently dividing cells, including germ cells, would make it an early form of germ-line gene therapy, albeit one that targets mtDNA rather than nuclear DNA. In addition, egg cell nuclear transfer relies on a procedure used in embryo or in somatic cell cloning, and it might present a relatively uncontentious setting for the refinement of procedures for cloning. Although it is not clear whether egg cell nuclear transfer is imminent, its proposal creates the opportunity to (1) identify categories of germ-line interventions, (2) explore whether ethical issues vary according to the category of germ-line intervention, and (3) craft more precise policy guidelines in which graduated levels of germ-line interventions are recognized. http://www.politicsandthelifesciences.org/Contents/Contents-1998-3/AbsBonn.html

-- Fertil Steril. 2003 Mar;79 Suppl 1:677-81

Microfilament disruption is required for enucleation and nuclear transfer in germinal vesicle but not metaphase II human oocytes. Tesarik J, Martinez F, Rienzi L, Ubaldi F, Iacobelli M, Mendoza C, Greco E. (Spain)

OBJECTIVE: To evaluate the usefulness of microfilament disruption before enucleation and nuclear transfer in human oocytes at different stages of maturation. DESIGN: Prospective experimental study. SETTING: Private clinics. PATIENT(S): Infertile couples undergoing assisted reproduction attempts. INTERVENTION(S): Oocyte enucleation and nuclear transfer, activation of reconstructed oocytes. CONCLUSION(S): Microfilament disruption before enucleation is required for germinal vesicle oocytes but not for metaphase II oocytes. [PMID: 12620476] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12620476

-- Hum Reprod. 2000 May;15(5):1149-54

Chemically and mechanically induced membrane fusion: non-activating methods for nuclear transfer in mature human oocytes. Tesarik J, Nagy ZP, Mendoza C, Greco E. (France)

Most current studies of nuclear transfer in mammalian oocytes have used electrofusion to incorporate donor cell nuclei into enucleated oocyte cytoplasts. However, the application of electrofusion to human oocytes is hampered by the relative ease with which this procedure induces oocyte activation. Here we tested a previously described chemical fusion technique and an original mechanical fusion procedure in this application. These techniques may be used in attempts to alleviate female infertility due to insufficiency of ooplasmic factors by nuclear transfer from patients' oocytes to enucleated donor oocyte cytoplasts. For eventual future use in human cloning, they would ensure prolonged exposure of transferred nuclei to metaphase promoting factor, which appears to be required for optimal nuclear reprogramming. [PMID: 10783368] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Display&dopt=pubmed_pubmed&from_uid=12620476

-- Hum Reprod. 1999 May;14(5):1312-7

A reliable technique of nuclear transplantation for immature mammalian oocytes. Takeuchi T, Ergun B, Huang TH, Rosenwaks Z, Palermo GD.

[PMID: 10325284] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10325284

-- Hum Reprod. 2000 Jul;15 Suppl 2:207-17

Spontaneous and artificial changes in human ooplasmic mitochondria. Barritt JA, Brenner CA, Willadsen S, Cohen J. (USA)

Our research has focused on promoting the development of compromised embryos by transferring presumably normal ooplasm, including mitochondria, to oocytes during intracytoplasmic insemination. Elimination of abnormal, rearranged mtDNA, such that the offspring inherit only normal mitochondria, is postulated to occur by a mtDNA 'bottleneck'. Among compromised human oocytes (n = 74) and early embryos (n = 137), investigations have shown the occurrence of deltamtDNA4977, the so-called common deletion, to be 33% among oocytes and 8% among embryos. In a total of 23 attempts in 21 women, eight healthy babies have been born and other pregnancies are ongoing. By examining the donor and recipient blood samples it is possible to distinguish differences in their mtDNA fingerprint. A small proportion of donor mitochondrial DNA was detected in samples with the following frequencies: embryos (six out of 13), amniocytes (one out of four), placenta (two out of four), and fetal cord blood (two out of four). Ooplasmic transfer can thus result in sustained mtDNA heteroplasmy representing both the donor and recipient. [PMID: 11041526] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12513856

-- Reprod Biomed Online. 2003 Dec;7(6):634-40

Micromanipulation of the human oocyte. Nagy ZP. (USA)

Intracytoplasmic sperm injection (ICSI) provides an excellent outcome in a consistent manner, and is therefore used worldwide as a routine procedure. Since its introduction, few modifications have been made to its methodology. Recently, a combination of ICSI with micro-hole drilling by laser (LA-ICSI) of the zona pellucida appeared to decrease oocyte degeneration rates and to improve embryo quality and implantation. Cytoplasmic transfer is a more recently introduced procedure where the objective is to improve the quality of patients' oocytes by transferring cytoplasm from a good quality donor oocyte, in cases where it is assumed that cytoplasm is compromised. Nuclear transfer, involving exchange of nuclei between donor and receptor oocytes, is still an experimental procedure, the objective being similar to cytoplasmic transfer in improving oocyte/embryo quality. A nuclear transfer procedure involving somatic cells for reproductive purposes should not be used in humans, for ethical and technical considerations. On the other hand, nuclear transfer for therapeutic purposes to obtain stem cells may be considered in respect of its unique potential in medicine. Finally, the most recently emerged new concept under investigation is the haploidization of somatic cells for the purpose of creating artificial gametes. PMID: 14748960 [PubMed - in process] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14748960

-- Chromosome Res. 2000;8(3):183-91

Generation of transgenic mice and germline transmission of a mammalian artificial chromosome introduced into embryos by pronuclear microinjection. Co DO, Borowski AH, Leung JD, van der Kaa J, Hengst S, Platenburg GJ, Pieper FR, Perez CF, Jirik FR, Drayer JI. (Canada)

We have generated transgenic mice by pronuclear microinjection of a murine satellite DNA-based artificial chromosome (SATAC). As 50% of the founder progeny were SATAC-positive, this demonstrates that SATAC transmission through the germline had occurred. FISH analyses of metaphase chromosomes from mitogen-activated peripheral blood lymphocytes from both the founder and progeny revealed that the SATAC was maintained as a discrete chromosome and that it had not integrated into an endogenous chromosome. To our knowledge, this is the first report of the germline transmission of a genetically engineered mammalian artificial chromosome within transgenic animals generated through pronuclear microinjection. We have also shown that murine SATACs can be similarly introduced into bovine embryos. The use of embryo microinjection to generate transgenic mammals carrying genetically engineered chromosomes provides a novel method by which the unique advantages of chromosome-based gene delivery systems can be exploited. [PMID: 10841045] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10841045

-- Hum Reprod. 2004 May;19(5):1189-94. Epub 2004 Apr 07

Chromosome number and development of artificial mouse oocytes and zygotes. Heindryckx B, Lierman S, Van der Elst J, Dhont M. (Belgium)

Infertility due to the absence of gametes is one of the last frontiers in reproductive medicine. Sperm or oocyte donation is currently the only treatment option but this approach lacks the genetic contribution of both partners. Artificial production of gametes through haploidization may offer an alternative strategy. The aim of this study was to evaluate the efficiency of producing artificial oocytes and zygotes with correct enucleated mature mouse oocytes to produce artificial oocytes. These observations question the possibility of obtaining chromosomally normal embryos from artificial oocytes or zygotes. PMID: 15070880 [PubMed - in process] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15070880

-- Methods Mol Biol. 2004;256:141-58

Microinjection of BAC DNA into the pronuclei of fertilized mouse oocytes. Vintersten K, Testa G, Stewart AF.

European Molecular Biology Laboratory, Heidelberg, Germany. [PMID: 15024165] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15024165

-- Methods Mol Biol. 2004;256:123-39

BAC engineering for the generation of ES cell-targeting constructs and mouse transgenes. Testa G, Vintersten K, Zhang Y, Benes V, Muyrers JP, Stewart AF. (Germany)

[PMID: 15024164] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15024164

-- Reprod Biomed Online. 2003 Dec;7(6):634-40

Micromanipulation of the human oocyte. Nagy ZP. (USA)

Intracytoplasmic sperm injection (ICSI) provides an excellent outcome in a consistent manner, and is therefore used worldwide as a routine procedure. Recently, a combination of ICSI with micro-hole drilling by laser (LA-ICSI) of the zona pellucida appeared to decrease oocyte degeneration rates and to improve embryo quality and implantation. Cytoplasmic transfer is a more recently introduced procedure where the objective is to improve the quality of patients' oocytes by transferring cytoplasm from a good quality donor oocyte, in cases where it is assumed that cytoplasm is compromised. Nuclear transfer, involving exchange of nuclei between donor and receptor oocytes, is still an experimental procedure, the objective being similar to cytoplasmic transfer in improving oocyte/embryo quality. A nuclear transfer procedure involving somatic cells for reproductive purposes should not be used in humans, for ethical and technical considerations. On the other hand, nuclear transfer for therapeutic purposes to obtain stem cells may be considered in respect of its unique potential in medicine. Finally, the most recently emerged new concept under investigation is the haploidization of somatic cells for the purpose of creating artificial gametes. [PMID: 14748960] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14748960

-- The New Zealand Medical Journal, Vol 115 No 1162 ISSN 1175 8716

Fertility hope for chemo patients as doctors grow eggs outside the body Most mammals produce only a few eggs at a time. If immature precursor cells could be matured outside the body, far more eggs could be obtained. Now Izuho Hatadafs team at Gunma University in Japan has managed to grow mouse eggs from their very earliest stages and produce healthy offspring from them.

If Hatadafs technique works with human eggs, it would provide a new way to preserve the fertility of female patients facing treatments such as radiotherapy or chemotherapy that damage their eggs. Eggs could be grown from slices of frozen ovaries. [T]herefs a catch. Hatadafs team managed to get some mouse eggs to start to mature by taking whole ovaries from fetuses and growing them for 28 days. But the eggs stalled at the final stage of development. To get them to complete their development, the researchers had to transfer their genetic material to mature eggs taken from adult mice - the same nuclear transfer technique used in cloning. That means any human treatments based on the technique would still have to rely on donor eggs, which are in short supply. (quoting from New Scientist 3, 2002) http://www.nzma.org.nz/journal/115-1162/190/content.pdf

-- Biol Reprod. 2004 Mar;70(3):752-8. Epub 2003 Nov 12

Nuclear and microtubule dynamics of G2/M somatic nuclei during haploidization in germinal vesicle-stage mouse oocytes. Chang CC, Nagy ZP, Abdelmassih R, Yang X, Tian XC. (USA)

During the haploidization process, it is expected that diploid chromosomes of somatic cells will be reduced to haploid for the generation of artificial gametes. In the present study, we aimed to use enucleated mouse oocytes ... The reconstructed oocytes were then induced to undergo meiosis in vitro ... Following oocyte activation, more than half (21/33, 63.6%) of the reconstructed oocytes with pseudo-PBs formed separated pseudopronuclei (PN), suggesting formation of functional spindles. In summary, this study demonstrated that a high proportion of G2/M somatic nuclei appear to undergo meiosis-like division, in two successive steps, forming a pseudo-PB and two separate pseudo-PN upon in vitro maturation and activation treatment. Moreover, the enucleated geminal vesicle cytoplast retained its capacity for meiotic division following the introduction of a somatic G2/M nucleus. [PMID: 14613892] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14613892

-- Reprod Biomed Online. 2001;3(3):205-211

Fertilization of mouse oocytes using somatic cells as male germ cells. Lacham-Kaplan O, Daniels R, Trounson A.

The Monash Institute of Reproduction and Development, Centre for Early Human Development, Melbourne, Australia. Female and male mouse somatic cells were injected into mouse F(1) oocytes. The cells used included cumulus cells (female) and muscle derived fibroblasts (male). The ability of the cells to fertilize oocytes and support embryonic development was examined. Following activation of the injected oocytes, two second polar bodies were extruded and two pronuclei were formed, one derived from the oocyte chromosomes and the other from the somatic cell chromosomes in a similar way to that observed following fertilization with secondary spermatocytes. Most (80-90%) of the 'zygotes' produced by somatic cells cleaved to two cells in culture and ~50% reached the morula stage. However, the developmental competence of the embryos to reach blastocysts was limited. The present study demonstrates that mouse somatic cells undergo haploidization when injected into metaphase II oocytes, fertilize oocytes as diploid male germ cells and support preimplantation development to a degree. [PMID: 12513856] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11041526

Next Page: B. Artificial Spermatogonia, Spermatocytes, and Sperms
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