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Haploidization to produce human embryos: a new frontier for micromanipulation
RBMOnline - Vol 8. No 5. 2004 492-495 Reproductive BioMedicine Online; www.rbmonline.com/Article/1329 on web 19 March 2004
Symposium: Clinical prospects of nuclear transfer and somatic cell haploidization Guest Editor: Zsolt Peter Nagy
Haploidization to produce human embryos: a new frontier for micromanipulation Zsolt Peter Nagy obtained his MD degree (1986) and the speciality degree in Obstetrics and Gynaecology (1996) at the Semmelweis Medical University in Budapest. The PhD degree was granted to him at the Free University of Brussels (VUB) in 1997. He worked for 8 years at the Centre for Reproductive Medicine of the VUB in the team that developed the ICSI procedure (1991–1997). He developed a new technique for fusion between karyoplast and cytoplast for nuclear transfer (2000). He is author or co-author of more than 100 publications. At present he is the scientific and laboratory director of Reproductive Biology Associates in Atlanta (USA).
Zsolt Peter Nagy Reproductive Biology Associates, 1150 Lake Hearn Drive, Suite 600, Atlanta, GA 30342, USA Correspondence: Tel: +1 404 4593557; Fax: +1 404 2573314; e-mail: [email protected] or [email protected] The technologies of gamete manipulation used in assisted reproduction are rapidly proliferating, and in some instances are adopted for clinical application before adequate research proves their efficiency or safety. Since micromanipulation techniques in humans are developing far beyond assisting fertilization by intracytoplasmic sperm injection (ICSI), there is a strong need to assess the impact of these methods on gamete/embryo development and, most importantly, for effects that may not be manifested until later in life. One of the ultimate goals of assisted reproduction has been to obtain offspring in infertile couples, a goal that was first achieved a little more than 25 years ago (Steptoe and Edwards, 1978). With the help of another breakthrough – ICSI – the success of IVF treatment was extended to patients suffering more severe male factor infertility (Palermo et al., 1992; Van Steirteghem et al., 1993); however, for couples of advanced reproductive age, success rates remained low. To address this problem, a new technique has been investigated, referred to as ‘haploidization’ or ‘artificial gamete production’. It was hoped that this process might help overcome one of the last barriers of infertility (Palermo et al., 2002a; Tesarik et al., 2001; Chang et al., 2004).
492
After some encouraging preliminary results (Palermo et al., 2002b; Lacham-Kaplan et al., 2001; Tesarik et al., 2001) it was demonstrated that somatic cell nuclear transfer into enucleated oocytes generates more problems than the established but technically challenging process of cloning (Tateno et al., 2003; Eichenlaub-Ritter, 2003; Chang et al., 2004). In the meantime, trying to develop further this exigent technique can provide us new insight on some of the most intriguing topics in cell biology: epigenetic remodelling and reprogramming of the genetic material that play a crucial role during natural gametogenesis as well as after nuclear transfer using adult somatic or embryonic cells.
Epigenetic alterations refer to diverse mechanisms of gene regulations/expressions that defy Mendelian rules. Undoubtedly gametogenesis, fertilization and embryo development are the phases when many of these events occur (El-Maarri et al., 2001), as represented schematically in Figure 1, although they also arise in embryo stem (ES) cells in vitro (Rasmussen, 2003). It was shown that during gametogenesis a complete erasing of any imprinting occurs, which is essential since some genes are imprinted differentially depending on gender (ovary/testis-specific imprinting; Fedoriw et al., 2004). During early development, a dramatic reduction in methylation levels occurs in the early preimplantation embryo (Monk et al., 1987), with a different rate in the male genome compared with the female genome (see Figure 1) and perhaps not independently the onset of the paternally derived regulatory mechanism (Szollosi and Yotsuyanagi, 1985). This is followed by an upsurge of de-novo methylation involving most CpG residues but leaving the CpG islands unmethylated at the time of implantation (Kafri et al., 1992). After implantation, most of the genomic DNA is methylated, whereas tissue-specific genes undergo demethylation in their tissues of expression (Razin and Cedar, 1991). Epigenetic events and mechanisms are extremely complex and much research is needed to enhance our knowledge of the processes involved; however, some of the proposed modes of actions are shown in Figure 2 including DNA methylation, histone modifications (methylation, acetylation), X chromosome inactivation (Xue et al., 2002), and non-coding micro RNA (miRNA) interference (Verdel et al., 2004). Not surprisingly, due to the complexity of epigenetic action on the genome (Edwards, 2003), there are many possibilities for pathological alterations (Humpherys et al., 2002) resulting mostly in the arrest of embryonic development, which may explain the extremely low efficiency of cloning and fetal abnormalities (Hill et al., 2000; Kang et
Symposium - Haploidization to produce human embryos - ZP Nagy
Figure 1. Genetic reprogramming through methylation in germ cells and embryo.
a
c
b
Figure 2. Mechanisms for (a) genetic imprinting via methylation/acetylation; (b) genetic imprinting involving the XIST gene for X chromosome inactivation where the RNA product alone or together with a nuclear factor binds to the chromosome and induces a conformational change; (c) regulating gene end product through microRNAs (miRNAs). These short length RNAs are thought to regulate gene expression primarily by inhibiting translation.
Figure 3. A theoretical model of producing artificial gametes with techniques that already exist.
493
Symposium - Haploidization to produce human embryos - ZP Nagy
al., 2001), though in some cases efficiency may be improved by taking into account physiological features of the species involved, e.g. altered timing for nuclear transfer (NT) in rabbit embryos (Chesne et al., 2002). Alarmingly, on some occasions diseases related to epigenetic alterations are manifested only in later life (such as Beckwith–Wiedemann syndrome; Gosden et al., 2003). When appreciating this highly orchestrated function of the chromatin, it may not come as a surprise that even just slightly altered environmental conditions, such as (extended) in-vitro culture, could cause epigenetic modifications that can perhaps be observed as behavioural changes only later in life (Ecker et al., 2004). In view of these recent developments, gametogenesis is more a focus of research now than ever before. Extending our understanding of how gametes are produced naturally, the regulating and organizing factors involved can reveal information that may help to develop new diagnostic and therapeutic measures. The accompanying review by Sarah Kimmins and colleagues (2004) on the gamete- and developmental–stage-specific gene regulator CREM gives an excellent demonstration of the importance of this topic. Nuclear transfer studies remain increasingly important not only for improving efficiency rates of the technique or success in various species but also as a tool/methodology to examine epigenetic organization/function of the chromatin (Wrenzycki and Niemann, 2003). Additionally, cloning techniques provide the methodological background to stem cell research that is expected to play a central role in disease treatments in the near future (Hochedlinger and Jaenisch, 2003; Edwards, 2004). Although recent nuclear transfer experiments failed on primates (Simerly et al., 2003), most recently it was successful on humans (Hwang et al., 2004) demonstrating both the extreme rapid progression of science and that rules derived from other species must be applied sparingly to man. Another paper in this symposium by Cindy Tian (2004) reviews the experimental knowledge gained from nuclear transfer in this regard. The final paper in the symposium provides a review on the present standing of ‘haploidization’ or ‘artificial gamete production’ by Takeuchi and Palermo (2004). It is clear from the most recent research results that haploidization does not perform in the way it was hoped. It was proposed earlier that haploidization may work similarly to cloning: transferring a somatic cell nucleus to enucleated (mature or maturing) egg cytoplasm, then performing oocyte activation (in a somewhat altered way compared with cloning) may result in semimeiotic reduction of the DNA content. Not totally unexpectedly, this approach was not fully successful, not only since the same problems emerged as encountered during cloning and relating to impaired nuclear remodelling and reprogramming, but also because of alterations in spindle formation and in chromosome alignment/segregation (Tateno et al., 2003; Chang et al., 2004).
494
However, the production of artificial gametes can be envisioned using an alternative approach, and a successful outcome is possibly nearer than expected. As Figure 3 depicts a combination of existing techniques that might provide a solution to this problem: (i) somatic cell nuclear transfer to enucleated oocyte (cloning; Cibelli et al., 2001; Hwang et al., 2004); (ii) in-vitro culture to blastocyst stage;
(iii) establishment of a stem cell line from the inner cell mass (ICM; Hwang et al., 2004); (iv) tissue-specific culture of stem cells to obtain gametes (oocyte or spermatozoon; Hubner et al., 2003; Geijsen et al., 2004). All of these techniques have been demonstrated to work separately, the ‘only’ action required being to channel them together in a single chain of testing research. Although this approach can be expected to provide functional gametes in the near future, yet another approach to somatic cell haploidization may be developed in coming years that may mimic better the true in-vivo conditions of meiosis. Such a technique might be technically more efficient and ethically more acceptable. This alternative technique ‘in-vitro meiosis’ requires the identification of cellular factors guiding the path of cell division between mitosis and meiosis and factors that govern meiotic cell division (Marston et al., 2004). With the use of more advanced methods such as microarrays it should be possible to accelerate this process of identifying the related gene products and then applying these factors to somatic cell(s) to help achieve this goal.
References Chang CC, Nagy ZP, Abdelmassih R et al. 2004 Nuclear and microtubule dynamics of G2/M somatic nuclei during haploidization in germinal vesicle-stage mouse oocytes. Biology of Reproduction 70, 752–758. Chesne P, Adenot PG, Viglietta C et al. 2002 Cloned rabbits produced by nuclear transfer from adult somatic cells. Nature Biotechnology 20, 366–369. Cibelli JB, Kiessling AA, Cunniff K et al. 2001 Rapid communication: somatic cell nuclear transfer in humans: pronuclear and early embryonic development. e-biomed: Journal of Regenerative Medicine 2, 25–31. Ecker DJ, Stein P, Xu Z et al. 2004 Long-term effects of culture of preimplantation mouse embryos on behaviour. Proceedings of the National Academy of Sciences of the USA 101, 1595–1600. Edwards RG 2003 Aspects of the molecular regulation of early mammalian development. Reproductive BioMedicine Online 6, 97–113. Edwards RG 2004 Stem cells today: A. Origin and potential of embryo stem cells. Reproductive BioMedicine Online 8, 275–306. Eichenlaub-Ritter U 2003 Reproductive semi-cloning respecting biparental origin. Reconstitution of gametes for assisted reproduction. Human Reproduction 18, 473–475. El-Maarri O, Buiting K, Peery E et al. 2001 Maternal methylation imprints on human chromosome 15 are established during or after fertilization. Nature Genetics 27, 341–344. Fedoriw AM, Stein P, Svoboda P et al. 2004 Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting. Science 303, 238–240. Geijsen N, Horoschak M, Kim K et al. 2004 Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427, 148–154. Gosden R, Trasler J, Lucifero D et al. 2003 Rare congenital disorders, imprinted genes, and assisted reproductive technology. Lancet 361, 1975–1977. Hill J, Burghardt R, Jones K et al. 2000 Evidence of placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biology of Reproduction 63, 1787–1794. Hochedlinger K, Jaenisch R 2003 Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. New England Journal of Medicine 349, 275–286. Hubner K, Fuhrmann G, Christenson LK et al. 2003 Derivation of oocytes from mouse embryonic stem cells. Science 300, 1251–1256.
Symposium - Haploidization to produce human embryos - ZP Nagy
Humpherys D, Eggan K, Akutsu H et al. 2002 Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei. Proceedings of the National Academy of Sciences of the USA 99, 1289–1294. Hwang WS, Ryu YJ, Park JH et al. 2004 Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science 12 February [Epub ahead of print] Lacham-Kaplan O, Daniels R, Trounson A 2001 Fertilization of mouse oocytes using somatic cells as male germ cells. Reproductive BioMedicine Online 3, 205–211. Kafri T, Ariel M, Brandeis M et al. 1992 Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes and Development 6, 705. Kang YK, Koo DB, Park JS et al. 2001 Aberrant methylation of donor genome in cloned bovine embryos. Nature Genetics 28, 173–177. Kimmins S, Kotaja N, Fienga G 2004 A specific programme of gene transcription in male germ cells. Reproductive BioMedicine Online 8, 496–500. Marston AL, Tham WH, Shah H et al. 2004 A genome-wide screen identifies genes required for centromeric cohesion. Science 303, 1367–1370. Monk M, Boubelik M, and Lehnert S 1987 Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99, 371–382. Palermo G, Joris H, Devroey P et al. 1992 Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340, 17–18. Palermo GD, Takeuchi T, Rosenwaks Z 2002a Technical approaches to correction of oocyte aneuploidy. Human Reproduction 17, 2165–2173. Palermo GD, Takeuchi T, Rosenwaks Z 2002b Oocyte-induced haploidization. Reproductive BioMedicine Online 4, 237–242. Rasmussen TP 2003 Embryonic stem cell differentiation: A
chromatin perspective. Reproductive Biology and Endocrinology 1, 100. Razin A and Cedar H 1991 DNA methylation and gene expression. Microbiological Reviews 55, 451–458. Simerly C, Dominko T, Navara C et al. 2003 Molecular correlates of primate nuclear transfer failures. Science 300, 297. Steptoe PC and Edwards RG 1978 Birth after the reimplantation of a human embryo. Lancet 2, 366. Szollosi D, Yotsuyanagi Y 1985 Activation of paternally derived regulatory mechanism in early mouse embryo. Developmental Biology 111, 256–259. Takeuchi T, Palermo GD 2004 Cloning and related techniques in reproductive medicine. Reproductive BioMedicine Online 8, 509–515. Tateno H, Akutsu H, Kamiguchi Y et al. 2003 Inability of mature oocytes to create functional haploid genomes from somatic cell nuclei. Fertility and Sterility 79, 216–218. Tesarik J, Nagy ZP, Sousa M et al. 2001 Fertilizable oocytes reconstructed from patient’s somatic cell nuclei and donor ooplasts. Reproductive BioMedicine Online 2, 160–164. Tian XC 2004 Reprogramming of epigenetic inheritance by somatic cell nuclear transfer. Reproductive BioMedicine Online 8, 501–508. Van Steirteghem AC, Nagy Z, Joris H et al. 1993 High fertilization and implantation rates after intracytoplasmic sperm injection. Human Reproduction 8, 1061–1066. Verdel A, Jia S, Gerber S et al. 2004 RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672–676. Wrenzycki C, Niemann H 2003 Epigenetic reprogramming in early embryonic development: effects of in-vitro production and somatic nuclear transfer. Reproductive BioMedicine Online 7, 649–656. Xue F, Tian XC, Du F et al. 2002 Aberrant patterns of X chromosome inactivation in bovine clones. Nature Genetics 31, 216–220.
495
Symposium: Clinical prospects of nuclear transfer and somatic cell haploidization Guest Editor: Zsolt Peter Nagy
Haploidization to produce human embryos: a new frontier for micromanipulation Zsolt Peter Nagy obtained his MD degree (1986) and the speciality degree in Obstetrics and Gynaecology (1996) at the Semmelweis Medical University in Budapest. The PhD degree was granted to him at the Free University of Brussels (VUB) in 1997. He worked for 8 years at the Centre for Reproductive Medicine of the VUB in the team that developed the ICSI procedure (1991–1997). He developed a new technique for fusion between karyoplast and cytoplast for nuclear transfer (2000). He is author or co-author of more than 100 publications. At present he is the scientific and laboratory director of Reproductive Biology Associates in Atlanta (USA).
Zsolt Peter Nagy Reproductive Biology Associates, 1150 Lake Hearn Drive, Suite 600, Atlanta, GA 30342, USA Correspondence: Tel: +1 404 4593557; Fax: +1 404 2573314; e-mail: [email protected] or [email protected] The technologies of gamete manipulation used in assisted reproduction are rapidly proliferating, and in some instances are adopted for clinical application before adequate research proves their efficiency or safety. Since micromanipulation techniques in humans are developing far beyond assisting fertilization by intracytoplasmic sperm injection (ICSI), there is a strong need to assess the impact of these methods on gamete/embryo development and, most importantly, for effects that may not be manifested until later in life. One of the ultimate goals of assisted reproduction has been to obtain offspring in infertile couples, a goal that was first achieved a little more than 25 years ago (Steptoe and Edwards, 1978). With the help of another breakthrough – ICSI – the success of IVF treatment was extended to patients suffering more severe male factor infertility (Palermo et al., 1992; Van Steirteghem et al., 1993); however, for couples of advanced reproductive age, success rates remained low. To address this problem, a new technique has been investigated, referred to as ‘haploidization’ or ‘artificial gamete production’. It was hoped that this process might help overcome one of the last barriers of infertility (Palermo et al., 2002a; Tesarik et al., 2001; Chang et al., 2004).
492
After some encouraging preliminary results (Palermo et al., 2002b; Lacham-Kaplan et al., 2001; Tesarik et al., 2001) it was demonstrated that somatic cell nuclear transfer into enucleated oocytes generates more problems than the established but technically challenging process of cloning (Tateno et al., 2003; Eichenlaub-Ritter, 2003; Chang et al., 2004). In the meantime, trying to develop further this exigent technique can provide us new insight on some of the most intriguing topics in cell biology: epigenetic remodelling and reprogramming of the genetic material that play a crucial role during natural gametogenesis as well as after nuclear transfer using adult somatic or embryonic cells.
Epigenetic alterations refer to diverse mechanisms of gene regulations/expressions that defy Mendelian rules. Undoubtedly gametogenesis, fertilization and embryo development are the phases when many of these events occur (El-Maarri et al., 2001), as represented schematically in Figure 1, although they also arise in embryo stem (ES) cells in vitro (Rasmussen, 2003). It was shown that during gametogenesis a complete erasing of any imprinting occurs, which is essential since some genes are imprinted differentially depending on gender (ovary/testis-specific imprinting; Fedoriw et al., 2004). During early development, a dramatic reduction in methylation levels occurs in the early preimplantation embryo (Monk et al., 1987), with a different rate in the male genome compared with the female genome (see Figure 1) and perhaps not independently the onset of the paternally derived regulatory mechanism (Szollosi and Yotsuyanagi, 1985). This is followed by an upsurge of de-novo methylation involving most CpG residues but leaving the CpG islands unmethylated at the time of implantation (Kafri et al., 1992). After implantation, most of the genomic DNA is methylated, whereas tissue-specific genes undergo demethylation in their tissues of expression (Razin and Cedar, 1991). Epigenetic events and mechanisms are extremely complex and much research is needed to enhance our knowledge of the processes involved; however, some of the proposed modes of actions are shown in Figure 2 including DNA methylation, histone modifications (methylation, acetylation), X chromosome inactivation (Xue et al., 2002), and non-coding micro RNA (miRNA) interference (Verdel et al., 2004). Not surprisingly, due to the complexity of epigenetic action on the genome (Edwards, 2003), there are many possibilities for pathological alterations (Humpherys et al., 2002) resulting mostly in the arrest of embryonic development, which may explain the extremely low efficiency of cloning and fetal abnormalities (Hill et al., 2000; Kang et
Symposium - Haploidization to produce human embryos - ZP Nagy
Figure 1. Genetic reprogramming through methylation in germ cells and embryo.
a
c
b
Figure 2. Mechanisms for (a) genetic imprinting via methylation/acetylation; (b) genetic imprinting involving the XIST gene for X chromosome inactivation where the RNA product alone or together with a nuclear factor binds to the chromosome and induces a conformational change; (c) regulating gene end product through microRNAs (miRNAs). These short length RNAs are thought to regulate gene expression primarily by inhibiting translation.
Figure 3. A theoretical model of producing artificial gametes with techniques that already exist.
493
Symposium - Haploidization to produce human embryos - ZP Nagy
al., 2001), though in some cases efficiency may be improved by taking into account physiological features of the species involved, e.g. altered timing for nuclear transfer (NT) in rabbit embryos (Chesne et al., 2002). Alarmingly, on some occasions diseases related to epigenetic alterations are manifested only in later life (such as Beckwith–Wiedemann syndrome; Gosden et al., 2003). When appreciating this highly orchestrated function of the chromatin, it may not come as a surprise that even just slightly altered environmental conditions, such as (extended) in-vitro culture, could cause epigenetic modifications that can perhaps be observed as behavioural changes only later in life (Ecker et al., 2004). In view of these recent developments, gametogenesis is more a focus of research now than ever before. Extending our understanding of how gametes are produced naturally, the regulating and organizing factors involved can reveal information that may help to develop new diagnostic and therapeutic measures. The accompanying review by Sarah Kimmins and colleagues (2004) on the gamete- and developmental–stage-specific gene regulator CREM gives an excellent demonstration of the importance of this topic. Nuclear transfer studies remain increasingly important not only for improving efficiency rates of the technique or success in various species but also as a tool/methodology to examine epigenetic organization/function of the chromatin (Wrenzycki and Niemann, 2003). Additionally, cloning techniques provide the methodological background to stem cell research that is expected to play a central role in disease treatments in the near future (Hochedlinger and Jaenisch, 2003; Edwards, 2004). Although recent nuclear transfer experiments failed on primates (Simerly et al., 2003), most recently it was successful on humans (Hwang et al., 2004) demonstrating both the extreme rapid progression of science and that rules derived from other species must be applied sparingly to man. Another paper in this symposium by Cindy Tian (2004) reviews the experimental knowledge gained from nuclear transfer in this regard. The final paper in the symposium provides a review on the present standing of ‘haploidization’ or ‘artificial gamete production’ by Takeuchi and Palermo (2004). It is clear from the most recent research results that haploidization does not perform in the way it was hoped. It was proposed earlier that haploidization may work similarly to cloning: transferring a somatic cell nucleus to enucleated (mature or maturing) egg cytoplasm, then performing oocyte activation (in a somewhat altered way compared with cloning) may result in semimeiotic reduction of the DNA content. Not totally unexpectedly, this approach was not fully successful, not only since the same problems emerged as encountered during cloning and relating to impaired nuclear remodelling and reprogramming, but also because of alterations in spindle formation and in chromosome alignment/segregation (Tateno et al., 2003; Chang et al., 2004).
494
However, the production of artificial gametes can be envisioned using an alternative approach, and a successful outcome is possibly nearer than expected. As Figure 3 depicts a combination of existing techniques that might provide a solution to this problem: (i) somatic cell nuclear transfer to enucleated oocyte (cloning; Cibelli et al., 2001; Hwang et al., 2004); (ii) in-vitro culture to blastocyst stage;
(iii) establishment of a stem cell line from the inner cell mass (ICM; Hwang et al., 2004); (iv) tissue-specific culture of stem cells to obtain gametes (oocyte or spermatozoon; Hubner et al., 2003; Geijsen et al., 2004). All of these techniques have been demonstrated to work separately, the ‘only’ action required being to channel them together in a single chain of testing research. Although this approach can be expected to provide functional gametes in the near future, yet another approach to somatic cell haploidization may be developed in coming years that may mimic better the true in-vivo conditions of meiosis. Such a technique might be technically more efficient and ethically more acceptable. This alternative technique ‘in-vitro meiosis’ requires the identification of cellular factors guiding the path of cell division between mitosis and meiosis and factors that govern meiotic cell division (Marston et al., 2004). With the use of more advanced methods such as microarrays it should be possible to accelerate this process of identifying the related gene products and then applying these factors to somatic cell(s) to help achieve this goal.
References Chang CC, Nagy ZP, Abdelmassih R et al. 2004 Nuclear and microtubule dynamics of G2/M somatic nuclei during haploidization in germinal vesicle-stage mouse oocytes. Biology of Reproduction 70, 752–758. Chesne P, Adenot PG, Viglietta C et al. 2002 Cloned rabbits produced by nuclear transfer from adult somatic cells. Nature Biotechnology 20, 366–369. Cibelli JB, Kiessling AA, Cunniff K et al. 2001 Rapid communication: somatic cell nuclear transfer in humans: pronuclear and early embryonic development. e-biomed: Journal of Regenerative Medicine 2, 25–31. Ecker DJ, Stein P, Xu Z et al. 2004 Long-term effects of culture of preimplantation mouse embryos on behaviour. Proceedings of the National Academy of Sciences of the USA 101, 1595–1600. Edwards RG 2003 Aspects of the molecular regulation of early mammalian development. Reproductive BioMedicine Online 6, 97–113. Edwards RG 2004 Stem cells today: A. Origin and potential of embryo stem cells. Reproductive BioMedicine Online 8, 275–306. Eichenlaub-Ritter U 2003 Reproductive semi-cloning respecting biparental origin. Reconstitution of gametes for assisted reproduction. Human Reproduction 18, 473–475. El-Maarri O, Buiting K, Peery E et al. 2001 Maternal methylation imprints on human chromosome 15 are established during or after fertilization. Nature Genetics 27, 341–344. Fedoriw AM, Stein P, Svoboda P et al. 2004 Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting. Science 303, 238–240. Geijsen N, Horoschak M, Kim K et al. 2004 Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427, 148–154. Gosden R, Trasler J, Lucifero D et al. 2003 Rare congenital disorders, imprinted genes, and assisted reproductive technology. Lancet 361, 1975–1977. Hill J, Burghardt R, Jones K et al. 2000 Evidence of placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biology of Reproduction 63, 1787–1794. Hochedlinger K, Jaenisch R 2003 Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. New England Journal of Medicine 349, 275–286. Hubner K, Fuhrmann G, Christenson LK et al. 2003 Derivation of oocytes from mouse embryonic stem cells. Science 300, 1251–1256.
Symposium - Haploidization to produce human embryos - ZP Nagy
Humpherys D, Eggan K, Akutsu H et al. 2002 Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei. Proceedings of the National Academy of Sciences of the USA 99, 1289–1294. Hwang WS, Ryu YJ, Park JH et al. 2004 Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science 12 February [Epub ahead of print] Lacham-Kaplan O, Daniels R, Trounson A 2001 Fertilization of mouse oocytes using somatic cells as male germ cells. Reproductive BioMedicine Online 3, 205–211. Kafri T, Ariel M, Brandeis M et al. 1992 Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes and Development 6, 705. Kang YK, Koo DB, Park JS et al. 2001 Aberrant methylation of donor genome in cloned bovine embryos. Nature Genetics 28, 173–177. Kimmins S, Kotaja N, Fienga G 2004 A specific programme of gene transcription in male germ cells. Reproductive BioMedicine Online 8, 496–500. Marston AL, Tham WH, Shah H et al. 2004 A genome-wide screen identifies genes required for centromeric cohesion. Science 303, 1367–1370. Monk M, Boubelik M, and Lehnert S 1987 Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99, 371–382. Palermo G, Joris H, Devroey P et al. 1992 Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340, 17–18. Palermo GD, Takeuchi T, Rosenwaks Z 2002a Technical approaches to correction of oocyte aneuploidy. Human Reproduction 17, 2165–2173. Palermo GD, Takeuchi T, Rosenwaks Z 2002b Oocyte-induced haploidization. Reproductive BioMedicine Online 4, 237–242. Rasmussen TP 2003 Embryonic stem cell differentiation: A
chromatin perspective. Reproductive Biology and Endocrinology 1, 100. Razin A and Cedar H 1991 DNA methylation and gene expression. Microbiological Reviews 55, 451–458. Simerly C, Dominko T, Navara C et al. 2003 Molecular correlates of primate nuclear transfer failures. Science 300, 297. Steptoe PC and Edwards RG 1978 Birth after the reimplantation of a human embryo. Lancet 2, 366. Szollosi D, Yotsuyanagi Y 1985 Activation of paternally derived regulatory mechanism in early mouse embryo. Developmental Biology 111, 256–259. Takeuchi T, Palermo GD 2004 Cloning and related techniques in reproductive medicine. Reproductive BioMedicine Online 8, 509–515. Tateno H, Akutsu H, Kamiguchi Y et al. 2003 Inability of mature oocytes to create functional haploid genomes from somatic cell nuclei. Fertility and Sterility 79, 216–218. Tesarik J, Nagy ZP, Sousa M et al. 2001 Fertilizable oocytes reconstructed from patient’s somatic cell nuclei and donor ooplasts. Reproductive BioMedicine Online 2, 160–164. Tian XC 2004 Reprogramming of epigenetic inheritance by somatic cell nuclear transfer. Reproductive BioMedicine Online 8, 501–508. Van Steirteghem AC, Nagy Z, Joris H et al. 1993 High fertilization and implantation rates after intracytoplasmic sperm injection. Human Reproduction 8, 1061–1066. Verdel A, Jia S, Gerber S et al. 2004 RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672–676. Wrenzycki C, Niemann H 2003 Epigenetic reprogramming in early embryonic development: effects of in-vitro production and somatic nuclear transfer. Reproductive BioMedicine Online 7, 649–656. Xue F, Tian XC, Du F et al. 2002 Aberrant patterns of X chromosome inactivation in bovine clones. Nature Genetics 31, 216–220.
495