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1 RBMOnline - Vol 10. No Reproductive BioMedicine Online; on web 15 December 2004 Article Cytogenetic analysis of human somatic cell haploidization Dr Vasiliy Galat received his PhD in 1997 from the Moscow Biotechnology Institute. He spent 2 years in the Max-Delbruck Center for Molecular Medicine, Berlin as a post-doctoral fellow then moved to the Reproductive Genetics Institute, Chicago. His research interests include fertilization, early mammalian development and cell differentiation. Dr Vasiliy Galat V Galat, S Ozen, S Rechitsky, A Kuliev 1, Y Verlinsky Reproductive Genetics Institute, 2825 North Halsted Street, Chicago, IL 60657, USA 1 Correspondence: Tel: ; Fax: ; anverkuliev@hotmail.com Abstract Despite recent interest in the derivation of female and male gametes through somatic cell nuclear transfer, there is still insufficient data on chromosomal analysis of these gametes resulting from haploidization, especially involving a human nuclear donor and recipient oocytes. The objective of this study was to investigate the fidelity of chromosomal separation during haploidization of human cumulus cells by in-vitro matured human enucleated MII oocytes. A total of 129 oocytes were tested 4 7, 8 14, or h after nuclear transfer (NT) followed by electro-stimulation, resulting in 71.3% activation efficiency on average. Haploidization was documented by the formation of two separate groups of chromosomes, originating from either polar body/pronucleus (PB/PN), or only 2PN, which were tested by 5-colour FISH, or DNA analysis for copy number of chromosomes 13, 16, 18, 21, 22 and X. Two PN were formed more frequently than PB/PN, irrespective of incubation time. In agreement with recent reports on mouse oocytes, as many as 90.2% of the resulting haploid sets tested showed abnormal chromosome segregation, suggesting unsuitability of the resulting artificial gametes for practical application at the present time. Keywords: chromosomal aneuploidy, haploidization, human oocytes, nuclear transfer, oocyte activation Introduction Somatic nuclei transferred (NT) into the cytoplasm of nonactivated eggs normally undergo premature chromosome condensation (PCC), with the condensed chromosomes organized into spindle-like structures resembling a metaphase plate (Tarkowski and Balakier, 1980). Activation of these reconstructed oocytes results in the formation of two sets of chromosomes, one of which separates with the extrusion of the polar body (PB). This is the phenomenon of haploidization of somatic cell nuclei induced by meiotic cytoplasm, representing a possible tool for artificial gamete production for infertility treatment (Tsai et al., 2000; Kaneko et al., 2001; Lacham- Kaplan et al., 2001; Tesarik et al., 2001; Tesarik, 2002; Palermo et al. 2002a,b; Nagy, 2004; Takeuchi and Palermo, 2004). The study of chromosomal distribution of cumulus cell nuclei haploidized by mouse oocytes has shown a correct haploid number of chromosomes in the range of 6 23% (Tateno et al., 2003a; Chen et al., 2004; Heindrycks et al., 2004). Fluorescence in-situ hybridization (FISH) analysis of haploidization outcome was also performed in a small sample of six human cumulus cell nuclei transferred into enucleated human oocytes, which showed normal chromosomal distribution (Tesarik et al., 2001). The present paper describes the results of a study on fidelity of chromosome separation resulting from haploidization of human cumulus cells by human oocytes, and demonstrates an extremely high frequency of chromosomal errors in the derived gametes. Materials and methods The method used was based on inducing nuclei of somatic cells to skip the S-phase of the cell cycle and undergo haploidization through introduction into enucleated MII oocytes, allowing the formation of artificial gametes from somatic cells. A total of 129 immature oocytes were used, which were donated by IVF patients who signed an informed consent form approved by the IRB of Reproductive Genetics Institute. These oocytes were matured in vitro and enucleated using UV-luminescence to ensure that all analysed 199
2 chromosomes resulting from haploidization belonged to the somatic cell nuclei transferred. Cumulus cells, presumably at G0 cell stage, were used as the nucleus donors. The cumulus masses were released into HTF medium (Specialty Media, Phillipsburg, NJ, USA) with hyaluronidase (100 IU/ml; type I-S; Sigma, St Louis, MO, USA) for approximately 5 min, and nuclei were mechanically isolated and introduced into ooplasts by injection. The reconstructed oocytes were cultured in 30 µl drops of HTF in 60 mm Petri dishes covered with the embryo tested mineral oil (Sigma), equilibrated by mixing 50 ml of the appropriate medium with 150 ml of oil incubated at 37 C in a 5% CO 2 /air atmosphere. The oocytes were activated at different times after NT, to evaluate the impact of prolonged nucleus remodelling in the ooplast cytoplasm on the accuracy of chromosome separation to 2PN. Group 1 was activated 4 7, group 2, 8 14, and group 3, h after NT. Activation was performed using an electrofusion device (XRONOS-1; Reproductive Genetics Institute, Chicago, IL, USA) in a medium consisting of 0.3 mol/l mannitol, 0.1 mmol/l MgSO 4, 0.05 mmol/l CaCl 2, and 0.1% polyvinylpyrrolidone. The outcome of haploidization was evaluated by testing either 2PN or PB/PN formed after activation. PB/PN were removed and fixed, or deposited into tubes with lysis buffer containing Protenase K (Invitrogen, Carsband, CA, USA) for molecular testing. The reconstructed oocytes were fixed for FISH analysis in a cold 3:1 solution of methanol and acetic acid and air-dried, while PB/PN were placed on slides and fixed in drops of a 3:1 solution of methanol and acetic acid. The fidelity chromosome distribution in PB/PN was tested by fivecolour FISH probes (Vysis, Downers Grove, IL, USA) or DNA analysis, using short tandem repeats (STR) markers for chromosomes 13, 16, 18, 21, 22 and X, in a multiplex nested PCR system. To prove the donor origin of the resulting haploid cells DNA profiles of the donor cumulus cells and recipient oocytes were also tested. For each oocyte nucleus donor pairs, different sets of STR markers were used depending on the genotype of each donor. Statistical analysis was performed using a Fisher exact test of differences between proportions. Results As seen from Table 1, 92 (71.3%) of a total of 129 oocytes surviving microsurgery were activated by electric stimulation, resulting in haploidization with the extrusion of PB in 12 (13%), and formation of 1, 2 or multiple PN in 80 (87%) oocytes. Overall, 41 (31.8%) of these oocytes were with 2PN, which is significantly higher than the number of oocytes with PB/PN (12; 9.3%; P < 0.01). However, this difference was less obvious for group 1 (12.1 versus 24.3%, not significant), compared with groups 2 and 3: (6.8 versus 41.4% for group 2; P < 0.01; 3.8 versus 42.4% for group 3; P < 0.01). Although the resulting pronuclei were similar in appearance to those derived from normal fertilization (Figure 1), there were positional differences from the early stages of development. While pronuclei resulting from fertilization migrate from the periphery towards each other and to the centre (Kunkle and Longo, 1975), the haploidization-induced pronuclei remained closely connected despite growing in volume. However, the pronuclei may be separated from each other microsurgically and used for either subsequent analysis or nuclear transfer. Table 2 presents the results of FISH analysis in the resulting haploid sets performed in 41 reconstructed oocytes, only four of which showed a normal distribution of chromosomes; the remaining 37 (90.2%) were abnormal, with one, two or three errors. Group 2 demonstrated a tendency towards higher fidelity of chromosomal separation compared with group 1 (18.2 versus 11.1%), while no euploidy and generally complex errors were observed in group 3. Although the sample is not sufficiently large to demonstrate statistical significance, there was higher fidelity of chromosomal segregation in the haploidization resulting in PB extrusion in groups 1 and 2 compared with the 2PN pattern (28.6 versus 9.0%). Polymerase chain reaction-based testing of the resulting haploid sets, performed in four reconstructed oocytes, showed no DNA residue from the recipient oocytes, confirming the donor genotype of both pronuclei (Figure 2). However, all the resulting pronuclei showed chromosomal errors, involving unequal numbers of chromatids at least for two chromosomes, with the X chromosome being involved in all the errors detected. Discussion These data demonstrate a relatively high activation rate of 71.3% obtained by electrical stimulation, despite the use of in- Table 1. Efficiency of different activation times after incubation following nuclear transfer (NT)(%). Experimental groups Not activated PB/PN PN 2PN >2PN Total NT (time of nuclear remodelling, h) Group 1 (4 7) 25 (33.8) g 9 (12.1) a 14 (18.9) 18 (24.3) d 8 (10.9) 74 Group 2 (8 14) 6 (20.7) h 2 (6.8) b 8 (27.6) 12 (41.4) e 1 (3.5) 29 Group 3 (15 21) 6 (23.0) i 1 (3.8) c 5 (19.3) 11 (42.4) f 3 (11.5) 26 Groups (4 14) 31 (30.0) 11 (10.7) 22 (21.5) 30 (29.1) 9 (8.7) 103 Groups (4 21) 37 (28.7) 12 (9.3) 27 (20.9) 41 (31.8) 12 (9.3) Superscripts indicate statistical significance of comparisons: PB extrusion versus 2PN formation group 1, not significant (a,d); group 2, P < 0.01 (b,e); group 3, P < 0.01 (c,f). PB extrusion rate between groups, not significant (a,b,c). 2PN formation rate between groups, not significant (d,e,f). NT oocyte activation rate between groups, not significant (g,h,i).
3 a b c d Figure 1. In-vitro development of reconstructed oocytes following haploidization. (a c) Reconstructed by somatic cell nuclei injection MII oocytes 10 h after electrostimulation: two haploidization pronuclei position close to each other from the very beginning, despite volume growth. (d) Isolated pronuclei ( 400). Table 2. Chromosomal aneuploidies (%) after haploidization. Experimental groups Normal 1 error 2 errors 3 errors Total NT (time of nuclei haploid remodelling, h) Group 1 (4 7) 2 (11.1) 9 (50.0) 7 (38.9) 0 18 Group 2 (8 14) 2 (18.2) 1 (9.0) 4 (36.4) 4 (36.4) 11 Group 3 (15 21) 0 4 (33.3) 4 (33.3) 4 (33.3) 12 Groups (4 14) 4 (13.8) 10 (34.5) 11 (37.9) 4 (13.8) 29 Groups (4 21) 4 (9.8) 14 (34.1) 15 (36.6) 8 (19.5)
4 202 Figure 2. Capillary electrophoresis: results of cumulus cell haploidization. (a) Normal segregation of chromosomes 21. Each of the pronuclei obtained from cumulus cells injected into the enucleated recipient MII oocyte has one chromosome 21, based on testing for the STR marker D21S290 located on chromosome 21 (PN1 and PN2), evident from the presence of a single allele 225 or 233 for these markers in each pronucleus. The DNA profiles of donor cumulus cell nuclei (donor nucleus), and recipient oocyte (oocyte nucleus) are presented below, demonstrating the evidence that both resulting pronuclei originated from haploidization. (b) Abnormal segregation of X chromosomes. None of the somatic cell alleles for chromosome X marker is present in a resulting pronucleus (PN1), which is in accordance with both of the chromosome X STR marker DXS8061 alleles 160/162 being detected in the corresponding sister pronucleus (PN2). The DNA profiles of donor cumulus cell nuclei (donor nucleus), and recipient oocyte (oocyte nucleus) are presented below, demonstrating the evidence that both of the resulting pronuclei originated from haploidization.
5 vitro matured oocytes, which would be expected to have lower activation and fertilization rates compared with naturally ovulated oocytes. The activation rate varied between the groups depending on the incubation time after NT, which may be extrapolated as the oocyte age, with the lowest activation rate observed in Group 1, stimulated only 4 7 h after NT. The fact that activation ability of oocytes may increase with oocyte ageing and with MII oocyte cytoplasm maturation to a competent state has previously been reported for other species (Ware et al., 1989). The evidence of haploidization, which occurred in 41.1% of activated oocytes in this study, was documented not only by PB extrusion, but also by 2PN-formation, the remaining activation patterns, including 1PN and multiple PN, obviously not representing haploidization (Table 1). The observed morphological appearance of activated oocytes may depend on the method of activation and the time of nuclear remodelling after NT, with relevant changes of spindle structures described in ageing mouse eggs (Webb et al., 1986). Initially, when the spindle moves towards the centre, reshaping metaphase to an anaphase-like structure, 2PN are formed in the activated eggs, but multiple PN are formed with the spindle becoming scattered and dissolved. In contrast to PB/PN haploidization events reported previously (Tesarik et al. 2001), the majority of the present series are represented by 2PN formation, which may be due to the increased time of nuclear remodelling or electrical stimulation used in this study compared with a sperm stimulation protocol. The fact that the PB extrusion rate after NT may differ depending on activation protocol used has also been shown in other species (Motlik et al., 2002). As seen from the DNA fingerprinting results, the resulting PN originated from the donor nuclei (Figure 2), but the majority (90.3%) were chromosomally abnormal. Therefore, although haploidization of somatic cells may be achieved consistently using MII oocyte cytoplasm, the aneuploidy rate is unacceptably high compared with normal meiosis. It may also be noted that haploidization failure in some instances may be associated with the fact that not all cumulus nuclei may be exactly at the G0 stage, as they were taken from cumulus oocyte complexes of IVF patients. As shown in mice, ovulation induction may induce some cumulus granulosa cells to enter a cell cycle (Schuetz, 1996). It will be necessary to improve the fidelity of chromosome distribution, if the resulting gametes are to be of any practical future use. For example, drug-induced arrest at G2 M cell phase was shown to dramatically increase the fidelity of chromosome separation in budding yeast with mutation in spindle checkpoint (Shonn et al., 2000). A reduced aneuploidy rate with incubation for 8 14 h was also observed in group 2 compared with group 1 (18.2 versus 11.1%), but no further improvement was observed with h post-nt incubation in group 3, leading to a contrasting increase in aneuploidy rate. The possible improvement was also observed in the haploidization resulting in PB/PN compared with 2PN (28.6 versus 11.1%), although the data are too preliminary to confirm this. In conclusion, the available data show that despite previous hopes for using nuclear transfer technology to produce female and male gametes through haploidization of somatic cells, the majority of the resulting haploid cells are chromosomally abnormal. Even if somatic cell haploidization could produce haploids with euploid chromosome sets, the probability of imprinting disorders cannot be excluded (Tateno et al., 2003b). Therefore, despite the feasibility of somatic cell haploidization using MII oocyte cytoplasm, clinical use of the resulting artificial gametes cannot be considered at the present time. References Chen SU, Chang CY, Lu CC et al Microtubular spindle dynamics and chromosome complements from somatic cell nuclei haploidization in mature mouse oocytes and developmental potential of the derived embryos. Human Reproduction 19, Heindrycks B, Lierman S, Van der Elst J, Dhont M 2004 Chromosome number and development of artificial mouse oocytes and zygotes. Human Reproduction 19, Kaneko M, Takeuchi T, Veek LL et al Haploidization enhancement to manufacture human oocytes. Human Reproduction 16 (suppl. 1), 4 5. Kunkle M, Longo FJ 1975 Cytological events leading to the cleavage of golden hamster zygotes. Journal of Morphology 146, Lacham-Kaplan O, Daniels R, Trounson A 2001 Fertilization of mouse oocytes using somatic cells as male germ cells. Reproductive BioMedicine Online 2, Motlik J, Alberio R, Zakhartchenko V et al The effect of activation of mammalian oocytes on remodeling of donor nuclei after nuclear transfer. Cloning Stem Cells 4, Nagy ZP 2004 Clinical prospects of nuclear transfer and somatic cell haploidization: haploidization to produce human embryos: a new frontier for micromanipulation. Reproductive BioMedicine Online 8, Palermo GD, Takeuchi T, Rosenwaks Z 2002a Oocyte-induced haploidization. Reproductive BioMedicine Online 4, Palermo GD, Takeuchi T, Rosenwaks Z 2002b Technical approaches to correction of oocyte aneuploidy. Human Reproduction 17, Schuetz AW, Whittingham DG, Snowden R 1996 Alterations in the cell cycle of mouse cumulus granulosa cells during expansion and mucification in vivo and in vitro. Reproductive Fertility Development 8, Shonn MA, McCarroll R, Murray AW 2000 Requirement of the spindle checkpoint for proper chromosome segregation in budding yeast meiosis. Science 289, Takeuchi T, Palermo G 2004 Clinical prospects of nuclear transfer and somatic cell haploidization: implications of cloning technique for reproductive medicine Reproductive BioMedicine Online 8, Tarkowski AK, Balakier H 1980 Nucleo cytoplasmic interactions in cell hybrids between mouse oocytes, blastomeres and somatic cells. Journal of Embryology and Experimental Morphology 55, Tateno H, Akutsu H, Kamiguchi Y et al. 2003a Inability of mature oocytes to create functional haploid genomes from somatic cell nuclei. Fertility and Sterility 79, Tateno H, Latham KE, Yanagimachi R 2003b Reproductive semicloning respecting biparental origin. A biologically unsound principle. Human Reproduction 18, Tesarik J 2002 Reproductive semi-cloning respecting biparental origin: embryos from syngamy between a gamete and a haploidized somatic cell. Human Reproduction 17, Tesarik J, Nagy ZP, Sousa M et al Fertilizable oocytes reconstructed from patient s somatic cell nuclei and donor ooplasts. Reproductive BioMedicine Online 2, Tsai MC, Takeuchi T, Bedford JM et al Alternative sources of gametes: reality or science fiction? Human Reproduction 15,
6 Ware CB, Barnes FL, Maiki-Laurila M, First NL 1989 Age dependence of bovine oocyte activation. Gamete Research 22, Webb M, Howlett SK, Maro B 1986 Parthenogenesis and cytoskeletal organization in ageing mouse eggs. Journal of Embryology and Experimental Morphology 95, Received 19 October 2004; refereed 5 November 2004; accepted 29 November
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