Article Poor development of human nuclear transfer embryos using failed fertilized oocytes

Transcription

1 RBMOnline - Vol 11. No Reproductive BioMedicine Online; on web 10 October 2005 Article Poor development of human nuclear transfer embryos using failed fertilized oocytes Prof. Roger A Pedersen Professor Pedersen has devoted his research for the past decade to the area of embryonic stem cells, contributing early on to the functional assessment of numerous genes developmental roles through gene targeting in mouse embryonic stem cells. In the late 1990s, however, he undertook studies of human embryonic stem (hes) cells, resulting in the derivation of hes cell lines that are currently being distributed worldwide for use by stem cell researchers. Professor Pedersen s team now focuses on the analysis of differentiation in hes cells. The group has established a robust approach for transfection of hes cells, established stable transgenic sublines expressing naturally fluorescent proteins, and used these lines to demonstrate the effectiveness of small interfering (si)rna in knocking down both transgene and endogenous gene expression. In addition, the Pedersen laboratory is studying mechanisms for maintenance of pluripotency in hes cells and the earliest steps in their differentiation, namely formation of the precursors of mesodermal and endodermal cell lineages, in collaboration with other Cambridge researchers. Marie-Cecile Lavoir 1, Jingly Weier, Joe Conaghan 2, Roger A Pedersen 3,4 Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA 94143, USA 1 Present address: Origen Therapeutics, Burlingame, CA, USA 2 Present address: San Francisco Fertility Centres, 55 Francisco Street, Suite 500, San Francisco, CA 94133, USA 3 Present address: Department of Surgery and Cambridge Institute for Medical Research, Addenbrooke s Hospital, Hills Road, University of Cambridge, UK CB2 2XY 4 Correspondence: Tel: ; Fax: ; ralp2@cam.ac.uk Abstract Failed fertilized human oocytes from IVF were enucleated and used as recipients for somatic cell nuclear transfer. The reconstructed embryos frequently formed an expanded nucleus from the injected genome after activation. However, subsequent development beyond the 1-cell stage was poor, and the resulting embryos showed chromosomal abnormalities. Poor development of oocytes after nuclear transfer contrasted with that of control, sperm-injected oocytes, which often progressed to cleavage stages. These results suggest that failed fertilized oocytes are not effective recipients for somatic cell nuclear transfer. Keywords: embryonic stem cells, human oocytes, preimplantation embryonic development, somatic cell nuclear transfer 740 Introduction Studies in domestic and laboratory species have shown that the diploid nucleus of a differentiated mammalian cell can recapitulate embryonic and fetal development when inserted into the cytoplasm of a mature oocyte, replacing the egg s own chromosomes (Wilmut et al., 2002). Moreover, mouse embryos generated by such a nuclear transfer procedure can be used to derive embryonic stem (ES) cells (Kawase et al., 2000; Munsie et al., 2000; Wakayama et al., 2001) and such cells can be used in treating genetic diseases (Rideout et al., 2002; Barberi et al., 2003). Human ES cell lines can also be derived from NT blastocysts, and very recently it has been shown that patient-specific embryonic stem cell lines can be established with efficient frequencies (Hwang et al., 2004, 2005). For transplantation therapy, the use of patient-specific ES cell lines would potentially circumvent the problem of immune matching (reviewed in Bradley et al., 2002) and could be a new avenue for obtaining gametes (Nagy and Chang, 2005). An ethical issue that accompanies the production of human NT embryos is the procurement of oocytes. Healthy women need to undergo ovarian stimulation, a procedure that can be uncomfortable and is not without risk (Magnus and Cho, 2005). These issues would be circumvented if oocytes that remain unfertilized after IVF could be used. These oocytes can undergo normal development if rescued by intracytoplasmic sperm injection (ICSI), albeit at a lower frequency and with a lower pregnancy success rate than freshly retrieved oocytes (Nagy et al., 1995; Lundin et al., 1996; Morton et al., 1997). Whilst the development of failed-fertilized oocytes may be reduced as compared with freshly retrieved oocytes, this has been attributed to polyspermy or to abnormalities

2 of the spindle, which have deleterious effects on genomic integrity (Szollosi, 1975; Juetten and Bavister, 1983). As the oocyte ages, there is a decrease in fertilization rates and an increase in 3PN formation after rescue ICSI (Nagy et al., 1995). However, it is well established that aged cytoplasm, following reconstitution with donor, somatic cell nuclei, can support development of bovine embryos to term (Ware et al., 1989; Barnes et al., 1993; Stice et al., 1994). Alternatively, bovine and rabbit materials have been used as oocyte sources in initial attempts to achieve early development following transfer of somatic nuclei of humans or other species (Dominko et al., 1999; Chen et al., 2003; Zavos, 2003). Recently, Stojkovic et al. (2005) reported on the absence of development after nuclear transfer to failed fertilized oocytes using human ES cells as nuclear donors. Although mouse ES cells undergo more effective reprogramming than somatic nuclei, as shown by a higher rate of term pregnancies per transferred blastocyst, the percentage of such oocytes that develop to the blastocyst stage is much lower (Wakayama et al., 1998, 1999). The reason is that the majority of ES cells are in S-phase of the cell cycle, and it has been established that fusing S-phase nuclei to an MII oocyte results in a high incidence of chromosomal abnormalities (Campbell et al., 1993) and poor development to the blastocyst stage. The low overall development rate arising from human ES cell nuclear transfer (Stojkovic et al., 2005) could therefore be attributed to chromosomal abnormalities due to the use of unsynchronized ES cells. Accordingly, the present study used serum-starved and confluent fibroblast cells as nuclear donors. These conditions induce a G0 or G1 state, which is conducive to successful development after transfer to an MII oocyte (Campbell et al., 1993). Such synchronized donor nuclei provide an alternative means of assessing the capacity of failed fertilized oocytes to support early development. Nevertheless, the study found poor early development following nuclear transfer compared with that after rescue ICSI. Moreover, the reconstituted oocytes showed extensive karyotypic abnormalities, suggesting that a chromosomal mechanism may underlie their developmental arrest. Materials and methods Oocytes All research oocytes were donated with informed consent by patients undergoing treatment in the IVF Program at the University of California, San Francisco, according to protocols approved by the Committee on Human Research. Oocytes selected for micromanipulation were meiotically mature, as assessed by the metaphase II configuration of chromosomes removed during enucleation, or by the presence of a polar body (rescue ICSI). The donor oocytes were retrieved from 13 donors with an average age of 33 years (range 26 36). Oocytes of consenting patients were evaluated h postinsemination by an embryologist not involved in the study, and if judged to be without pronuclei, they were transferred to the research laboratory. In certain cases, oocytes that were assessed as having failed to fertilize when evaluated h after insemination were injected with spermatozoa in an effort to rescue fertilization as part of the patients infertility treatment. In such cases, not all the resulting embryos could be evaluated for cleavage because some were used clinically. Development of such embryos beyond early cleavage stages occurred at a low frequency. In other studies using rescue ICSI, pregnancies have been obtained (Morton et al., 1997; Yuzpe et al., 2000). Genomic donor cells Donor cells were fibroblasts, either CRL 7895, a diploid female cell line (ATCC, Rockville, MD, USA), or BJ5ta, a diploid male telomerized cell line (obtained from Geron Corporation, Menlo Park, CA, USA). Cells were either grown to confluency or cultured in <0.1% serum for at least 4 days before use (thereby inducing the cells to enter G0 or G1, then trypsinized for approximately 3 4 min), washed and resuspended in 12% polyvinylpyrrolidone (MW 360,000) in 1% sodium citrate. Nuclei were isolated by aspirating and expelling the cells into the injection pipette in FHM, a HEPESbuffered modification of KSOM (Specialty Media, Lafayette, NJ, USA) + 4 μg/ml cytochalasin D (Sigma, St Louis, MO, USA) + 2 μg/ml Hoechst (Calbiochem, San Diego, CA, USA) as described (Wakayama et al., 1998). ICSI Failed fertilized oocytes were injected with a single immobilized spermatozoon h after the initial insemination, using standard ICSI procedures (Palermo et al., 1992). Injected oocytes were further cultured in P1 medium supplemented with 10% v/v synthetic serum substitute (both from Irvine Scientific, Santa Ana, CA, USA) in a gas phase of 5% CO 2 in air at 37 C for h before being examined for the presence of pronuclei. The quality of embryos was graded according to a 5-point grading system, with grade I being flawless (Dawson et al., 1987) Nuclear transfer A slit was made in the oocyte s zona pellucida adjacent to the polar body, and a suction pipette was used to remove the oocyte spindle and associated chromosomes, which were examined under fluorescence. Such enucleated oocytes were quickly scanned under fluorescence to exclude the potential presence of spermatozoa. Donor karyoplasts were injected with a micropipette (8 10 μm) attached to a piezo impact drive unit (PMM-140 FU; Prime Tech Ltd, Tokyo, Japan) as described (Kimura and Yanagimachi, 1995). Two to 3 h after injection, oocytes were activated as described (Levron et al., 1995). Briefly, the oocytes were placed in 0.3 M mannitol medium containing 0.05 mmol/l CaCl 2 and 0.01 mmol/l MgSO 4 in water for 20 min followed by an electrical pulse, (AC pulse: 8 s at 0.4 kv/cm or 4 s at 0.2 kv/cm + 1 DC pulse: 99 μs at 1.2 kv/cm) (BTX electro cell manipulator; Genetronix Inc., San Diego, CA, USA). Embryos reconstituted by nuclear transfer were co-cultured with BRL cells (ATCC) in 50 μl droplets of KSOM (potassium-enriched simplex optimization medium obtained from Specialty Media) under paraffin oil in 5% CO 2 in air at 37 C (Lavoir et al., 1998). 741

3 Fluorescence in-situ hybridization After reconstituted embryos ceased to progress developmentally, their zona pellucida was removed by incubation in 0.5% pronase. The embryo was then placed in 1% sodium citrate for 5 min, placed on a clean slide, and ~10 μl of methanol/ acetic acid (1:1) was dropped on it. Slides were then placed in 3:1 (vol:vol) methanol:acetic acid for 24 h. Fluorescence insitu hybridization (FISH) was performed using probes specific for repeated DNA on chromosomes X, Y, 21 and 18 (Vysis, Downer s Grove, IL, USA). The hybridization mixture was prepared following the probe manufacturer s protocol using 1 μl of each probe per 10 μl of hybridization mixture (Vysis). Prior to FISH, the slides were denatured at 76 C for 5 min in 70% formamide/standard saline citrate, ph 7.0, dehydrated in successive concentrations of ethanol, and allowed to air dry. The hybridization mixture was denatured at 76 C for 7 min before application to the slides. The hybridization proceeded at 37 C overnight in a moisture chamber. After hybridization the slides were washed and mounted in 4,6-diamino-2- phenylindole) (DAPI; 0.05 μg/ml; Calbiochem) in antifade solution (0.1% p-phenylenediamine dihydrochloride (Sigma) to counterstain genomic DNA. Signals were visualized using fluorescence microscopes equipped with filters for DAPI, fluorescein isothiocyanate (FITC), Spectrum Orange/ rhodamine, and Spectrum Aqua excitation and detection. Results Embryos produced through nuclear transfer survived micromanipulation and formed interphase nuclei at similar frequencies as those produced by rescue ICSI (Table 1). No differences were seen in the use of the two activation pulses (see Materials and methods). Neither the survival of oocytes after nuclear transfer nor the development of nuclei was significantly worse than that obtained using rescue ICSI (Fisher s Exact test, one-tailed). Control embryos produced by rescue ICSI using similar oocytes were capable of morphologically normal early cleavage. However, only a few of the embryos produced by nuclear transfer progressed beyond the 1-cell stage. Early cleavage of oocytes was significantly lower after nuclear transfer than after rescue ICSI (Fisher s Exact test, one-tailed: P = ). Those that did develop were delayed and underwent only one or two early cleavage divisions. Embryos produced by nuclear transfer were examined by FISH to determine their chromosomal status (Table 2). Chromosomal aneuploidy (classified as hyperdiploidy or hypodiploidy) was present in all but one of the reconstituted embryos (8/9) and in most of the resulting nuclei (10/13) analysed by FISH. Neither of the two embryos that progressed to early cleavage stages was diploid, and a third oocyte that appeared to have reached the 6-cell stage was classified as fragmented, as no chromosomal signals were detected in its analysis by FISH. Table 1. Early development of failed-fertilized oocytes after nuclear transfer or rescue ICSI. Figures in parentheses are percentages. Total Oocytes Survived Nuclear Total oocytes oocytes injected injection development evaluated for development Not Cleaved Cleavage Stage Reached cleaved Nuclear transfer 2-cell 3/4-cell CRL 16 a BJ5ta 23 a Total a 30 * (77) 14 * (47) b (86) 2 (14) 1 c 1 c Rescue ICSI 2-cell 3-cell 4-cell Oocytes * (86) 50 * (57) 27 d 14 (52) 13 e (48) with PB 742 a Number of oocytes undergoing genomic replacement that survived the enucleation procedure. b Oocytes scored as uncleaved were at the 1-cell stage (n = 11) or had been classified as fragmented (n = 1, oocyte 35A; see Table 2). c Oocytes scored as cleaved after genomic replacement included one whose chromosomal status was indeterminate (oocyte 26B) and another with hyperdiploid status (oocyte 35B; see Table 2). Scoring these oocytes as cleaved thus represents the best-case interpretation. d Not all oocytes undergoing rescue ICSI were subsequently evaluated for their in-vitro development. e The majority of oocytes undergoing rescue ICSI that subsequently cleaved showed minimal or modest fragmentation (9/13, grades I III). A few (4/13) showed substantial fragmentation, grade IV. *Survival of microinjection procedure and nuclear development of oocytes were not significantly different between genome replacement and rescue ICSI groups (Fisher s Exact test: oocytes surviving injection, P = , one-tailed; oocytes undergoing nuclear development, P = , one-tailed). Early cleavage of oocytes was significantly lower after genome replacement than after rescue ICSI (Fisher s Exact test, P = , one-tailed).

4 Table 2. Chromosomal status of oocytes developing after nuclear transfer. Oocyte Evaluation Source of Nuclear status Chromosomal status Characterization designation of oocyte somatic after fi xation based on FISH results development nucleus 26A 1PN CRL 7895 Lost during fixation None Indeterminate 26B 3 4 cells CRL 7895 Lost during fixation None Indeterminate 33A 2 big + CRL nuclei (96 h) x,x,21,21,21; x,21; 21 Hyperdiploid; hypodiploid; 1 small PN hypodiploid 33B 1 big PN CRL nucleus (96 h) x,x, 21,21; y Diploid (with sperm remnant) (micronucleus) 35A 6 cells CRL 7895 no nuclei found None Fragmentation 35B 2 cells CRL nucleus (144 h) x,x,x,x,21,21 Hyperdiploid 45A 3PN BJ5ta 2 nuclei (72 h) XY18,18; X,18,18 Diploid; hypodiploid 45B 1PN BJ5ta 1 nucleus (72 h) Y,18 Hypodiploid 52A 1PN BJ5ta 1 nucleus (48 h) X,X,Y,Y,18,18,18,18 Hyperdiploid 52B 1PN BJ5ta 1 nucleus (48 h) X,X,Y,Y,Y,18,18,18, 18,18 Hyperdiploid 54 0PN (24 h) BJ5ta 2 nuclei (204 h) X,X,18; X,Y,18,18 Hypo/hyperdiploid; diploid 56 0PN BJ5ta 1 nucleus (204 h) X,X,Y,Y,18,18,18 Hyperdiploid Discussion The efficiency of enucleation and nuclear reconstitution using failed fertilized oocytes was similar to efficiencies obtained in studies using freshly collected oocytes (Cibelli et al., 2001; Hwang et al., 2004, 2005; Takeuchi et al., 2005). In the present protocols, a piezo injector was used to insert the nucleus into the enucleated oocyte. Use of this procedure may account for the satisfactory survival of manipulative procedures, especially considering the fragility of aged oocytes, thus suggesting nuclear injection as a promising alternative to fusion in human nuclear transfer. Early development after activation of reconstituted oocytes (as assessed by the extent of cleavage) was limited, with few embryos progressing beyond the 1-cell stage, while embryos produced by rescue ICSI progressed at significantly higher frequency. This disparity may be attributed to the deposition of kinesin proteins onto the meiotic spindle of mature human and non-human primate oocytes (Simerly et al., 2003). Accordingly, as a result of enucleation, primate oocytes are rendered incapable of undergoing chromosomal segregation essential for normal preimplantation and subsequent stages of development (Simerly et al., 2003). It should be noted, however, that embryos resulting from rescue ICSI can also possess abnormal numbers of chromosomes (e.g. Nagy et al., 1995), suggesting that oocyte age per se could contribute to karyotypic errors. However, neither a comparison of development using freshly donated oocytes, nor an analysis of the chromosomal status of rescue ICSI was undertaken in the present study. Alterations have recently been made in the nuclear transfer procedures that can substantially improve the efficiencies of blastocyst production and ES cell derivation in humans and non-human primates (Simerly et al., 2004; Hwang et al., 2005). The main differences between those procedures and this nuclear transfer protocol involve their use of freshly donated oocytes and a squeezing procedure for enucleation. The enucleation of freshly matured oocytes is considered essential for proper development of the reconstituted embryo, presumably thereby limiting sequestration of the oocyte s kinesins by its meiotic spindle (Simerly et al., 2004; Hwang et al., 2005). Although gently squeezing the metaphase spindle out of the oocyte seems to improve the development of the reconstituted embryo (Hwang et al., 2004, 2005; Simerly et al., 2004), interphase nuclei were also obtained by one of the same groups in over 60% of reconstituted embryos after enucleation using suction (Simerly et al., 2003), indicating that initial nuclear development, at least, should proceed independently of the enucleation technique used. In addition, donor age appears to influence the efficiency of successful nuclear transfer and ES cell derivation (Hwang et al., 2005), and the majority of patients undergoing IVF (thereby providing the failed fertilized oocytes in this study) are older than 30 years of age (average in this study, 33 years). Moreover, failed fertilized oocytes are by necessity matured and arrested at MII stage when they are obtained the day after fertilization, thereby precluding early enucleation. These considerations, together with the observations of poor early development, suggest that nuclear transfer using failed fertilized human oocytes most likely will not provide an alternative to the use of oocytes retrieved in conjunction with the SCNT procedure itself. A possible alternative might be oocytes retrieved during follicle reduction (Stojkovic et al., 2005), since these oocytes can be collected before maturation. In conclusion, the successful application of nuclear transfer to generate embryonic stem cells from human oocytes might necessitate the fresh retrieval of oocytes from younger donors, rather than the use of failed fertilized oocytes that emerge from IVF treatment for infertility. 743

5 744 Acknowledgements We thank the physicians, staff and patients of the UCSF IVF Program for their support of this study. This work was funded by the University of California BioSTAR program with Geron Corporation. References Barberi T, Klivenyi P, Calingasan NY et al Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nature Biotechnology 21, Barnes F, Endebrock M, Looney C et al Embryo cloning in cattle: the use of in vitro matured oocytes. Journal of Reproduction and Fertility 97, Bradley J, Bolton EM, Pedersen R 2002 Stem cell medicine encounters the immune system. Nature Reviews Immunology 2, Campbell K, Ritchie W, Wilmut I 1993 Nuclear cytoplasmic interactions during the first cell cycle of nuclear transfer reconstructed bovine embryos: implications for deoxyribonucleic acid replication and development. Biology of Reproduction 49, Chen Y, He ZX, Liu A, Wang K et al Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes. Cell Research 13, Cibelli JB, Kiessling AA, Cunniff K et al Somatic cell nuclear transfer in humans: pronuclear and early embryonic development. e-biomed Journal of Regenerative Medicine 2, Dawson K, Margara R, Hillier S et al Factors affecting success at the time of embryo transfer. Abstracts from the Third Meeting of ESHRE. Human Reproduction 2, p Dominko T, Mitalipova M, Haley B et al Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species. Biology of Reproduction 608, Hwang WS, Roh SI, Lee BC et al Patient-specific embryonic stem cells derived from human SCNT blastocysts. Science 308, Hwang WS, Ryu YJ, Park JH et al Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science 303, Juetten J, Bavister B 1983 Effects of egg aging on in vitro fertilization and first cleavage division in the hamster. Gamete Research 8, Kawase E, Yamazaki Y, Yagi T et al Mouse embryonic stem (ES) cell lines established from neuronal cell-derived cloned blastocysts. Genesis 28, Kimura Y, Yanagimachi R 1995 Intracytoplasmic sperm injection in the mouse. Biology of Reproduction 52, Lavoir M-C, Conaghan J, Pedersen R 1998 Culture of human embryos for studies on the derivation of human pluripotent cells: a preliminary investigation. Reproduction Fertility and Development 10, Levron J, Cohen J, Willadsen S 1995 Highly effective method of human oocyte activation. Zygote 3, Lundin K, Sjogren A, Hamberger L 1996 Reinsemination of one-dayold oocytes by use of intracytoplasmic sperm injection. Fertility and Sterility 66, Magnus D, Cho MK 2005 Issues in oocyte donation for stem cell research. Science 308, Morton PC, Yoder CS, Tucker MJ et al Reinsemination by intracytoplasmic sperm injection of 1-day-old oocytes after complete conventional fertilization failure. Fertility and Sterility 68, Munsie MJ, Michalska AE, O Brien CM et al Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei. Current Biology 10, Nagy ZP, Chang C-C 2005 Current advances in artificial gametes. Reproductive BioMedicine Online 11, Nagy ZP, Staessen C, Liu J et al Prospective, auto-controlled study on reinsemination of failed-fertilized oocytes by intracytoplasmic sperm injection. Fertility and Sterility 64, Palermo G, Joris H, Devroey P et al Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340, Rideout WM 3rd, Hochedlinger K, Kyba M et al Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 109, Simerly C, Navara C, Hyun SH et al Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction. Developmental Biology 276, Simerly C, Dominko T, Navara C et al Molecular correlates of primate nuclear transfer failures. Science 300, 297. Stice S, Keefer C, Matthews L 1994 Bovine nuclear transfer embryos: oocyte activation prior to blastomere fusion. Molecular Reproduction and Development 38, Stojkovic M, Stojkovic P, Leary C et al Derivation of a human blastocyst after heterologous nuclear transfer to donated oocytes. Reproductive BioMedicine Online 11, Szollosi D 1975 Mammalian eggs aging in the fallopian tubes. In: Blandau RJ (ed.) Aging Gametes. Karger, Seattle. Takeuchi T, Neri QV, Palermo GD 2005 Construction and fertilization of reconstituted human oocytes. Reproductive BioMedicine Online 11, Wakayama T, Tabar V, Rodriguez I et al Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 292, Wakayama T, Rodriguez I, Perry AC et al Mice cloned from embryonic stem cells. Proceedings of the National Academy of Science USA 96, Wakayama T, Perry AC, Zuccotti et al Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, Ware C, Barnes F, Maiki-Laurila M et al Age dependence of bovine oocyte activation. Gamete Research 22, Wilmut I, Beaujean N, de Sousa PA et al Somatic cell nuclear transfer. Nature 419, Yuzpe AA, Liu Z, Fluker MR 2000 Rescue intracytoplasmic sperm injection (ICSI)-salvaging in vitro fertilization (IVF) cycles after total or near-total fertilization failure. Fertility and Sterility 73, Zavos PM Human reproductive cloning: the time is near. Reproductive BioMedicine Online 6, Received 27 May 2005; refereed 24 June 2005; accepted 5 September 2005.