Embryogenesis and Blastocyst Development After Somatic Cell Nuclear Transfer in Nonhuman Primates: Overcoming Defects Caused by Meiotic Spindle Extraction$

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Developmental Biology 276 (2004) 237–252 www.elsevier.com/locate/ydbio

Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction$

Calvin Simerlya,b,1, Christopher Navaraa,b,1, Sang Hwan Hyuna,b,1, Byeong Chun Leec, Sung Keun Kangc, Saverio Capuanoa,b, Gabriella Gosmana,b, Tanja Dominko2, Kowit-Yu Chonga,b, Duane Comptond, Woo Suk Hwangc, Gerald Schattena,b,*

aDepartment of Obstetrics-Gynecology-Reproductive Sciences, Pittsburgh Development Center, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, 204 Craft Avenue, Pittsburgh, PA 15213, USA bDepartment of Cell Biology-Physiology, Pittsburgh Development Center, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, 204 Craft Avenue, Pittsburgh, PA 15213, USA cDepartment of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea dDepartment of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA

Received for publication 13 August 2004, revised 28 September 2004, accepted 12 October 2004

Abstract

Therapeutic cloning or nuclear transfer for stem cells (NTSC) seeks to overcome immune rejection through the development of embryonic stem cells (ES cells) derived from cloned blastocysts. The successful derivation of a human embryonic stem cell (hESC) line from blastocysts generated by somatic cell nuclear transfer (SCNT) provides proof-of-principle for btherapeutic cloning,Q though immune matching of the differentiated NT-hES remains to be established. Here, in nonhuman primates (NHPs; rhesus and cynomologus macaques), the strategies used with human SCNT improve NHP-SCNT development significantly. Protocol improvements include the following: enucleation just prior to metaphase-II arrest; extrusion rather than extraction of the meiotic spindle-chromosome complex (SCC); nuclear transfer by electrofusion with simultaneous cytoplast activation; and sequential media. Embryo transfers (ET) of 135 SCNT-NHP into 25 staged surrogates did not result in convincing evidence of pregnancies after 30 days post-ET. These results demonstrate that (i) protocols optimized in humans generate preimplantation embryos in nonhuman primates; (ii) some, though perhaps not yet all, hurdles in deriving NT-nhpES cells from cloned macaque embryos (therapeutic cloning) have been overcome; (iii) reproductive cloning with SCNT-NHP embryos appears significantly less efficient than with fertilized embryos; (iv) therapeutic cloning with matured metaphase-II oocytes, aged oocytes, or bfertilization failuresQ might remain difficult since enucleation is optimally performed prior to metaphase-II arrest; and (v) challenges remain for producing reproductive successes since NT embryos appear inferior to fertilized ones due to spindle defects resulting from centrosome and motor deficiencies that produce aneuploid preimplantation embryos, among other anomalies including genomic imprinting, mitochondrial and cytoplasmic heterogeneities, cell cycle asynchronies, and improper nuclear reprogramming. D 2004 Elsevier Inc. All rights reserved.

Keywords: Embryogenesis; Blastocyst; Meiotic spindle extraction; Nuclear transfer; Stem cells; Molecular motors; NuMA

$ In accordance with the International Society for Stem Cell Research’s (ISSCR) Nomenclature Statement (September 2, 2004), bnuclear transfer Introduction (NT)Q is used instead of btherapeutic cloningQ and bNT stem cellsQ or NTSC describe stem cells derived from NT embryos. The terms breproductive Human therapeutic cloning, in which human embryonic Q b Q cloning and therapeutic cloning are used herein as well for clarity. stem cells (hESCs) are derived from somatic cell nuclear * Corresponding author. Fax: +1 412 641 2410. E-mail address: [email protected] (G. Schatten). transfer (SCNT) blastocysts, represents an innovative strategy 1 Authors contributed equally to this work. for overcoming, in principle, immune rejection of differ- 2 Current address: CellThera, Worcester, MA 01605, USA. entiated hESCs after transplantation. Until this year, therapeu-

0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.10.006 238 C. Simerly et al. / Developmental Biology 276 (2004) 237–252 tic cloning in non-murine species encountered seemingly al., 2001); bnuclear reprogrammingQ by which even terminally insurmountable scientific challenges—let alone nonscientific differentiated somatic nuclei are restored back to pluripotency ones facing clinical investigators. Hwang et al. (2004) reported through chromatin remodeling (Jeanisch et al., 2002; Shi et al., the first human therapeutic cloning success, with the derivation 2003; Tamada and Kikyo, 2004), and mitochondrial hetero- of a pluripotent hESC line from 20 SCNT blastocysts produced plasmy (Hiendleder et al., 2004; St John and Schatten, 2004; by the autologous transfer of the donor woman’s own cumulus Steinborn et al., 2000), among others. Furthermore, the cells into her own enucleated oocyte (Hwang et al., 2004). research prowess enabled by mouse embryonic stem (ES) Because of the profound implications of human therapeutic cells, including chimeric offspring production, is swiftly being cloning, and the unlikelihood of swiftly and reliably confirm- extrapolated to primate ES cells—human (Hwang et al., 2004; ing, refuting, and/or extending these findings in clinical Thomson et al., 1998) and nonhuman (Suemori et al., 2001; laboratories outside the Republic of Korea (due to laws and Thomson and Marshall, 1998; Wolf et al., 2004) alike. Clonal regulations of various other nations), this study investigates propagation of NHP offspring has been achieved by embry- SCNT in fertile non-human primates (NHPs) examining each onic cell nuclear transfer (Meng et al., 1997) in which an experimental parameter, including enucleation procedures and unrelated male and female were born following ECNTwith an timing as well as autologous (i.e., transfer of the egg’s own 8- to 12-cell blastomere nucleus, and by artificial twinning by cumulus cells) versus heterologous donor cell transfer. disaggregation and later reaggregation of uncompacted Comparisons are made between the developmental fates of embryos (Chan et al., 2000). Notwithstanding media reports, SCNT-NHP embryos after embryo transfer (ET; reproductive human SCNT claims are now considered hoaxes (Schatten et cloning) and with embryonic stem cell derivations (therapeutic al., 2003). cloning). Simerly et al. (2003) reported last year on unanticipated Somatic cell nuclear transfer in NHPs is well justified to obstacles from NHP-SCNT resulting from the extraction of the answer several basic and preclinical challenges in devel- metaphase-II spindle-chromosome complex (SCC) during opmental biology (Schatten, 2002; Wolf, 2004) including the cloning’s first step of benucleation,Q that is, removal and following: fundamental discoveries on the mechanisms of discarding of the meiotic chromosomes along with the spindle nuclear reprogramming in primates and the developmental and its adherent structures and proteins (Simerly et al., 2003). origins of primate embryos both before and after implanta- We reported that despite seemingly normal preimplantation tion; the potential to produce limited numbers of NHPs, development SCNT-NHP pregnancies did not result, and that identical in nuclear genetics, for biomedical and behavioral SCNT-NHP constructs showed disarrayed microtubule patterns research; as well as answering urgent problems in stem cell and displayed misaligned chromosomes on multipolar spindles, biology (Thomson et al., 1998), especially whether SCNT- largely resulting from the depletion of microtubule motors (e.g., NHP ESCs are indeed immune matched with the donor NHP kinesins) and centrosome (e.g., nuclear mitotic apparatus; in a model with more direct clinical implications than mice. NuMA) proteins during SCC extraction, among other pre- Fundamentally, development by SCNT conception enables viously defined reasons. Hwang et al. (2004) introduced several investigations into the essential mechanisms of fertilization, alternative procedures for SCNT, including the extrusion versus genomic imprinting, preimplantation development, embryo- the extraction of the SCC; enucleation prior to metaphase-II genesis, placentation, and organogenesis, as well as prenatal arrest; autologous SCNT; fructose instead of glucose as the and postnatal normalcy. energy source; sequential culture media; synthetic albumin Since the birth of bDollyQ (Wilmut et al., 1997), SCNT replacer; and 2-h reprogramming duration; among others offspring have been cloned from over a dozen species and nine (Hwang et al., 2004). By investigating NHP-SCNT into genera (Wilmut, 2002), including many sheep (Willadsen, enucleated NHP oocytes, as well as NHP-SCNT into 1986; Wilmut et al., 1997), mice (Wakayama et al., 1998), pigs enucleated bovine oocytes, we present evidence confirming (Onishi et al., 2000; Polejaeva et al., 2000), cattle (both in NHPs that preimplantation embryonic developmental rates domestic and endangered) (Cibelli et al., 1998; Kato et al., are significantly improved by employing the technological 1998; Vogel, 2001; Wells et al., 1999), and goats (Baguisi et innovations of Hwang et al. (2004) to overcome difficulties, al., 1999). Successes have been reported with the cloning of a including met-II SCC extraction, that NHP-SCNT produces cat (Shin et al., 2002b), a rat (Zhou et al., 2003), a mule (Woods cloned embryos routinely and reliably, and that the NuMA and et al., 2003), and a horse (Galli et al., 2003). Births of SCNT kinesin behaviors during NHP-SCNT differ fundamentally offspring, including transgenic SCNT offspring (Matsuda et when transferred into primate oocytes versus bovine oocytes. al., 2002), are rare, with average efficiency rates just 1–3% of SCNT constructs (Wakayama and Perry, 2002). However, these figures vary depending on species, technical expertise, Materials and methods seasonality, and other factors. Regardless of the rarity of SCNT births, the very achievement of mammalian SCNT during the Oocyte collection past several years has stimulated new insights for deciphering developmental mechanisms, including vital genomic imprints Procedures for superstimulation, oocyte staging, and in both the fetus and the placenta (Mann et al., 2003; Rideout et intracytoplasmic sperm injection (ICSI) fertilization of C. Simerly et al. / Developmental Biology 276 (2004) 237–252 239 nonhuman primate eggs were described by Hewitson et al. selected under an inverted microscope and transferred into (1998). Immature bovine oocytes were obtained from the perivitelline space of enucleated oocytes. These couplets Bomed (Madison, WI) and overnight express shipped in were equilibrated with 0.3 M mannitol solution containing modified TC199 maturation media for the collection of 0.5 mM Hepes, 0.1 mM CaCl2, and 0.1 mM MgCl2 for 4 mature oocytes as described previously (Navara et al., 1994). min and transferred to a chamber containing two electrodes that were overlaid with the mannitol solution. Couplets were Enucleation fused with two DC pulses of 2.7 kV/cm2 for 15 As using a BTX Electro-Cell Manipulator 2001 (BTX, Inc., San Diego, Removal of the meiotic spindle and chromosomes was CA). Successful fusion was confirmed 45–60 min after accomplished in two ways. Using the bsquishQ enucleation electroporation by absence of the donor cell in the method (Hwang et al., 2004; depicted in animation in perivitelline space. supplemental material) for pre-metaphase-II spindle aspira- For embryonic blastomere cell nuclear transfer (ECNT), tion, a cumulus-free oocyte is held with a holding donor blastomeres were isolated from 16- to 32-cell stage micropipette (110 Am outer diameter) and the zona embryos after zona pellucida removal with 0.5% pronase pellucida is partially dissected with a fine glass needle to (Boehringer Mannheim, Indianapolis IN) and culturing in make a slit near the first polar body. The first polar body Ca+- and Mg+-free TALP-Hepes with 1 mM each EDTA and and adjacent cytoplasm containing the meiotic spindle, EGTA. A single blastomere was pipetted into the perivitel- ranging from telophase-I to pro-metaphase-II, are extruded line space of an enucleated oocyte and after a 30- to 60-min by squeezing with the needle. Oocytes are enucleated in recovery fused with two DC pulses as described above. For Hepes-buffered TALP supplemented with 0.3% BSA and some cumulus SCNTs, enucleated oocytes were directly 7.5 Ag/ml cytochalasin B (Sigma-Aldrich, St. Louis, MO). injected with a single donor cell using a 9-Am pipette, Alternatively, for metaphase-II spindle aspiration, mature following the mouse SCNT procedures of intracytoplasmic unfertilized oocytes were first exposed to TALP Hepes with nuclear injection (ICNI) (Simerly and Navara, 2003; 7.5 Ag/ml cytochalasin D (CCD; Sigma) prior to suction Wakayama and Perry, 2002). Bovine SCNT and ECNT were being applied with a 22-Am-I.D. pipette (Humagen, performed as previously described (Navara et al., 1994). Charlottesville, VA) to aspirate the first polar body and underlying cytoplasm, including the second meiotic spindle. Activation protocols After enucleation, karyoplasts were stained with 5 Ag/ml bisbenzimide (Hoechst 33342, Sigma-Aldrich) for 5 min Reconstructed embryos were activated (Table 1) by (i) and observed under an inverted microscope equipped with simultaneous electrical fusion treatment (EFA); (ii) chemical epifluorescence. Oocytes still containing DNA materials activation 2 h after NT fusion by exposure to 5 AM were excluded from further experiments. ionomycin (CalBiochem, La Jolla, CA) for 4 min followed by a 4-h incubation in 1.9 mM 6-dimethylaminopurine Nuclear transfer (EFIAD), or (iii) injection of NHP sperm extract (EFSFA; 120–240 pg mlÀ1; Simerly et al., 2003). Activated oocytes The somatic cell nuclear donor source included disso- were washed three times with TALP-HEPES (Bavister et al., ciated cumulus cells (both autologous and heterologous 1983) supplemented with 4 mg/ml fatty acid-free BSA sources) and primary rhesus fibroblast cell lines (passage 2– (Sigma-Aldrich) and placed in 25-Al microdrops (five to 4). Trypsinized single cells from the confluent fibroblast line seven oocytes per drop) of G1.1 culture media (Vitrolife, demonstrating a smooth surface or cumulus cells were Boulder, CO) under mineral oil at 378Cin5%CO2.

Table 1 Development of NHP clones reconstructed with fresh cumulus cells or skin fibroblasts Donor cells Fusion No. No. of oocytes (%) Number of cloned embryos to develop (% of fused) (source) activation Enucleated Fused PN 2- to 4-cell 8- to 16-cell NT + Fert Morula Blastocyst formation chimera Cumulus EFAa 15 12 (80) 6 (50) 3 (50) 2 (33) 2 (33) 1 (17) 0 (homologous) EFIADb 14 10 (71) 6 (60) 5 (83) 4 (67) 3 (50) 0 0 EFSFAc 12 10 (83) 5 (50) 5 (100) 3 (60) 1 (20) 0 0 Fibroblast EFAa 12 9 (67) 7 (77) 5 (71) 5 (71) 5 (71) 3 (43) 3 (43) (heterologous) EFIADb 18 15 (80) 12 (75) 11 (92) 11 (92) 9 (75) 3 3d (25) 1d EFSFAc 18 15 (83) 13 (86) 9 (69) 6 (46) 3 (23) 1 (8) 0 a EFA: Electrical fusion and activation simultaneously. b EFIAD: Electrical fusion followed by activation 2 h later. c EFSFA: Electrical fusion followed by sperm factor activation 2 h later. d One morula was the NT + Fert chimera, with one resulting blastocyst. 240 C. Simerly et al. / Developmental Biology 276 (2004) 237–252

Fertilization achieved by mimicking cloning procedures centrosomes. Slides were examined using conventional immunofluorescence and/or laser-scanning confocal micro- To dissect the adverse consequences of SCNTs many scopy. Conventional fluorescence microscopy was per- harsh physical and chemical procedures (e.g., cytochalasin, formed first using a Nikon Eclipse E1000 microscope with physical enucleation, electrical fusion, chemical activation, high numerical aperture objectives. Data were recorded etc.) from the biological incompatibilities of SCNT itself, digitally using a cooled CCD camera (Hamamatsu Instru- we reconstructed fertilized eggs using the procedures of ments Inc., Japan) and Metamorph software (Universal cloning. These FertClones were produced by the fusion of a Imaging, West Chester, PA). Laser-scanning confocal micro- donor nucleus into an intact oocyte followed by ICSI scopy was performed using a Leica SP-2 equipped with fertilization 2–3 h later. SCNT+ met-II intact constructs argon and helium–neon lasers for the simultaneous excita- were produced by fusion of the donor nucleus into an intact tion of Alexa-conjugated secondary antibodies and Toto-3 oocyte followed by artificial activation with either sperm DNA stain. factor or ionomycin/DMAP as described above.

In vitro culturing Results

All nuclear transfer constructs were developed in Experimental strategy sequential G1-G2-mSOF media as described by Hwang et al. (2004). G1 media were employed for 48 h after NT prior Notwithstanding the fundamental and clinical importance to transfer to G2 media for an additional 48 h. Around the of understanding the early nuclear and cytoskeletal events morula stage, embryos were transferred to mSOF with following NT for hES cell derivations, appropriate ethical fructose until blastocyst stage. concerns as well as federal and/or state restrictions prohibit such investigations on human embryos in the United States. Embryo transfer Because NHP eggs closely mimic the fertilization events observed in humans (Simerly et al., 1995; Wu et al., 1996), For embryo transfer, surrogate macaque females were NT constructs were generated specifically from fertile selected on the basis of serum estradiol and progesterone macaques for these experiments; i.e., the best oocytes were levels. Pregnancies were ascertained by endocrinological studied here. The strategy was first to mirror the techniques profiles and fetal ultrasound performed between days 16 and successfully employed in producing human cloned blasto- 30 (Hewitson et al., 1999). cysts for the derivation of hESCs in rhesus and cynomologus macaques. Next, we sought information on NHP-NT con- Imaging structs and embryo arrests, tracing the microtubule and DNA configurations during interphase, first mitosis, and embryonic Live oocyte imaging was accomplished using a Nikon TE development. Finally, we investigated specific centrosome- 2000 inverted microscope equipped with polarization optics associated proteins and molecular motors at mitosis and (SpindleView; CRI, Cambridge, MA) and a thermoplate meiosis, including their fate upon removal of the meiotic temperature control stage (Tokai HIT Inc., Japan) to maintain spindle-chromosome complex (SCC), as well as the cortex 378C. Methods for immunocytochemistry were performed as overlying the meiotic spindle region and adherent cytoplasm. described (Simerly and Schatten, 1993). NuMA, human spleen embryonic tissue and testis (HSET), and Eg5 were bSquishQ enucleation and in vitro development of SCNT detected using rabbit polyclonal antibodies as described NHP constructs (Mountain et al., 1999; Simerly et al., 2003). Microtubules were immunolabeled with E7 mouse monoclonal antibody Dynamic imaging of the spindle-chromosome complex in (Simerly et al., 1995). BrDU labeling was performed using a live unfertilized NHP oocytes by SpindleView polarization kit according to the manufacturer’s instructions as previously optics is useful for the accurate assessment of meiosis prior to published (Hewitson et al., 1999). Primary antibodies were enucleation. The second meiotic spindle is seen as a bright, detected using Alexa secondary antibodies (Alexa-546 goat birefringent bipolar structure against the dark cytoplasm (Fig. anti-mouse IgG; Alexa-488 goat anti-rabbit IgG; Molecular 1A, arrow). Squish enucleation of oocytes that nearly Probes, Eugene, OR). Each primary and secondary antibody complete first polar body extrusion often removes the SCC was applied for 40 min at 378C. Rinses were performed with at telophase-I stage (Fig. 1B). Following enucleation by PBS+ 0.1% Triton. DNA was fluorescently detected with 10 extrusion at this pre-metaphase-II stage, a single rhesus Ag/ml Hoechst 33342 (Sigma) and 1 AM Toto-3 (Molecular fibroblast cell demonstrating a normal karyotype (Fig. 1C, Probes) added to the penultimate rinse. Coverslips were inset) is transferred by electrofusion, which also serves to mounted in Vectashield antifade (Vector Labs, Burlingame, simultaneously activate the constructs. By 8 h postfusion and CA). Controls included nonimmune and secondary anti- activation, a single decondensed nucleus is visible in the bodies alone, which did not detect spindle microtubules or smooth cytoplasm of the 1-cell construct (Fig. 1C). C. Simerly et al. / Developmental Biology 276 (2004) 237–252 241

Fig. 1. SCNT-NHP embryo preimplantation development in vitro. (A) SpindleView imaging of the just formed metaphase-II spindle (arrow) in a living NHP oocyte. The first polar body (Pb) is visible just above the bipolar spindle structure. (B) Karyoplast formed from bsquishQ enucleation of an oocyte just after polar body extrusion. The telophase-I spindle is visible after immunolabeling with HSET antibody (green; inset: microtubules, red) and Hoechst DNA stain of the meiotic chromosomes (blue). (C) SCNT construct from 8-h postactivation following nuclear transfer by electrofusion, showing the single nucleus in the activated cytoplasm. Inset: Representative normal karyotype from the donor rhesus fibroblast cell line used for somatic cell nuclear transfer (SCNT). (D–K) In vitro development of SCNT embryos through the cleavage stages; 2-cell (D), 3-cell (E), 8-cell (F; arrow: slight fragmentation), 16-cell (G), the compacting morula stage (H), early blastocyst (I; arrow: early blastocoel), expanded blastocyst (J), and the hatched blastocyst stage (K). (L) NHP-ES cells derived from an NT Â ICSI fertilized chimeric blastocyst, as imaged by HMC optics 4 days post-outgrowth on nonhuman primate embryonic feeder (nhpEF) cells. Numbers represent time postactivation, except panel L: days post-outgrowth. Scale bars = 20 Am.

Seemingly normal in vitro preimplantation development stage is observed by 72 h postactivation (Fig. 1G; focused on from first cleavage to expanded, hatched blastocyst stage is upper plan to show single nuclei in each blastomere) and by shown in Figs. 1D–K and Table 1. The in vitro development 96 h postactivation, the compacted morula with flattened, of SCNT-derived constructs appears accelerated compared to undistinguished blastomeres is observed (Fig. 1H). The first ICSI fertilized embryo development (Hewitson et al., 1998). signs of the early blastocyst (Fig. 1I) containing a con- This difference is perhaps related to the use of a defined spicuous blastocoel (arrow) are found by 110 h postactiva- three-stage sequential culture medium employed for NT tion and the expanded blastocyst with a large, distended embryos in contrast to the traditional use of co-culture in a blastocoel cavity observed by 125 h postactivation (Fig. 1J). complex medium employed after fertilization (S-H Hyun, The fully expanded, hatched blastocyst is observed by 154 h personal observation). By 24 h postactivation, the SCNT postactivation, the stage typically selected for isolation of the constructs have undergone first cleavage, showing even ICM to derive stem cells (Fig. 1K). blastomeres with a single nucleus apparent in each daughter Table 1 presents NHP-SCNT development with either cell (Fig. 1D). By 36 h postactivation, second division fresh, homologous cumulus cells or confluent heterologous commences (Fig. 1E; 3-cell embryo with a single daughter rhesus skin fibroblast cells. Generally, higher successful NT nucleus visible in upper left blastomere). At 50 h postfusion, nuclear reformation, and embryonic development are activation, 8-cell SCNT embryos are observed, with even observed using the fibroblast cells as a donor source sized, smooth blastomeres, and single nuclei within each cell compared to cumulus cells. Furthermore, when the electro- (Fig. 1F). Slight fragmentation, a common occurrence in fusion intensity permits simultaneous fibroblast fusion and NHP embryos, is also visible in the perivitelline space of cytoplast activation (EFA), significantly more expanded some embryos (Fig. 1F, arrow). Development to the 16-cell blastocysts are produced compared to either chemical or 242 C. Simerly et al. / Developmental Biology 276 (2004) 237–252 sperm factor activation after 2 h of incubation post-NT. Of the three SCNT blastocysts produced by simultaneous fusion/activation, only two had identifiable inner cell mass (ICM) cells following immunosurgery for NT-nhpES cell derivations. A fourth chimeric blastocyst was derived by the reaggregation of an SCNT morula (electrofused followed by 2 h ionomycin/DMAP isolation) with a fertilized morula (Fig. 1L). All of these putative NT-nhpES cells stopped growing in vitro after 1 week. Despite 135 embryo transfers into 25 timed NHP recipients, no evidence of a sustained pregnancy was confirmed (Table 2). Early pregnancy indicators, e.g., ultrasonography and serum nhpCG measurements (Munro et al., 1997) ~ day 16, are known to be inconclusive predictors for on-going pregnancies. Table 2 reports pregnancy establishments as verified by fetal heartbeats imaged at day 30. Whereas control NHP ETs result in pregnancy rates as high as 66% (Hewitson et al., 1999), as in ART clinics, these pregnancy rates vary seasonally and annually due to both defined (e.g., embryo quality, surrogates, environmental, and genetic parameters) and still undefined factors. In parallel studies, contemporaneous with these experiments, transfer of 147 transgenic and control ETs into another 41 surrogates led to only 6 confirmed fetuses, a modest 14.6% pregnancy rate. For these reasons, definitive conclusions remain premature and await further investigations. As previously reported in NHPs, few SCNT embryos (b1%; Mitalipov et al., 2002) develop to the blastocyst stage. Here, despite SCNT embryos appearing morpholog- ically normal (Figs. 1C–K; Table 1), many NT constructs Fig. 2. Abnormal preimplantation development of ECNT-derived NHP generated by aspirated enucleation still demonstrate aneu- embryos. (A) First mitotic telophase clone (green), showing atypical chromosome segregation (blue) at the end of first mitosis. Arrow points to a ploidy (Fig. 2). DNA missegregation is already prevalent by lagging chromosome (blue) located within the interzonal microtubules first mitotic telophase (Fig. 2A), as evident by lagging (green). (B–F) Abnormal chromosome segregation (blue) and microtubule chromosomes and loosely organized DNA at the spindle organization (green) in ECNT cloned embryos are apparent at the 2-cell (B), poles (Fig. 2A, blue, DNA; arrow: lagging chromatin). At 4-cell (C), 6-cell (D), and 8-cell stages (E), where most of embryonic second division (Fig. 2B), numerous karyomeres in daugh- development arrests. (F) Control 8-cell stage parthenogenote demonstrating normal chromosome (blue) and interphase microtubule patterns (green). All ter blastomeres (blue) and abnormal assembly of the second images double labeled for microtubules (green) and DNA (blue). Pb = polar mitotic spindles are observed (green). By the 4-cell stage body. Scale bars = 20 Am. (Fig. 2C), the optimum stage for embryo transfer to timed recipients for pregnancy establishment in macaques (Chan typical microtubule and DNA configurations for this stage et al., 2000), the DNA configurations (blue) vary widely (Fig. 2F). within each daughter blastomere. As development proceeds to the 8-cell stage (Figs. 2D–E), monastral interphase Abnormal first interphase microtubule organization in microtubule patterns (green) either lacking DNA or with NHP-SCNT or ECNT constructs is distinct from bovine NT inappropriate chromosomes (blue) are observed. A control clones or NHP androgenotes parthenogenetic embryo at the 8-cell stage demonstrates The somatic cell’s nuclear DNA and cytoplasmic Table 2 constituents (i.e., mitochondria and the centrosome, the Outcomes after SCNT transfers into timed recipients cell’s major microtubule organizing center; MTOC) enter Embryo type No. of embryos Recipient Pregnancy rates, the oocyte’s cytoplasm after SCNT by fusion. Analysis of transferred number i.e., fetal heartbeats microtubule patterns in SCNT constructs, made by aspira- at day 30 tion of mature metaphase-II meiotic spindles, suggests that SCNT 135 25 0 the somatic centrosome ineffectively assembles cytoplasmic Transgenic and 147 41 6 (14.6%) microtubules near the incorporated somatic nucleus by the ICSI controls end of interphase (Figs. 3A–B: microtubules, green; DNA, C. Simerly et al. / Developmental Biology 276 (2004) 237–252 243

Fig. 3. Abnormal microtubule patterns after nuclear transfer in NHP, but not bovine, constructs. (A–B) Disarrayed microtubules (green) assembled near the transferred somatic cell nucleus (blue) indicate a dysfunctional somatic centrosome following intracytoplasmic nuclear injection (ICNI) and activation by either sperm factor or ionomycin/DMAP. (C) Cortical microtubule patterns (green) similar to parthenogenetically activated oocytes observed after somatic cell nuclear transfer (blue) and activation. (D) An NHP-NT construct derived by embryonic nuclear transfer using a dissociated 16-cell stage rhesus blastomere. Multiple microtubule organizing centers (green, arrows) within the cytoplasm are detected distal to the transferred blastomere nucleus (blue). (E) NHP-NT construct derived by the transfer of a male pronucleus (MPn, blue) into an enucleated oocyte. Microtubules (green) are tightly focused at the transferred nucleus and radiate into the cytoplasm. Pb: polar body; FPN: female pronucleus. (F) DNA synthesis onset in the transferred somatic cell nucleus (sc, blue) as well as male (MPN, blue) and female (FPN, blue) pronuclei is detected by BrDU incorporation (green) 20 h post-ICSI. Neither the first nor second polar bodies (Pb) incorporate BrDU. Inset: Microtubules (red) and DNA (blue). (G–H) Tightly focused microtubule arrays (green) emanating from the transferred nucleus in activated bovine enucleated cytoplasts following either somatic cell or embryonic cell nuclear transfer. (I) Normal anastral, bipolar spindles (green) with aligned chromosomes assemble at metaphase (blue) following embryonic nuclear transfer. Similar mitotic spindle morphologies have been observed after somatic cell nuclear transfer. (J) A focused microtubule array (green) from a rhesus fibroblast cell (blue) transferred into a bovine enucleated oocyte suggests that normal somatic centrosomal function at interphase is dependent on the maternal cytoplasmic source more than the donor nuclear source. All images double labeled for microtubules (green) and DNA (blue), except panel F: triple labeled for BrDU (green), microtubules (red), and DNA (blue). Scale bars = 10 Am. blue), if at all. Typically, a cortical disarrayed microtubule cycle progression in the transferred somatic nucleus, as pattern is observed after SCNT construct activation, observed in the decondensed male and female pronuclei, suggestive of microtubule patterns in parthenogenetically respectively (Fig. 3F; BrDU, green). activated oocytes (Fig. 3C: microtubules, green; DNA, blue; Wu et al., 1996). After ECNT, the contributed blastomere Interspecific SCNT centrosome(s) consistently assembles cytoplasmic microtubules, albeit from multiple MTOC sites (ECNT; Fig. 3D, Because bovine SCNT succeeds at significantly higher arrows). Conversely, when NT is performed using a male rates than NHP-SCNT, we performed interspecific SCNT to pronucleus from an ICSI fertilized zygote as the donor, a address the issue of whether the bovine cytoplasm was more more typical symmetrical microtubule array emanating from conducive to NT-spindle formation than the NHP ooplasm, the sperm centrosome is observed (Fig. 3E; MPn, arrow). or whether the bovine somatic cell’s centrosome was more Following SCNT into a metaphase-II intact oocyte and resilient than the NHP somatic centrosome for NT-spindle activation by ICSI fertilization (Fert+ SCNT), BrDU nuclear organization. Unlike non-human primate clones, bovine X labeling indicates successful DNA synthesis onset and cell bovine SCNT results in the formation of a single radially 244 C. Simerly et al. / Developmental Biology 276 (2004) 237–252 arrayed aster juxtaposed to the nucleus, akin to fertilization’s sperm aster (ECNT: Fig 3G; SCNT: Fig. 3H; microtubules, green). Bovine NT constructs produce normal bipolar spindles with aligned chromosomes at first mitosis (Fig. 3I). Furthermore, the bovine egg cytoplasm appears to tolerate exogenous centrosome transfer better than NHP constructs, as evident by a focused microtubule astral array adjacent to the reformed monkey fibroblast nucleus after SCNT into the bovine enucleated cytoplast (Fig. 3J). These results suggest that the primate ooplasm might recognize adventitious centrosomes, other than the sperm centrosome, and perhaps a mechanism for centrosome destruction, vestigial from oogenesis, might persist longer in primates eggs versus their domestic species counterparts.

Androgenesis

To investigate if interphase microtubule defects observed in NHP activated SCNT and ECNT constructs are the result of the removal of the SCC during the enucleation process, we examined microtubule and DNA patterns in androgenotes, derived by ICSI fertilization of enucleated oocytes (Fig. 4). As the introduced sperm DNA decondenses in the enucleated cytoplast, a microtubule astral array assembles from the introduced paternal centrosome (Fig 4A, arrow; arrowhead: incorporated sperm axoneme). The microtubules expand within the cytoplasm (Fig. 4B, green) and at late interphase are observed emanating from the duplicated, split paternally derived centrosomes adjacent to the fully formed male pronucleus (Fig. 4C, arrows). At late prometaphase, a bipolar spindle with asters (centrosomes) at each pole and aligning metaphase chromosomes assembles (Fig. 4D, arrows; arrowhead: sperm axoneme). Androgenotes typically progress through first mitosis, completing accurate chromosome segregation at the first cell division (Fig. 4E). Eighty percent (8/10) of androgenotes examined at the pronuclear stage demonstrated normal microtubule and chromosome patterns, though only 24% (8/33) produced normal 8-cell stage embryos. Collectively, these data suggest that interphase microtubule organization after primate NT has a stricter requirement for the sperm centrosome than observed in species where nuclear transfer succeeds.

Fig. 4. Microtubule patterns are normal in NHP androgenotes. (A–B) A mature spermatozoa (blue) microinjected into an enucleated rhesus oocyte assembles tightly focused microtubule arrays (green) from the sperm centrioles (arrowhead: sperm axoneme) that extend into the cytoplasm within 8 h post-ICSI. (C) Centrosome duplication, splitting, and microtubule assembly (green) are observed on opposite sides of the male pronucleus by 20 h post-ICSI. (D) A bipolar spindle (green) with small astral arrays (green, arrows) at the spindle poles assembles at metaphase (blue) in androgenotes. Arrowhead shows the incorporated sperm axoneme. (E) A 2-cell-stage androgenote demonstrating normal DNA segregation (blue) and microtubule assembly (green) near the daughter nuclei following cell division. All images double labeled for microtubules (green) and DNA (blue). Scale bars = 20 Am. C. Simerly et al. / Developmental Biology 276 (2004) 237–252 245

SCNT clones, missing spindle molecular motors and matrix that nucleate microtubules are generally lacking (Fig. 5C, proteins, form multipolar spindles with misaligned green). chromosomes Key centrosomal and molecular motor proteins are deficient in mitotic spindles after nuclear transfer, compared At first mitosis, NHP NT constructs, generated after with spindle assembly in constructs produced by combining aspiration of the mature metaphase-II meiotic spindle, nuclear transfer with either fertilization (Simerly et al., 2003) develop disarrayed spindles with misaligned chromosomes or artificial activation of intact oocytes (i.e., no enucleation of (Figs. 5A–C; Simerly et al., 2003). Following blastomere the SCC). NuMA, a spindle pole assembly protein found in NT, the MTOCs observed nucleating microtubules, pre- mammalian oocytes (Lee et al., 2000; Tang et al., 2004), sumably from the introduced somatic centrosome(s), are concentrates in the reconstituted interphase SCNT or ECNT distinctly misaligned on the assembled tri- or tetrapolar nucleus following activation (Fig. 5D, green). However, by metaphase spindles containing scattered chromosomes first mitosis, NuMA protein is either not detected (Simerly et (Figs. 5A–B, green, arrows). Similar aberrant mitotic al., 2003) or at barely detectable concentrations on the NT spindle assembly and chromosomal anomalies are found mitotic spindle poles (Fig. 5E; green). Conversely, mitotic in mitotic SCNTs, though definitive spindle pole MTOCs constructs produced from the activation of intact oocytes after SCNT (SCNT+ Met-II) generate spindles with more aligned chromosomes and abundant NuMA on the poles, but still with misaligned MTOCs (Fig. 5F, green; arrows: centrosomes). Similarly, the mitotic kinesin HSET, which traffics to the microtubule minus-ends in meiotic and mitotic spindles (Simerly et al., 2003), is undetected in mitotic nuclear transfer spindles displaying misaligned chromosomes (Fig. 5G; green; inset: microtubules, red; DNA, blue). HSET protein detection is somewhat restored at spindle poles of mitotic SCNT+ Met-II constructs (Fig. 5H, green), though the chromosomes remain scattered in the microtubule spindle lattice (Fig. 5H, blue). The oppositely directed kinesin mitotic

Fig. 5. Dysfunctional somatic cell centrosomes and microtubule-based molecular motors are evident in mitotic metaphase NHP constructs. (A–B) Mitotic metaphase ECNT construct with tripolar spindles (green), abnormal centrosome localization (arrows) at the poles, and misaligned chromosomes at the equator (blue). (C) A first mitotic metaphase SCNT clone with poor bipolar spindle morphology (green), no discernible somatic cell centrosome at the spindle poles, and misaligned chromosomes at the equator (blue). (D– F) NuMA detection in interphase and mitotic NT constructs. (D) NuMA (green) is detected in the ECNT reconstructed nucleus at late interphase. Inset: Random disarrayed microtubule pattern (red) and DNA (blue) are observed in this ECNT clone. Similar observations were made for interphase SCNT constructs. (E) An SCNT construct at first mitotic metaphase showing a multipolar spindle (red) with misaligned chromosomes (blue) and diminished NuMA detection at the poles (green). (F) A first mitotic metaphase spindle produced from activation of a metaphase-II spindle intact oocyte after SCNT (SCNT+ Met-II). Four misplaced centrosomes (arrows) are present within the tetrapolar spindle (red) as the chromosomes align at the equator (blue). NuMA (green) is strongly detected at the centrosomes and four spindle poles. (G) The minus-end-directed kinesin HSET (green) is not detected in SCNT first mitotic constructs. Inset: Spindle microtubules (red) and misaligned DNA (blue). (H) A first mitotic metaphase spindle in an SCNT+ Met-II intact construct. HSET is strongly detected at the metaphase spindle poles (green). Inset: Spindle microtubules (red) and DNA (blue). (I) The plus-end-directed kinesin Eg5 detection in a first mitotic SCNT spindle. The multipolar metaphase spindle (red) shows Eg5 present at the centromere region on the misaligned chromosomes (blue). (J–K) Mitotic metaphase and telophase spindles in control parthenogenetic embryos. Eg5 (green) is detected at aligned chromosomes (blue) on bipolar metaphase spindles (red) but translocates to interzonal microtubules (K: red) and the developing midbody apparatus by telophase (K: blue). (A–C) Double-labeled images for microtubules (green) and DNA (blue). All other images tripled labeled for NuMA (D–F), HSET kinesin (G and H), or Eg5 kinesin (I–K), microtubules (red), and DNA (blue). Scale bars = 10 Am. 246 C. Simerly et al. / Developmental Biology 276 (2004) 237–252 microtubule motor protein Eg5, observed at the centromeres spindle pole formation in NT constructs. Here, HSET and and on the kinetochore-microtubule bundles in meiotic and NuMA detection in the enucleated karyoplast and in the mitotic spindles (Simerly et al., 2003), remains paired at sister remaining cytoplast following removal of the SCC is kinetochores on multipolar spindles with misaligned chro- investigated. Following successful enucleation of the SCC mosomes in mitotic nuclear transfers (Fig. 5I). This pattern of (Fig. 6A, inset, DNA), no assembled microtubules are Eg5 is similar to kinesin protein detection in parthenogeneti- observed in the cytoplast (Fig. 6A, green). A large cally activated control mitotic embryos at metaphase and monastral interphase microtubule array assembles follow- telophase (Figs. 5J–K, green). In summary, when NuMA and ing sperm factor or ionomycin/DMAP chemical activation HSET are missing, dysfunctional mitotic spindles form in NT of the enucleated cytoplast (Fig. 6B, green; inset: DNA). constructs. Rarely, activated cytoplasts will produce atypical, astral bipolar spindle-like structures without DNA at first mitosis HSET concentrates on the second meiotic spindle in human (Fig. 6C, green; inset: DNA). The SCC-containing and nonhuman primate oocytes karyoplast, produced by squish enucleation, retains the intact metaphase-II spindle (Fig. 6D; inset: red, micro- Previous results suggest that meiotic spindle removal tubules; blue, DNA) with HSET protein detected in the depletes the egg cytoplasm of vital proteins for first mitotic spindle lattice (Fig. 6D, green). Cumulus and fibroblast

Fig. 6. The minus-end-directed kinesin HSET assembles exclusively at the second meiotic spindle in NHPs and not in Paclitaxel-induced cytoplasmic microtubules. (A–C) Microtubule patterns (green) observed in rhesus cytoplast after enucleation (A) or following artificial activation at 24 (B) or 48 h (C) post- ionomycin/DMAP. Following enucleation, no assembled cytoplasmic microtubules (A: green) are observed. After activation, abundant disarrayed microtubules (B: green) assemble in the DNA-less cytoplast. Rarely, a microtubule structure resembling a bipolar spindle (C: green) assembles in the cytoplasm. Insets: DNA imaging confirming successful removal of the SCC. (D) Microtubules (red, inset), HSET (green), and DNA (blue) imaging of the intact meiotic spindle and chromosomes in a karyoplast following squish enucleation. (E–F) Cytoplasmic HSET (green) detection in NHP cumulus (E: green; blue, DNA) and rhesus fibroblast cells (F: green; red, microtubules; blue, DNA) suggests that some somatic cell HSET may be transferred during NT. (G) A mature oocyte treated for 30 min with 10 AM paclitaxel demonstrating HSET (green) localization at the spindle pole microtubules (red), though not at assembled cytoplasmic microtubule bundles (arrows). Second meiotic chromosomes, blue. (H–J) A rhesus enucleated cytoplast treated 30 min with 10 AM paclitaxel prior to fixation and detection of DNA (H, blue), microtubules (I, red), and HSET (J, green). The assembled cytoplasmic microtubule bundles are not labeled by HSET antibody. (A, B, C, and E) Double-labeled images for microtubules (green) and DNA (blue). (D–G) Triple-labeled images for HSET (green), microtubules (red), and DNA (blue). (H–J) Single-labeled images for DNA (blue), microtubules (red), and HSET (green). Scale bars = 10 Am. C. Simerly et al. / Developmental Biology 276 (2004) 237–252 247

SCNT donor cells retain somatic HSET at the cell cycle supports conclusions that HSET distribution is restricted to stage used for NT (Figs. 6E–F, green), though perhaps at the SCC. insufficient concentrations or inappropriate locations to augment spindle assembly in mitotic NT constructs after Cytoplasmic NuMA after removal of the SCC fusion. Likewise, this cytoplasmic protein would largely be removed due to cytoplasmic stripping in the needle prior to NuMA resides at the spindle poles of the SCC following NT by ICNI into the recipient’s cytoplasm. enucleation by extrusion (Fig. 7A, green). The remaining enucleated cytoplast has neither assembled microtubules Paclitaxel-induced microtubule recruitment (Fig. 7B, upper inset, red) or NuMA detection in the cytoplasm (Fig. 7B, green; lower inset: DNA). Paclitaxel Paclitaxel, an effective microtubule disassembly inhib- treatment of SCC-intact oocytes suggests NuMA local- itor, was employed to investigate cytoplasmic HSET ization at disorganized spindle poles (Fig. 7C, green, lower recruitment to augmented microtubules in the presence or arrow) and the assembled cytoplasmic microtubule asters absence of the SCC. Exposure of intact oocytes to Paclitaxel (Fig. 7C, green, arrowhead). Both cumulus and fibroblast enhances HSET protein only at the second meiotic spindle SCNT donor cells demonstrate intranuclear NuMA prior to poles (Fig. 6G: arrowheads, HSET, green: arrows: cyto- NT (Figs. 7D–E, green). Yet, abundant maternal NuMA plasmic microtubule asters). When the enucleated cytoplast protein remains after SCC extrusion and is available for lacking the SCC (Fig. 6H, blue, DNA) is treated with nuclear importation, as seen within an activated enucleated paclitaxel, numerous cytoplasmic microtubule bundles oocyte that failed SCNT fusion but contains the decon- assemble in the cytoplasm (Fig. 6I, red), but without HSET densed sperm nucleus (SCNT+ Fert; Fig. 7F, green; MPn: protein detection (Fig. 6J, green). Taken together, this male pronucleus). Furthermore, the amount of somatic

Fig. 7. The spindle pole matrix protein NuMA is not restricted to the second meiotic spindle but also resides in the cytoplasm of NHP oocytes after SCC enucleation. (A) NuMA (green), microtubules (red), and DNA (blue) detection of the intact second meiotic spindle removed by squish enucleation. (B) The enucleated cytoplast, formed after removal of the SCC by squish enucleation, shows no NuMA (green) or microtubule assembly (top inset: red; bottom inset: DNA, blue) in the cytoplasm. (C) A mature NHP oocyte treated for 20 min with 10 AM paclitaxel shows NuMA (green) accumulation within the second meiotic spindle (lower arrow; red, microtubules; blue, DNA) and the cytoplasmic microtubule bundles (upper arrow; red, microtubules; blue, DNA). (D–E) NuMA detection in the somatic cell nuclei of cumulus (D: green) and rhesus fibroblast cells (E: green; red, microtubules). Perhaps these somatic cells contribute additional NuMA following SCNT. (F) A dFertCloneT failure derived from an oocyte that failed nuclear transfer of the fibroblast cell by electrofusion (arrowhead) but was successfully activated by intracytoplasmic sperm injection (ICSI). NuMA (green) is strongly detected in the decondensed male pronucleus (MPn) but diminished in the unsuccessful fibroblast cell (arrowhead) attached at the oocyte surface. Upper inset: Red, microtubules; blue, DNA. Lower inset: The sperm centrosome organizes a microtubule astral array (arrow) near the decondensed male pronucleus. (G) A dFert-CloneT failure produced by SCNT into an intact second meiotic metaphase-II arrested oocyte that unsuccessfully activated following ICSI (arrow). NuMA (green) is found at both poles on the intact metaphase-II spindle (red) with aligned chromosomes (blue) as well as at the base of the condensed sperm head (blue, arrow). However, NuMA (green) is missing from the disorganized multipolar NT spindle microtubules (red) assembling around the scattered somatic cell chromosomes (blue). (H) ECNT into a metaphase-II intact oocyte that failed subsequent activation by sperm factor microinjection. NuMA (green) is detected at the poles in both the NT spindle (lower arrow; red, microtubules; blue, DNA) and the intact second meiotic metaphase spindle (upper arrow; red, microtubules; blue, DNA). All images triple labeled for NuMA (green), microtubules (red), and DNA (blue), except panel D: single labeled for NuMA (green), and panel E: double labeled for microtubules (red) and NuMA (green). Scale bars = 10 Am, except panel D: 1 Am. 248 C. Simerly et al. / Developmental Biology 276 (2004) 237–252 intranuclear NuMA provided by a fibroblast cell at fusion is primates demand perhaps the only two structures missing relatively small compared to the maternal protein available during cloning—the sperm’s centrosome and the egg’s for nuclear importation (Fig. 7F; green, arrowhead: non- meiotic spindle-chromosome complex. In cattle, the somatic fused fibroblast cells). Nevertheless, NuMA recruitment to centrosome is transferred during SCNT (Navara et al., 1994; NT spindles following SCNT appears compromised. Failed- Shin et al., 2002a) while mice rely on the maternal to-activate SCNT+ fertilization constructs that remain firmly centrosome in the ooplasm (Schatten, 1994). Unlike cloning arrested at second meiosis often display NuMA at both in domestic species (centrosome transfer along with nuclear meiotic spindle poles (Fig. 7G; arrowheads) and at the transfer; Navara et al., 1994) and in mice (exceptional condensed sperm head (Fig. 7G; arrow), but with NuMA reliance on maternal centrosome), primate cloning appears missing from the chaotic spindle surrounding the transferred further hindered by aneuploidies generated after enucleation nucleus (Fig. 7G, NT). Conversely, SCNT combined with (Simerly et al., 2003). aging metaphase-II intact oocytes (Fig. 7H: upper arrow) Hwang et al. (2004) have devised procedural innovations that subsequently fail to activate assemble bipolar NT that succeed in human SCNT embryogenesis (depicted in the spindles that recruit cytoplasmic NuMA to their poles, appended animation). NT stem cells, i.e., ESC derivations though chromosomes do not align properly (Fig. 7H, lower from SCNT blastocysts, appear less successful than from arrow: green, NuMA). Taken together, the data suggest that blastocysts conceived by fertilization. A single hESC line meiotic spindle removal depletes the egg cytoplasm of was derived from 20 SCNT blastocysts, while three hESC HSET, a vital protein for first mitotic spindle pole lines were successfully derived from 17 fertilized blastocysts. formation. Also the residual NuMA, retained within the NHP blastocysts with distinct inner cell mass cells are found oocyte’s cytoplasm following spindle extrusion and in 50% of embryos after fertilization, but only 20% following imported into the interphase nucleus following SCNT, is SCNT. While these estimates are based on limited data sets, not effectively targeted to the spindle poles in mitotic perhaps ESC derivations after SCNT (therapeutic cloning) constructs or is below threshold concentrations, perhaps are 80% less successful than ESC derivations from fertilized indicating interference with mechanisms that recruits blastocysts. Whether NHP-SCNT embryos are equally useful NuMA to the microtubule minus-ends (Compton, 1998). for either therapeutic cloning or reproductive cloning awaits further studies, though preliminary evaluations suggest that ESC derivations from SCNT blastocysts are favored. Discussion Regardless, human reproductive cloning, already considered unsafe, unethical, and unwarranted, demands enforceable Cloning, already inefficient, is achieved routinely only in international consensus precluding any attempts (Annas and those well-studied species (domestic species, mice) that Elias, 2004; Aschwanden, 2003; Kass, 2004). provide vast numbers of both oocytes and surrogates, systems The assembly and organization of the zygote’s first often capable of multiple deliveries and amenable to transfers mitotic spindle—the zygotic spindle—differ between fertil- of supernumerary embryos. SCNT now succeeds in nine ization and after NT (Fig. 8). During fertilization (left mammals (Wilmut, 2002). Primate SCNT research is panel), the sperm enters the egg either by fusion with the advancing knowledge regarding hESC utility and accept- oolemma or by ICSI (shown), and the mature oocyte is ability, including ESC derivations without compromising arrested at second meiotic metaphase. The meiotic spindle is embryo viability (e.g., embryo splitting and blastomere organized by centrosomal molecules including NuMA biopsy; Chan et al., 2000) as well as from parthenogenotes (green dumbbells) as well as minus-microtubule end with intact meiotic spindles (Cibelli et al., 1998). NHP directed motors (e.g., HSET kinesin). The metaphase offspring generated by SCNT, if successful, might provide chromosomes align at the equator of the second meiotic the founding breeding NHPs to produce identical preclinical spindle, and other kinesins, in this case Eg5, are localized to models so that crucial investigations requiring NHPs might the centromere/kinetochore region. Eg5 is typically a be completed faster, with greater accuracy and with fewer spindle pole-associated kinesin, and while it is unusually animals studied, since genetic variation confounders in the localized at the centromere in primate eggs, it is detected currently outbred populations would be minimized. only at the spindle poles in mitotic embryonic stem cells Notwithstanding the research importance of both repro- (unpublished results). The egg cortex region adjacent to the ductive and therapeutic cloning in NHPs, and human meiotic spindle is different from the rest of the cortex in therapeutic cloning, cloning in primates is challenged by primate oocytes, as in other species (cortical cross hatch), understandable limits on oocyte availabilities, as well as by with concentrations of fodrin (Schatten et al., 1986), WAVE cellular requirements in assembling the primate’s first 1(Rawe et al., 2004), myosin IIA/B (Simerly et al., 1998), mitotic spindle that appears stricter than in the species and dense microfilaments (Maro et al., 1990). cloned successfully. SCNT hurdles include problems in The sperm aster (middle left panel) assembles from the bnuclear reprogramming,Q genomic imprinting, cell cycle centriole-containing centrosome contributed by the sperm, asynchronies, egg activation, and mitochondrial and cyto- subjacent to the male pronucleus (insert) as the female plasmic incompatibilities. Ironically, NHP and human pronucleus decondenses. The cortical specification is C. Simerly et al. / Developmental Biology 276 (2004) 237–252 249 depicted by the cross hatch and both the first and second metaphase-II are extruded (upper panel, right). Extrusion polar bodies are excluded for clarity. The zygotic spindle of these pre-metaphase-II spindles extracts a smaller spindle (lower panel, left), that is, the zygote’s first mitotic spindle centrosome complex (SCC), discarding fewer microtubule- after fertilization, contains centrioles within either spindle and centrosome-associated molecules and adhering cyto- poles, or at least the spindle pole with the incorporated plasm, and less specialized cortex. During NT, the somatic sperm tail. While it is assumed that the centrioles in the cell contributes its own centrosome along with the somatic spindle pole contralateral to the sperm tail results from the centriole (middle right). However, unlike bovine NT, either replication of the sperm centriole, experimental proof is still the somatic centriole/centrosome complex is inactive in lacking. The zygotic spindle poles also contain NuMA, NHP oocytes or the primate oocytes retain a centriole/ HSET, and other centrosome molecules, for example, centrosome destruction mechanism that might persist longer gamma-tubulin (Palacios et al., 1993; Stearns et al., 1991), in primate oogenesis than in other animals. Immature pericentrin (Doxsey et al., 1994), and PCM-1 (Balczon et oocytes destroy centrioles (Szollosi et al., 1972), yet the al., 1994, 2002; Dammermann and Merdes, 2002). mechanism is neither understood nor conserved among Nuclear transfer involves artificial activation and avoids different mammals (Schatten et al., 1994). exposure to sperm (right panel). Removal of the maternal DNA is performed earlier than metaphase-II arrest by following the procedures of Hwang et al. (2004). Here we Fig. 8. Centrosome transmission during primate nuclear transfer (right) and show that spindles ranging from telophase-I to pro- fertilization (left). NT begins with squish enucleation (top right), the removal of the unfertilized oocyte’s pre-metaphase-II meiotic spindle- chromosome complex (SCC), leaving some NuMA (Green cross-linker) and HSET (Red pacman) molecular motor protein remaining in the ooplasm. Better retention of vital proteins (i.e., myosin-II, fodrin, Wave-1; actin filaments: cross hatches) in the meiotic spindle cortex perhaps required for cleavage furrow formation at first mitosis is also anticipated following squish enucleation. Right inset: Enucleation of the pre- metaphase-II SCC removes less of the minus-end-directed spindle proteins NuMA (green cross-linker) and HSET motors (Red pacman). Middle right: Nuclear transfer by electrofusion (yellow lightening bolt) introduces an embryonic or somatic nucleus and centrioles (red orthogonal cylinders) containing g-tubulin and pericentrin (red lattice) into the enucleated cytoplast, as well as providing the simultaneous activating stimulus to initiate development. The fate of the somatic centriole/centrosome complex in the developing NT construct is not currently known. Bottom right: Enucleation by squish extrusion along with simultaneous fusion/activation results in more organized bipolar first mitotic spindles, although some chromosome misalignments that produce aneuploid embryos are still evident. Inset: NT-mitotic spindles display mostly aligned chromosome pairs (blue) with Eg5 at their centromere/kinetochore regions (yellow pacman). Microtubules assemble (green) into organized spindles with both NuMA (green cross-linker) and HSET (red pacman) present at their spindle poles. Fertilization left: sperm entry, by either IVF or ICSI (shown), activates the egg’s metabolism and contributes the paternal haploid genome to the now fertilized zygote. Left inset: The typical meiotic spindle arrested at second metaphase (blue chromosomes) is unusual since it lack centrioles at the poles. The plus-end-directed kinesin motor, Eg5, concentrated at the centromeres (yellow pacman), anchored at the growing end (+) of the polarized microtubules (green). Centrosome molecules NuMA (green cross-linker) and HSET (red pacman), responsible for meiotic spindle organization, are concentrated at the converging microtubule (À) ends and would be removed during enucleation at this stage. Middle left: The fertilizing sperm contributes the centriole pair (red orthogonal cylinders) containing paternal g-tubulin and pericentrin (red lattice). This sperm centrosome complex recruits maternal g-tubulin from which sperm aster microtubules assemble (green). Bottom left: First mitotic spindle assembly after fertilization. The sperm centrosome duplicates during first interphase, with the sperm tail-centriole complex visible at one pole of the bipolar, anastral spindle. The bipolar mitotic spindle contains aligned chromosomes (blue) with Eg5 at each kinetochore pair (yellow pacman). NuMA (green cross-linker) and HSET (red pacman) are found at the spindle poles along with the centrioles and g-tubulin/pericentrin (red lattice). The other spindle pole (demarked by a question mark) is either organized without a centriole pair or contains centrioles of unknown derivation (either coming from the duplicated, split paternal centriole or perhaps derived from de novo maternal centriole formation). 250 C. Simerly et al. / Developmental Biology 276 (2004) 237–252

The zygotic spindle after NT in primates is organized Appendix A. Supplementary data around centrosomes that contain NuMA and HSET, but without centriole-containing centrosomes. While these Supplementary data associated with this article can zygotic spindles are bipolar and barrel shaped, they may be found, in the online version, at doi:10.1016/ be less accurate in mitotic chromosome alignment and j.ydbio.2004.10.006. anaphase chromosome segregation (right bottom) than centriole-containing zygotic spindles after fertilization. SCNT affords a novel strategy for investigating cyto- References plasmic inheritance. For example, mitochondria are mater- nally inherited (Giles et al., 1980) naturally and after SCNT Annas, G.J., Elias, S., 2004. Politics, morals and embryos. Nature 431, (ovine unimaternal; Evans et al., 1999; bovine; bimaternal; 19–20. Steinborn et al., 1998; murine bimaternal tissue specific; Aschwanden, C., 2003. Nations fail to agree on extent of human cloning Inoue et al., 2004). Recently, triparental heteroplasmy has ban. Bull. World Health Organ 81, 850–851. Baguisi, A., Behboodi, E., Melican, D.T., Pollock, J.S., Destrempes, M.M., been shown after ECNT-NHP (St John and Schatten, 2004). Cammuso, C., Williams, J.L., Nims, S.D., Porter, C.A., Midura, P., MtDNA in both ECNT offspring are derived from three Palacios, M.J., Ayres, S.L., Denniston, R.S., Hayes, M.L., Ziomek, lineages—the oocyte and both embryonic lineages (the C.A., Meade, H.M., Godke, R.A., Gavin, W.G., Overstrom, E.W., anticipated maternal mtDNA and unexpectedly paternal Echelard, Y., 1999. Production of goats by somatic cell nuclear transfer. mtDNA from the donor blastomere). Heteroplasmic trans- Nat. Biotechnol. 17, 456–461. Balczon, R., Bao, L., Zimmer, W.E., 1994. PCM-1, a 228-kD centrosome mission has been reported in ART clinics (Barritt et al., autoantigen with a distinct cell cycle distribution. J. Cell Biol. 124, 2002; Schultz and Williams, 2002) and might confound 783–793. ESC or offspring (Inoue et al., 2002) phenotypes. Balczon, R., Simerly, C., Takahashi, D., Schatten, G., 2002. Arrest of cell Reproduction is error prone: Clinically, fewer than a cycle progression during first interphase in murine zygotes micro- quarter of natural conceptions succeed (Edwards, 2001), and injected with anti-PCM-1 antibodies. Cell Motil. Cytoskeleton 52, 183–192. even in favorable systems, somatic cell nuclear transfer is at Barritt, J.A., Kokot, M., Cohen, J., Steuerwald, N., Brenner, C.A., 2002. least 10-fold worse. Multiple defects are likely to account Quantification of human ooplasmic mitochondria. Reprod. Biomed. for cloning failures (Rideout et al., 2001): for example, Online 4, 243–247. nuclear reprogramming; telomere shortening; cell cycle Bavister, B.D., Boatman, D.E., Leibfried, L., Loose, M., Vernon, M.W., asynchrony; gene misexpression during development; 1983. Fertilization and cleavage of rhesus monkey oocytes in vitro. Biol. Reprod. 28, 983–999. genomic imprinting errors; suboptimal culture conditions; Chan, A.W., Dominko, T., Luetjens, C.M., Neuber, E., Martinovich, C., mitochondrial heteroplasmy (St John and Schatten, 2004); Hewitson, L., Simerly, C.R., Schatten, G.P., 2000. Clonal propagation mitotic spindle defects resulting from centrosome and motor of primate offspring by embryo splitting. Science 287, 317–319. extractions during enucleation (Simerly et al., 2003); Cibelli, J.B., Stice, S.L., Golueke, P.J., Kane, J.J., Jerry, J., Blackwell, exaggerated placenta: fetus ratios; and technical damage. C., Ponce de Leon, F.A., Robl, J.M., 1998. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280, Notwithstanding these reproductive cloning hurdles, ther- 1256–1258. apeutic cloning for human and NHP stem cell research has Compton, D.A., 1998. Focusing on spindle poles. J. Cell Sci. 111 (Pt. 11), been dramatically accelerated by the findings of Hwang et 1477–1481. al. (2004). Determining whether ESC derived after SCNT Dammermann, A., Merdes, A., 2002. Assembly of centrosomal proteins are indeed immune matched to the donor is now possible and microtubule organization depends on PCM-1. J. Cell Biol. 159, 255–266. with nonhuman primates. Doxsey, S.J., Stein, P., Evans, L., Calarco, P.D., Kirschner, M., 1994. Pericentrin, a highly conserved centrosome protein involved in microtubule organization. Cell 76, 639–650. Acknowledgments Edwards, R.G., 2001. IVF and the history of stem cells. Nature 413, 349–351. Evans, M.J., Gurer, C., Loike, J.D., Wilmut, I., Schnieke, A.E., Schon, We thank Drs. S. Doxsey (U. Mass) and T. Stearns E.A., 1999. Mitochondrial DNA genotypes in nuclear transfer-derived (Stanford) for antibodies; Dr. Mark Tepper (Serono; r-hLH, cloned sheep. Nat. Genet. 23, 90–93. r-hCG, and/or r-hFSH) and Drs. Shin-Ho Kang and Won-Bae Galli, C., Lagutina, I., Crotti, G., Colleoni, S., Turini, P., Ponderato, N., Kim of the Dong-A SEETECH Co. LTD (r-hFSH) for Duchi, R., Lazzari, G., 2003. Pregnancy: a cloned horse born to its dam hormone donations; and our fabulous laboratory and veter- twin. Nature 424, 635. Giles, R.E., Blanc, H., Cann, H.M., Wallace, D.C., 1980. Maternal inarian colleagues, including Jodi Mich-Basso, Ethan Jacoby, inheritance of human mitochondrial DNA. Proc. Natl. Acad. Sci. David McFarland, Hina Qidwai, Carrie Redinger, and Jamie U. S. A. 77, 6715–6719. Tomko. We appreciate comments from Dr. Tony Plant Hewitson, L., Takahashi, D., Dominko, T., Simerly, C., Schatten, G., 1998. (Pittsburgh’s Center for Research in Reproductive Physiology) Fertilization and embryo development to blastocysts after intracyto- and Betsy True for the illustration. All protocols were IACUC plasmic sperm injection in the rhesus monkey. Hum. Reprod. 13, 3449–3455. approved. We gratefully acknowledge NIH research support. Hewitson, L., Dominko, T., Takahashi, D., Martinovich, C., Ramalho- SHH was sponsored by a postdoctoral fellowship from the Santos, J., Sutovsky, P., Fanton, J., Jacob, D., Monteith, D., Neuringer, Korea Science and Engineering Foundation (KOSEF). M., Battaglia, D., Simerly, C., Schatten, G., 1999. Unique checkpoints C. Simerly et al. / Developmental Biology 276 (2004) 237–252 251

during the first cell cycle of fertilization after intracytoplasmic sperm nuclear trafficking is essential for genomic and cytoskeletal dynamics injection in rhesus monkeys. Nat. Med. 5, 431–433. during fertilization: cell-cycle-dependent shuttling between M-phase Hiendleder, S., Bebbere, D., Zakhartchenko, V., Reichenbach, H.D., and interphase nuclei. Dev. Biol. 276, 253–267. Wenigerkind, H., Ledda, S., Wolf, E., 2004. Maternal-fetal trans- Rideout III, W.M., Eggan, K., Jaenisch, R., 2001. Nuclear cloning and placental leakage of mitochondrial DNA in bovine nuclear transfer epigenetic reprogramming of the genome. Science 293, 1093–1098. pregnancies: potential implications for offspring and recipients. Cloning Schatten, G., 1994. The centrosome and its mode of inheritance: the Stem Cells 6, 150–156. reduction of the centrosome during gametogenesis and its restoration Hwang, W.S., Ryu, Y.J., Park, J.H., Park, E.S., Lee, E.G., Koo, J.M., Jeon, during fertilization. Dev. Biol. 165, 299–335. H.Y., Lee, B.C., Kang, S.K., Kim, S.J., Ahn, C., Hwang, J.H., Park, Schatten, G.P., 2002. Safeguarding ART. Nat. Med. 8 (Suppl), S19–S22. K.Y., Cibelli, J.B., Moon, S.Y., 2004. Evidence of a pluripotent human Schatten, H., Cheney, R., Balczon, R., Willard, M., Cline, C., Simerly, C., embryonic stem cell line derived from a cloned blastocyst. Science 303, Schatten, G., 1986. Localization of fodrin during fertilization and early 1669–1674. development of sea urchins and mice. Dev. Biol. 118, 457–466. Inoue, K., Ogura, A., Hayashi, J., 2002. Production of mitochondrial DNA Schatten, G., Prather, R., Wilmut, I., 2003. Cloning claim is science fiction, transgenic mice using zygotes. Methods 26, 358–363. not science. Science 299, 344. Inoue, K., Ogonuki, N., Yamamoto, Y., Takano, K., Miki, H., Mochida, K., Schultz, R.M., Williams, C.J., 2002. The science of ART. Science 296, Ogura, A., 2004. Tissue-specific distribution of donor mitochondrial 2188–2190. DNA in cloned mice produced by somatic cell nuclear transfer. Genesis Shi, W., Zakhartchenko, V., Wolf, E., 2003. Epigenetic reprogramming in 39, 79–83. mammalian nuclear transfer. Differentiation 71, 91–113. Jeanisch, R., Eggan, K., Humpherys, D., Rideout, W., Hochedlinger, K., Shin, M.R., Park, S.W., Shim, H., Kim, N.H., 2002a. Nuclear and 2002. Nuclear cloning, stem cells, and genomic reprogramming. microtubule reorganization in nuclear-transferred bovine embryos. Cloning Stem Cells 4, 389–396. Mol. Reprod. Dev. 62, 74–82. Kass, L., 2004. We don’t play politics with science. Minn. Med. 87, 47–48. Shin, T., Kraemer, D., Pryor, J., Liu, L., Rugila, J., Howe, L., Buck, S., Kato, Y., Tani, T., Sotomaru, Y., Kurokawa, K., Kato, J., Doguchi, H., Murphy, K., Lyons, L., Westhusin, M., 2002b. A cat cloned by nuclear Yasue, H., Tsunoda, Y., 1998. Eight calves cloned from somatic cells of transplantation. Nature 415, 859. a single adult. Science 282, 2095–2098. Simerly, C.R., Navara, C.S., 2003. Nuclear transfer in the rhesus monkey: Lee, J., Miyano, T., Moor, R.M., 2000. Spindle formation and dynamics of opportunities and challenges. Cloning Stem Cells 5, 319–331. gamma-tubulin nuclear mitotic apparatus protein distribution during Simerly, C., Schatten, G., 1993. Techniques for localization of spe- meiosis in pig and mouse oocytes. Biol. Reprod. 62, 1184–1192. cific molecules in oocytes and embryos. Methods Enzymol. 225, Mann, M.R., Chung, Y.G., Nolen, L.D., Verona, R.I., Latham, K.E., 516–553. Bartolomei, M.S., 2003. Disruption of imprinted gene methylation and Simerly, C., Wu, G.J., Zoran, S., Ord, T., Rawlins, R., Jones, J., Navara, C., expression in cloned preimplantation stage mouse embryos. Biol. Gerrity, M., Rinehart, J., Binor, Z., 1995. The paternal inheritance of the Reprod. 69, 902–914. centrosome, the cell’s microtubule-organizing center, in humans, and Maro, B., Kubiak, J., Gueth, C., De Pennart, H., Houliston, E., Weber, M., the implications for infertility. Nat. Med. 1, 47–52. Antony, C., Aghion, J., 1990. Cytoskeleton organization during Simerly, C., Nowak, G., de Lanerolle, P., Schatten, G., 1998. Differential oogenesis, fertilization and preimplantation development of the mouse. expression and functions of cortical myosin IIA and IIB isotypes during Int. J. Dev. Biol. 34, 127–137. meiotic maturation, fertilization, and mitosis in mouse oocytes and Matsuda, J., Takahashi, S., Ohkoshi, K., Kaminaka, K., Kaminaka, S., embryos. Mol. Biol. Cell 9, 2509–2525. Nozaki, C., Maeda, H., Tokunaga, T., 2002. Production of transgenic Simerly, C., Dominko, T., Navara, C., Payne, C., Capuano, S., Gosman, G., chimera rabbit fetuses using somatic cell nuclear transfer. Cloning Stem Chong, K.Y., Takahashi, D., Chace, C., Compton, D., Hewitson, L., Cells 4, 9–19. Schatten, G., 2003. Molecular correlates of primate nuclear transfer Meng, L., Ely, J.J., Stouffer, R.L., Wolf, D.P., 1997. Rhesus monkeys failures. Science 300, 297. produced by nuclear transfer. Biol. Reprod. 57, 454–459. St John, J.C., Schatten, G., 2004. Paternal mitochondrial DNA transmission Mitalipov, S.M., Yeoman, R.R., Nusser, K.D., Wolf, D.P., 2002. Rhesus during nonhuman primate nuclear transfer. Genetics 167, 897–905. monkey embryos produced by nuclear transfer from embryonic Stearns, T., Evans, L., Kirschner, M., 1991. Gamma-tubulin is a highly blastomeres or somatic cells. Biol. Reprod. 66, 1367–1373. conserved component of the centrosome. Cell 65, 825–836. Mountain, V., Simerly, C., Howard, L., Ando, A., Schatten, G., Compton, Steinborn, R., Zakhartchenko, V., Jelyazkov, J., Klein, D., Wolf, E., Muller, D.A., 1999. The kinesin-related protein, HSET, opposes the activity M., Brem, G., 1998. Composition of parental mitochondrial DNA in of Eg5 and cross-links microtubules in the mammalian mitotic cloned bovine embryos. FEBS Lett. 426, 352–356. spindle. J. Cell Biol. 147, 351–366. Steinborn, R., Schinogl, P., Zakhartchenko, V., Achmann, R., Schernthaner, Munro, C.J., Laughlin, L.S., Illera, J.C., Dieter, J., Hendrickx, A.G., Lasley, W., Stojkovic, M., Wolf, E., Muller, M., Brem, G., 2000. Mitochondrial B.L., 1997. ELISA for the measurement of serum and urinary chorionic DNA heteroplasmy in cloned cattle produced by fetal and adult cell gonadotropin concentrations in the laboratory macaque. Am. J. cloning. Nat. Genet. 25, 255–257. Primatol. 41, 307–322. Suemori, H., Tada, T., Torii, R., Hosoi, Y., Kobayashi, K., Imahie, H., Navara, C.S., First, N.L., Schatten, G., 1994. Microtubule organization in Kondo, Y., Iritani, A., Nakatsuji, N., 2001. Establishment of embryonic the cow during fertilization, polyspermy, parthenogenesis, and nuclear stem cell lines from cynomologus monkey blastocysts produced by IVF transfer: the role of the sperm aster. Dev. Biol. 162, 29–40. or ICSI. Dev. Dyn. 222, 273–279. Onishi, A., Iwamoto, M., Akita, T., Mikawa, S., Takeda, K., Awata, T., Szollosi, D., Calarco, P., Donahue, R.P., 1972. Absence of centrioles in the Hanada, H., Perry, A.C., 2000. Pig cloning by microinjection of fetal first and second meiotic spindles of mouse oocytes. J. Cell Sci. 11, fibroblast nuclei. Science 289, 1188–1190. 521–541. Palacios, M.J., Joshi, H.C., Simerly, C., Schatten, G., 1993. Gamma-tubulin Tamada, H., Kikyo, N., 2004. Nuclear reprogramming in mamma- reorganization during mouse fertilization and early development. J. Cell lian somatic cell nuclear cloning. Cytogenet. Genome Res. 105, Sci. 104 (Pt 2), 383–389. 285–291. Polejaeva, I.A., Chen, S.H., Vaught, T.D., Page, R.L., Mullins, J., Ball, S., Tang, C.J., Hu, H.M., Tang, T.K., 2004. NuMA expression and function in Dai, Y., Boone, J., Walker, S., Ayares, D.L., Colman, A., Campbell, mouse oocytes and early embryos. J. Biomed. Sci. 11, 370–376. K.H., 2000. Cloned pigs produced by nuclear transfer from adult Thomson, J.A., Marshall, V.S., 1998. Primate embryonic stem cells. Curr. somatic cells. Nature 407, 86–90. Top. Dev. Biol. 38, 133–165. Rawe, V.Y., Payne, C., Navara, C., Schatten, G., 2004. WAVE 1 intra- Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, 252 C. Simerly et al. / Developmental Biology 276 (2004) 237–252

G., Marshall, V.S., Jones, J.M., 1998. Embryonic stem cell lines derived Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J., Campbell, K.H., 1997. from human blastocysts. Science 282, 1145–1147. Viable offspring derived from fetal and adult mammalian cells. Nature Vogel, G., 2001. Endangered species. Cloned gaur a short-lived success. 385, 810–813. Science 291, 409. Wolf, D.P., 2004. Assisted reproductive technologies in rhesus macaques. Wakayama, T., Perry, A.C., 2002. Cloning of mice. In: Cibelli, J.B., Lanza, Reprod. Biol. Endocrinol. 2, 37. R.P., Campbell, K.H., West, M.D. (Eds.), Principles of Cloning. Wolf, D.P., Kuo, H.C., Pau, K.Y., Lester, L., 2004. Progress with non- Academic Press, San Diego, pp. 301–341. human primate embryonic stem cells. Biol. Reprod. Wakayama, T., Perry, A.C., Zuccotti, M., Johnson, K.R., Yanagimachi, R., Woods, G.L., White, K.L., Vanderwall, D.K., Li, G.P., Aston, K.I., Bunch, 1998. Full-term development of mice from enucleated oocytes injected T.D., Meerdo, L.N., Pate, B.J., 2003. A mule cloned from fetal cells by with cumulus cell nuclei. Nature 394, 369–374. nuclear transfer. Science 301, 1063. Wells, D.N., Misica, P.M., Tervit, H.R., 1999. Production of cloned calves Wu, G.J., Simerly, C., Zoran, S.S., Funte, L.R., Schatten, G., 1996. following nuclear transfer with cultured adult mural granulosa cells. Microtubule and chromatin dynamics during fertilization and early Biol. Reprod. 60, 996–1005. development in rhesus monkeys, and regulation by intracellular calcium Willadsen, S.M., 1986. Nuclear transplantation in sheep embryos. Nature ions. Biol. Reprod. 55, 260–270. 320, 63–65. Zhou, Q., Renard, J.P., Le Friec, G., Brochard, V., Beaujean, N., Cherifi, Y., Wilmut, I., 2002. Are there any normal cloned mammals? Nat. Med. 8, Fraichard, A., Cozzi, J., 2003. Generation of fertile cloned rats by 215–216. regulating oocyte activation. Science 302, 1179.