Microtubule and mitotic spindle defects in clones

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The embryo formed during nuclear transfer must successfully complete many of the same functions during the first cell cycle that an embryo formed by normal fertilization must perform. While knowledge of the cytoplasmic events of the mammalian zygotic first cell cycle is far from complete, we do know a great deal about the necessary microtubule regulation during the first cell cycle of the fertilized zygote. Except for the mouse, all mammals studied41-46 adhere to the paternal inheritance of the dominant microtubule organizing center (MTOC). Briefly, the only microtubules present in the mature unfertilized oocyte are those found in the meiotic spindle. After insemination, microtubules are found in the cytoplasm as a small tuft adjacent to the incorporated sperm nucleus and nucleated from the sperm centrosome.47 As this microtubule aster develops, the microtubules contact the female pronucleus and are necessary for pronuclear apposition. By the time of mitosis, the sperm centrosome duplicates and splits to form the poles for the first mitotic spindle. As cytokinesis takes place, the asters fill the cytoplasm and become the interphase microtubule array with each pole of the mitotic spindle serving as the centrosome for the daughter blastomeres. The organization of the microtubules relative to the incorporated sperm plays a critical role in development as poor organization of these micro-tubules is related to poor developmental outcome, as shown in analysis of return to estrous after embryo transfer in the bovine.38 Few studies have been conducted to determine microtubule patterns and centrosome fate during nuclear transfer. Most of these zygotes have been examined after transfer of embryonic cells (ECNT) rather than somatic cell transfer (SCNT): bovine44 (ECNT),48 (SCNT); rabbit49'50 (ECNT); porcine51 (SCNT) and non-human primate52 (ECNT and SCNT),53 (SCNT).

In the case of bovine ECNT zygotes, an astral array of microtubules (Fig. 2A, green) is associated with the donor nucleus (Fig. 2A, blue) nucleated from the centrosome of the donor cell. Occasionally, multiple asters (Fig. 2B, green) form associated with the donor nucleus (Fig. 2B, blue). This latter may represent defects in microtubule formation or a donor cell with a replicated centrosome. These results serve as a reminder that nuclear transfer is a misnomer and that in the case of cloning utilizing cell fusion (the vast majority of non-murine cloning), all of the donor cell's organelles are transferred into the recipient oocyte. In the case of the centrosome, this organelle remains active in the zygote in domestic species. Similar results have been observed during bovine somatic cell nuclear transfer (Navara etal., unpublished results).48 At a similar time point during rabbit ECNT, Pinto-Correia et al. (1995) described no microtubule formation associated with the pronucleus (pronuclei) in the cytoplasm of the nuclear transfer zygote. In this same study, Pinto-Correia et al. (1995) examined the mitotic spindle structure of rabbit nuclear transfer embryos generated from embryonic

Figure 2. Laser-Scanning Confocal Microscopy of two zygotes after Nuclear Transfer. At 6.5 hours after activation, a large microtubule aster (A, green) is seen associated with the donor nucleus (A, blue). Two microtubule asters are sometimes associated with the donor nucleus. Fusion of the 32-cell stage donor blastomere with an enucleated oocyte activated with ionomycin and DMAP occurred at 1.5 hours after activation. Green = microtubules; blue = DNA; Bar= 10 ^m. Reprinted with permission from Navara etal., Dev Biol 162:29-40 (1994).

blastomeres. They reported the very striking result that 5/7 nuclear transplant zygotes had misaligned chromosomes on the metaphase plate and spindle errors. While only a small number of zygotes were examined, if this percentage holds true, this represents a potential for gross errors in development. Normal mitotic spindles are predominant during bovine cloning (Navara et al. unpublished results),48 but spindle abnormalities are commonplace during non-human primate somatic cell nuclear transfer.52'53 Ng et al. (2004) examined the spindle formed during premature chromatin condensation immediately after donor cell fusion. They found only 13.5% of these formed normal bipolar spindle structures in the meiotic cytoplasm. These authors did not examine the organization of mitotic spindles. In our experience,52'54 primate NT embryonic and somatic constructs do not differ from control embryos when imaged using Hoffman optics. However, when microtubule patterns are examined at interphase, primate NTs display either unfocused microtubules or multiple arrays emanating from several foci. At first mitosis, all 116 ECNTs and all 30 SCNTs examined by immuno-cytochemistry displayed abnormal spindle morphology and poorly aligned chromosomes.52 Despite these mitotic spindle defects, NT constructs cleave but unequal division and aneuploidy result.

Aberrant mitotic spindle assembly during nuclear transfer (Fig. 3A) suggests key mitotic proteins are absent entirely, not localized correctly or otherwise non-functional in the NT construct cytoplasm. NuMA (Nuclear-Mitotic Apparatus), a nuclear matrix protein also responsible for spindle pole assembly in somatic cells,55'56 is observed at meiotic (Fig. 3B) and mitotic spindle (Fig. 3C) poles in primate eggs and zygotes. The somatic donor cell nucleus contains NuMA (not shown) but it is not observed in enucleated oocytes nor is it typically detected at disorganized mitotic spindles after NT (Fig. 3D). The kinesins HSET and Eg5, oppositely directed mitotic microtubule motors, play key roles in proper assembly of the mitotic spindle. HSET, the human homologue of the Kar3 kinesin-like family of minus end directed motors, is found at the minus ends of the microtubules, while Eg5, a bimC kinesin-like protein with plus end directionality, is typically found at the spindle poles.57'58 HSET, which is observed at the spindle poles of meiotic and mitotic spindles in primates, is not detected in NT mitotic spindles (Fig. 3E). Conversely, Eg5 detects centromere pairs at meiosis and mitosis, and remains present on misaligned chromosomes on NT spindles (Fig. 3F). Collectively, these observations suggest that meiotic spindle removal either depletes the ooplasm of NuMA and HSET or results in their inaccurate positioning during mitotic spindle pole formation.

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Figure 3. Faulty Mitotic Spindles Produce Aneuploid Embryos After Primate Nuclear Transfer. A. Defective NT mitotic spindle with misaligned chromosomes. Centrosomal NuMA at meio-sis B. and mitosis C., but not NT-mitosis D. Centrosomal kinesin HSET is also missing after NT E., but not centromeric Eg5 F. Bipolar mitotic spindles with aligned chromosomes and centrosomal NuMA after NT into fertilized eggs G. Blue (DNA); red (^-tubulin); green (B, C, G: NuMA; D: HSET; F: Eg5). Bar= 10^m. Reprinted with permission from: Simerly etal., Science 300:297 (2003).

Figure 3. Faulty Mitotic Spindles Produce Aneuploid Embryos After Primate Nuclear Transfer. A. Defective NT mitotic spindle with misaligned chromosomes. Centrosomal NuMA at meio-sis B. and mitosis C., but not NT-mitosis D. Centrosomal kinesin HSET is also missing after NT E., but not centromeric Eg5 F. Bipolar mitotic spindles with aligned chromosomes and centrosomal NuMA after NT into fertilized eggs G. Blue (DNA); red (^-tubulin); green (B, C, G: NuMA; D: HSET; F: Eg5). Bar= 10^m. Reprinted with permission from: Simerly etal., Science 300:297 (2003).

To rule out the possibility that the invasive manipulations of enucleation were damaging the oocytes, the following experiment was performed. Oocytes were enucleated by needle aspiration and the autologous karyoplast carrying the meiotic spindle and maternal chromosomes was fused back into the oocyte = 95; 67.1% success; Simerly et al., 2003). Intracyto-plasmic sperm injection (ICSI) was then performed on the reconstituted oocyte to restore the normal diploid complement of DNA from both the sperm and the egg, along with the respective cytoplasmic components. These "FertClones" develop more successfully than either embryonic or somatic NTs, with one pregnancy established after 16 embryo transfers into eight surrogates. However, the pregnancy was "blighted" (an implantation attempt lacking fetal development). Perhaps not surprisingly, apop-totic rates were higher in these embryo constructs as compared with ICSI fertilized embryos.54

In a complementary series of experiments, the meiotic spindle was not removed and NT was performed concurrently with ICSI generating tetraploid constructs (55 oocytes; 54.4% success).52 These constructs, when cultured until first mitosis, organized aligned chromosomes on bipolar spindles with centrosomal NuMA (Fig. 3G). The NT mitotic spindles could be distinguished from the fertilized spindle by the presence of the sperm tail visible at one pole by DIC optics.

Taken together, normal bipolar spindles found in tetraploids suggest meiotic spindle removal as a primary source of NT anomalies at first mitosis. Since "FertClones" gave apparently normal divisions, the application of the enucleation step alone could not account for observed NT mitotic defects. Proper mitotic spindles can organize around somatic chromosomes when the meiotic spindle is not removed. However, current approaches using enucleation may remove vital mitotic spindle assembly components in primates.

It is not entirely clear why primate oocytes have a stricter requirement for recycled meiotic spindle proteins than other species. Experiments with cattle and mouse oocytes suggest that the mitotic kinesins and NuMA are not exclusively concentrated on the meiotic spindle in these species and thus may be available to more properly organize a mitotic spindle after enucleation (Simerly etal., 2004).59

Before considering the possibilities of "therapeutic cloning" as a potential source of tissue matched, ESCs the inefficiencies of NT must be overcome. These inefficiencies are observed across species and are manifested in failed or misguided reprogramming of somatic gene expression and in improper microtubule organization in the first cell cycle, resulting in aneuploidy and poor in vitro development. The extraordinary achievement of Hwang et al.37 encourages the endless possibilities "therapeutic cloning" might bring, but the vast experience of somatic cell cloning in other species details the numerous scientific problems remaining to be addressed.

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