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Publisher Summary This chapter discusses reproduction of centrosomes in vitro . The structural complexity of the animal centrosome, particularly the presence of the centriole pair as a conspicuous feature, suggests that a specific study of the animal centrosome is needed. An alternative approach to decipher the centrosome duplication pathway has been to use marine or amphibian eggs in which early development occurs without cell growth by segmentation, following each doubling of chromosomes and of the sperm-inherited centrosome. Xenopus egg extracts are the most characterized cell-free systems to observe not only centrosome assembly but also cell cycle-dependent regulation of this pathway. The chapter discusses a new strategy for developing a centrosome duplication assay in a Xenopus egg extract. The main feature of the assay is to use in situ centrosomes instead of isolated centrosomes. Triton X-100-extracted cytoplasts prepared from mouse L929 cells are used in this assay. This has several advantages: in the absence of nuclei, cytoplasts are very flat and are attached stably to the cover-slip on which the cells are grown. Their cytoplasmic compartments are distributed about the centrosome, which site at the geometric center of the cytoplast. Thus cytoplasts from early G1 cells provide a favorable starting material, as their centrosome does not initiate the structurally identifiable steps of duplication.

Telophase enucleation has been proven to be an efficient method for preparing recipient cytoplasts in bovine embryonic nuclear transfer (2, 11). This research was designed to study in vitro development of bovine oocytes containing transferred somatic cell nuclei, reconstructed by using enucleated in vitro-matured oocytes 32 h of age at telophase II stage as recipient cytoplasts, compared with those 24 h of age at metaphase II stage. Two protocols for donor cell injection were adopted, i.e., subzonal injection (SUZI) and intracytoplasmic injection (ICI). Bovine oviduct epithelial cells (BOECs) and bovine cumulus cells (BCCs) from an adult cow were used as nuclear donors for these experiments. In SUZI groups, the fusion rate of donor cells, both BOECs and BCCs, with MII enucleated oocytes were higher than those with TII enucleated oocytes (54% vs. 41% and 53% vs. 39%, respectively; P0.05), but the development rates to morula plus blastocyst stage in MII groups were lower than those in TII groups (22% vs. 39% and 21% vs. 41%, respectively; P0.05). In ICI groups, about 26% of enucleated MII oocytes injected with BOECs or BCCs cleaved and only small parts of them developed to blastocyst stage (4% and 3%, respectively; P0.05). When BOECs or BCCs were intracytoplasmically injected into oocytes enucleated at TII stage, no blastocyst was formed in either donor cell group and no cleavage occurred in BOEC group. Our data demonstrated that telophase enucleation is beneficial to early embryo development when bovine somatic nuclei are transferred by subzonal injection. However, it is harmful when donor cells are directly injected into the cytoplast of the enucleated oocytes.

Epithelial polarization and neuronal outgrowth require the assembly of microtubule arrays that are not associated with centrosomes. As these processes generally involve contact interactions mediated by cadherins, we investigated the potential role of cadherin signalling in the stabilization of non-centrosomal microtubules. Here we show that expression of cadherins in centrosome-free cytoplasts increases levels of microtubule polymer and changes the behaviour of microtubules from treadmilling to dynamic instability. This effect is not a result of cadherin expression per se but depends on the formation of cell–cell contacts. The effect of cell–cell contacts is mimicked by application of beads coated with stimulatory anti-cadherin antibody and is suppressed by overexpression of the cytoplasmic cadherin tail. We therefore propose that cadherins initiate a signalling pathway that alters microtubule organization by stabilizing microtubule ends.

The co-evolution of nuclear and mitochondrial genomes in vertebrates led to more than 100 specific interactions that are crucial for an optimized ATP generation. These interactions have been examined by introducing rat mtDNA into mouse cells devoid of mitochondrial DNA (mtDNA). When mtDNA-less cells derived from the common mouse (Mus musculus domesticus) were fused to cytoplasts prepared from Mus musculus, Mus spretus, or rat (Rattus norvegicus), a comparable number of respiring clones could be obtained. Mouse xenomitochondrial cybrids harboring rat mtDNA had a slower growth rate in medium containing galactose as the carbon source, suggesting a defect in oxidative phosphorylation. These clones respired approximately 50% less than the parental mouse cells or xenomitochondrial cybrids harboring Mus spretus mtDNA. The activities of respiratory complexes I and IV were approximately 50% lower, but mitochondrial protein synthesis was unaffected. The defects in complexes I and IV were associated with decreased steady-state levels of respective subunits suggesting problems in assembly. We also showed that the presence of 10% mouse mtDNA co-existing with rat mtDNA was sufficient to restore respiration to normal levels. Our results suggest that evolutionary distance alone is not a precise predictor of nuclear-mitochondrial interactions as previously suggested for primates.

An efficient method for freezing donor cells is necessary when using nucleus transfer of somatic cells for large-scale cloning. In the present study, we developed a method for freezing and thawing bovine cumulus cell-derived cultured cells to be used as nucleus donors. Cumulus cells were obtained from ovaries of living and slaughtered bovine and cultured in vitro. Cumulus cell-derived cultured cells were serum-starved for several days to induce a quiescent state and then frozen at -70 degrees C for at least 2 d. Immediately thereafter or 2 h after thawing, the cells were used as donor cells for nuclear transfer without additional in vitro culture. The fusion rate with recipient cytoplasts was not affected by the cumulus cell source (slaughtered or living) or time after thawing (0 and 2 h). The cleavage rate of frozen-thawed cumulus cell-derived cultured cells from slaughtered cows immediately after thawing (0 h) was highest (97%) and was significantly higher than that of controls (85%) or cells transferred 2 h after thawing (85%). There were no significant differences among any of the groups in the potential of the nuclear transfer embryos to develop into blastocysts (34 vs 44 and 44%, 39 vs 45 and 46%). Thus, storage of bovine cumulus cell-derived cultured cells in the quiescent state at -70 degrees C is effective and might be useful and convenient for large-scale cloning. The maximum storage periods and developmental potential of embryos after such nucleus transfers requires further examination.

Most current studies of nuclear transfer in mammalian oocytes have used electrofusion to incorporate donor cell nuclei into enucleated oocyte cytoplasts. However, the application of electrofusion to human oocytes is hampered by the relative ease with which this procedure induces oocyte activation. Here we tested a previously described chemical fusion technique and an original mechanical fusion procedure in this application. Enucleated metaphase II oocytes were first agglutinated with karyoplasts originating from other metaphase II oocytes and then induced to fuse with the use of polyethylene glycol or by micromanipulation with an intracytoplasmic sperm injection (ICSI) micropipette. Both techniques yielded a high frequency of fusion and did not cause oocyte activation. Moreover, the reconstructed oocytes were easily activated by subsequent treatment with ionophore A23187 and 6-dimethylaminopurine. These techniques may be used in attempts to alleviate female infertility due to insufficiency of ooplasmic factors by nuclear transfer from patients' oocytes to enucleated donor oocyte cytoplasts. For eventual future use in human cloning, they would ensure prolonged exposure of transferred nuclei to metaphase promoting factor, which appears to be required for optimal nuclear reprogramming.

Tripronuclear human zygotes were used to determine the feasibility of an enucleation procedure without the use of cytoskeleton inhibitors to obtain intact cytoplasts for nuclear transfer research. Of 61 tripronuclear human zygotes manipulated, 100% of the zygotes survived after removal of one pronucleus and 90.1% after complete enucleation, proving the reliability of the proposed microsurgical technique. Morphological changes were observed in the resulting cytoplasts during extended culture. At 36 h post-insemination, 87.5% of cultured cytoplasts showed morphological changes, which included cleavage into two 'blastomeres', three 'blastomeres' with various degrees of fragmentation, one cell with fragments or gross fragmentation. Comparison with the diploid human zygotes from the same harvests, which had undergone the first cleavage division by this time, showed similar timing in cleavage suggesting autonomous cytoplasmic activity in human zygotes.

We investigated the possibility of producing calves from transferable bovine embryos obtained by nuclear transfer using somatic cell-derived cell lines. Muscle cells obtained from 2 Japanese Black bulls were dispersed in Hank's solution supplemented with collagenase Type-I. The separated muscle cells were cultured in Dulbecco's Modified Eagle's medium (D-MEM) supplemented with 10% fetal bovine serum (FBS) at 39 degrees C in an atmosphere of 5% CO2 in air. Cells were passaged at least 4 times, and for 5 d prior to nuclear transfer they (donor cells: karyoplasts) were cultured in D-MEM supplemented with 0.5% FBS (to induce quiescence) or 10% FBS. Recipient oocytes were produced by in vitro culture of bovine oocytes that were obtained at a slaughterhouse and then enucleated in modified phosphate buffered saline supplemented with cytochalasin B. Embryos were reconstructed by 3 protocols using karyoplasts cultured in the medium with 0.5% FBS. 1) Group A: recipient oocytes (cytoplasts; n = 157) were treated with Ca ionophore A 23187, ethanol and cycloheximide, and then a karyoplast was fused to an activated cytoplast. 2) Group B: karyoplasts were transferred to cytoplasts (n = 117), and the couplets were treated with electric stimulation and then Ca ionophore A 23187 and cycloheximide. 3) Group C: cytoplasts (n = 104) were cultured for a further 12 h before fusion, and then the couplets were treated with electric stimulation and cycloheximide. 4) Group D: in addition to the above 3 groups, karyoplasts cultured in the medium with 10% FBS were transferred to recipient cytoplasts (n = 137) and treated as in Protocol 2. Reconstructed embryos were cultured in modified CR1aa for 8 d, and the development of embryos was assessed. In total 73 blastocysts were obtained, and the frequency of development to the blastocyst stage in Group A (2.5%) was lower than that of Groups B, C and D (20.5, 18.3 and 19.0%, respectively; P < 0.01). Of these the sex of 21 blastocysts was determined by rapid Y-chromosome detection assay, and all were male, suggesting that nuclear replacement had been achieved successfully. When 26 blastocysts were transferred to 20 recipient cows, 8 of them became pregnant; 4 cows subsequently aborted about 60 d after embryo transfer while the remaining 4 cows calved. These results indicate that reconstructed embryos obtained by nuclear transfer of muscle cell-derived cell lines can develop to the blastocyst stage, and some are sufficiently competent to develop to term. Particularly important was the finding that special culture protocols for somatic cells prior to nuclear transfer were not necessary in our system.

To improve the enucleation rate in newly matured bovine oocytes, we investigated the position of cytoplasmic chromatin in relation to the polar body and the consequent enucleation efficiency before and after sequential activation with calcium ionophore A23 187 and cycloheximide. Oocytes aspirated from the follicles of slaughterhouse-collected ovaries were cultured for 18 to 20 h. With Hoechst staining, only 40.7% of the chromatin material was found adjacent to the first polar body in metaphase II oocytes, while 100% was located adjacent to the second polar body in oocytes after the activation. Enucleation trials after activation showed a higher enucleation rate (91.5%) than that before activation (59.9%). The following experiment determined the effect of using both kinds of cytoplast on the in vitro development of nuclear transfer embryos. Blastomeres of the 32-cell-stage in vitro-produced embryos were transferred, fused to the activated cytoplasts and cultured in vitro. No significant difference was detected in fusion, cleavage or development to blastocysts obtained 7 d (174 h) post fusion. In conclusion, this study showed that young in vitro-matured bovine oocytes sequentially activated with calcium ionophore and cycloheximide have cytoplasmic chromatin material adjacent to the second polar body, leading to a high enucleation rate.

The duration of M-phase is largely determined by the time necessary for the formation of a functional metaphase spindle and the correct alignment of all chromosomes on the metaphase plate. The spindle assembly checkpoint prevents the exit from M-phase before the proper alignment of all chromosomes on a metaphase plate in many cell types. In the present paper we show that the first mitotic M-phase of the mouse embryo lasts about 119 min, while the second embryonic M-phase lasts only about 70 min. Histone H1 kinase is activated rapidly during nuclear envelope breakdown in both mitoses. Its maximum, however, is followed by a plateau only during the first mitosis. In the second mitosis, the inactivation of histone H1 kinase activity follows its maximum directly. Histone H1 kinase is more stable in the cytoplasts obtained from mouse embryos during the first embryonic M-phase than during the second one. The stability of histone H1 kinase is greatly increased by the presence of the mitotic apparatus in both M-phases. The mitotic spindle assembly during the first and the second mitoses differs and the first metaphase spindle is stabilised during the period of maximum histone H1 kinase activity. These data show that an unknown developmentally regulated mechanism controls the duration of the two first mitoses in the mouse embryo.

Factors affecting the efficiency of animal cloning remain to be elucidated1,2. Enucleation of recipient oocytes is a critical step in cloning procedures and typically is performed by aspirating a portion of the cytoplasm underlying the first polar body. Enucleation is evaluated using epifluorescence after Hoechst staining for DNA3,4,5,6, which may disrupt functions of the cytoplast, especially mitochondria7. Mitochondrial DNA in Dolly and other cloned sheep has been shown to derive exclusively from recipient oocytes8. Not only might evaluation of the aspirated karyoplast portion5 inadequately reflect the state of the cytoplast, it is also time consuming. Here we report a reliable, noninvasive technique for spindle imaging and enucleation of oocytes using a new microscope, the Pol-Scope9. The efficiency of enucleation was 100%, and only 5.5% of the oocytes' mitochondria entered the karyoplast upon Pol-Scope-directed removal of the spindle. Moreover, Pol-Scope imaging of spindles and micromanipulation did not compromise the developmental competence of reconstituted oocytes and cytoplasts.

Recent results in animal cloning have demonstrated that the events of cell differentiation can be reversed. This reversal necessarily requires genetic reprogramming of the transplanted nucleus. It has emerged from review articles that synthesis of nuclear and ribosomal RNA is blocked after fusion independently of cell cycle stage of the transferred nucleus, and that the cytoplasm condition of the cytoplast controls the speed of this inhibition. Transcription of the embryonic genome in nuclear transplant embryos begins either at the same stage as in normal development or even earlier. Expression analysis of individual genes showed up differences between nuclear transplant and normal embryos at the blastocyst stage. A certain degree of nuclear reprogramming after fusion is necessary, but exact timing and the qualitative sequence of the processes are probably not necessary for the successful embryonic development of the cloned embryo. In the future, observation of the expression of individual genes will bring more exact information about nucleus reprogramming.

Apoptosis may be defined as a regulated lethal process in which the cell activates catabolic processes which, within the limits of a near-to-intact plasma membrane, lead to a stereotyped ensemble of biochemical and morphological alterations. Such alterations include a reduction in cell size, a condensation of chromatin, and changes in the physicochemistry of the plasma membrane facilitating the recognition and heterophagic removal of the apoptotic cell by adjacent normal cells. The most striking morphological change in apoptotic cells concerns the nucleus which invariably exhibits chromatin condensation, mostly associated with enzymatic degradation of nuclear DNA. However, chromatin condensation is a sign of apoptosis rather than a mechanism leading to cell death, since non-nuclear apoptosis-associated alterations can be induced in anucleate cells (cytoplasts), as this has been shown in 1994 (Jacobson et al. 1994, Schulze-Osthoff et al. 1994).

Cytokineplasts (CKP) are granule-poor cytoplasts from human polymorphonuclear leukocytes (PMN) that retain motile function, even (unlike the parent PMN) after cryopreservation. Employing time-lapse videomicroscopy, we examined the chemotactic properties of CKP after cryopreservation toward erythrocytes lysed by laser microirradiation. Paths of locomotion were plotted for six CKP in the field, and velocities were calculated at 10-sec intervals. Mean velocities of the six fragments, ranging from 9.3 to 20.8 μm/min, are of the order of fresh, intact PMN, the fastest of locomoting cells.

Human neutrophils incubated for 1 h in vitro with 10% commercial pooled, human serum containing high levels of free fatty acids (1141 microM) displayed a distinct lipid signal, typical of triacylglycerol, in the 1H NMR spectrum. Concurrently their plasma membrane triacylglycerol mass increased 4.6-fold with a selective rise in the content of palmitic and linoleic acids. Although qualitatively similar, these effects were much greater than those observed after incubating neutrophils with 50 microg.mL-1 of lipopolysaccharide in the presence of 10% AB serum with normal free fatty acid content (345 microM, LPS/S). Incubation of neutrophils with an artificial mixture of free fatty acids at concentrations found in commercial serum, or with the fatty acid fraction isolated from commercial serum increased the 1H NMR-detectable triacylglycerol. The signal intensity of the 1H NMR-detectable triacylglycerol depended on the triacylglycerol composition, and correlated with increased membrane triacylglycerol mass. Cellular uptake of 3H-labelled palmitic or oleic acids increased in the presence of commercial serum but not with LPS/S, with little contribution in either case to the triacylglycerol pool that increased in mass. Pulse-chase experiments demonstrated that with LPS/S and commercial serum, radiolabelled palmitic acid was preferentially incorporated into triacylglycerol located in the plasma membrane. This process could occur at the plasma membrane, as cytoplasts efficiently convert exogenous fatty acids into triacylglycerol. We propose that LPS/S and serum containing high levels of free fatty acid, important in conditions of sepsis and inflammation, may facilitate the sequestration of palmitic acid into triacylglycerol by different pathways. This triacylglycerol originates from exogenous and endogenous free fatty acids, is 1H NMR-visible, and may have a role in regulating apoptosis.

CLONE 'a group of two or more individuals with identical genetic makeup derived, by asexual reproduction, from a single common parent or ancestor' (The Chambers Dictionary 1993, Chambers Harrap). The term clone was originally applied to plants but has subsequently been used in a much broader context to include a person or thing closely similar to another, a copy or replica. In animals, true clones, as defined above, may be produced by embryo splitting or blastomere separation either artificially, or as occurs naturally in the production of identical twins. In these individuals all of the components making up the individual, including nuclear genetic material (the genome) and other maternally derived factors are derived from a single unique embryo which is the result of sexual reproduction. The term clone has been applied to animals produced by the technique of nuclear transfer. In this asexual process, nuclear genetic material is transferred from a donor cell (karyoplast) into a recipient cell (cytoplast) from which the genetic material has been removed. In farm animals the cytoplast of choice is the matured oocyte (or unfertilised egg) thus the animals developing from this technique are not true clones as each cytoplast is often derived from a different animal. The resultant animals may therefore be more aptly described as 'genomic copies'. In mammals, successful development of embryos reconstructed by nuclear transfer was originally restricted to using early embryos as nuclear donors, however, recent progress has demonstrated successful development using nuclei from embryonic, foetal and adult derived cell populations. Numerous factors affect the development of embryos reconstructed by nuclear transfer including; the cell cycle stage of the recipient cell, the cell cycle stage of the donor nucleus, the differentiated state of the donor nucleus, activation of the recipient cell, the culture method. In addition, there are variations in success between species, these may be related to differences in organisation of the cytoskeleton and/or the meiotic spindle in the recipient cell,differences in cell cycle control during early development, the onset of zygotic transcription or differences in the metabolic requirements of early embryos in vitro. The aim of this article is to describe and discuss some of these factors in relation to the successful development of nuclear transfer reconstructed embryos and in particular to the 'reprogramming' or 'remodeling' of the donor genetic material to attain successful development.

Cytoplasts from single spermatocytes of NZB/BinJ mice were separated from the nuclei and individually microinjected into B6D2F1 (C57BL/6 × DNBA/2J) hybrid embryos at the pronuclear stage (20 h after hCG injection). Of 363 zygotes injected, 311 (86%) survived and developed. From these experiments, we transferred 222 embryos into 20 pseudopregnant recipients. Eighteen (90%) became pregnant and 82 pups were born (37% of transfers). Mitochondrial DNA (mt DNA) from the NZB/BinJ strain lacks a RsaI restriction site and can thus be distinguished from the host embryo following PCR amplification. We were unable to detect the transferred mtDNA in blastocysts on day 4–5 after injection. Nor could we detect NZB/BinJ mtDNA in placentae, nor in tissues from mice born to host mothers following the transfer of blastocysts that developed from injected zygotes. Rejection of paternal mitochondria by the embryo normally occurs at the 4- to 8-cell stage in mice and is apparently dependent on mutual recognition between the mitochondria and the nuclear genome. We conclude that this mechanism has probably already developed by the time the germ cells have become committed to meiosis.

Nuclear transfer experiments in mammals have attempted to reprogram a donor nucleus to a state equivalent to the zygotic one. Reprogramming of the donor nucleus is, among other features, indicated by a synthesis of ribosomal RNA (rRNA). The initiation of rRNA synthesis is simultaneously reflected in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout all three cell cycles. In the eight-cell stage embryo, a primary vacuole appeared as an electron lucid area originating in the centre of the nucleolar precursor body. In nuclear transfer embryos reconstructed from nonactivated cytoplasts, the nuclear envelope was fragmented or completely broken down at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary vacuoles. A nucleolar precursor body typical for the two-cell stage control embryos was never observed. None of the reconstructed embryos of this group reached the eight-cell stage. Nuclear transfer embryos reconstructed from activated cytoplasts, in contrast, exhibited a complete nuclear envelope at all time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear transfer embryos, which was one cell cycle earlier than in control embryos. Only nuclear transfer embryos reconstructed from activated cytoplasts underwent complete remodelling of the nucleolus. The reorganisation of the donor nucleolar architecture into a functionally active nucleolus was observed as early as in the four-cell stage nuclear transfer embryo. These ultrastructural observations were correlated with our autoradiographic data on the initiation of RNA synthesis in nuclear transfer embryos. Mol. Reprod. Dev. 52:253–263, 1999. © 1999 Wiley-Liss, Inc.

Fully grown mouse oocytes isolated from large antral follicles and cultured in vitro complete their maturation up to the second metaphase with extrusion of the first polar body (1PB) with a 40/50 proportion (80%). When their cytoplasmic volume is, however, reduced before the onset of culture, the frequency of oocytes completing maturation gradually decreases. In the half oocytes, 66% (33/50) extruded 1PB, while in third oocytes the proportion was 57% (28/49) and in quarter oocytes no polar bodies were extruded. The time course of germinal vesicle breakdown was also delayed in comparison to the decreased cytoplasmic volume. Moreover, the isolated germinal vesicles surrounded with a thin cytoplasmic rim only remained intact after a prolonged culture. The full competence of complete maturation can be restored by fusion of an additional cytoplast to the manipulated nucleate parts. We postulate that a critical nucleocytoplasmic volume ratio is absolutely necessary for normal maturation in mammalian oocytes.

During apoptosis, DNA fragmentation and intracellular acidification occur concurrently. Previous results have shown that intracellular acidification is not required for DNA fragmentation, while the alternative, that acidification is a consequence of DNA fragmentation was analyzed here. To obviate the requirement of any nuclear function in acidification, apoptosis was induced by staurosporine in cytoplasts made from the breast tumor cell line MDA-MB-468. Both cells and cytoplasts demonstrated externalization of phosphatidylserine that was prevented by the pan-caspase inhibitor zVAD-fluoromethylketone or by expression of Bcl-2. Intracellular acidification was observed in both cells and cytoplasts and this was also inhibited by both zVAD-fluoromethylketone and Bcl-2. These results show that intracellular acidification and DNA fragmentation are independent consequences of caspase action during apoptosis.

Cryopreservation of cytoplasts would help to resolve the logistics of matching the availability of oocytes with embryo donors in nuclear transfer. Therefore, the developmental potential of nuclear transfer bovine embryos reconstructed using vitrified cytoplasts was investigated. In vitro matured oocytes were denuded, enucleated, activated with calcium ionophore (10 microM, 5 min) and cycloheximide (10 microg/mL, 6 h) and then vitrified by the open pulled straw (OPS) method. After immediate warming, the nuclear transfer embryos were reconstructed using blastomeres from nonvitrified,in vitro-produced embryo donors. Compared with control nuclear transfer embryos that were reconstructed using nonvitrified cytoplasts, fusion rates (% +/- SEM) were not affected (83.7+/-9.2 vs. 79.8+/-4.6; P>0.05), but cleavage (55.7+/-2.9 vs. 92.8+/-3.9; P = 0.0002) and blastocyst rates (7.2+/-5.0 vs. 32.6+/-7.8; P = 0.0025, vitrified vs. nonvitrified cytoplasts, respectively) per successful fusion were reduced. One nuclear transfer blastocyst reconstructed from a vitrified cytoplast was transferred to a synchronized recipient. After a normal length gestation (265 d), twin calves (21 and 26 kg) were delivered. Microsatellite analysis confirmed that the calves were homozygotic (the embryo split in utero), and were derived from the in vitro-produced embryo donor. The twins were dead at birth, but post-mortem analysis of the calves indicated no abnormalities or infections, suggesting that their death was related to the twin pregnancy and the known fragility of nuclear transfer calves. These data demonstrate that open pulled straw-vitrified cytoplasts are capable of supporting full-term development of nuclear transfer embryos.

We have investigated the transmission of parental mitochondrial DNA (mtDNA) in three clones of born cattle obtained by intraspecific cytoplast-blastomere fusion. Using allele-specific TaqMan PCR a low level transmission of blastomere mtDNA (DB mtDNA) into the cloned offspring was detected, thereby generating a heteroplasmic population of mtDNA. The amount of DB mtDNA was 13% and 18% in two animals of a clone which derived from a 24-cell morula and 0.6% and 0.4% in two calves of clonal origin derived from a 92-cell morula. These values are in accordance with the tendency expected for neutral mtDNA segregation that the fewer cell divisions that have occurred in the donor embryo, the higher the amount of DB mtDNA. We also found a strong decrease of DB mtDNA which was about three orders of magnitude in the third clone derived from a 52-cell morula stage.

The effects of different concentrations of etoposide and cycloheximide (ETO-CHXM), used for chemical enucleation of mouse oocytes, on polar body extrusion and chromatin expulsion were tested. The developmental ability of blastomeres of late 2-cell stage embryos fused to chemically enucleated oocytes of different ages or cytoplasts from different sources was also examined in vitro. Metaphase I oocytes cultured in different concentrations of ETO-CHXM (10-50 micrograms/ml/each) extruded polar bodies at rates similar to those cultured without ETO-CHXM (58.5-65.9% and 64.6%, respectively). However, low percent of the oocytes (1.7-6.2%) expressed signs of meiotic perturbation, which was manifested by blebbing of the cytoplasmic membrane and extrusion of two or more polar body-like fragments. Twenty-three percent of the chemically enucleated oocytes cultured in ETO-CHXM-free medium spontaneously fused to their polar bodies. The rates of total chromatin expulsion were similar when ETO-CHXM concentrations were 36 and 50 micrograms/ml (93.5 and 98%, respectively). The results also showed that the cleavage rates of reconstituted embryos were significantly (P0.001) affected by the age of the chemically enucleated oocytes. Cytoplasts of bisected oocytes that matured in vivo supported the development of 31.7% of the reconstituted embryos to the blastocyst stage. However, both cytoplasts of chemically enucleated oocytes and in vitro matured oocytes did not support the development to the blastocyst stage. A high percentage (85.5%) of the reconstituted embryos with chemically enucleated recipients displayed abnormality of the metaphase plate. These results suggest that concentrations of etoposide between 36 and 50 micrograms/ml are optimum for enucleation of mouse oocytes. Furthermore, increasing the age or reducing the cytoplasmic volume of the chemically enucleated oocytes did not improve the development of the reconstituted embryos to the blastocyst stage.

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulusoocyte complexes (COCs) were denuded before or after maturationin vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high-possibly due to nuclear repair mechanisms. Protein proles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specic proteins are synthesized in the nuclear region.

Publisher Summary This chapter provides a brief review of the expanding field of in vitro apoptosis studies. First, the chapter describes the cell-free systems reported thus far and provides some comments on the unique attributes of each system. Second, tentative criteria for validation of new apoptotic systems are proposed. These are based on the current understanding of the morphological and biochemical processes of apoptotic cell death. Finally, the methodologies that have been used for analyzing the systems are described together with a number of the more significant findings achieved from such analyses. The advantage of cell-free systems in the study of apoptosis is that they can overcome the stochastic nature of the onset of apoptotic events in cell populations. Several cell-free systems have been devised that recapitulate the morphological changes of apoptosis using components isolated from cells such as nuclei, cytoplasm, and organelles. These cell-free systems offer several advantages in the biochemical analysis of cell death pathways. The nuclei used as substrates for in vitro apoptosis reactions are typically isolated from healthy cells. Thus, the apoptosis-inducing signal comes from the extracts rather than the nuclei. These extracts seem to show no species or tissue specificity for substrate nuclei. The extracts can induce apoptotic changes in nuclei from a wide variety of sources such as human HeLa cervical carcinoma cells, GM3798 lymphoblastoid cells, Jurkat T cells, CEM T lymphoblastoid cells, mouse S49 cells, mouse liver, hamster liver, rat liver, rat thymocytes, and Xenopus sperm nuclei assembled in vitro. This apparently passive role of nuclei in the execution of apoptosis is consistent with experiments indicating that apoptotic changes can proceed even in cytoplasts, which lack nuclei.

Inhibitors of nitric oxide (NO) synthase are reported to inhibit both the adherence of polymorphonuclear leukocytes (PMN) to substrate and chemotaxis (directed locomotion) of PMN as determined in Boyden chamber assays. In the current study, we examined both human blood PMN and granule-poor motile cytoplasts derived from them (cytokineplasts, CKP), under direct microscopic observation with concomitant time-lapse video recording, for their ability to respond chemotactically to an erythrocyte destroyed by laser microirradiation. In this system we can observe directly and continuously the orientation and trajectory of PMN before, during, and after establishment of a chemotactic gradient. For both PMN and CKP we employed three different inhibitors of NO synthase (Nω-methyl-l-arginine, N-iminoethyl-l-ornithine, and diphenyleneiodonium) in at least twice the concentrations employed to inhibit chemotaxis of PMN in Boyden chambers or killing of bacteria in CKP. Although small differences in adhesion might not have been appreciated, treated PMN and CKP were each indistinguishable from untreated controls in their ability to orient in a newly created chemotactic gradient and in their trajectories toward the chemotactic target.

We summarize here the procedures for nuclear transfer using S-phase cytoplasts and describe a new method for avoiding loss of reconstructed embryos from the oviducts during in vivo culture. We obtained 2 clones of 5 genetically identical animals following the transfer of blastomeres from 16-cell embryos into enucleated preactivated cytoplasts. Metaphase II oocytes and embryos were surgically collected from superovulated Sarda breed ewes 54 and 120 h after sponge removal, respectively. Oocytes were exposed for 15 min to 5 mug/ml of Hoechst 33342 and were micromanipulated at room temperature. Efficiency in embryo reconstruction was 100% for enucleation and 98% for fusion. Embryos were embedded in agar as separate clones and transferred into the oviducts of temporary recipients. The fimbriae were closed with glass-nylon made filters. Embryo recovery from the temporary recipients was 97.3%, with a cleavage rate of 81.4%; development to morula-blastocyst stage was 70.6%. A total of 29 Grade 1 blastocysts corresponding to 5 clones were transferred into 13 naturally synchronous ewes, and scanning was performed at 30 and 90 d. Ten ewes were pregnant at the first scanning and nine at the second for a final pregnancy rate of 71.4%; the survival rate at term was 48%. Overall, we obtained 4 clones of identical lambs: two sets of 5 (one male set and one female set) and two sets of twins (both sets male). Pregnancy length in recipients carrying clones was longer than the standard period in Sarda breed (153 vs 150 d, respectively). Weight at birth was higher for male lambs obtained from nuclear transfer than for normal males (4.1 vs 3.6 kg), while the weight for females was normal.

The study explored a novel approach for preserving the maternal genome without the entire oocyte by air-drying the cat germinal vesicle (GV) in the presence of the disaccharide trehalose. Specifically, we examined GV structure and function after desiccation, storage at 4°C (up to 32 wk), and rehydration including the ability to resume meiosis after injection into a fresh, conspecific cytoplast. In experiment 1, DNA integrity was similar to fresh controls after 1 and 4 wk storage in the presence of trehalose, but was more fragmented at later time points (especially after 32 wk). Nuclear envelope integrity was sustained in >90% of oocytes stored for 0, 4, or 16 wk regardless of protective treatment. In experiment 2, compacted, air-dried GVs were stored for 2 or 4 wk, rehydrated, and injected into fresh cytoplasts. After culture for 24 h in vitro, up to 73% of oocytes reconstructed with desiccated GVs preserved in trehalose resumed meiosis compared to 30% of those dried in the absence of the disaccharide. At each storage time point, trehalose presence during air-drying was advantageous for resumption of meiosis, with >20% of oocytes completing nuclear maturation to metaphase II. This demonstrates a potential for preserving the female genome using the GV alone and for multiple weeks after desiccation. Trehalose enhanced the process by retaining the ability of a dried and rehydrated GV to resume communication with the surrounding cytoplasm of the recipient oocyte to permit reaching metaphase II and likely sustain subsequent embryo development.

CPT-11, a derivative of camptothecin, induced apoptotic cell death characterized by chromosomal DNA fragmentation in human hepatoma PLC cellsin vitro.Cell viability of PLC cells was significantly decreased by CPT-11 in a dose-dependent manner, and CPT-11-induced cytotoxic activity was completely prevented by an interleukin-1β converting enzyme-like protease inhibitor YVAD and a protein synthesis inhibitor cycloheximide. To examine the roles of the cytoplasm and the nucleus in CPT-11-induced apoptosis, we prepared cytoplasts from PLC cells and nuclei from rat hepatocytes (RHN). Apoptotic cell death as characterized by cell fragmentation was observed in whole PLC cells but not in PLC cytoplasts after incubation with CPT-11. In addition, CPT-11 could not induce chromosomal DNA fragmentation in RHN. In contrast, the extracts of CPT-11-treated PLC cells, which mainly contained cytosol proteins, induced chromosomal DNA fragmentation of RHN in a dose-dependent manner. These results indicate that CPT-11-induced apoptosis required the presence of both the cytoplasm and the nucleus and suggest that the apoptosis required thede novosynthesis of an apoptosis initiator.

Proton pump activity is not measurable in the plasma membrane of unstimulated neutrophils but becomes readily detectable upon activation by soluble agonists. The mechanism of pump activation was investigated in this report. V-type H+ pump activity, estimated as a bafilomycin A1-sensitive elevation of the cytosolic pH, was stimulated in suspended neutrophils by chemotactic peptides and by phorbol esters. Stimulation of pump activity induced by the agonists was greatly enhanced by cytochalasin B, an agent known to potentiate granular secretion in neutrophils. We therefore compared the rate and extent of pump activation with the pattern of exocytosis of the four types of secretory organelles present in neutrophils, using flow cytometry and enzyme-linked immunosorbent assay. The kinetics of exocytosis of secretory vesicles and secondary and tertiary granules but not primary granules paralleled the appearance of pump activity. The subcellular localization of the pump was defined by cellular fractionation and immunoblotting using an antibody to the C subunit of the V-type ATPase. The pump was abundant in tertiary granules, with significant amounts present also in primary granules and secretory vesicles. The pump was scarce in secondary granules and not detectable in the cytosol. Finally, the agonists failed to stimulate pump activity in neutrophil cytoplasts, which are intact cell fragments devoid of acidic granules. Together, our results suggest that the V-type H+-ATPase is not constitutively present in the plasma membrane of neutrophils but is delivered to the surface membrane by exocytosis during cellular activation. Tertiary granules and secretory vesicles are the most likely source of V-ATPases. Following insertion in the plasma membrane, the pump is poised to effectively extrude the excess metabolic acid that is generated during chemotaxis and bacterial killing.

To investigate the role that aging-dependent accumulation of mitochondrial DNA (mtDNA) mutations plays in the senescence processes, mitochondria from fibroblasts of 21 normal human individuals between 20 weeks (fetal) and 103 years of age were introduced into human mtDNA-less (rhoo) 206 cells by cytoplast x rhoo cell fusion, and 7-31 transformant clones were isolated from each fusion. A slight cell donor age-dependent decrease in growth rate was detected in the transformants. Using an O2 consumption rate of 1 fmol/min/cell, which was not observed in any transformant among 158 derived from individuals 20 weeks (fetal) to 37 years of age, as a cut-off to identify respiratory-deficient clones, 11 such clones were found among 198 transformants derived from individuals 39-103 years of age. Furthermore, conventional and nonparametric analysis of the respiratory rates of 356 clones revealed a very significant decrease with donor age. In other analyses, a very significant age-dependent decline in the mtDNA content of the clones was observed, without, however, any significant correlation with the decrease in O2 consumption rate in the defective transformants. These observations clearly indicate the occurrence in the fibroblast-derived transformants of two independent, age-related functional alterations of mtDNA, presumably resulting from structural damage to this genome.

Nucleus transfer for the production of multiple embryos derived from a donor embryo relies upon the reprogramming of the donor nucleus so that it behaves similar to a zygotic nucleus. One indication of nucleus reprogramming is the RNA synthetic activity. In normal bovine embryogenesis, the embryo relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time it begins to transcribe its own RNA. In this experiment, RNA synthesis was detected in nucleus transfer embryos (NTE) and control embryos by pulsing with 3H-uridine, fixation, and autoradiography on semithin sections. NTE were produced using either a MII phase (nonactivated) cytoplasts at 32 hr of maturation or S-phase (activated) cytoplasts activated with calcium ionophore A23187 and cycloheximide treatment ∼8 hr prior to fusion with a blastomere from an in-vitro-produced morula stage embryo at 32 hr of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell stages, whereas in all late 8-cell stages, it was present. In NTE from nonactivated (MII phase) cytoplasts, there was a sharp decline in RNA synthesis at 1 hr and 3 hr after fusion and a total absence by 20 hr after fusion. In contrast, NTE from activated (S phase) cytoplasts exhibited continued high levels of RNA synthesis at 1 hr and moderate levels at 3 hr after fusion, although it had ceased by 20 hr after fusion. In all NTE (activated and nonactivated cytoplasts), there was no RNA synthesis seen at the 2-cell stage. However, at the 4-cell stage, weak RNA synthesis was seen in all NTE from activated cytoplasts, whereas none was observed in those from MII nonactivated cytoplasts. At the 8-cell stage, nearly all NTE from S-phase cytoplasts showed weak to moderate levels of RNA synthesis. We conclude that the nucleus reprogramming differs between NTE reconstructed from activated and nonactivated cytoplast with the former undergoing a slower cessation of RNA synthesis after fusion and earlier resumption of RNA synthesis, occurring as early as the 4-cell stage. © 1996 Wiley-Liss, Inc.

When in vitro-matured oocytes were enucleated, aged and kept at 10°C before reconstitution, the in vitro development of nuclear transfer embryos to the blastocyst stage did not differ from that obtained with in vitro fertilization. This suggests that these recipient cytoplasts constitute a suitable environment for the development of the nuclear transplant. The aim of the present study was to investigate, at the biochemical level, the result of the preparation of recipient oocytes, including enucleation, ageing and cooling. For this purpose the phosphorylation profiles of four groups of in vitro-matured bovine oocytes (aged oocytes, aged-cooled oocytes, enucleated-aged oocytes and enucleated-aged-cooled oocytes (recipient cytoplasts)) were analyzed. These recipient cytoplasts exhibited a phosphorylation profile similar to that of activated oocytes. Maturation promoting factor (MPF) activity, which was high in young metaphase II oocytes, in aged oocytes, in enucleated-aged oocytes and in aged-cooled oocytes, dropped to the basal level in enucleated-aged-cooled oocytes (recipient cytoplasts), while mitogen-activated protein kinase (MAPK) activity remained elevated. The combination of enucleation, ageing and cooling following oocyte in vitro maturation resulted in an interphase-like stage cytoplasm having a phosphorylation profile and low MPF activity similar to activated oocytes, but exhibiting high MAPK activity.

Publisher Summary This chapter describes platelet-mediated transformation of human mitochondrial DNA-less ( ρ ˚)cells. The isolation of human cell lines completely devoid of mitochondrial DNA (mtDNA) and the demonstration that these cell lines can be repopulated with exogenous mtDNA by mitochondria-mediated transformation represents a major advance for human mitochondrial genetics studies. mtDNA-less ( ρ o ) cells have proved to be very useful for the study of mtDNA-linked diseases. The method for introducing mitochondria derived from cells of patients or normal human individuals into ρ ˚ cells involves fusion of enucleated cells (cytoplasts) with the ρ ˚ cells. An alternative method for introducing mitochondria into human ρ ˚ 0 cells has been developed, which involves the fusion with these cells of platelets derived from the individual's blood. The advantage of this method is that there is no need to establish cultures derived from the cells of the patient or the normal individual.

Publisher Summary This chapter describes methods for the transfer of mitochondria from one cellular environment to another, focusing on methods for the transfer of mitochondria into human cells lacking mtDNA ( ρ ˚ cells). The relatively large sizes of these organisms has facilitated the introduction into them of subcellular fractions containing mitochondria by microinjection. These investigations utilize mtDNA-encoded mutations specifying morphological or growth characteristics, or associate with specific drug resistance markers, for isolating mitochondrial transformants among the injected cells. The development of efficient, large-scale methods to obtain enucleated mammalian cells, termed “cytoplasts,” provides a basis for the transfer of mitochondria into cells by the fusion of cytoplasts with the cells of interest to form cytoplasmic hybrids, termed “cybrids.” The methods described in the chapter require donor mitochondria containing a selectable marker. The requirement for a selectable marker in donor mitochondria limits the utility of these systems for mitochondrial transfer, as there are only a small number of selectable markers associated with resistance to specific drugs available.

One-cell parthenogenetic haploid embryos and blastomeres of the 2- and 4-cell diploid mouse embryos were observed in vitro for the occurrence of two cytoplasmic activities: the cortical activity and the chromatin condensation activity. For this purpose anucleated halves (AHs) and nucleated halves (NHs) were produced by bisection of one-cell embryos and of blastomeres. The cortical activity (manifested by surface deformations) was observed only during the first cleavage cycle. In AHs the surface activity began at the same time as in NHs and disappeared before the time of the cleavage division of nucleated halves. Anucleate fragments of blastomeres from 2- and 4-cell embryos did not exhibit any cortical activity. In the absence of the native nucleus the chromatin condensation activity (assayed by premature chromatin condensation of interphase thymocyte nuclei introduced into cytoplasts by cell fusion) could also have been detected only in the first cleavage cycle. In AHs this activity appeared at the time when NHs started to cleave and disappeared after the NHs finished the first cleavage division. AHs obtained from 2-cell and 4-cell stage blastomeres did not reveal condensation activity.

Activation of the membrane-associated NADPH oxidase of neutrophils requires several cytosolic factors including p47phox, p67phox and p21rac2. We compared NADPH oxidase activity with the membrane translocation of p47phox, p67phox, and p21rac2. In a cell-free system, GTP gamma S stimulated translocation of p47phox and p67phox to the plasma membrane only in the presence of arachidonate, and this translocation correlated with NADPH oxidase activity of the reisolated plasma membranes (R = 0.94 and 0.97, respectively). In contrast, GTP gamma S-stimulated p21rac2 translocation with or without arachidonate, and the extent of translocation did not correlate with oxidase activity (R = 0.17). Neutrophil cytoplasts were used to relate membrane translocation of p47phox, p67phox and p21rac2 to membrane oxidase activity in response to the inflammatory agonists. Whereas N-formyl- methionyl-leucyl-phenylalanine stimulated equimolar, transient membrane translocation of p47phox and p67phox which kinetically paralleled NADPH oxidase activity, relatively little p21rac2 translocated (moles of p47phox/p21rac2 = 16.6). Moreover, although phorbol 12-myristate 13- acetate stimulated both the stable translocation of p47phox and p67phox and sustained NADPH oxidase activity, it did not stimulate p21rac2 translocation. From these data we conclude that membrane translocation of p21rac2 does not regulate NADPH oxidase activity stoichiometrically.

The ploidy of single-pronucleated human zygotes obtained after conventional in vitro fertilization was determined by fluorescent in situ hybridization (FISH) using multiple simultaneous probes for gonosomes and autosomes. After zona removal the single-pronucleated zygotes were exposed to cytochalasin B, and the pronucleus, surrounded by scant cytoplasm and the plasma membrane (karyoplast), was divided from the rest of the egg (cytoplast). The karyoplasts and the corresponding cytoplasts were analyzed separately by FISH. Of the 16 zygotes analyzed, 10 had haploid pronuclei and 6 were diploid. Four diploid pronuclei contained XY chromosomes, and 2 contained XX chromosomes. These results suggest that during the course of their interaction, human gamete nuclei can associate together and form diploid, single-pronucleated zygotes. These findings confirm a newly recognized variation of human pronuclear interaction during syngamy.

We investigated the effects of lipopolysaccharide (LPS) on the subcellular location of the integrin receptor CD11b/CD18 (Mac-1). Cytoplast and subcellular fractionation experiments were performed to distinguish between receptor shedding and intracellular receptor transport as mechanisms involved for the effects of LPS on CD11b/CD18 expression. Cytoplast preparations demonstrated the same percentage of cell-associated receptors +/- LPS. Subcellular fractionation experiments demonstrated a shift from primarily plasma membrane fractions to azurophilic granules. Protein tyrosine kinase inhibition with genistein (50 microM) inhibited the LPS-induced translocation of CD11b/CD18 receptors to azurophilic granules. The effects of LPS (10 ng/mL) alone were reproduced with LPS (.1 ng/mL) plus heat-inactivated pooled normal human serum. Preincubation of PMN with anti-CD14 monoclonal antibodies prevented the effects of LPS+serum on the translocation of CD11b/CD18 receptors. These results demonstrate that LPS regulates CD11b/CD18 expression by inducing intracytoplasmic translocation of this receptor to azurophilic granules. This process involves activation of protein tyrosine kinase, and endosomal acidification contributes to the degradation of these receptors within azurophilic granules.

The influence of oocyte age and presence of oocyte meiotic apparatus on the behaviour of introduced blastomere nuclei was evaluated. Blastomeres from 4-cell mouse embryos were fused to intact (metaphase II) oocytes, demi-oocytes (nucleate) or cytoplast (anucleate). Fusion and simultaneous activation of the recipient oocytes were accomplished by a single electrical pulse at 20 or 24 h post human chorionic gonadotrophin (hCG) administration. The hybrids were fixed for evaluation 2 h after fusion. There was no difference in the behaviour of blastomere nuclei in whole oocytes and demi-oocytes. Most nuclei fused to the nucleate recipients at 20 h underwent breakdown of nuclear membrane (NMBD), chromosome condensation and consequently proceeded to telophase, in parallel with the resident meiotic chromosomes. Following fusion to cytoplasts, only a small portion of the blastomere nuclei underwent chromosome condensation and the vast majority (83%) of the nuclei remained in interphase. The influence of oocyte age on nuclear behaviour was assessed in oocyte-blastomere hybrids prepared by simultaneous fusion and activation at 20 and 24 h post-hCG administration. The introduced nuclei proceeded to telophase in 63% of the hybrids constructed at 20 h, but in only 28% of those constructed at 24 h. We conclude that nuclei introduced into aged or enucleated oocytes at the time of activation are predominantly remodelled in their interphase configuration.

The heteroplasmic tRNA LYS mutation in the mitochondrial DNA (mtDNA) is responsible for the phenotypic expression and the transmission of MERRF syndrome. However, the genetic behaviors of the mutant and wild-type mtDNA molecules within a cell are still unknown. We demonstrated a clear genetic complementation of the mutant and wild-type mtDNAs, with a sharp threshold around 10% in the wild-type, in the MERRF transformants, and in their subclones by a cytoplast transfer of the mitochondria into an mtDNA-less cell line, p 0 cell. By contrast, no interaction was observed between the two functionally complementary mtDNAs that were originally located in distinct organelles and sequentially introduced into a p 0 cell line (genetic independence). These results imply that the sorting of the mtDNA molecules among mitochondria plays a crucial role in the phenotypic expression and transmission of the disease. © 1995 John Wiley & Sons, Inc.

Publisher Summary This chapter discusses the progress of nuclear transplantation studies in mammals, the important biological factors influencing the development of eggs and embryos reconstituted by nuclear transplantation, and the prospects for future studies in this field. The transplantation of a nucleus to an appropriate cytoplast and the subsequent analysis of its embryonic development provide the most vigorous and direct tests of genomic totipotency of a cell nucleus. The most advanced studies are those carried out in amphibia. Totipotency, the capacity to direct the formation of fertile frogs, has been demonstrated for the nuclei of many kinds of embryonic cells; pluripotency, as judged by the development of heart-beating larvae, has been proved for all the cell nuclei tested to date.

The ability to compact and inject the cat germinal vesicle (GV) into a recipient cytoplast allows exploration of a new fertility preservation strategy that avoids whole oocyte freezing and could enable drying and storage of GV at supra-zero temperatures. Previous studies demonstrated that (1) 60% of GVs can survive simple air-drying in the presence of trehalose followed by 4 weeks of storage at 4 °C and (2) 10% of reconstructed oocytes with rehydrated GV can achieve nuclear maturation (Graves-Herring et al., 2011). However, air-drying is an uncontrolled process that often results in non-uniform distributions of water within and between samples. Compared to existing techniques, microwave assisted dehydration appears to be a simple and reliable approach to dry biological samples at ambient temperatures (Chakraborty et al., 2008). The objective of the study therefore was to (1) explore the feasibility of drying GV exposed to trehalose using microwave processing and (2) assess the impact on GV structural integrity (chromatin configuration and incidence of DNA damage). Immature cat oocytes from adult ovaries were denuded from the cumulus cells and permeabilized with hemolysin. Groups of five denuded oocytes were exposed to Tris–HCL EDTA (pH 8.0) buffer containing 200 mM trehalose for 10 min and then transferred into 10-μl droplets placed on small pieces of Millipore Isopore Membrane filters (n = 182 total oocytes in 5 replicates). A maximum of eight droplets were arranged on the turntable of a CEM SAM 255 microwave system. Drying was conducted at 20% power for 5, 8, 20, 25 or 30 min. After each time point, samples were weighed immediately to establish the extent of water loss and then were rehydrated before fixation in 4% paraformaldehyde followed by GV chromatin configuration assessment (Hoechst staining) and DNA fragmentation assay (TUNEL). Fresh denuded oocytes (n = 60 total oocytes) were fixed as controls. The average ambient humidity during all measurements was 43.2 ± 1.1%. From time 0 through 30 min of microwave treatment, moisture contents (g water/g dry weight) progressively decreased (0 min: 12.3 ± 0.5; 5 min: 8.3 ± 1.2; 8 min: 8.9 ± 1.2; 20 min: 5.0 ± 0.5; 25 min: 1.9 ± 0.3; 30 min: 1.3 ± 0.4). GV chromatin configuration was not affected by the microwave treatment. The proportion of degenerated oocytes (absence of GV) ranged from 10% to 15% after the different time points and was not different (P > 0.05; Chi-square test) from the fresh controls. Interestingly, pooled proportions of GV with DNA damage following 5–8 min of microwave dehydration were not different (P > 0.05) from fresh controls (14.3% and 10.9%, respectively). However, the incidence of DNA fragmentation was higher (43.5%; P

Objective To demonstrate step-by-step micromanipulation procedures required for transfer of spindle-chromosomal complexes between mature oocytes. Design Video presentation of reproductive biology study. Setting In vitro fertilization and embryo manipulation laboratory. Animal(s) Rhesus ( Macaca mulatta ) primates. Intervention(s) Transplantation of the genetic material between mammalian oocytes offers many opportunities to study various aspects of nuclear-cytoplasmic interactions during oogenesis, fertilization and embryo development. We demonstrate the feasibility of isolation and transfer of chromosomes between mature metaphase II (MII) primate oocyte. After fertilization, manipulated oocytes were capable of producing healthy offspring or embryonic stem cells. Main Outcome Measure(s) In this video, we show micromanipulation procedures required for isolation and transfer of spindle-chromosomal complexes between rhesus MII oocytes. In brief, the spindle is visualized using a polarized microscope and extracted into a membrane enclosed karyoplast. Karyoplasts are then reintroduced into an enucleated recipient oocyte (cytoplast, derived from an another female) by karyoplast-cytoplast membrane fusion. Result(s) Newly reconstructed oocytes consist of nuclear genetic material from one female and cytoplasmic components, including mitochondria and mitochondrial DNA from another. Conclusion(s) This video demonstrates the protocol developed for primate oocytes that successfully allowed of isolation and transfer of chromosomes between mature metaphase II (MII) oocytes. Potential clinical applications include mitochondrial gene replacement therapy to prevent transmission of mtDNA mutations and treatment of infertility caused by cytoplasmic defects in oocytes. Video is available at http://fertstertforum.com/2012974tachibana/.