Microfilaments and microtubules control the shape, motility, and subcellular distribution of cortical mitochondria in characean internodal cells

Summary. The shape, motility, and subcellular distribution of mitochondria in characean internodal cells were studied by visualizing fluorescent dyes with confocal laser scanning microscopy and conducting drug-inhibitor experiments. Shape, size, number, and distribution of mitochondria varied according to the growth status and the metabolic activity within the cell. Vermiform (sausage-shaped), disc-, or amoeba-like mitochondria were present in elongating internodes, whereas very young cells and older cells that had completed growth contained short, rodlike organelles only. Mitochondria were evenly distributed and passively transported in the streaming endoplasm. In the cortex, mitochondria were sandwiched between the plasma membrane and the stationary chloroplast files and distributed in relation to the pattern of pH banding. Highest mitochondrial densities were found at the acid, photosynthetically more active regions, whereas the alkaline sites contained fewer and smaller mitochondria. In the cortex of elongating cells, small mitochondria moved slowly along microtubules or actin filaments. The shape and motility of giant mitochondria depended on the simultaneous interaction with both cytoskeletal systems. There was no microtubule-dependent motility in the cortex of nonelongating mature cells and mitochondria only occasionally travelled along actin filaments. These observations suggest that mitochondria of characean internodes possess motor proteins for microtubules and actin filaments, both of which can be used either as tracks for migration or for immobilization. The cortical cytoskeleton probably controls the spatiotemporal distribution of mitochondria within the cell and promotes their association with chloroplasts, which is necessary for exchange of metabolites during photosynthesis and detoxification.Correspondence and reprints: Arbeitsgruppe Pflanzenphysiologie, Fachbereich Zellbiologie, Universität Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.     

Microtubule orientation and dynamics in elongating characean internodal cells following cytosolic acidification, induction of pH bands, or premature growth arrest

Summary Cortical microtubules (MTs) at indifferent zones in immatureNitella internodes were investigated by injection of fluorescently tagged sheep brain tubulin into living cells and by immunofluorescence on fixed material. Nearly identical MT patterns and numbers were detected with the two techniques, indicating that sheep brain tubulin incorporated into all cortical MTs. MTs were aligned transversely to the long axis of the cell and approximately one MT was present every micrometer of longitudinal cell distance. Treatment of internodes with propionic acid to acidify cytosolic pH caused depolymerization of MTs and an increase in the unpolymerized tubulin pool. Transfer of young, vigorously elongating cells to media inducing premature growth cessation resulted in a slight decrease in microtubule numbers but did not significantly alter microtubule orientation patterns or microtubule lifespans. MTs remained transverse for days following growth cessation before finally assuming a more random alignment characteristic of mature, non-growing internodes. No differences in MT numbers, orientation, or dynamics were detected between acid and alkaline bands in internodes incubated in a band-inducing medium. Thus, properties of cortical MT arrays were not closely coupled to growth status or to regional differences in cellular physiology associated with pH banding.Abbrevations BIM band-inducing medium - CCM Chara culture medium - CF carboxyfluorescein - FRAP fluorescence redistribution after photobleaching - MT microtubule     

Actin-dependent deposition of putative endosomes and endoplasmic reticulum during early stages of wound healing in characean internodal cells

We investigated the behaviour of organelles stained with FM1-43 (putative endosomes) and/or LysoTracker Red (LTred; acidic compartments) and of the endoplasmic reticulum (ER) during healing of puncture and UV-induced wounds in internodal cells of Nitella flexilis and Chara corallina. Immediately after puncture, wounds were passively sealed with a plug of solid vacuolar inclusions, onto which a bipartite wound wall was actively deposited. The outer, callose-containing amorphous layer consisted of remnants of FM1-43- and LTred-labelled organelles, ER cisternae and polysaccharide-containing secretory vesicles, which became deposited in the absence of membrane retrieval (compound exocytosis). During formation of the inner cellulosic layer, exocytosis of secretory vesicles with the newly formed plasma membrane is coupled to endocytosis via coated vesicles. Migration of FM1-43- and LTred-stained organelles, ER and secretory vesicles towards the cell cortex and deposition of a bipartite wound wall could also be induced by spot-like irradiation with ultraviolet light. Cytochalasin D reversibly inhibited the accumulation and deposition of organelles. Our study indicates that active actin-dependent deposition of putative recycling endosomes is required for wound healing (plasma membrane repair) and supports the hypothesis that deposition of ER cisternae helps to restore wounding-disturbed Ca(2+) metabolism.     

Induction of exocytosis in characean internodal cells by locally restricted application of chlortetracycline and the effect of cytochalasin B,depolarizing and hyperpolarizing agents*

Abstract. Local exocytosis with concomitant deposition of cell wall material (plugs) was induced in mature characean internodal cells by application of Sephadex beads loaded with a chlortetracycline (CTC)/CaCl₂ solution. Plugs can be formed anywhere on the cells independently of structural and/or functional plasma membrane domains, provided that the pH of the CTC/CaCl₂ solution is above 5.5. Plug formation is inhibited by cytochalasin B and membrane depolarizing agents. Treatment with fusicoccin enhances exocytosis. The results suggest that actin filaments are involved in the transport of vesicles and endoplasmic reticulum cisternae towards the cell membrane, and that the membrane potential determines the extent of CTC mediated Ca²⁺ influx.     

Probing cytoskeletal pre-stress and nuclear mechanics in endothelial cells with spatiotemporally controlled (de-)adhesion kinetics on micropatterned substrates

The mechanical properties of living cells reflect their propensity to migrate and respond to external forces. Both cellular and nuclear stiffnesses are strongly influenced by the rigidity of the extracellular matrix (ECM) through reorganization of the cyto- and nucleoskeletal protein connections. Changes in this architectural continuum affect cell mechanics and underlie many pathological conditions. In this context, an accurate and combined quantification of the mechanical properties of both cells and nuclei can contribute to a better understanding of cellular (dys-)function. To address this challenge, we have established a robust method for probing cellular and nuclear deformation during spreading and detachment from micropatterned substrates. We show that (de-)adhesion kinetics of endothelial cells are modulated by substrate stiffness and rely on the actomyosin network. We combined this approach with measurements of cell stiffness by magnetic tweezers to show that relaxation dynamics can be considered as a reliable parameter of cellular pre-stress in adherent cells. During the adhesion stage, large cellular and nuclear deformations occur over a long time span (>60 min). Conversely, nuclear deformation and condensed chromatin are relaxed in a few seconds after detachment. Finally, our results show that accumulation of farnesylated prelamin leads to modifications of the nuclear viscoelastic properties, as reflected by increased nuclear relaxation times. Our method offers an original and non-intrusive way of simultaneously gauging cellular and nuclear mechanics, which can be extended to high-throughput screens of pathological conditions and potential countermeasures.     

Nuclear division,nuclear distribution and cytokinesis in filamentous fungi

Nuclear division, nuclear distribution and cytokinesis are fundamental processes of all eukaryotic organisms, and filamentous fungi, specificallyAspergillus nidulans andNeurospora crassa, have provided sophisticated genetic systems for identification of the genes required for these processes. Mutational analyses have led to identification of novel proteins that have subsequently been found to be conserved components required for nuclear-specific functions. Formation of the mitotic spindle inA. nidulans has been shown to be dependent onγ-tubulin, a central element of all microtubule organizing centres, and two kinesin-related proteins. Analysis ofA. nidulans mitotic mutants has led to identification of two important cell-cycle regulators, NIMA and BIME. The NIMA kinase is required for entry into mitosis, and BIME has recently been identified as a subunit of an anaphase-promoting complex that targets cyclins for proteolysis. The microtubule-associated motor protein cytoplasmic dynein has been discovered in bothA. nidulans andN. crassa, and it has been proposed that it provides motive force for the distribution of nuclei within hyphae. Future studies of nucleus-specific processes in filamentous fungi are likely not only to identify additional novel structural and regulatory proteins, but also lead to an understanding of how the processes of nuclear division, nuclear distribution and septation are altered to meet the developmental needs of the organism.     

Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC₆ indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene.     

Life-history traits associated with fragmentation vulnerability of lizards in the Thousand Island Lake, China

Following habitat fragmentation, the remnant faunal community will undergo a period of species loss or 'relaxation.' Theory predicts that species with particular life-history traits, such as a small population size, small geographical range, low fecundity and large body size, should be more vulnerable to fragmentation. In this study, we investigated the relationships between the above life-history traits and the fragmentation vulnerability index (the number of islands occupied) of five lizard species inhabiting recently isolated land-bridge islands in the Thousand Island Lake, China. Data on life-history traits were collected from field surveys (population density) and from the literature (body size, clutch size and geographical range size). The species–area relationships for lizards sampled from the mainland versus on the islands differed significantly (i.e. the number of species inhabiting islands was decreased relative to similar-sized areas on the mainland), indicating that species extinction has occurred on all of the study islands following isolation. For the fragmentation vulnerability index, model selection based on Akaike's information criterion identified natural density at mainland sites as the best correlate of vulnerability to fragmentation, supporting the hypothesis that rare species are most vulnerable to local extinction and will be lost first from fragmented landscapes. In contrast, there was little evidence for an effect of lizards' snout–vent length, clutch size or geographical range size on fragmentation vulnerability. Identification of species traits that render some species more vulnerable to fragmentation than others has important implications for conservation and can be used to aid direct management efforts.     

Tissue- and development-specific distributions of cytoskeletal elements in growing cells of the maize root apex

ABSTRACT

Indirect immunofluorescence performed using sections of actively growing maize root apices fixed and then embedded in low-melting-point Steedman's wax has proved efficient in revealing the arrangements and reorganizations of motility-related cytoskeletal elements which are associated with root cell development and tissue differentiation. This powerful, yet relatively simple, technique shows that specific rearrangements of both microtubular (MT) and actin microfilament (MF) arrays occur in cells as they leave the meristem and traverse the transitional region interpolated between meristem and elongation region. Cytoskeletal and growth analyses have identified the transition zone as critical for both cell and root development; it is in this zone that cell growth is channelled, by the cytoskeleton, into a strictly polarized mode which enables root tips to extend rapidly through the soil in search of water and nutrients. An integrated cytoskeletal network is crucial for both the cytomorphogenesis of individual cells and the overall morphogenesis of the plant body. The latter process can be viewed as a reflection of the tight control which cytoskeletal networks exert not only over cell division planes in the cells within meristematic apices but also over the orientation of cell growth in the meristem and elsewhere. Endoplasmic MTs interconnecting the plasma membrane with the nucleus are suggested to be involved in cell division control; they may also act as a two-way cytoskeletal communication channel for signals passing to and fro between the extracellular environment and the genome. Moreover, the dynamism of endoplasmic MTs exerts direct effects on chromatin structure and the accompanying nuclear architecture and hence can help exert a cellular level of control over cell growth and cell cycle progression. Because the inherent dynamic instability of MTs depends on the concentration of tubulin dimers within the cytoplasm, we propose that when asymmetric cell division occurs, it will result in two daughter cells which differ in the turnover rates of their MTs. This phenomenon could be responsible for different cell fates of daughter plant cells produced by such cell divisions.     

Nuclear transfer in cattle: Effect of nuclear donor cells,cytoplast age,co-culture,and embryo transfer

There are many factors affecting the efficiency of nuclear transfer technology. Some are evaluated here using our novel approach by enucleating oocytes at 20–22 hr after in vitro maturation (IVM), culturing the enucleated oocytes (cytoplasts) for 8–10 hr or 18–20 hr to gain activation competence and then conducting nuclear transfer. In the first experiment, we demonstrated that cumulus cell (CC) monolayer can support some cloned embryos to develop into morulae or blastocysts. Co-culture with CC and bovine oviduct epithelial cell (BOEC) monolayers resulted in no differences (P 0.05) in supporting the development of cloned embryos (Experiment 2). When in vitro matured oocytes were enucleated at 22 hr after IVM followed by nuclear transfer 18–20 hr later, cleavage and morula or blastocyst development of the cloned embryos were similar to those resulting from the enucleated oocytes which had been matured in vivo (Experiment 3). Frozen embryos as nuclear donor cells worked equally well as fresh embryos for cloning in embryo development which was superior to IVF embryos (Experiment 4). However, fresh embryos resulted in a higher proportion (P < 0.05) of blastomere recovery than did frozen or IVF ambryos. Finally, embryo transfer of cloned embryos from our procedure produced a viable calf, demonstrating the commercial value of this novel approach of the technology. © 1993 Wiley-Liss, Inc.     

Cell density-dependent decrease in cytoskeletal actin and myosin in cultured osteoblastic cells: correlation with cyclic AMP changes.

During bone development, osteoblasts form a contiguous layer along recently deposited osteoid and their morphology changes from fibroblast-like to cuboidal. In culture, similar changes occur with increased cell density. We examined the possible role of cyclic AMP in this process since cyclic AMP was reported to increase in fibroblasts with increased cell density and similar shape changes were seen in response to parathyroid hormone, which also increases cellular cyclic AMP in osteoblastic cells. Osteoblast-enriched rat calvaria cells were seeded at increasing density. The distribution between Triton X-100 extractable and nonextractable actin and myosin was estimated by polyacrylamide gel electrophoresis. Intracellular cyclic AMP was estimated by prelabeling the cellular ATP pool with 3H-adenine, followed by extraction and separation of 3H-cAMP by high-performance liquid chromatography. We found that osteoblastic cells contain about 40 pg actin and 5.3 pg myosin per cell. Around 60% of the actin and 70% of the myosin were in the nonextractable (crosslinked) form at cell densities of 10,000 to 50,000 cells per cm2. Above 50,000 cells/cm2, there was a cell density-dependent reduction in crosslinked actin and myosin and a concomitant increase in cellular cyclic AMP. A comparable rise in cyclic AMP, produced by incubation with phosphodiesterase inhibitors, and treatment with other agents that increase cyclic AMP produced a similar decrease in the level of cytoskeletal actin and myosin. Cytochalasin B treatment, through its effect on actin polymerization, produced similar changes in cell shape and cytoskeletal actin. The findings suggest that an elevation in intracellular cyclic AMP may play a role in the density-dependent changes in cell shape and microfilament organization observed in osteoblasts.     

Whitening,thallus decay and fragmentation in Gracilaria chilensis associated with an endophytic amoeba

Whitening of Gracilaria chilensis, accompanied by tissue softening and thallus fragmentation, was found to be associated with the presence of an endophytic amoeba. Although the symptoms developed originally in green mutant thalli, subsequent infections in the laboratory also affected normal, wild-type G. chilensis. Ultrastructural evidence indicates that the amoebae perforate the host cell walls of both cortical and medullary cells and digest their protoplasm. Feeding by the amoeba appears to involve both phagocytosis and enzymatic digestion of the host tissue. Destruction of the host tissue resulted in large cavities first, followed by thallus fragmentation. No other organism was found during the early stages of thallus invasion by the amoeba, although bacteria may appear once the amoeba reaches the inner tissues of the host.     

Structural changes induced in capillary endothelial cells by growth factors: Altered distribution of cytoskeletal elements and organelles in cells spreading in the presence of tumor conditioned medium and hypothalamus derived growth factor

Using high-voltage and conventional electron microscopy of cell whole mounts, we have investigated the effects of tumor-conditioned medium and hypothalmus-derived growth factor on the structure of capillary endothelial cells during their attachment and spreading in tissue culture. Cells were cultured in A, Dulbecco's Modified Eagle's Medium (DMEM) and 10% calf serum; B, equal parts of A and 48 hr mouse sarcoma conditioned medium; and C, A containing 10 units of hypothalamus-derived growth factor. Cells cultured in all three media were fully spread, and to the same extent, by 4 hr after plating. While spreading, cells cultured in DMEM alone developed prominent stress fibers and contained numerous bundles of microtubules which formed radical tracts along which mitochondria and other organelles rapidly moved to the cell periphery. Stress fibers were thinner and microtubule tracts fewer in number in cells cultured in tumor-conditioned medium. In 4 hr, organelles moved only part of the distance to the cell margin. Stress fibers were rudimentary and microtubules randomly orientated in cells exposed to hypothalamus-derived growth factor. Most organelles remained near the cell nucleus. The dramatic decrease in stress fibers and microtubule tracts in cells grown in tumor-conditioned medium and hypothalamus-derived growth factor and the subsequent decreased capacity of the cells to move organelles toward their periphery could have some functional significance relative to the growth-promoting activity of these substances.     

Nucleotides in rat liver cells: sedimentation and fluorescence analysis; DNA, associated with residual nuclear structures

Rat hepatocyte nucleoids, obtained at different conditions, have been studied by sedimentation and fluorescence methods. Divalent metal ions have been found to play an important role in the superhelical organization of nuclear DNA. Depending on nucleoid isolation conditions, different DNA fragments become associated with the residual nuclear structure. In rapidly sedimenting nucleoids, where nuclear DNA exhibits greater compaction, two types of DNA fragments, differing is size, are associated. In slowly sedimenting nucleoids, where nuclear DNA is in a more relaxed state, one type of DNA fragments is associated. It is assumed that nuclear DNA compaction is conditioned by the formation of additional DNA binding sites with residual nuclear structures and involves divalent metal ions. It has been shown with the aid of restrictase analysis that both types of DNA fragments contain different repeated nucleotide sequences.     

Immunocytochemical localization of nuclear antigens in the unicellular green alga,Chlamydomonas reinhardtii,processed by cryofixation and freeze-substitution

Summary We describe the preparation of monoclonal antibodies to nuclear antigens in the green alga,Chlamydomonas reinhardtii, and their localization at the light and electron microscope level. Supernatants from hybridomas were screened by the ELISA method and the four antibodies giving the strongest signal were subjected to further analysis. At the LM level immunogold silver staining was used on semi-thick resinless sections. We have examined at the EM level the distribution of these antigens by post-embedding immunocytochemical techniques on sections of conventionally fixed specimens compared to cryofixed and freeze-substituted ones. Enhanced ultrastructural preservation was observed in cells which were cryofixed, freeze-substituted and embedded at –35°C in Lowicryl K4M. Different preparative procedures involving cryofixation and substitution are described. Of the four antibodies three were localized under light and electron microscopy. All three were distributed in the interchromatin space. One of these antigens (QUL4D2, 54 kDa) is also found in the dense fibrillar component and fibrillar centers of the nucleolus.Abbreviations DFC dense fibrillar component - EM electron microscope - FC fibrillar center - GAM5 goat anti-mouse IgM coupled to 5 nm colloidal gold - Ig immunoglobulin - LM light microscope - MAb monoclonal antibody - PAG protein A-gold - PBS phosphate buffered saline - PEG polyethylene glycol     

Variation in reproductive modes and population genetic structures of a monocarpic perennial herb,Cardiocrinum cordatum,in relation to habitat fragmentation

Reproductive systems are recognized as having a profound influence on the extent and structure of genetic variation in plant populations. To investigate the spatiotemporal variation in the reproductive modes (sexual and vegetative reproduction) and population genetic structures of a monocarpic perennial herb, Cardiocrinum cordatum (liliaceae), we selected a variety of habitats (e.g. large forested area including primeval forest, small fragmented secondary forest, and so on) around Sapporo City, Japan. We conducted breeding experiments, monitored the fate and growth of marked individuals for 3 years, and also analyzed the spatiotemporal genetic variation of flowering plants within the populations using allozyme variation. Plants emasculated prior to anthesis produced mature fruits in all populations examined. However, seed production was significantly lower in the small fragmented populations, possibly because of the low availability of pollinators and subsequent pollen limitation. In these fragmented populations, the mature flowering plants tended to be more dependent on vegetative reproduction for their recruitment, because they can only produce flowers once in their lifetime. Genetic diversity using samples from mature flowering plants in each population was lower in the small fragmented populations than in the large populations. In addition, although genotypic compositions in the fragmented populations were more or less similar during the 3 years of the study, the dominant genotypes changed temporally and spatially every year in the large populations. The present study demonstrated that the reproductive features of C. cordatum can be altered in various environmental conditions, such as habitat fragmentation, and these changes considerably affected the population genetic structures and vice versa.     

Microtubules associated with nuclear pore complexes and coated pits in the CNS

Summary Using a new albumin prefixation technique, microtubules have been observed in close association with the nuclear pores of neurons and glia. Thus, microtubules may be involved in such phenomena as anchoring, migration or rotation of the nucleus or in chemical messenger transport between nucleus and cytoplasm. Microtubules are also seen running close to the coated pits of dendrites. The implications are discussed.The authors gratefully acknowledge the support of grants from the British Medical Research Council (E.G.G.) and USPHS grants NS 09678 and NS 04053; National Institutes of Neurological and Communicative Disorders and Stroke (L.E.W.). We thank Hilary Samson for excellent technical collaboration, Julie Barron and Trevor King for technical assistance and Ann Harris for secretarial help     

Terminology and nomenclature of the cytoskeletal elements associated with the flagellar/ciliary apparatus in protists

Summary The terminology used to describe the cytoskeleton of protists is sometimes redundant and confusing. The independent origins of protistology from studies on algae, fungi, and protozoans led to these problems. Additionally, recent studies indicate that seemingly unrelated taxa form new, monophyletic groups. However, homologous structures that support monophyly are difficult to identify when the literature is published in journals restricted to specific fields or when the terminology is in duplicate or triplicate. The purpose of this paper is to establish clear definitions, identify synonyms, and indicate homologies where possible.     

Mixed-solvothermal syntheses, structures and luminescence properties of two new Zn(II) phosphonates with layered and 3D framework structures

Two new zinc phosphonates with 2-hydroxyphosphonoacetic acid (HPAA) and 1-hydroxyethylidenediphosphonic acid (hedpH₄), [Zn₂{HO₃PCH(OH)CO₂}₃]·2NH₂(CH₃)₂·3H₂O (1) and [Zn₃{CH₃C(OH)(PO₃)₂}₂]·2NH₂(CH₃)₂·H₂O (2) have been synthesized under mixed-solvothermal conditions at 160 °C and structurally characterized by X-ray single-crystal diffraction, X-ray powder diffraction, infrared spectroscopy and elemental analysis. The structure of compound 1 comprises Zn1O₆ and Zn2O₆ octahedra connected by [HO₃PCH(OH)CO₂]²⁻ to form a 2D layered structure with one-dimensional channel system along c-axis direction, and the protonated dimethylamine cations are being located between two adjacent layers. Interestingly the layers of 1 arranged in an alternative sequence (ABAB). Compound 2 features a 3D framework structure with channels along the b- and c-axis, respectively. The charge-compensating protonated Hdma⁺ cations and solvate water molecules are located inside the channels along the c-axis. A notable feature for compound 2 is the presence of the alternate left- and right-handed helical chains in the structure. The luminescence properties of compounds 1 and 2 have also been studied.     

Nuclear transfer in the bovine embryo: a comparison of 5-day, 6-day, frozen-thawed, and nuclear transfer donor embryos

Micromanipulation and electrofusion were utilized for nuclear transfer in bovine embryos. Embryonic blastomeres from 5-day (estrus = day 0), 6-day, frozen-thawed 5-day, and first-generation nuclear transfer embryos (embryos were themselves a product of nuclear transfer with the original donor being a 5-day embryo) were transferred into bisected bovine oocytes by electrofusion. The percentage of donor cells fusing with the recipient oocytes was compared between different types of donor embryos. The percentage of embryos developing normally into morula or blastocysts following 6 days culture in the sheep oviduct was also recorded and compared between different donor embryo types. No significant differences were found between donor blastomeres for the percent successfully fused to oocytes: 5-day, 294 of 513 (57.3%); 6-day, 252 of 405 (62.2%); frozen-thawed 5-day, 111 of 144 (77.1%); nuclear transfer, 142 of 223 (63.7%); or the percent developing normally following nuclear transfer: 5-day, 92 of 444 (20.7%); 6-day, 84 of 357 (23.5%); frozen-thawed 5-day, 32 of 127 (25.2%); nuclear transfer, 31 of 199 (15.6%). These data suggest that a variety of donor embryos can successfully be utilized for bovine embryo cloning. Also, development of blastomeres from frozen-thawed 5-day donors and from donors that are themselves the product of nuclear transfer suggest that the production of multiple identical offspring is possible by frozen storage of seed stock and serial recloning.