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Cytoplasts prepared from L929 or Hepa-2 cells were separated from whole cells using density gradients of renografin. Using this technique, cytoplasts can be isolated from cell lines which cannot be routinely enucleated with an efficiency of 100%. The purified cytoplasts excluded the vital dye trypan blue and were utilized in nuclear transplantation experiments to reconstruct whole viable cells capable of division. In addition, the renografin gradient technique was used to separate the newly reconstructed cells from any contaminating "non-renucleated" cytoplasts. This will permit immediate biochemical characterization of cytoplasmic-nuclear hybrid cells without interference from contaminating cytoplasts.

Pigment migration in cultured erythrophores of the squirrel fish Holocentrus ascensionis, after manipulation with K+, epinephrine, 3',5'-dibutyryl cyclic adenosine monophosphate, theophylline, and caffeine, is essentially identical to that observed in this chromatophore in situ. For such observations, the erythrophores are dissociated from the scales with hyaluronidase and collagenase, and allowed to spread on an amorphous collagen substrate, where they resemble the discoid erythrophore in situ. In this state, they are readily fixed by glutaraldehyde and osmium tetroxide, and are then critical-point dried for whole-cell viewing in the high voltage electron microscope. The organization and fine structure of the erythrophore cytoplast was stereoscopically examined after fixation of the pigment granules in four experimental states: pigment dispersed, pigment aggregated, pigment aggregating, and pigment dispersing. In the dispersed cell, granules are contained in an extensive three-dimensional lattice composed of radially oriented microtubules and a network of fine filaments 3-6 nm in diameter (microtrabeculae), whereas in the aggregated cell, the microtrabecular system is absent, and the majority of the microtubules appear displaced into the cortices on the cytoplasmic surface of the plasma membrane. In cells fixed while aggregating, few microtrabeculae are observed, although formless thickenings are observed in the cortices, on granules, and between clumped granules. In dispersing cells, the microtrabecular system is reformed from material stored in the cortices and with the granules in the centrosphere. These observations suggest that the granules are suspended in a dynamic microtrabecular system that withdraws during pigment aggregation and is restructured during pigment dispersion. The microtubules guide linear granule motion not by defining physical channels, but by a recognizable affinity of microtubules, microtrabeculae, and granules for one another.

Enucleation techniques combining mild centrifugation in the presence of cytochalasin B permit cells to be separated into nuclear fragments (karyoplasts) and cytoplasmic fragments (cytoplasts). These fragments, though stable for a short time, will ultimately degenerate by the procedures described in this report. One can, however, fuse cytoplasts to karyoplasts by using polyethylene glycol and obtain viable reconstituted cells whose properties may be useful for understanding some aspects of the nuclear-cytoplasmic interactions associated with tumorigenicity and steroidogenesis. However, the presence of cybrids, hybrids, and parental whole cell contaminants along with the reconstituted cell population make it necessary to have genetic markers that reside in both the nucleus and cytoplasm in order to preferentially identify reconstituted cells derived from a karyoplast fused to a cytoplast. By utilizing the Y-1 cell line, which is tumorigenic and responds to corticotropin by secreting steroids, and the AMT-BU-A1 (AMT) cell line, which is nontumorigenic and does not respond to corticotropin but has a nuclear marker, BrdUrd r , and a cytoplasmic marker, CAP r , we have reconstituted cells containing Y-1 karyoplasts and AMT cytoplasts. In this report we extend our previous techniques by describing an identification procedure that allowed us to isolate cells reconstituted from AMT karyoplasts fused to Y-1 cytoplasts. The results of these experiments support the concept that with these cell lines the nucleus (karyoplast) is ultimately sufficient to control the phenotypic expression or suppression of tumorigenicity and steroidogenesis.

The frequency of cybrid colony formation was measured in fusions between enucleated chloramphenicol (CAP)-resistant mouse cells and CAP-sensitive mouse cells in varying ratios. By labeling the CAP-resistant cytoplasts with polystyrene beads and then performing the same fusions with CAP-sensitive cells, the frequency of cybrid fusions could be measured. Comparison of the frequency of viable cybrids (cybrid colonies) with the frequency of cybrid fusions showed that, with increasing fusion ratios of cytoplasts to cells, the proportion of cells fused to cytoplasts increased. Further, the viability of cybrid fusions increased from about 1 in 500 to nearly 1 in 60 over the range of cytoplast-to-cell ratios studied.

The present research presents a metholodogy for the bulk production of cytoplasts from suspension-culture-derived protoplasts of Solanurn nigrum (L.) containing cytoplasmically carried resistance to the herbicide atrazine. The ultimate purpose of this research is to use such cytoplasts in somatic fusion with protoplasts of atrazine—sensitive crop plants in an effort to transfer the herbicide resistance without concommitant transferance of nuclear genes of the weed species. Such a method is in contrast to those with similar aims such as the use of iodoacetate or irradiation inactivation of the donor nuclei.

The neutrophil activators phorbol 12-myristate 13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine, serum-treated zymosan, and IgG-coated latex cause an increase in protein phosphorylation in human neutrophil cytoplasts, concomitantly with an increase in oxygen consumption. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, phosphorylation was apparent in many proteins, must abundantly in 42-, 47-, 50-, 60-, and 80-kDa proteins. In neutrophil cytoplasts from autosomal chronic granulomatous disease (CGD) patients that were stimulated with PMA, the phosphorylation of a 47-kDa protein is absent. The localization of this protein in PMA-activated control cytoplasts is mainly in the cytosol and, to a lower and more variable extent, in the membrane. After addition of purified protein kinase C to lysates of nonstimulated control cytoplasts, phosphorylation occurred at the 47-kDa level in both the cytosol and the membrane fraction. With lysates of autosomal CGD cytoplasts, in vitro phosphorylation of the 47-kDa protein was completely absent. After separation of cytoplast proteins on a sodium dodecyl sulfate-polyacrylamide gel and excision of the 47-kDa protein(s), phosphorylation of the isolated 47-kDa band was observed in the presence of purified protein kinase C. This reaction was again absent when autosomal CGD cytoplasts were used as starting material. Our studies have identified the 47-kDa protein in neutrophil cytoplasts as a true substrate for protein kinase C and indicate that the defect in phosphorylation at the 47-kDa level in autosomal CGD cytoplasts is due to a defective protein.

Murine Balb/3T3 and murine A-MT-BU-A1 mammary tumor cells were separated in the presence of cytochalasin B into enucleated cytoplasmic components (cytoplasts) and nucleated subcellular components (karyoplasts). Karyoplasts were derived from 3T3 cells, while cytoplasts were derived from A-MT-BU-A1 cells that were both chloramphenicol-resistant (CAP r ) and sensitive to hypoxanthine/aminopterin/thymidine (HAT s ). CAP r has been shown to be cytoplasmically transmitted (possibly a mitochondrial gene mutation), while sensitivity to medium containing HAT has been shown to be transmitted by the nucleus (i.e., nuclear gene mutation). Such CAP r cytoplasts derived from A-MT-BU-A1 cells were then fused, using polyethylene glycol, to HAT-resistant 3T3 karyoplasts. The mononucleated reconstituted cells produced by such procedures were cloned in medium containing both HAT and CAP. Some of the reconstituted cells survived, because they were resistant to both drugs, while the nuclear and cytoplasmic whole cell contaminants were killed by one or the other of the two drugs. The results of these experiments indicate that reconstituted cells that are derived from two different cell lines are viable, as indicated by their ability for long-term proliferation in culture. Most of the clones derived resembled morphologically the 3T3 nuclear donor parent cells, but some of the clones did not resemble either parental cell line. It is anticipated that such selection techniques will permit more complete analysis of interrelationships between nucleus and cytoplasm.

Cybrid, reconstituted and hybrid cells were formed by fusing cytoplasts with whole cells, cytoplasts with nucleoplasts, or whole cells with whole cells, respectively. The cells or cellular fragments were derived from murine melanomas expressing melanocytic functions and from tumorigenic or non-tumorigenic fibroblasts. The cybrids and reconstituted cells resembled the nuclear donor parent in the expression of melanocytic activity and tumorigenicity. The hybrid cells resembled the fibroblastic parent when comparable numbers of chromosomes were present from each parent. A ratio of 1.5:1 in favor of the melanoma genome resulted in hybrids of either phenotype. The expression and extinction of melanocytic functions and the expression and suppression of tumorigenicity were transmitted only through the nucleus. The stable expression of A-type viral particles, on the other hand, may have been inherited also by cytoplasmic transfer.

basic problems in research on eukaryotic cells. The enucleation technique with cytochalasin B (CB) makes it possible to isolate viable reconstituted cells or cytoplasmic hybrids (cybrids) by fusing karyoplasts (or intact cells) to cytoplasts. Chloramphenicol-resistant (CAP’) cytoplasts from the double-mutant cells which are sensitive to a medium containing hypoxanthine, aminopterin and thymidine (HAT medium) and resist chloramphenicol (CAP) can be fused with HAT-resistant, CAP-sensitive cells (or karyoplasts). Such fusions have led to the isolation of CAP’-reconstituted cells or cybrids essentially free of contaminating parental cells that were eliminated by selection with both HAT and CAP [l]. We have thus isolated many CAP’-interspecific reconstituted cells and cybrids by fusing karyoplasts (or intact cells) of HAT-resistant, CAP-sensitive mouse melanoma Bl6 cells with cytoplasts of HAT-sensitive, CAP-resistant rat L,jTG l CAP’ cells. Expression of HGPRT activity and CAP-resistance are essential for the survival of cells in this selection medium. This paper describes the epigenetic modulation of the mouse HGPRT gene in cells reconstituted by fusing rat cytoplasts with mouse karyoplasts. By gel electrophoretic analysis, we found 2 components of HGPRT, a main and a minor component, in several mouse cell lines. Epigenetic modulation of the mouse HGPRT gene occurred in some of the reconstituted cells or cybrids isolated and grown as clones. In these clones the main band of HGPRT migrated much faster than the HGPRT of the parental Bl6 cells.

The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14- to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer than 200 species of polypeptides were detected; the major polypeptides made at this time were identified as the alpha A-, alpha D-, and beta-globin chains. The dormant erythrocyte nuclei were next reactivated to transcriptional competence by transplantation into enucleated mouse or chicken embryo fibroblasts, with frequencies of cytoplast renucleation of about 50 and 90%, respectively. Since large numbers of hybrid cells could be constructed, a biochemical analysis was possible. Electrophoretic analysis of the [35S]methionine-labeled polypeptides made in the hybrid cell types showed that polypeptides having the mobilities of only two (alpha A and alpha D) of the three major adult globin chains were made as major constituents of the hybrid cells. However, analysis of 14C-amino acid-labeled polypeptides revealed that a beta-like polypeptide that lacked methionine was also synthesized in large amounts. This polypeptide was tentatively identified as the early embryonic globin species rho. Globin synthesis was detected as early as 3 h after nuclear transplantation and as late as 18 h, the last time measured in these experiments. It appeared that globin polypeptides made at very early times were translated at least partially from chicken messenger ribonucleic acid introduced into the hybrid cells during fusion, whereas those made at later times were translated primarily from newly synthesized globin messenger ribonucleic acid. The potential usefulness of this hybrid cell system in analyzing mechanisms regulating globin gene expression is discussed.

Murine spleen and lymphoma cells were almost completely enucleated in suspension by centrifugation in discontinuous Ficoll gradients in the presence of cytochalasin-B. Viability of cytoplasts (cells lacking nuclei) and karyoplasts (nuclei) was over 90%. Pure fractions of cytoplasts or karyoplasts were then obtained by fractionation of the cytochalasin-B treated cells with unit gravity sedimentation in bovine serum albumin. Protein synthesis was seen in cytoplasts when they were stimulated with mitogens, such as lipopolysaccharide (LPS). This method can consistently yield pure fractions of morphologically and functionally intact karyoplasts or cytoplasts from normal lymphocytes with very low contamination of nucleated cells. Pure karyoplasts or cytoplasts obtained in this way could be extremely useful for subcellular analysis of lymphocyte activation.

A variety of novel methods have been developed in recent years for the genetic modification of plant cells. These include the techniques of protoplast and cytoplast fusion, uptake of cell organelles, and molecular techniques involving the uptake and integration of foreign DNA. Each of these aspects is adequately-covered elsewhere in this volume.

A method is described in which cytochalasin B is used to fractionate Ehrlich ascites tumor cells into cytoplasts and (nucleated) karyoplasts. The plasma membrane and cytoplasm are selectively removed from these cells by this method such that the cytoplasts rarely contain membranous organelles (e.g., mitochondria) which are retained in the karyoplast during fractionation. ATP concentrations similar to those found in whole cells and glycolytic activity were measured in cytoplasts in the presence but not the absence of glycose. Cytoplasts also actively transport Na+, K+, and α-aminoisobutyric acid to steadystate distribution ratios similar to those found in whole cells. It was concluded that these cytoplasts are a simplified model system for the study of active transport in Ehrlich cells.

Splenic lymphocytes of C3H/HeJ mice are defective in responding to the mitogenic activity of bacterial lipopolysaccharide (LPS), which has been attributed to a mutation at a single gene locus on chromosome 4 (ref. 5). It was suggested that C3H/HeJ mice lack a cell-surface receptor necessary for LPS recognition. However, specific binding of 125I-labelled lipid-A of LPS to B cells was found to be equivalent in LPS-responder and LPS-nonresponder C3H/HeJ, C57BL/10 ScCR and C57BL/10 ScN mice. This suggests that the genetic defect in C3H/HeJ mice is related to triggering mechanisms rather than to LPS binding. We recently established a method for separating mouse splenic lymphocytes into karyoplasts (minicells) and cytoplasts (enucleated cells) with high purity. This method makes possible the exchange of nuclei from one type of lymphocyte to another. In the present study, karyoplasts were purified from LPS-nonresponder C3H/HeJ mice and transferred to mitomycin C-treated or X ray-irradiated splenic lymphocytes taken from LPS-responder C3H/HeN mice by fusion with polyethyleneglycol. These reconstituted hybrid cells could respond to LPS, suggesting that nuclei from C3H/HeJ mice could be activated by LPS if proper signals were provided from cell-surface receptors. Moreover, responsiveness to concanavalin A (Con A) of mitomycin C-treated or X ray-irradiated lymphocytes was also recovered by the microinjection of karyoplasts from untreated lymphocytes.

The techniques of somatic cell hybridization have provided a valuable means of studying mechanisms of regulation of mammalian cell differentiation and transformation. Most previous studies have indicated that fusions between tumorigenic and nontumorigenic cells result in hybrid cells that are usually tumorigenic. In recent years it has been demonstrated that the phenotypic expression of tumorigenicity is at least partially due to the extensive chromosome loss that occurs in most interspecific and some intraspecific hybrid cells. In the present study we have utilized enucleation techniques that permit cells to be divided into nuclear (karyoplast) and cytoplasmic (cytoplast) cell fragments. Even though these nuclear and cytoplasmic fragments are metabolically stable for short periods of time, in our hands they ultimately degenerate. Viable cells can be reconstructed by PEG-induced fusion of karyoplasts to cytoplasts. Since reconstructed cells apparently do not segregate chromosomes, they may provide a clearer understanding of the interactions between the nucleus and the cytoplasm in the control of the expression of tumorigenicity. We have reconstructed cells using karyoplasts from the tumorigenic Y-1 cell line and cytoplasts from a nontumorigenic cell line, A-MT-BU-A1. In addition we have reconstructed cells containing Y-1 cytoplasts and A-MT-BU-A1 karyoplasts. The reconstructed cells porduced were assayed for tumorigenicity by their ability to grow in soft agar and in nude mice. The results of these experiments indicate that the reconstructed cells containing a tumorigenic nucleus and a nontumorigenic cytoplasm ultimately are tumorigenic and conversely the reconstructed cells containing a nontumorigenic nucleus and a tumorigenic cytoplasm are nontumorigenic. These experiments support the concept that with these cell lines the nucleus (karyoplast) is sufficient to control the phenotypic expression of tumorigenicity.

Mammalian cells can be separated into enucleated (cytoplast) and nucleated (karyoplast) fragments by subjecting cells to a centrifugal force in the presence of the fungal metabolite cytochalasin B. Cytoplasts and karyoplasts have been partially characterized regarding their ultrastrueture, enzymatic constitution, and their physiological behavior. The isolated cellular components can be fused to whole cells or to each other. The fusion of cytoplasts to whole cells provided the first demonstration of cytoplasmic inheritance in mammalian cells. Viable cells are formed by fusing the components to each other.

According to the current model of steroid hormone action oestrogen is thought to bind to its receptor in the cytoplasm of target cells1,2 and the oestrogen–receptor complex is then translocated into the nucleus2–4. This model is based on evidence obtained in homogenized cell preparations in which free receptor is associated with the cytosol, whereas steroid-bound receptor is associated with the nuclear fraction. Some data suggest, however, that the unfilled receptor may reside in the nucleus, and that cytosolic localization represents an extraction artefact. We have now reinvestigated the subcellular distribution of unfilled oestrogen receptor using cytochalasin B-induced enucleation to obtain cytoplast and nucleoplast fractions from receptor-containing GH3 cells derived from rat pituitary tumours. We found that cytoplasts prepared from GH3 cells contain little oestrogen-binding activity and that most of the unfilled oestrogen receptors are associated with the nuclear fraction. We therefore suggest that the standard model is in error and that the unoccupied receptor is nuclear in the intact cell.

Fusions in which one of the partners is a cytoplasmic fragment of a cell, or cytoplast, have been extensively used in recent years to investigate a variety of problems in biology. For example, cytoplasmic inheritance can be observed. Properties of a cell that can be transferred and maintained through its cytoplasm to a recipient cell are presumed to be controlled by cytoplasmic hereditary factors. Thus, cytoplasmic transfer of chloramphenicol (CAP) resistance in mouse and human cells suggested that mitochondrial DNA (mtDNA) controlled CAP resistance (Bunn et al., 1974; Wallace et al., 1975), and this suggestion has proved correct (Clark and Shay, 1980; Blanc et al., 1981). Similarly, the cytoplasmic transfer of murine intracisternal A particles (Malech and Wivel, 1976) and of microtubule-organizing centers (Shay et al., 1978) suggest at least a partial replicative autonomy for these particles.

An intracellular activity, which is induced by dimethyl sulfoxide (DMSO) or hexamethylenebisacetamide (HMBA) and leads to erythroid differentiation in mouse Friend cells, was characterized by cell fusion between genetically marked intact cells and cytoplasts. For this, a procedure for rapid selection of cybrids was devised by sensitizing non-fused cells with oligomycin. We were able to demonstrate that cytoplasts derived from DMSO- (or HMBA)-treated cells trigger erythroid differentiation upon fusion with UV-irradiated cells. The activity in the cytoplasts remained only transiently and its induction was inhibited by biologically active phorbol esters or cycloheximide. The activity, however, was not induced in cytoplasts by directly treating them with DMSO (or HMBA). These results indicate that (1) the intracellular erythroid-inducing activity is located in cytoplasts, (2) it acts in trans and induces erythroid differentiation as a dominant factor and (3) its production requires de novo nuclear protein synthesis. The mechanisms of the induction of the intracellular activity and of how it triggers erythroid differentiation are discussed.

Publisher Summary This chapter draws attention to the most significant recent advances in research on Acetabularia and related Dasycladales. Progress in research on Acetabularia and related Dasycladales has been particularly rapid in the past few years. The foundation of an “International Research Group on Acetabularia” and the organization of three international symposia have greatly stimulated investigations on these giant unicellular marine algae. New ideas have arisen from these meetings and what is equally important, a sincere wish for international collaboration. Acetabularia is certainly one of the most useful organisms for studies on cellular growth and differentiation in the absence of the nucleus. It is particularly suitable for investigating the subtle relationships, which exist between nucleus and cytoplasm and for examining the intergenomic cooperation inside the eukaryotic cell. The possibility of obtaining anucleate cells or cell fragments, regenerating nucleate basal parts, cytoplasts, isolated nuclei and organelles permits various types of in vivo and in vitro biological and biochemical experiments. Moreover, elegant intra- and interspecific grafts are possible with different species of Acetabularia.

The fine structure of Squalus acanthias (Elasmobranchii) semen was investigated to determine the cellular component responsible for the steroidogenic activity previously demonstrated in the seminal plasma of this species. Semen was found to consist of bundles of spermatozoa, many of which were encased in a sleeve of cytoplasm restricted to the tail region; large, dense bodies tacking a limiting membrane and numerous anuclear cytoplasmic remnants containing lipid droplets, mitochondria, areas of agranular reticulum, and possibly unreleased spermatozoa. These remnants, which we have termed cytoplasts, morphologically resemble in appearance Sertoli cells of S. acanthias at the time of spermiation. This structural similarity, plus the fact that many elasmobranch species slough the apical regions of Sertoli cells during the release of spermatozoa, indicates that the cytoplasts present in the semen of S. acanthias originate from Sertoli cells. Furthermore, the occurrence in these cytoplasts of organelles normally associated with steroid synthesis strongly suggests these structures are the source of steroidogenic enzymes in the semen of S. acanthias . The steroidal contribution to the semen by Sertoli cell cytoplasts may be necessary for either maturation or maintenance of spermatozoa in the excurrent reproductive ducts of S. acanthias .

Publisher Summary Prescott et al . reported an efficient, relatively simple technique for the enucleation of large numbers of cultured mammalian cells. The method entails plating cells on a glass coverslip or some other surface and centrifuging the surface, cell-side down in medium containing the mold metabolite cytochalasin B. Prescott's method or variations of it have found widespread application. The use of enucleated cells, or cytoplasts, thus prepared is justified by the findings that cytoplasts retain many of the morphological and behavioral characteristics of whole cells. The enucleation method of Prescott nevertheless has certain drawbacks. These include the fact that the yield is limited by the surface area that can be exposed to centrifugal forces, and the method cannot be used on cells that adhere poorly or not at all to solid surfaces. Authors have therefore explored the possibility that cells can be enucleated while in suspension by isopycnic centrifugation in the presence of cytochalasin B, and have developed a procedure of high efficiency and yield that can theoretically be applied to almost any eukaryotic cell lacking a cell wall.

Cytochalasin B (CB) was used to enucleate cells (cytoplasts) and to obtain karyoplasts (nuclei) from the human diploid fetal lung fibroblast strain WI-38. Fusion of cytoplasts and nuclei from young and old cells was accomplished with the aid of inactivated Sendai virus. Viable nuclei may be obtained from the karyoplast pellet after passage through a layer of bovine albumin which retains any contamination cytoplasts. The majority of successful fusions forming “whole cells” occurred when cytoplast from “old” cultures (PDL 40–51) and karyoplasts from “young” cultures were used (PDL 12–22), but almost always resulted in limited division of the viable reconstructed cells. When successful fusion occurred between “young” cytoplasts and “young” karyoplasts the number of cell divisions obtained was comparable to control cells kept under similar conditions.

Cytoplasmic hybrids (cybrids) were made by fusing the chloramphenicol resistant (CAP-r) cytoplast of one mouse species with the chloramphenicol sensitive (CAP-s) whole cell of another mouse species or subspecies. The mitochondrial DNA (mt-DNA) of parents and cybrids was analysed by restriction endonuclease digestion and electrophoresis. Two types of cybrids were found: 1) those that maintained a balanced combination of endogenous, CAP-s and alien, CAP-r mt-DNA, and 2) those that showed repopulation by the alien CAP-r mt-DNA. When cybrids of the first type were returned to medium without CAP, the CAP-r phenotype segregated concordantly with the mt-DNA of the CAP-r subspecies, indicating that they are linked. In cybrids that showed repopulation, no segregation was observed, indicating that substitution was probably complete. No evidence of recombination was found. The repopulated heterospecific cybrids may be useful in determining which polypeptides are encoded by the mt-DNA. When CAP-s cells were treated with purified mt-DNA from CAP-r strains, CAP-s cells were efficiently “transformed” to CAP-r. Only the endogenous CAP-s mt-DNA was detected in these transformants. Fine structure restriction analysis suggests that small regions of the CAP-s mt-DNA may be altered, perhaps by incorporation of some CAP-r mt-DNA.

We have examined the effects of enucleation and of inhibitors of mRNA synthesis (actinomycin D and cordycepin) on protein turnover of HeLa cells. Enucleation markedly inhibited the rate of protein degradation for short-lived proteins. However, cells centrifuged in the absence of cytochalasin B at the speed required to obtain cytoplasts showed protein degradation rates identical to those of cytoplasts, while inhibitors of mRNA synthesis did not affect the process. Although enucleation may affect degradation of specific proteins, these results suggest that centrifugation is largely responsible for the inhibition of protein degradation in cytoplasts.

The number and shape of pollen nuclei in cytologically refractive plants can be determined from anthers fixed in FAA, preserved in 70% ethanol, treated 10–30 min with 50% HCl in 95% ethanol, and stained with iron acetocarmine saturated with chloral hydrate, 1–16 hr. Pollen is then pressed from the anther into a drop of plain acetocarmine, warmed, examined and permanently mounted in Venetian turpentine medium. This procedure clears the cytoplasm of many opaque obstructions and frequently separates the exine from the cytoplast. It may be used with flowering material which has been stored several years at room temperature.

Summary 1. The cells of yeasts and filamentous fungi are surrounded by a cell wall. The cytoplast is bounded by a plasmalemma and contains nuclei, mitochondria, food granules and vacuoles. 2. Cell walls of vegetative fungal cells usually possess a microfibrillar ‘skeleton’, architecturally similar to that of plants, but composed of chitin or hemicellulose. True cellulose predominates only in the Oomycetes. Spore walls are often differently constructed from those of vegetative cells of the same species. 3. The cell contents (plasmalemma, endoplasm and endoplasmic reticulum, nucleus, mitochondria, vacuoles and miscellaneous granular inclusions), as seen in sections and in centrifuged material, are in general similar to those of plants. Some minor differences are indicated. The structure of the flagella of motile cells of the Lower Fungi is fundamentally similar to that of the flagella of lower plants and animals. 4. The electron microscope has contributed to our knowledge of growth and development of fungal structures. It has extended previous knowledge of the process of budding in yeasts, of the development of haustoria and of the formation and germination of fungal spores. 5. Poor nutrition, anaerobic conditions and sublethal irradiation cause abnormalities in the structure of mitochondria. Toxic amounts of irradiation prevent normal nuclear division in budding yeast cells. 6. Electron microscope studies have aided taxonomy by demonstrating fundamental structural differences between Fungi and Actinomycetes and between certain subdivisions of the fungi.

This study compared the developmental potential of bovine nuclear transfer embryos with varying amounts of cytoplasm. Embryos formed from single cytoplasts fused to blastomeres by a single electrical pulse or from double cytoplasts using a double electrical pulse resulted in reconstituted embryos containing 75% and 150% of the original oocyte volume. No differences in fusion, cleavage, or development rates to blastocysts were observed between the groups. Mean cell numbers 2 days after fusion were significantly lower in single-cytoplast clones. Cell numbers of resulting blastocysts were likewise significantly lower in single-cytoplast clones. Embryos formed by fusion of blastomeres with single cytoplasts using a single electrical pulse or from double cytoplasts using either a single or a double pulse resulted in reconstituted embryos containing 50%, 100% and 100% of the original oocyte volume. Again, no differences in fusion or cleavage rates were observed between groups, but the development to blastocysts at day 7 was significantly higher in double cytoplasts constructed with one fusion pulse than in single cytoplasts (P < 0.05). Mean cell numbers 2 days after fusion were significantly lower in single-cytoplast clones (P < 0.05), but at the blastocyst stage, no statistically significant differences in cell numbers were observed. The results of this study show that cytoplasmic volume plays a role in the development of nuclear transfer embryos. When using crude enucleation methods such as oocyte bisection, normal cytoplasmic volumes can be achieved by fusing double cytoplasts with embryonic blastomeres.

The developmental competence of mouse and human early embryos appears to be directly related to the metabolic capacity of a finite complement of maternally inherited mitochondria that appear to begin to replicate after implantation. Mitochondrial dysfunctions resulting from a variety of intrinsic and extrinsic influences, including genetic abnormalities, hypoxia and oxidative stress, can profoundly influence the level of ATP generation in oocytes and early embryos, which in turn may result in aberrant chromosomal segregation or developmental arrest. Deletions and mutations in oocyte mitochondrial DNA may subtend metabolic deficiencies or replication disorders in some infertile women and in women of increased reproductive age. Here, we describe methods for (i) the compartmentalization of mouse and human oocyte mitochondria into unique cytoplasts enriched for these organelles, and (ii) their transfer by microinjection into intact recipient oocytes. Metabolically active mitochondria in donor and recipient metaphase II stage oocytes were labelled with mitochondria-specific fluorescent probes, and the fate and location of donated mitochondria in recipient oocytes were followed by conventional epifluorescence and scanning laser confocal fluorescence microscopy. The net ATP content of undisturbed and recipient oocytes from the same cohort(s) was measured quantitatively at timed intervals after mitochondrial injection. The results demonstrate the feasibility of isolating and transferring mitochondria between oocytes, an apparent increase in net ATP production in the recipients, and the persistence of activity in the transferred mitochondria. The findings are discussed with respect to mitochondrial function and dysfunction in mammalian oocytes and embryos, and to the potential clinical applications of mitochondrial donation as they relate to the creation of heteroplasmic embryos.

The development of a somatic cell nuclear transfer procedure for the production of blastocyst stage cattle embryos is described. Bovine fetal fibroblasts were used for fusion experiments with surgically enucleated oocytes (cytoplasts) following the establishment of optimal parameters for electrofusion from isofusion contours. Fusion rates were increased by decreasing size of the cytoplasts used but cleavage was decreased by decreasing size of the cytoplast used (quarter, half and whole cytoplasts). The use of double cytoplasts did not improve cleavage, and development to blastocysts could not be achieved. In a comparison of electrofusion of fibroblasts with cytoplasts in the subzonal perivitelline space with intracytoplasmic injection of nuclei and parthenogenetically activated oocytes, 2%, 14% and 24% developed to blastocysts respectively. In the group injected with isolated nuclei, the passage number (4 to 9) had no apparent influence on developmental competence to blastocysts. The embryos produced by nuclear injection of somatic cell nuclei showed the normal pattern of cell surface appearance of TEC-3 and TEC-4 stage-specific epitopes during development, as seen in fertilized oocytes. We conclude that the nuclear injection of somatic cell nuclei is a relatively efficient way to clone bovine embryos.

The Nuclear Mitotic Apparatus (NuMA) protein is a multifunctional protein that is localized to the nucleus in interphase and to the poles of the mitotic apparatus during mitosis. In unfertilized porcine oocytes, NuMA is localized to the meiotic spindle. NuMA is removed along with the meiotic spindle during the enucleation process before reconstructing the egg by introducing the donor cell nucleus to produce cloned embryos. Questions have been raised regarding the source for NuMA in cloned embryos, as the enucleated oocyte does not contain detectable NuMA in the cytoplasm. To determine the source of NuMA in porcine nuclear transfer (NT) embryos, we conducted an immunofluorescence microscopy study with antibodies against NuMA to investigate the appearance and distribution of NuMA before and after reconstructing NT embryos with porcine skin fibroblasts. We used donor cells from a confluent culture with all cells in interphase. For comparative studies, we also determined the immunofluorescence pattern of NuMA, gamma-tubulin, and alpha-tubulin in porcine fibroblasts, parthenogenetic embryos and in vitro fertilized (IVF) embryos. Results show that NuMA was localized in nuclei of 33.5% (163/456) of the serum-deprived fibroblasts used as donor cells. No NuMA staining was detected in enucleated pig oocytes. Immediately after nuclear transfer, NuMA staining was absent in all donor cell fibroblast nuclei (0 h) but staining was detected by 6 h within the reconstructed eggs, at which time the transferred somatic cell nucleus swelled in most cells (19/27) and became a pronucleus-like structure. NuMA was localized exclusively within the pronucleus-like structures (15/27). At 25 h, NuMA was detected inside the nucleus (16/25) either in one-cell or in 2-cell stage embryos. Interestingly, in parthenogenetic embryos, NuMA staining was not detected in all 42 eggs examined at 1 h, and evident NuMA staining was only detected inside a few (4/51 at 6 h; 6/48 at 25 h) of the nuclei. In IVF embryos, NuMA was detected within the nucleus at 6 h (5/20) and 25 h (13/16). These results show that the donor cell nucleus contains NuMA that is contributed to the reconstructed embryo and possibly activated by mechanisms in the oocyte's cytoplast.

Mitochondrial DNA content remains constant between the mature egg and the blastocyst stage in mammals, making this the only period in development when genotypes segregate to daughter cells without the confounding effect of genotype replication. To analyze the segregation patterns of mitochondrial DNA during preimplantation development, we introduced polymorphic mitochondria either peripherally (cytoplast transplantation) or in the perinuclear vicinity (karyplast transplantation) into zygotes. Genotype ratios were significantly more variable among blastomeres from cytoplast (coefficient of variation = 83.8%) than karyoplast (coefficient of variation = 34.7%) reconstructed zygotes. These results suggest that heteroplasmy caused by polymorphic mitochondria positioned in the periphery of oocytes at the time of fertilization shows a more stringent segregation pattern than when the organelle is in the vicinity of the nucleus. Moreover, donor-to-host mitochondrial genotype ratios in karyoplast-derived groups increased significantly during development, particularly in the C57BL/6 group, where the ratio practically doubled between the four-cell (17.3%) and the blastocyst stage (29.6%). Although the mechanisms controlling this preferential replication of nuclear-type mitochondrial DNA are unknown, it is suggested that access to nuclear-derived transcription and replication factors could lead to the preferential replication of perinuclear mitochondrial genotypes during morula and blastocyst formation.

Until now, micromanipulators have been regarded as indispensable for somatic cell nuclear transfer. This paper describes an improved zona-free nuclear transfer procedure with manual bisection of oocytes, selection of cytoplasts by Hoechst staining, and two-step fusion of somatic cells from primary granulosa cell cultures with two cytoplasts. Blastocyst rates in the three systems tested for zona-free embryo culture were 0%, 18%, and 36% for microdrops, well of the wells (WOW system), and microcapillaries (GO system), respectively. This simple, rapid, and inexpensive procedure may become a useful alternative to the existing techniques for somatic cell nuclear transfer for large-scale application of the technology.

BACKGROUND: We assessed the maturational competence and the chromosomal pattern of mouse oocytes reconstructed by germinal vesicle (GV) transfer technique using nuclear and/or cytoplasmic components from cryopreserved GV stage oocytes. METHODS: From 657 GV oocytes (326 fresh and 331 frozen/thawed), four groups of reconstructed oocytes were obtained by micromanipulation and electrofusion: fresh GV-fresh cytoplast (FF), thawed GV-thawed cytoplast (TT), fresh GV-thawed cytoplast (FT), thawed GV-fresh cytoplast (TF). All reconstructed oocytes were cultured in vitro to metaphase II. RESULTS: Survival rate after manipulation and electrofusion, as well as progression to metaphase II, did not differ significantly among the four groups. Comparing reconstructed oocytes with fresh and thawed control pools, the only difference was a slightly but significantly higher maturation rate in the TT pool versus matched controls (P < 0.01). Cytogenetic analysis of 25 reconstructed oocytes showed the expected number of 20 chromosomes in 88% of them. CONCLUSIONS: We conclude that both nuclear and cytoplasmic components derived from cryopreserved immature oocytes are suitable for GV transfer procedure, and generate chromosomally normal oocytes able to progress to metaphase II in vitro. The possibility of using cryostored immature oocytes as a source of nuclei and cytoplasm could help in applying GV transfer procedure, both in research and clinical settings.

The denucleation of L cells and human lymphocytes in suspension was carried out by incubation with cytochalasin D25 and 50 microgram/ml and with ultracentrifugation over a discontinuous Ficoll gradient. Ultrastructural examination of the separate layers confirmed the presence of the anucleate cytoplasm (cytoplasts) with intact membrane and the separated nuclei (karyoplasts).

We have documented the presence of neutral endopeptidase 24.11 (NEP), on the surface of human neutrophils (PMN) and PMN cytoplasts. Cytoplasts are whole cell preparations which contain cytomatrix, but lack internal membranes and organelles ,such as nuclei and lysosomal granules. These structures have been extracted mechanically, leaving the plasma membrane “outside-out” topology intact. Cytoplasts are very useful in correlative studies of cell surface structure and function. Biochemically, the membrane component of cytoplasts is predominantly plasma membrane; structurally, chemical activity may be localized to domains of the intact cell surface. NEP is a membrane-bound metalloendopeptidase present in human PMN' s. We have marked NEP on the plasma membranes of PMNs and PMN cytoplasts via pre-embedding iramunocytochemistry. We used scanning electron microscopy (SEM) with backscattered electron imaging (BEI) to visualize Au labelled anti-NEP on the surface of a large number of cells. Transmission electron microscopy (TEM) was used to confirm the presence of the enzyme on PMN's and PMN cytoplasts.Suspensions of PMN or PMN cytoplasts (2 x 106 cells/ml) were fixed for 8 min at room temp. in 0.25% glutaraldehyde in phosphate buffered saline (PBS) pH 7.2 rinsed in PBS, treated with 0.1% glycine in PBS for 10 rain and then incubated for 15 min in 5% normal goat serum (NGS) in 0.1% bovine serum albumin dissolved in PBS (BSA/PBS). Following this step, cells were incubated for 20 min in anti- NEP antibody, rinsed in BSA/PBS, incubated in goat anti-rabbit IgG coupled to 15nm colloidal Au particles (GARG15) for 1 h and again rinsed in PBS. Postfixation for 30 min in 2.5% glutaraldehyde and PBS rinsing followed. For SEM a drop of cell suspension was put on a polylysine- treated Formvar-carbon-coated Au grid and cells were allowed to settle and attach for 30 min. The grid was rinsed in water, dehydrated and critical point dried. Cells were coated with carbon before viewing by SEM. For TEM, following immunolabelling, cells were post-fixed in OsO4, rinsed, dehydrated and embedded in Epon for sectioning.

Employing catalytic methods, the Mn- and Cu-content was determined in different cell fractions (chloroplasts, cytoplasts) of freeze-dried leaves ofValerianella olitoria isolated with a new dry method byBehrens et al.1. Cu was found essentially in the fractions with high chloroplast content; Mn was found mainly in the fractions with low chloroplast content.

Red Cell Membrane: Structure and Function. Edited by G. A. Jamieson and Tibor J. Greenwalt. (The American National Red Cross Second Annual Scientific Symposium, Washington, DC, May 1969.) Pp. 384. (Lippincott: 1969. Distributed in UK by Blackwell: Oxford.) £8.25.

Mammalian Cell Membranes. Vol. 4: Membranes and Cellular Functions. Pp. 262. Vol. 5: Responses of Plasma Membranes. Pp. 260. Edited by G. A. Jamieson and D. M. Robinson. (Butterworths: London and Boston, Massachusetts, 1977.) £18 each volume.