Isolation,Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.     

Substructure of inner mitochondrial membranes of myocardial cells as shown by cryo-ultramicrotomy

Summary The substructure of the inner mitochondrial membranes has been studied by cryo-ultramicrotomy under conditions during which denaturation of proteins by treatment with chemical solutes has been totally avoided. In such preparations, the inner membrane has a substructure consisting of globular subunits. These subunits have an average diameter of ca. 20Å–ca. 62Å and are fairly regularly spaced. Intracristal space is absent in the unstained, freeze-dried preparations, whereas a space of ca 40Å is seen in preparations lightly treated by OsO₄-vapour. It is concluded that the subunits of the inner mitochondrial membranes probably consist either of single protein molecules or of complexes of protein molecules.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

Immunolocalization of the substances P- and FMRFamide in the atrium of the snail <Emphasis Type="Italic">Achatina fulica</Emphasis>

The methods of immunohystochemistry and electron microscopic immunocytochemistry were applied for the investigation of the localization of the substances P (SP)- and FMRFamide in the auricle of the mollusc Achatina fulica. Nerve fibers that innervate a snail auricle are in close contact with the granular cells (GC) disposed among muscular and endocardial cells, forming the neuroendocrinal complexes. Both neuromediators were detected in the cells of these auricular neuroendocrinal complexes. The method of immunoperoxidase histochemistry has shown the localization of SP- and FMRFamide immunoreactive material in granules of auricular GCs. Electron microscopic immunocytochemistry has confirmed the presence of SP- and FMRFamide-immunoreactive material in GC granules; moreover, it has also shown its presence in the neurosecretory granules of nerve fibers, both located in neuroendocrinal complexes in contact with cardiomyocytes.     

Changes in the fine structure of the testicular Leydig cells of the seasonally-breeding bat,Myotis adversus

Summary Leydig cells of the bat, Myotis adversus, have been examined by electron microscopy throughout fourteen months. During the breeding season the Leydig cells become hypertrophied and are characterised by prominent areas of agranular endoplasmic reticulum and numerous small, membrane-bound granules. Microperoxisomes are also observed. During the period of testicular regression. Leydig cell size and the number of membrane-bound granules are greatly reduced. Lipid droplets and dense bodies are more numerous.     

Calcium and magnesium levels in isolated mitochondria from human cardiac biopsies

Summary A non-enzymatic method is presented for isolating mitochondria from small-sized human cardiac samples, including ventricular needle biopsies of 15–25 mg of wet weight. Electron microscopy demonstrates that these fractions are rich in structurally well preserved mitochondria. Calcium and magnesium levels of fractions are determined by atomic absorption flame spectroscopy. Comparative analyses are made in similar fractions of the mouse ventricle. Calcium concentrations of mitochondria isolated in the presence of ruthenium red do not differ significantly between the human auricle and ventricle, averaging 61 nmol Ca/mg protein and 68 nmol Ca/mg protein, respectively. Mitochondrial calcium level is lower in the mouse ventricular fractions, averaging 7 nmol Ca/mg protein. Mitochondrial magnesium amounts to slightly less than 60% of the calcium levels in the human heart, while it exceeds the calcium level by more than 100 per cent in the mouse heart. There is no significant difference of mitochondrial calcium between normal auricles, and, auricles of patients with increased right atrial mean pressure and/or volume overload.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

On the origin of Z-band material and myofilaments in myoblasts from the human atrial wall

Summary The origin of cardiac myofibrils in cells from the atrial wall in human embryos was studied. Z-band substance appears throughout the cytoplasm as irregular electron dense patches in a network of thin filaments. The thin and thick filaments are synthesized as separate units in the sarcoplasm and are later aggregated into myofibrils. Complexes of Z substance and thin filaments occur numerously at different stages of myofibrillar organisation. Thick filaments are formed in close proximity to free ribosomes and are later incorporated in an hexagonal pattern into the Z-band/thin filament complex.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy

The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity-- suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.     

Calcium containing particles in mitochondria of heart muscle cells as shown by cryo-ultramicrotomy and X-ray microanalysis

Summary Mitochondria of normal myocardial cells of the sand rat and the mouse as well as of the left ventricle of man, have been examined for their content of calcium. Ultrahistochemistry and X-ray microanalysis revealed two basically different inclusions: Osmiophilic mitochondrial granules and Spherical mitochondrial particles. Osmiophilic mitochondrial granules were found in conventionally fixed and plastic embedded tissues as well as in cryosections of chemically fixed and sucrose infused tissues. Such granules lacked inert electron density and probably consisted mainly of unsaturated lipids. X-ray spectra obtained from these tissues revealed no peaks for calcium. Spherical mitochondrial particles were present in dry-cut cryo-sections of N₂-frozen tissues not treated by fixatives and/or cryoprotectants. These particles were deeply electron dense in unstained, freeze-dried cryo-sections. They usually measured from 600Å–900Å in diameter in the normal myocardium of the sand rat and the mouse and from 250 Å–400Å in diameter in the left ventricular myocardium of man. Significant calcium peaks could be identified in the X-ray spectra of these particles, whereas none occurred in the analyses of other tissue regions. Potassium was detected with about equal frequency in the particles and in other parts of the tissue. On the basis of the inert electron density of the particles and their absence in chemically fixed tissues as well as of the results of the X-ray analysis, it is concluded that they contain precipitates of extremely labile ions of mitochondrial calcium.We should like to thank Mrs. Trine Jensen for skillful technical assistance. This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from the Norwegian Research Council for Science and the Humanities     

Outgrowth in vitro of cervical epithelium separated from the uterovaginal anlage of newborn mice

Summary After gentle trypsinization, the pseudostratified columnar Müllerian epithelium that lines the uterine cervix of newborn mice could be separated from the enclosing stromal tissue. Pure epithelial tubes explanted in vitro and were allowed to grow in a standard medium for 3–4 days forming a confluent colony of rather closely-fitting cells. The cell sheet was studied by a preparatory technique that allows examination of a large number of cells with preserved intercellular spatial orientation. Attempts were made to identify cultured cells according to the morphology of cell types in the cervicovaginal epithelium in vivo.Electron micrographs revealed that, close to the explant, the cultured cell sheet exhibited several features similar to the Müllerian epithelium in vivo. Outside these central areas of the colony was a broad transitional zone consisting of thin platelike cells distinguished by an abundance of microfilaments. At the periphery of the colonies, bulky cells possessing microvilli and a vacuolated cytoplasm tended to overlap adjoining platelike cells. These bulky cells had a morphology resembling that of the superficial cells seen in the upper vagina and common cervical canal of immature and diestrous animals. The epithelial development in the cultures apparently simulated the transformation in vivo from a pseudostratified Müllerian epithelium in the newborn to a stratified epithelium resembling that of the uppermost vagina and common cervical canal of immature animals. Judged by morphological and cytochemical criteria, the Müllerian cells in the outgrowth obviously had many changed features. It thus seems questionable whether the cells grown in vitro are comparable with the corresponding cells in vivo when used for experiments requiring the controlled conditions of the culture environment.Supported by grants from the Norwegian Research Council for Science and the Humanities and from the Norwegian Cancer Society     

Immunocytological studies on an estradiol sensitive antigen in the cervicovaginal epithelium of neonatal mice

Summary Cells of the cervicovaginal epithelium of neonatal mice underwent morphological changes in response to estradiol injection. On the luminal border, estradiol treatment caused development of distinct microvilli and a prominent surface coat of delicate filamentous material. Very deep nuclear folds appeared, and the border between adjacent cells became strongly interdigitated. The cells developed a pronounced smooth and rough endoplasmic reticulum, and dark-stained membrane-bounded granules accumulated in the apical part of the cells.Estradiol promoted increased production of an antigenic material specific for the cervicovaginal epithelium (CVA). Immunofluorescence studies demonstrated CVA in the most apical part of the cells, in the extracellular material on the epithelial surface, and in the intercellular spaces between adjacent epithelial cells. This was confirmed by immunoferritin methods, which revealed that the antigen was localized to the surface coat and to material adhering closely to the exterior of the cell membrane, the part facing the lumen and also the part facing intercellular spaces. Within the cells, ferritin tagging was recognized around the membranes enclosing the dark-stained granules in the apical part of the cells and also on the inside of the luminal cell membrane. This is so interpreted that CVA acquires its antigenic properties when passing out from the dark-stained granules through the surrounding envelope. CVA apparently forms part of the glycocalyx of the cervicovaginal cells.This investigation was supported by grants from the Norwegian Cancer Society (Landsforeningen mot Kreft) and the Norwegian Research Council.     

Insulin-like growth factors,hepatocyte growth factor and transforming growth factor-alpha in mouse tongue myogenesis

Many reports have shown that tongue striated muscles have several unique characteristics not found in other skeletal muscles such as limb and trunk. Several peptide growth factors are reported to play important roles in skeletal myogenesis. In this article, the roles of insulin-like growth factors (IGF), hepatocyte growth factor (HGF) and transforming growth factor (TGF)-alpha in mouse tongue myogenesis were studied using an organ culture system of the mandible or tongue obtained from mouse embryos. It was found that IGF-I promotes the differentiation of tongue myoblasts. HGF plays an essential role in the migration and proliferation of tongue myogenic cells, and inhibits the differentiation of tongue myoblasts. TGF-alpha does not play an essential role in the proliferation of tongue myogenic cells, but does promote the early differentiation of tongue myoblasts. The role of IGF-I in the differentiation of tongue myoblasts, and that of HGF in the migration, proliferation and differentiation of tongue myogenic cells appear to be almost identical to their roles in the myogenesis of limb and cultured myogenic cell lines. However, the role of TGF-alpha in the proliferation and differentiation of tongue myogenic cells appears to be different from its role in the myogenesis of limb and cultured myogenic cell lines such as C2 and L6.     

Sod2 overexpression preserves myoblast mitochondrial mass and function, but not muscle mass with aging

Mice lacking superoxide dismutase-2 (SOD2 or MnSOD) die during embryonic or early neonatal development, with diffuse superoxide-induced mitochondrial damage. Although stem and progenitor cells are exquisitely sensitive to oxidant stress, they have not been well studied in MnSOD2-manipulated mouse models. Patterns of proliferation and differentiation of cultured myoblasts (muscle progenitor cells), PI3-Akt signaling during differentiation, and the maintenance of mitochondrial mass with aging using myoblasts from young (3–4 week old) and aged (27–29 months old) MnSOD2-overexpressing ( Sod2- Tg) and heterozygote ( Sod2 ^{+/ −}) mice were characterized by us. Overexpression of MnSOD2 in myoblasts had a protective effect on mitochondrial DNA abundance and some aspects of mitochondrial function with aging, and preservation of differentiation potential. Sod2 deficiency resulted in defective signaling in the PI3-Akt pathway, specifically impaired phosphorylation of Akt at Ser473 and Thr308 in young myoblasts, and decreased differentiation potential. Compared with young myoblasts, aged myoblast Akt was constitutively phosphorylated, unresponsive to mitogen signaling, and indifferent to MnSOD2 levels. These data suggest that specific sites in the PI3K-Akt pathway are more sensitive to increased superoxide levels than to the increased hydrogen peroxide levels generated in Sod2 -transgenic myoblasts. In wild-type myoblasts, aging was associated with significant loss of mitochondrial DNA relative to chromosomal DNA, but MnSOD2 overexpression was associated with maintained myoblast mitochondrial DNA with aging.     

Ultrastructure of atrial and ventricular myocardium in the pike Esox lucius L. and mackerel Scomber scombrus L. (Pisces)

Summary Atrial and ventricular muscle in the pike and mackerel hearts consists of narrow, branching cells. The atrial cells in the two species are similar whereas the ventricular cells differ. The sarcolemma is attached to the Z and M lines of the sarcomere. Intercalated discs are common, and the transverse parts display desmosomes and intermediate junctions. Nexuses are uncommon and only occur in the longitudinal parts of the intercalated discs. The sarcoplasmic reticulum forms a regular hexagonal network on the myofibrillar surface. Subsarcolemmal cisternae form peripheral couplings at the I-A level. Junctional processes are usually inconspicuous, but an electron dense substance is present between the sarcolemma and the Junctional sarcoplasmic reticulum. Specific heart granules are common in atrial cells of both species and in ventricular cells of the pike, but are very scarce in mackerel ventricular muscle.This work was supported by grants from the Norwegian Research Council for Sciences and the Humanities     

Reduced myotube diameter,atrophic signalling and elevated oxidative stress in cultured satellite cells from COPD patients

The mechanisms leading to skeletal limb muscle dysfunction in chronic obstructive pulmonary disease (COPD) have not been fully elucidated. Exhausted muscle regenerative capacity of satellite cells has been evocated, but the capacity of satellite cells to proliferate and differentiate properly remains unknown. Our objectives were to compare the characteristics of satellite cells derived from COPD patients and healthy individuals, in terms of proliferative and differentiation capacities, morphological phenotype and atrophy/hypertrophy signalling, and oxidative stress status. Therefore, we purified and cultivated satellite cells from progressively frozen vastus lateralis biopsies of eight COPD patients and eight healthy individuals. We examined proliferation parameters, differentiation capacities, myotube diameter, expression of atrophy/hypertrophy markers, oxidative stress damages, antioxidant enzyme expression and cell susceptibility to H₂O₂ in cultured myoblasts and/or myotubes. Proliferation characteristics and commitment to terminal differentiation were similar in COPD patients and healthy individuals, despite impaired fusion capacities of COPD myotubes. Myotube diameter was smaller in COPD patients (P = 0.015), and was associated with a higher expression of myostatin (myoblasts: P = 0.083; myotubes: P = 0.050) and atrogin‐1 (myoblasts: P = 0.050), and a decreased phospho‐AKT/AKT ratio (myoblasts: P = 0.022). Protein carbonylation (myoblasts: P = 0.028; myotubes: P = 0.002) and lipid peroxidation (myotubes: P = 0.065) were higher in COPD cells, and COPD myoblasts were significantly more susceptible to oxidative stress. Thus, cultured satellite cells from COPD patients display characteristics of morphology, atrophic signalling and oxidative stress similar to those described in in vivo COPD skeletal limb muscles. We have therefore demonstrated that muscle alteration in COPD can be studied by classical in vitro cellular models.     

Development and fate of the secretory granules of juxtaglomerular epithelioid cells

Summary The development and fate of the secretory granules in murine, rat and human juxtaglomerular epithelioid cells were examined using ultrastructural and immunocytochemical methods. The formation of mature renin granules occurs by fusion of rhomboid protogranules followed by coalescence of their paracrystalline contents, and by the fusion of roundish juvenile granules having an amorphous internum. Protogranules with paracrystalline contents are prominent in animals with stimulated renin synthesis, indicating an overcharge in processing and/or packaging of the secretory product, renin, under these conditions. Various similarities between lysosomes/multivesicular bodies (MVBs) and juvenile renin granules have been observed. With the exception of small MVBs, no renin-negative organelles that could be regarded as lysosomes were found in epithelioid cells of mice and rats. Therefore, we suggest that renin granules are modified lysosomes. Immunocytochemical findings indicate that juvenile secretory granules of epithelioid cells represent the converting and activating compartment for prorenin. Endocytosed foreign tracers such as HRP or cationized ferritin are preferentially internalized by juvenile renin granules, which hence appear to be outstanding by their fusogeneity. Consequently, juvenile granules are probably responsible for the secretion of prorenin, and mature granules for that of active renin.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg     

Season-Induced Changes in the Liver Cells and Ovarian Tissues During Growth,Maturation, Spawning and Post-spawning Phases in Spiny Eel <Emphasis Type="Italic">Mastacembelus</Emphasis> <Emphasis Type="Italic">armatus</Emphasis> (Lacepède, 1800)

Cytological status of hepatocytes in respect to seasons influencing ovarian activities has been studied in Mastacembelusarmatus (Hamilton). Histologically, the hepatocyte of female liver was provided with distinct nucleus and basophilic cytoplasmic granules. In M. armatus different germ line cells were recognized on the basis of size and histoarchitecture of the cells. By examining seasonal changes in relation to hepatocytes and ovary, it was found that during growth and maturation phases the density of cytoplasmic granules of hepatocytes was increased in number as well as the nucleus become hypertrophied. These features were well correlated with the occurrence of cortical alveolus and yolk granule stage in the ovary. During spawning phase the cytoplasmic granules were sparse in the hypertrophied hepatic cells. This was due to the dynamic cytological activities like vitellogenesis and occurrence of mature oocytes in the ovary. However, no significant alterations were noticed in the hepatocytes during post spawning phase due to the appearance of new germ line cells in the ovary. Cytological changes in the hepatocytes were correlated with the reproductive phases in female M. armatus.     

Effect of scavengers of active oxygen species and pretreatment with acetyl-salicylic acid on the injury to cultured endothelial cells by thrombin-stimulated platelets

Summary Thrombin-stimulated human platelets adhere to and injure cultured human endothelial cells. We hypothesize that generation of active oxygen species by the stimulated platelets are involved in the injury. To confirm this, catalase [final concentration (8.25 μg/ml)], superoxide dismutase (SOD) (10 μg/ml), ofd-mannitol (9 mg/ml) were added to the cell culture medium before the experiments. Platelet suspension (200.000/μl) and thrombin (4 U/ml) were added and the culture dishes shaken for 15 min at room temperature. In separate experiments the endothelial cells were pretreated with acetylsalicylic acid (0.05, 0.1, or 0.5 mM) to test whether the arachidonic acid metabolism of the endothelial cells is involved in the injury process. In preliminary experiments we were able to confirm that platelets, when stimulated by thrombin, produce chemiluminescence which was suppressed by mannitol but not by catalase or SOD. The degree of injury to cultured endotheial cells by thrombin-stimulated platelets, as measured by release of⁵¹Cr from prelabeled endothelial cells, was reduced significantly with the presence of mannitol, but only moderately when catalase or SOD had been added. Morphometric quantification based on scanning electron micrographs of the endothelial cells after exposure to thrombin-stimulated platelets in the presence of catalase or mannitol showed a reduced number of injured cells. Pretreatment of the endothelial cells with acetylsalicylic acid did not cause any significant change in the degree of endothelial cell injury as measured by the⁵¹Cr release. It is concluded that active oxygen species, in particular hydroxyl radicals, may be generated during thrombin stimulation of platelets and cause injury to the endothelial cells. This work was supported by the Norwegian Research Council for Science and the Humanities and the Norwegian Council for Cardiovascular Diseases. We express our gratitude for their grants.     

Cytological studies of developing muscle with special reference to myofibrils,mitochondria, Golgi material and nuclei

Summary Developing striated muscle in the chick embryo was studied with special reference to the cytological changes which occur as the myofibrils are being elaborated. Observations were made on the mitochondria, cytoplasmic granules, Golgi material, neutral red bodies, nuclei, and nucleoli, and on the developing fibrils. Particular attention was directed to the early part of the histogenetic process when a few primary myofibrils are being formed and becoming striated. This period is characterized by hypertrophy of the Golgi material, nuclei, and nucleoli.The myofibrils were found to arise in close association with filamentous mitochondria, apparently at the expense of the numerous small cytoplasmic granules which fill the early myoblasts. The mitochondria change staining reaction during this time and to a certain extent disappear. Many ring-shaped mitochondria, the significance of which is not known, were found among the unchanged mitochondria which remain near the nucleus.At first the myofibrils are homogeneous and do not take the stain well. Later they stain heavily, although they remain homogeneous, and finally striation appears. Evidence was presented that the Z-membrane begins development before striation is visible.When the primary fibrils have become striated, formation of myofibrils by the above method apparently ceases and further increase in the number of fibrils is probably brought about by longitudinal splitting of those formed first.At this time, also, the Golgi material decreases in amount, the nuclei become smaller, and the nucleoli break up and lose their distinct form.A secondary fibril system was also described which, it is believed, is distinct from the myofibril system.Mitotic division followed by cleavage of the cytoplasm occurs in the earliest myoblasts, probably for the purpose of increasing the number of these cells. However, as soon as fibrillization sets in, mitotic division ceases and the nuclei multiply by amitosis not followed by cleavage of the cytoplasm.The nucleoli divide before amitotic division of the nuclei. Evidence was presented that certain nuclear granules may be associated with the amitotic division process.The Golgi material in embryonic muscle is situated at the two poles of the nuclei. This furnishes evidence in favor of the belief that the Golgi material of adult muscle has a similar location and is not represented by the Cajal-Fusari network.Neutral red granules were demonstrated near the nucleus and in the axial cytoplasm, but they did not coincide in position with the Golgi material.It was noted that the cytological changes which take place when the myofibrils are being elaborated in the developing myoblasts resemble those in gland cells at the time secretory materials are being formed.