An electron microscope cytochemical study of calcium ion localization in the honey-bee flight muscle.

The authors studied the sarcomere ultrastructure and distribution of calcium ions in the cross striated flight muscles (longitudinal-dorsal and tergosternal) of the honey bee after being fixed in osmium tetraoxide and buffered to 7.4 pH and also according to the method of Carasso and Favard (1966). they ascertained an intensive and constant reaction to the presence of calcium on the level of myofilaments within the periphery of the sarcomeres and light and dark mitochondria. Calcium concentrations are not detectable in the Z line and M line. The authors carried out a control of the results using the method of Carasso and Favard (1966).     

Morphometric in vitro analysis of the control of the activity of the neurosecretory dark green cells in the freshwater snail Lymnaea stagnalis (L.)

Summary The intracellular localization of calcium by means of cytochemical techniques was studied in smooth muscle cells of mouse intestine. When the lead acetate method according to Carasso and Favard (1966) was used calcium was found in mitochondria and sarcoplasmic reticulum and occasionally between the myofilaments. The active ATP-dependent accumulation of calcium into cell structures was investigated by the oxalate method (Heumann and Zebe, 1967). After appropriate treatment the only structures of smooth muscle cells which contained calcium oxalate (identified by microprobe analysis) were elements of the sarcoplasmic reticulum.The results are discussed in relation to the role of calcium in the control of muscle activity during the contraction-relaxation cycle.The electron probe microanalysis was carried out at SIEMENS (Berlin) in collaboration with Dr. von Muschwitz. I thank Miss M. Schlatter for her skillful assistance. The investigation was supported by the Deutsche Forschungsgemeinschaft.     

Ultrastructural changes in the muscle fibers of the rat diaphragm and the dynamics of intracellular calcium redistribution as affected by an anticholinesterase substance--chlorophos

Ultrastructural changes of the rat diaphragm muscle fibers and electron histochemical distribution of calcium ions were studied following chlorophos administration in 5, 15 and 45 minutes (dose - 300 mg/kg, intraperitoneally). The local swelling of mitochondrial matrix and the appearance of contractures were found first in postsynaptical region. Then the postsynaptical alterations increased; the swelling and fragmentation of sarcoplasmic reticulum were observed in addition to desorganization of mitochondrial ultrastructure. Granules of the histochemical product were revealed in mitochondria, in the sarcoplasmic reticulum and in filaments. Changes in distribution of calcium ions in the rat diaphragm muscle fibres after chlorophos administration and the role of Ca++ the in the mechanism of muscle alteration discussed.     

Distribution of calcium ions in the muscle fibers of the rat diaphragm depending upon their state and the method of histochemical treatment

Distribution of calcium ions in the rat diaphragm muscle fibers has been studied electron histochemically using various fixation techniques and chemical treatment of the tissue. When potassium pyroantimonate in water solution is used after a short perfusate fixation with aldehydes, the reaction product granules are revealed in mitochondria, in the disk I, in the center of the disk A, more seldom the precipitate is found in the sarcoplasmic reticulum (SR) and in the T-system. The presence of calcium ions in the precipitate is proved by means of treatment the preparations with ethylenglycol- and ethylen-diamine-tetra-acetic acids. When contracture is resulted from potassium rhodanide administration, in mitochondria the reaction product granules decrease in their number, the precipitate disappears from the central part of the disk A, while the number of the granules increases in the SR terminal cisterns. The data obtained are compared with calcium ions distribution observed at the freezing-substitution method without an additional chemical fixation, as well as the histochemical fixations after Oschman method and at a usual fixation with OsO4. Certain similarity is revealed in distribution of the calcium pyroantimonate granules at aldehyde fixation and when the freezing-substitution method is used.     

A 45Ca autoradiographic and stereological study of freeze-dried smooth muscle of the guinea pig vas deferens

In an effort to more clearly elucidate the role of cellular structures as calcium sinks and sources in smooth muscle cells, the intracellular distribution of radioactive calcium was evaluated by a new method based on freeze-drying. The guinea pig vas deferens was exposed to a physiological salt solution that contained 45Ca. The muscle was then freeze-dried and prepared for electron microscope autoradiography. The grain density over the plasma membrane, mitochondria, and sarcoplasmic reticulum (SR) was significantly greater than that of the matrix. These results suggest that the plasma membrane, mitochondria and SR have the capacity to accumulate calcium. Which of these structures serve as a source of calcium for contraction remains to be determined. A stereological comparison between freeze-dried and conventionally prepared smooth muscles revealed several differences. The cross- sectional area of freeze-dried cells was about twice that of conventionally prepared cells. Moreover, mitochondria and sub-surface vesicles occupied a significantly smaller percentage of the cell in the freeze-dried tissue than they did in the conventionally prepared tissue.     

Calixarene C-91 stimulates Ca2+ accumulation in the myometrium mitochondria

Calixarenes, owing to the ability to form supramolecular complexes with biologically important molecules and ions, can influence a course of biochemical processes and, accordingly, be considered as perspective molecular platforms for creation of physiologically active compounds. The work purpose is to study calixarene C-91 influence on systems of active Ca ions transport which are localized in subcellular membrane structures (mitochondria, sarcoplasmic reticulum, plasma membrane) of myometrial cells. It has been shown, that calixarene C-91 addition to incubation medium led to an increase in Ca2+ accumulation level in mitochondria. The maximal stimulating effect was 173% and it was observed at 100 microM concentration. It is suggested, that calixarene C-91 can enter mitochondria with the subsequent precipitation of Ca ions in a matrix therefore calcium capacity increases, and as a consequence, higher Ca2+ accumulation in these structures is observed. In a wide range of concentration (1-100 microM) calixarene C-91 did not influence a level of Ca2+ accumulation in sarcoplasmic reticulum of myometrial cells. Titration of solubilized Ca2+, Mg2+-ATPase by calixarene C-91 (0,1-100 microM) did not cause changes in its activity. Thus, calixarene C-91 increases Ca2+ accumulation level in mitochondria, but practically does not influence calcium pumps activity of a plasma membrane and sarcoplasmic reticulum of myometrial cells.     

Ultrastructural and gene-expression changes in the calcium regulation system of rat skeletal muscles under exhausting exercise

The expression of genes responsible for the synthesis of essential proteins regulating the calcium-ion balance and ultrastructural characteristics of fast-twitch (m. extensor digitorum longus, EDL) and slow-twitch (m. soleus, SOL) skeletal muscles under prolonged exercise were studied in an experimental model of forced-swimming rats. A day after the end of the exercise, no significant changes in any of the five investigated genes were revealed in the SOL. A few triad elements (T-tubules and cisternae of sarcoplasmic reticulum) were revealed. A small number of excitation-contraction coupling (ECC) structures in the control and a slight increase in their amount after exercises were noticed. Polymorphism and mitochondrial defects within SOL muscles indicate the importance of these structures in the regulation of calcium balance. In EDL muscles, adaptation mechanisms are aimed mainly at pumping Ca²⁺ ions to the sarcoplasmic reticulum, where the main calcium buffer is calsequestrin. Expression of SERCA1 gene increased by an order of magnitude, and that of CASQ1 increased by three times. Electron microscopy showed a major role of triads in the maintenance of calcium homeostasis in the EDL muscles, as well as a greater destruction of these muscles compared to SOL after exhausting exercise. The high level of triads and a possible activation of the CICR (calcium-induced calcium release) mechanism in fast-twitch muscles can cause damage to them during exhausting exercise. Adaptation of SOL muscles is associated with structural rearrangements of the mitochondrial apparatus, while adaptation of the EDL muscles is caused by calcium removal from the sarcoplasm with Ca-ATPase and its retention in the sarcoplasmic reticulum by calsequestrin.     

Characteristics of functioning of electromechanical coupling in striated muscles of higher and lower vertebrates

A comparative pharmacological analysis of relative contributions of different signal transduction pathways in the activation of contraction (excitation-contraction coupling, ECC) in intact fast striated muscles of frog and lamprey was performed. It was found that the major mechanism responsible for the ECC in muscles of both animals is Ca2+ release from the sarcoplasmic reticulum through the ryanodine-sensitive channels. However, the ECC in lamprey muscle displays some important differences in the units of electromechanical coupling, which precede the calcium release from sarcoplasmic reticulum. The maximum contraction force in frog muscle develops during caffeine-induced contracture, which indicates that all Ca2+ stored in sarcoplasmic reticulum is released through ryanodine-sensitive channels. In contrast, in lamprey muscle, the maximum force develops not in response to high caffeine concentration, but in response to repetitive electrical stimulation. Hence, in addition to stores liberated by ryanodine-sensitive channels, some other sources of calcium ions should exist, which contribute to the contraction activation. A source of this additional Ca2+ ions can be external medium, because acetylcholine contracture is abolished in a calcium-free medium. In frog muscle, the acetylcholine contracture was abolished in a Na(+)-free solution. It was concluded that in frog muscle ECC can be triggered by changes in the transmembrane potential (depolarization-induced calcium release), while in lamprey muscle the entry of calcium ions into myoplasm as the trigger in ECC (calcium-induced calcium release). The lamprey muscle was found to be more resistant to tetrodotoxin and tetracaine, which is indicative of a role in the activation of contraction of tetrodotoxin-resistant Na+ and/or Ca2+ channels. It was concluded, that ECC mechanism in striated muscles of low vertebrates is not limited by the generally accepted scheme of depolarization-induced calcium release but can include some other schemes, which require the Ca2+ influx into the cell.     

Effect of Mg ions and spermine on ATP-dependent Ca2+ transport in myometrial intracellular structures. I. Comparative study of Ca2+ accumulation in mitochondria and sarcoplasmic reticulum

In experiments, which were carried out with the use of a radioactive label (45Ca2+) on the suspension of rat uterus myocytes treated by digitonin solution (0.1 mg/ml), influence of Mg ions and spermine on Mg2+, ATP-dependent Ca2+ transport in mitochondria and sarcoplasmic reticulum was investigated. Ca2+ accumulation in mitochondria (1324 +/- 174 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to thapsigargin (100 nM) and was blocked by ruthenium red (10 microM). Oxalate-stimulated Ca2+ accumulation in sarcoplasmic reticulum (136 +/- 17 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to ruthenium red and was blocked by thapsigargin. It has been shown, that initial speed and level of energy-dependent Ca2+ accumulation in mitochondria considerably exceeded the values of these parameters for sarcoplasmic reticulum Ca2+-accumulation system. Ca2+ accumulation kinetic in mitochondria was characterized by a steady-state phase (for 5-10 min. of incubation) while accumulation kinetic of this cation in sarcoplasmic reticulum corresponded to zero order reaction. Increase of Mg2+ concentration up to 5 mM led to activation of Ca2+-accumulation systems in mitochondria and sarcoplasmic reticulum (values of activation constants K(Mg) for Mg2+ were 2.8 and 0.6 mM, accordingly). Concentration dependence of spermine action on Ca2+ accumulation in mitochondria was described by a dome-shaped curve with a maximum at 1 mM spermine. In case of sarcoplasmic reticulum Ca2+ pump only the inhibition phase was tested at spermine concentration above 1 mM. However values of inhibition constants for both transporting systems were practically identical--5.2 +/- 0.6 and 5.7 +/- 0.7 mM, accordingly. Hence, Mg ions carry out the important role in regulation of energy-dependent Ca2+ transporting systems both in uterus smooth muscle mitochondria and sarcoplasmic reticulum. Spermine acts first of all on mitochondrial calcium uniporter.     

Ultrastructural localisation of calcium deposits in pig ovarian follicles

Calcium intracellular signaling regulates many intracellular events including oocyte maturation. This signaling is strongly dependent on the influx of calcium ions from extracellular spaces and on the state of intracellular calcium stores. In this study, intracellular calcium deposits were detected in follicle-enclosed pig oocytes using the combined oxalate-pyroantimonate method. These deposits were observed in the nucleus, the mitochondria, the cytoplasm, and on the surface of lipid droplets. The amount of calcium deposits was expressed as a percentage of the area of the respective cellular compartment, which is covered with calcium deposits on ultrathin sections. The distribution of calcium deposits in oocytes changed during folliculogenesis. The amount of calcium deposits in nuclei (1.11% of the area of oocyte nuclei) and cytoplasm (1.02%) in oocytes from secondary and early antral follicles (0.90% nuclei; 0.99% cytoplasm) is significantly lower (P < 0.05) than the amount of calcium deposits in these compartments in oocytes from primary follicles (2.51% nuclei; 2.34% cytoplasm) or antral follicles with growing oocyte (2.91% nuclei; 2.21% cytoplasm). The amount of calcium deposits in mitochondria of oocytes from primary follicles (1.27%) or antral follicles with growing oocyte (1.14%) is significantly lower (P < 0.05) than in the nucleus (2.51% in oocytes from primary follicles; 2.91% in growing oocytes from antral follicles) or cytoplasm (2.34% in oocytes from primary follicles; 2.21% in growing oocytes from antral follicles). The amount of calcium deposits in the cytoplasm of fully-grown oocytes (1.46%) dropped to levels significantly lower (P < 0.05) than those observed in the oocyte nucleus (2.29%). On the basis of these data, we can conclude that the population of follicles on pig ovaries differs in the distribution and concentration of calcium deposits in oocytes, and these changes may be involved in the regulation of the meiotic competence of oocytes.     

Mitochondrial and extramitochondrial Ca2+ pools in the perfused rat liver. Mitochondria are not the origin of calcium mobilized by vasopressin

A nondisruptive technique developed by Bellomo et al. (Bellomo, G., Jewell, S. A., Thor, H., and Orrenius, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6842-6846) has been used to examine the distribution of calcium ions between mitochondrial and extramitochondrial compartments in the perfused rat liver. The amount of calcium released by the uncoupler 2,4-dinitrophenol from the mitochondrial compartment was 19 +/- 2 nmol X g-1, wet weight, which is equivalent to a total calcium concentration of 3.5 X 10(-4) M in the mitochondria and is by several orders of magnitude smaller than the concentration thought to be present in these organelles. The amount of calcium released from the liver in the presence of the divalent cation ionophore A 23187 was 96 +/- 7 nmol X g-1, wet weight, which is of the same order of magnitude as the amount released by the calcium-dependent hormone vasopressin (97 +/- 11 nmol X g-1, wet weight). Experiments with different sequential combinations of hormone with uncoupler or ionophore reveal that in the perfused liver, in contrast to isolated hepatocytes or isolated mitochondria, the amount of calcium attributable to the mitochondria is too small to account for the calcium released during hormonal stimulation. Consequently extramitochondrial calcium stores are the main source of cellular calcium mobilized under this condition. In addition these findings imply that in the liver several mitochondrial enzymes, e.g. alpha-oxoglutarate dehydrogenase, can be effectively regulated by calcium and that the role of mitochondria in buffering the cytosolic free calcium in vivo has to be reconsidered.     

The use of a model water-lipid system for the study of the electric function of Ca2+, Mg2+-ATPase of the sarcoplasmic reticulum

The method of dynamic capacity in the model organic phase-water system was used to investigate a possibility of studying the electrical function of Ca2+,Mg2+-ATPase from sarcoplasmic reticulum of the rabbit hind limb skeletal muscles. Decane and decane solution of azolectin were used as an organic phase. It is stated that in the model systems the sarcoplasmic reticulum Ca2+,Mg2+-ATPase did not cause ATP-dependent changes in the boundary Volta potential (delta phi) irrespective of the presence of polyvalent cation chelates in the organic phase. The fragmented sarcoplasmic reticulum is able of realizing Mg-ATP, Ca2+-dependent generation of delta phi only with phospholipids present in the organic phase. It is supposed that generation of delta phi of the fragmented sarcoplasmic reticulum is due to the active transport of calcium ions by the reticulum Ca2+,Mg2+-ATPase.     

Programmed cell death: Cytochemical and X-ray microanalytical characterization of calcium compartments in neuromuscular junctions during the normal breakdown of the intersegmental muscles in the giant silkmothAntheraea polyphemus

Summary Calcium stores were cytochemically demonstrated using a combined oxalate—pyroantimonate method in the neuromuscular junctions of the degenerating intersegmental muscles in the giant silkmothAntheraea polyphemus. The elemental composition of punctate precipitates of the reaction product was determined by electron probe X-ray microanalysis of unstained thin sections by energy-dispersive spectrometry and wavelength-dispersive spectrometry. The wavelength-dispersive spectra collected over terminal axons demonstrate a significant calcium signal and a trace of antimony.During the rapid lytic phase of spontaneous muscle degeneration, the calcium punctate deposits were detected in presynaptic terminals in the following sites: the synaptic vesicles and the mitochondria. Calcium precipitates were also found in the dense bodies and the mitochondria encountered in the glial convolutions. No calcium deposit was seen in the synaptic clefts and intercellular spaces of the subsynaptic reticulum of type I and type II. A comparison of calcium to antimony ratios between the terminal axons and the sarcoplasmic lysosomes revealed highly significant differences (P<0.001). Such a variability of the calcium to antimony ratio may be related to different conditions of precipitation or antimony diffusion in the different cell compartments. It was concluded that such synaptic terminals do not appear damaged in spite of the muscle degeneration and presumably continue to perform vital functions while the muscles are no longer contractile 20 h after adult ecdysis.     

Ultrastructural organisation and the sites of calcium localisation in the lamprey striated muscle

The present paper examines the ultrastructure of the sarcoplasmic recitulum (SR) and the T system in the striated muscle of the lamprey. The pyroantimonate method was used to visualise the sites of intracellular calcium localisation. Characteristic for the muscle studied are the presence of numerous intricately shaped invaginations on the surface membrane of muscle fibres and peripheral contacts between SR cisternae and the sarcolemma. In addition to calcium localised in the terminal cisternae of SR and N-bands of the I-disk, as typical of vertebrate muscles, a great amount of calcium is present in the subsarcolemmal region, corresponding to the area of invaginations, and in longitudinal elements of SR.     

小鼠卵母细胞成熟和受精过程中Ca^2＋分布变化的研究

用焦锑酸钾原位定位法、膜结合Ｃａ＾２＋荧光探针金霉素标记法,分别在电镜和光镜水平对小鼠卵成熟和卵受精过程中结合态Ｃａ＾２＋的分布及其变化进行了研究,发现：１）Ｃａ＾２＋分布于线粒体、胞质、内质网囊泡、微绒毛和透明带等部位,其中以线粒体基质中分布密度为最大;２）减数分裂Ｉ中、后期于纺锤体极区结合有较多的Ｃａ＾２＋;３）生发泡、纺锤体和原核内膜结合态Ｃａ＾２＋含量很少,但纺锤体和原核周围分布较多;４）     

A muscle mutant of Drosophila melanogaster: the electron microscopic study of the indirect flight musculature]

Dominant autosomal mutation l(2)M66 DCS induced in Drosophila melanogaster by ethyl-methane-sulfonate was studied. Electron-microscopic studies of asynchronous (fibrillar) and synchronous (tubular) muscles in 24-hour old mutants showed pathological changes in their fine structure. All systems were affected: the fragmentation of the Z-lines, disappearance of protofibrils, degenerative changes of mitochondria, sarcoplasmic reticulum, and the T-system, the appearance of membrane aggregates and lysosomes, the presence of a large amount of glycogen were detected. These changes in the ultrastructure of the flight muscles in mutants are similar to those observed in the process of physiological degeneration of insect muscles.     

Cytosolic free Ca2+ concentration and intracellular calcium distribution of Ca2+-tolerant isolated heart cells

The cytosolic free Ca2+ concentration of calcium-tolerant rat myocytes has been measured by the null point titration technique using arsenazo III as a Ca2+ indicator and digitonin to permeabilize the plasma membrane. The mean value obtained for 8 separate preparations was 270 +/- 35 nM. The distribution of releasable calcium between the mitochondrial and sarcoplasmic reticular compartments was measured by the successive additions of uncoupler and A23187 to cells pretreated with ruthenium red. The relative distribution of calcium in each pool was independent of the cell calcium content up to the maximum value of releasable calcium investigated (4.5 nmol/mg of cell dry weight) and was distributed in the approximate ratio of 2:1 in favor of the sarcoplasmic reticulum. The cells contained 1 nmol of calcium/mg of cell dry weight in a form nonreleasable by A23187, which was independent of the total cell calcium content as measured by atomic absorption spectroscopy. It is calculated that the calcium content of mitochondria in heart under physiological conditions is about 5 nmol/mg of mitochondrial protein. At this level, the mitochondria are likely to provide effective buffering of the cytosolic free Ca2+ concentration of quiescent heart cells. The corresponding intramitochondrial free Ca2+ is in a range above values needed to regulate the activity of Ca2+-dependent enzymes of the citric acid cycle in heart. The physiological calcium content of the sarcoplasmic reticulum in heart cells is estimated to be about 2.5 nmol/mg of cell dry weight, which is at least 5-fold greater than the amount of calcium release calculated to cause maximum tension development of cardiac muscle.     

Mitochondria in cardiomyocyte Ca signaling

Ca²⁺ signaling is of vital importance to cardiac cell function and plays an important role in heart failure. It is based on sarcolemmal, sarcoplasmic reticulum and mitochondrial Ca²⁺ cycling. While the first two are well characterized, the latter remains unclear, controversial and technically challenging.In mammalian cardiac myocytes, Ca²⁺ influx through L-type calcium channels in the sarcolemmal membrane triggers Ca²⁺ release from the nearby junctional sarcoplasmic reticulum to produce Ca²⁺ sparks. When this triggering is synchronized by the cardiac action potential, a global [Ca²⁺]_i transient arises from coordinated Ca²⁺ release events. The ends of intermyofibrillar mitochondria are located within 20 nm of the junctional sarcoplasmic reticulum and thereby experience a high local [Ca²⁺] during the Ca²⁺ release process. Both local and global Ca²⁺ signals may thus influence calcium signaling in mitochondria and, reciprocally, mitochondria may contribute to the local control of calcium signaling. In addition to the intermyofibrillar mitochondria, morphologically distinct mitochondria are also located in the perinuclear and subsarcolemmal regions of the cardiomyocyte and thus experience a different local [Ca²⁺].Here we review the literature in regard to several issues of broad interest: (1) the ultrastructural basis for mitochondrion – sarcoplasmic reticulum cross-signaling; (2) mechanisms of sarcoplasmic reticulum signaling; (3) mitochondrial calcium signaling; and (4) the possible interplay of calcium signaling between the sarcoplasmic reticulum and adjacent mitochondria.Finally, this review discusses experimental findings and mathematical models of cardiac calcium signaling between the sarcoplasmic reticulum and mitochondria, identifies weaknesses in these models, and suggests strategies and approaches for future investigations.     

ULTRASTRUCTURAL ALTERATIONS PRODUCED IN MAMMALIAN MYOCARDIUM BY VARIATION IN PERFUSATE IONIC COMPOSITION

This study describes the changes produced in the subcellular morphology of mammalian myocardium when perfusate sodium, calcium, and chloride concentrations are varied. By means of a recently developed perfusion technique, functioning dog papillary muscles were perfused with isotonic solutions of varying ionic compositions. Examination of the tissue in the electron microscope revealed that control muscles showed satisfactory preservation of ultrastructure, demonstrating that the protocol itself did not create significant morphological artefact. Low sodium chloride perfusion produced dilatation of both transverse tubules and longitudinal sarcoplasmic reticulum elements. Low sodium or high calcium concentrations produced dilation of tubular elements of the longitudinal sarcoplasmic reticulum while leaving transverse tubules intact. High calcium perfusion produced mitochondrial swelling and vacuolization. Mitochondrial precipitate, both crystalline and amorphous in form, was observed and presumed to be calcium phosphate, either alone or mixed with calcium carbonate. The possibility that the morphological changes observed might indicate subcellular loci of specific ion permeability is discussed. A correlation of the known kinetic behavior of sodium and calcium ions in mammalian myocardium with the ultrastructural alterations produced is suggested.     

Ultrastructural localization of intracellular calcium stores by a new cytochemical method

We describe a new cytochemical method for ultrastructural localization of intracellular calcium stores. This method uses fluoride ions for in situ precipitation of intracellular calcium during fixation. Comparisons made using oxalate, antimonate, or fluoride showed that fluoride was clearly superior for intracellular calcium localization in eggs of the sea urchin Strongylocentrotus purpuratus. Whereas oxalate generally gave no intracellular precipitate and antimonate gave copious but random precipitate, three prominent calcium stores were detected using fluoride: the tubular endoplasmic reticulum, the cortical granules, and large, clear, acidic vesicles of unknown function. The mitochondria of these eggs generally showed no detectable calcium deposits. X-ray spectra confirmed the presence of calcium in the fluoride precipitates, although in some cases magnesium was also detected. Rat skeletal muscle and sea urchin sperm were used to test the reliability of the fluoride method for calcium localization. In rat skeletal muscle, most fluoride precipitate was confined to the sarcoplasmic reticulum. Using sea urchin sperm, which transport calcium into the mitochondria after exposure to egg jelly to induce the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria contain no detectable calcium-containing precipitate. Within 4 min after induction of the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria displayed many foci of calcium-containing precipitate. The use of fluoride for intracellular calcium localization therefore appears to be a substantial improvement over previous cytochemical methods.