Extracellular localization of proteasomes in human sperm

The proteasome, a multienzymatic protease complex is present in human sperm. Here we present evidence indicating that the proteasome has an extracellular localization, on the plasma membrane of the sperm head. Motile sperm (>90%) in PBS were incubated with the proteasome inhibitors clasto-lactacystin beta-lactone or epoxomicin. Then, the substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC) was added and the enzyme activity evaluated in a spectrofluorometer. Other aliquots were resuspended in Tyrode's medium and incubated at different concentrations for various times with or without inhibitors in the presence of 0.4% azocasein. Hydrolysis of azocasein was evaluated at 440 nm. In addition, sperm membrane proteins were obtained incubating the sperm with Triton X-114 or with 0.5 M KCl plus Triton X-100 and removing insoluble material by centrifugation at 5,000g for 40 min. Proteasomal activity was evaluated with SLLVY-AMC and its presence corroborated by Western blotting. Formaldehyde fixed, unpermeabilized sperm were incubated with anti-proteasome monoclonal antibodies and evaluated using indirect immunofluorescence. The effect of proteasome inhibitors upon the progesterone-induced acrosome reaction was also evaluated. Results indicated that (a) whole, intact sperm were able to hydrolyze the proteasome substrates SLLVY-AMC and azocasein; this activity was inhibited by proteasome inhibitors; (b) proteasomal activity was detected in soluble sperm membrane protein preparations and Western blotting revealed the presence of the proteasome in these fractions; (c) indirect immunofluorescence revealed staining of the head region, particularly of the post acrosomal region; and (d) the proteasome plays an important role during the acrosome reaction.     

Mouse Uterine 24p3 Protein as a Suppressor of Sperm Acrosome Reaction

Under in vitro conditions, incubation with 0.3% bovine serum albumin (BSA) and 1.8 mM CaCl₂ induces mouse sperm capacitation and increases the consequential acrosome-reaction. The effect of mouse uterine 24p3 protein on such stimulated sperm has been investigated to understand the biological function of the 24p3 protein. Variations in the intracellular pH (pHi), calcium concentration, cAMP levels and tyrosine phosphorylation in cytosol were determined and on in vitro mouse fertilization was evaluated. The presence of 24p3 protein reduced the response of sperm to BSA and calcium by suppressing the elevation of intracellular pH, calcium uptake, cAMP accumulation and protein tyrosine phosphorylation of BSA/calcium-stimulated sperm and showed inhibitory effect on mouse in vitro fertilization. The results indicated the inhibition of the BSA-stimulated sperm acrosome reaction by 24p3 protein then suppressed sperm fertilization. We suggested that the 24p3 protein acts as an in vitro inhibitor of the acrosome reaction in BSA stimulated sperm and this might be an anti-fertilization factor in vitro.     

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.     

Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis.     

Effects of metalloendoprotease substrates on the human sperm acrosome reaction

The substrates carbobenzyloxyserylleucylamide, carbobenzyloxyglycylleucylamide and carbobenzyloxyglycylphenylalanylamide were used as potential competitive inhibitors of endogenous metalloendoprotease activity. When the acrosome reaction was elicited by a potential physiological stimulus, human follicular fluid, each of the substrates (1-1.5 mM) inhibited exocytosis. Carbobenzyloxyserylleucylamide also inhibited the acrosome reaction when exocytosis was stimulated using the calcium ionophore ionomycin, but carbobenzyloxyglycylleucylamide was not inhibitory and carbobenzyloxyglycylphenylalanylamide actually enhanced exocytosis under these conditions. Experiments using the fluorescent indicator fura-2 revealed that the increase in intracellular, free calcium stimulated by follicular fluid in human spermatozoa was depressed by carbobenzyloxyglycylphenylalanylamide but not by carbobenzyloxyserylleucylamide. The peptide carbobenzyloxyglycylglycylamide, which is not a substrate for metalloendoproteases, had no effect on the acrosome reaction, whether stimulated by follicular fluid or ionomycin. While the results with carbobenzyloxyserylleucylamide suggest a possible involvement of a metalloendoprotease in the human sperm acrosome reaction, our other results demonstrate that these carbobenzyloxy peptides have complex effects on the process of exocytosis in human spermatozoa, and suggest caution in interpretation of data obtained using such peptides on intact cells.     

Increased activity of the human sperm tyrosine kinase SRC by the cAMP-dependent pathway in the presence of calcium

SRC-related tyrosine kinases are suggested to play a role in the increase of sperm protein phosphotyrosine content that occurs during capacitation. In our laboratory, we previously demonstrated that the SRC-related tyrosine kinase YES1 (also known as c-YES) is present in human spermatozoa. However, since it is negatively regulated by Ca(2+), whose intracellular concentration increases during capacitation, another kinase would most likely be involved in the capacitation-related increase in sperm protein tyrosine phosphorylation. The present study represents the first direct assessment of SRC tyrosine kinase activity in ejaculated mammalian sperm. By immunohistochemistry on human testis sections, it is clearly shown that SRC is expressed during spermatogenesis, mainly in round and elongating spermatids. Using an indirect immunofluorescence approach, SRC is detected in the acrosomal region of the head and in the sperm flagellum of ejaculated sperm. This tyrosine kinase is associated with the plasma membrane and with cytoskeletal elements, as suggested by its partial solubility in nonionic detergents. Despite its partial solubility, SRC kinase activity was assayed after immunoprecipitation using acid-denatured enolase as a substrate. It is clearly demonstrated that SRC activity is inhibited by SU6656 and PP1, selective SRC family tyrosine kinase inhibitors, and activated in a Ca(2+)-dependent manner. Furthermore, it is shown that SRC is activated in a cAMP/PRKA-dependent manner; SRC coimmunoprecipitates with the catalytic subunit of the cAMP-dependent protein kinase (PRKAC) and is phosphorylated by this latter kinase, resulting in an increase in enolase phosphorylation. All these results support the involvement of the tyrosine kinase SRC in the increase in sperm protein phosphotyrosine content observed during capacitation.     

Src kinases catalytic activity regulates proliferation, migration and invasiveness of MDA-MB-231 breast cancer cells

SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27(Kip1) expression and reduced c-Myc levels, restraining G1–S transition. In contrast, SU6656 did not modify p27(Kip1) expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.     

Differential sensitivity of progesterone- and zona pellucida-induced acrosome reactions to pertussis toxin

Pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins) have previously been shown to mediate the zona pellucida-induced acrosome reaction in mammalian sperm. In this study we compared the inhibitory effect of pertussis toxin on the zona-induced acrosome reaction in human spermatozoa with that on the reaction induced by progesterone, another physiological acrosome reaction-promoting stimulus associated with the ovulated oocyte. Up to the concentration of 1 μg/ml, pertussis toxin did not produce any direct effects on the acrosome reaction frequency nor did it influence sperm movement and viability. However, preincubation of spermatozoa with the toxin at a concentration of 100 ng/ml completely abolished the increase in the acrosome reaction frequency upon subsequent exposure to solubilized zona pellucida material. In contrast, the same treatment did not impair the ability of spermatozoa to initiate the acrosome reaction in response to progesterone. Moreover, the preincubation with pertussis toxin did not modify the changes in the intracellular concentration of calcium ions occurring after progesterone addition. These data suggest that different physiological stimuli may utilize different signal transduction pathways to induce the human sperm acrosome reaction. © 1993 Wiley-Liss, Inc.     

Capacitation induces tyrosine phosphorylation of proteins in the boar sperm plasma membrane.

Capacitation (activation) of mammalian spermatozoa is accompanied by protein phosphorylation, elevation of the intracellular calcium concentration and an increased plasma membrane fluidity. The subcellular localization of tyrosine phosphorylation during capacitation have not yet been elucidated. The aim of this study was to investigate whether boar sperm capacitation induces tyrosine phosphorylation of plasma membrane proteins. Capacitation induced tyrosine phosphorylation of 3 proteins (27, 37, and 40 kDa), which coincided with an increase in the plasma membrane fluidity. The importance of the induced tyrosine phosphorylation in sperm binding to the zona pellucida and the induction of the acrosome reaction is discussed.     

Hamster sperm capacitation: role of pyruvate dehydrogenase A and dihydrolipoamide dehydrogenase

Recently, we demonstrated that pyruvate dehydrogenase A2 (PDHA2) is tyrosine phosphorylated in capacitated hamster spermatozoa. In this report, using bromopyruvate (BP), an inhibitor of PDHA, we demonstrated that hamster sperm hyperactivation was blocked regardless of whether PDHA was inhibited prior to or after the onset of hyperactivation, but the acrosome reaction was blocked only if PDHA was inhibited prior to the onset of the acrosome reaction. Further, inhibition of PDHA activity did not inhibit capacitation-associated protein tyrosine phosphorylation observed in hamster spermatozoa. It is demonstrated that the essentiality of PDHA for sperm capacitation is probably dependent on its ability to generate effectors of capacitation such as reactive oxygen species (ROS) and cAMP, which are significantly decreased in the presence of BP. MICA (5-methoxyindole-2-carboxylic acid, a specific inhibitor of dihydrolipoamide dehydrogenase [DLD]), another component of the pyruvate dehydrogenase complex (PDHc), also significantly inhibited ROS generation and cAMP levels thus implying that these enzymes of the PDHc are required for ROS and cAMP generation. Furthermore, dibutryl cyclic adenosine monophosphate could significantly reverse the inhibition of hyperactivation observed in the presence of BP and inhibition of acrosome reaction observed in the presence of BP or MICA. The calcium ionophore, A23187, could also significantly reverse the inhibitory effect of BP and MICA on sperm acrosome reaction. These results establish that PDHA is required for hamster sperm hyperactivation and acrosome reaction, and DLD is required for hamster acrosome reaction. This study also provides evidence that ROS, cAMP, and calcium are involved downstream to PDHA.     

Sperm proteasomes are responsible for the acrosome reaction and sperm penetration of the vitelline envelope during fertilization of the sea urchin Pseudocentrotus depressus

The roles of sperm proteasomes in fertilization were investigated in the sea urchin Pseudocentrotus depressus. Two proteasome inhibitors, MG-132 and MG-115, inhibited fertilization at 100 microM, whereas chymostatin and leupeptin showed no inhibition. Among three proteasome substrates, Z-Leu-Leu-Glu-MCA showed the strongest inhibition toward fertilization. MG-132 inhibited the egg-jelly-induced, but not ionomycin-induced, acrosome reaction. In addition, MG-132, but not E-64-d, inhibited fertilization of dejellied eggs by acrosome-reacted sperm. MG-132 showed no significant inhibition toward the binding of reacted sperm to the vitelline layer. Proteasomes were detected by Western blotting in the acrosomal contents, which are partially released upon exocytosis. We also found that the inhibition pattern of the caspase-like activity of the proteasome in the acrosomal contents by chymostatin and proteasome inhibitors coincided well with their inhibitory abilities toward fertilization. Furthermore, the vitelline layer of unfertilized eggs appears to be ubiquitinated as revealed by immunocytochemistry and Western blotting. Extracellular ATP, required for the degradation of ubiquitinated proteins by the proteasome, was also necessary for fertilization. These results indicate that the sperm proteasome plays a key role not only in the acrosome reaction but also in sperm penetration through the vitelline envelope, most probably as a lysin, during sea urchin fertilization.     

Signals of seminal vesicle autoantigen suppresses bovine serum albumin-induced capacitation in mouse sperm

Capacitation is the prerequisite process for sperm to gain the ability for successful fertilization. Unregulated capacitation will cause sperm to undergo a spontaneous acrosome reaction and then fail to fertilize an egg. Seminal plasma is thought to have the ability to suppress sperm capacitation. However, the mechanisms by which seminal proteins suppress capacitation have not been well understood. Recently, we demonstrated that a major seminal vesicle secretory protein, seminal vesicle autoantigen (SVA), is able to suppress bovine serum albumin (BSA)-induced mouse sperm capacitation. To further identify the mechanism of SVA action, we determine the molecular events associated with SVA suppression of BSA's activity. In this communication, we demonstrate that SVA suppresses the BSA-induced increase of intracellular calcium concentration ([Ca2+]i), intracellular pH (pH(i)), the cAMP level, PKA activity, protein tyrosine phosphorylation, and capacitation in mouse sperm. Besides, we also found that the suppression ability of SVA against BSA-induced protein tyrosine phosphorylation and capacitation could be reversed by dbcAMP (a cAMP agonist).     

Cell surface localization of a novel non-genomic progesterone receptor on the head of human sperm

Cell surface receptors for progesterone were visualized in human sperm using fluorescein isothiocyanate-progesterone 3-(O-carboxymethyl) oxime-bovine serum albumin (FITC prog CMO BSA). The receptors were confined to the head and not the midpiece or tail. FITC prog CMO BSA was also an effective stimulus to elevate intracellular free calcium in human sperm as detected by fura-2 fluorescence. The elevation of intracellular free calcium is a stimulus for the acrosome reaction, a process which is necessary to occur for sperm to fertilize the egg. It is proposed that progesterone, which is present in the female reproductive tract, can bind to progesterone receptors located in the plasma membrane of the sperm head and elicit an influx of Ca2+ into the underlying cytoplasm and or acrosome and induce the acrosome reaction and facilitate fertilization.     

Nitric oxide regulates human sperm capacitation and protein-tyrosine phosphorylation in vitro.

The aim of the present study was to investigate whether the generation of nitric oxide by human spermatozoa is associated with human sperm capacitation and with the tyrosine phosphorylation of sperm proteins. Human spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of sperm proteins. A tight correlation between capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human sperm capacitation and emphasize the importance of oxidoreduction reactions in the fine control of sperm physiology.     

Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.     

Evidences for the presence of chymotrypsin-like activity in human spermatozoa with a role in the acrosome reaction

The effect of chymotrypsin inhibitors and substrates on the human sperm acrosome reaction stimulated by the human zonae pellucidae or follicular fluid were evaluated. Motile spermatozoa, selected by a Percoll gradient, were incubated at 1 × 10⁷ cells/ml, 37°C, and 5% CO₂, After 4.5 hr, the chymotrypsin inhibitor TPCK (N-Tosyl-L-Phenylalanine-Chloromethyl Ketone) or the substrate ATEE (N-Acetyl-L-Tyrosine Ethyl Ester) were added for 30 min. Then, four oocytes were added and the percentage of acrosome-reacted spermatozoa on the zona was determined. TPCK and ATEE inhibited the zona pellucida-induced acrosome reaction. The chymotrypsin inhibitors TPCK and chymostatin and the chymotrypsin substrates ATEE, BTEE (N-Benzoyl-L-Tyrosine Ethyl Ester), Succinyl-Ala-Ala-Phe-7-Amido-4-Methyl-Coumarin (Suc-Ala-Ala-Phe-AMC), and Succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methyl-Coumarin (Suc-Leu-Leu-Val-Tyr-AMC) inhibited the human follicular fluid-induced acrosome reaction. Sperm extracts exhibited hydrolytic activity toward Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. This enzyme activity was abolished by TPCK and chymostatin, was independent of Ca²⁺, and was not modified by 1,10 phenanthroline. In addition, the activity was present in the supernatant after the acrosome reaction was induced with calcium ionophore and in epididymal spermatozoa recovered from the cauda region. Electron microscopic observations indicated that the inhibitors prevented the membrane events of the acrosome reaction. These data suggest an association between human spermatozoa and chymotrypsin-like activity with a possible role in the acrosome reaction. © 1994 Wiley-Liss, Inc.     

An influx of extracelluar calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid

The role of Ca²⁺ in the human sperm acrosome reaction was investigated using the fluorescent calcium indicator fura-2. Previous experiments have shown that a Sephadex G-75 column fraction of human follicular fluid can stimulate the human sperm acrosome reaction [Suarez SS, Wolf DP, Meizel S (1986): Gamete Res 14:107–121]. Using fura-2, we demonstrated that this Sephadex G-75 fraction also stimulates a rapid, transient increase in intracellular free Ca²⁺. This Ca²⁺ transient is blocked either by chelation of extracellular calcium or by addition of the Ca²⁺ antagonist La³⁺. We have also been able to stimulate the acrosome reaction in human sperm without significant loss of motility, using the divalent cation ionophore ionomycin. Acrosome reactions stimulated by whole follicular fluid, the G-75 fraction, or ionomycin are all blocked by removal of extracellular Ca²⁺. These results strongly suggest that an influx of extracellular Ca²⁺ is responsible for intiating the acrosome reaction in human sperm treated with human follicular fluid. This is the first demonstration in mammalian sperm that a potentially physiological stimulus can cause an increase in intracellular Ca²⁺ concomitant with the acrosome reaction.     

Proteasome (multicatalytic proteinase) of sea urchin sperm and its possible participation in the acrosome reaction

The egg jelly-induced acrosome reaction of the sea urchin, Strongylocentrotus intermedius, was inhibited by succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA), but not by Suc-Ala-Ala-Pro-Phe-MCA. The proteases with hydrolytic activity toward the former were purified from sperm extract by DEAE-Sephacel and hydroxylapatite chromatographies, Sephacryl S-300 gel filtration, and heparin-Sepharose CL-6B chromatography. Two types of protease were separated, and the molecular weights were estimated to be 65 and 700 kDa, respectively, by gel filtration. The former was accompanied by hydrolytic activity toward Suc-Ala-Ala-Pro-Phe-MCA, which was not hydrolyzed by the latter. Polyacrylamide gel electrophoresis of 700 kDa protease gave a single protein band under nondenaturing conditions and at least eight bands in the range of 22-33 kDa in the presence of sodium dodecyl sulfate (SDS). The substrate specificity and the inhibitor sensitivity of 700 kDa protease indicate that it contains two types of the activity, one is chymotrypsin-type and the other trypsin-type. The former activity was enhanced by poly-L-lysine or SDS. These properties of 700 kDa protease are similar to those of proteasomes (multicatalytic proteinases) isolated from various eukaryotic sources. We had previously shown that inhibitors of chymotrypsin-like proteases inhibit the increase of intracellular Ca2+ concentration by egg jelly, resulting in the inhibition of the acrosome reaction of St. intermedius (Matsumura and Aketa, Gamete Res 23:255-266, 1989). Bringing these findings together, we suggest that the chymotrypsin-like activity of sperm proteasome participates in the onset of the acrosome reaction of St. intermedius.     

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.     

Activation of Proteasome in Sea Urchin Sperm by Lysophosphatidylinositol and by Sperm Lipids

Effects of phospholipids, their metabolites and endogenous sperm lipids on the chymotrypsin-like activity of proteasome purified from sperm of the sea urchin, Strongylocentrotus intermedius were examined. Some lysophospholipids remarkably enhanced the activity. The most prominent activation was found in lysophosphatidylinositol (LPI) which enhanced about 12-fold at 2.5 μg/ml. On the other hand, higher concentrations (above 250 μg/ml) were required for the enhancement of the activity by some saturated fatty acids and phospholipids. Lipids extracted from sperm also were effective in the enhancement, and those from sperm which were treated for 15 sec in egg jelly were more effective than those from untreated sperm. These results suggest that certain metabolites belonging to lysophospholipids are produced during the acrosome reaction and activate sperm proteasome. Also, they are not inconsistent with our view that the chymotrypsin-like activity of sperm proteasome participates in the acrosome reaction (23, 24).