Presence of a trypsin-like protease in starfish sperm acrosome.

Marthasterias glacialis sperm cells were treated with ionophore A23187, centrifuged, and the supernatants were assayed for esterase activity. With N-benzoyl-L-arginine ethyl ester-HCl (BAEE) as substrate, a net activity was determined which was not detectable when N-acetyl-L-tyrosine ethyl ester (ATEE) was used. The BAEE trypsin-like activity was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-L-lysine chloromethyl ketone-HCl (TLCK), and phenyl methyl sulfonyl fluoride (PMSF), but not by L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK). The presence of proteolytic activity in acrosomal exudates was further demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic zymography (gelatin-SDS-PAGE). The presence of several bands of low proteolytic activity and of one band of high proteolytic activity, which also has the lower molecular weight, together with the fact that all are inhibited by benzamidine, suggests the existence of a trypsin-like proteinase system. The effect of the acrosomal exudate on the oocyte jelly coat was investigated by SDS-PAGE analysis. All jelly proteins appeared to be digested by the acrosomal enzymes. Furthermore, if SBTI is added shortly after insemination, the sperm fail to fertilize the oocytes. These results indicate that the starfish sperm acrosomal vesicle contains a trypsin-like protease which may be involved in sperm penetration through the oocyte jelly coat.     

Presence of ATPase and alkaline phosphatase activities in the starfish sperm acrosome

ATPase activity was cytochemically detected in the peripheral acrosomal component of ionophore-reacted sperm, while alkaline phosphatase activity was demonstrated in the upper and central components of the acrosome and, at fertilization, at the site of sperm-oocyte binding. Supernatants of ionophore treated sperm suspensions were assayed for ATPase, alkaline and acid phosphatase activities. Results suggest that alkaline phosphatase may be involved both in the acrosomal reaction and oocyte jelly lysis but the function of the acrosomal ATPase remains unknown.     

Cyclic AMP-dependent PKA phosphorylates starfish sperm proteins during acrosome reaction

The induction of acrosome reaction (AR) happens when starfish spermatozoa encounter the egg jelly (EJ). This complex process involves different signal transduction pathways, such as elevation of cAMP and the activation of protein kinase A (PKA). The specific inhibitors of PKA (H89 and KT5720) have been shown to inhibit the EJ-induced Ca²⁺ elevation and AR. By using a Phospho-Ser/Thr PKA substrate antibody, we have detected an increased phosphorylation of 150, 200 and 220-kDa protein bands when starfish spermatozoa treated with EJ. The specific PKA inhibitors effectively inhibit phosphorylation of these proteins, suggesting an involvement of PKA on EJ-induced AR.     

Guanylyl cyclase and cGMP-specific phosphodiesterase participate in the acrosome reaction of starfish sperm

In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, i.e. ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for inducing the acrosome reaction. Experimentally, ARIS and asterosap are sufficient for the induction. However, when sperm are treated only with asterosap, they become unresponsive to the egg jelly to undergo the reaction. In this study, we analysed the mechanism of the acrosome reaction, using sperm inactivation by asterosap as a clue. Asterosap causes a rapid and transient increase in intracellular cGMP through the activation of the asterosap receptor, a guanylyl cyclase, and causes an increase in intracellular Ca(2+). When sperm were pretreated with asterosap, the guanylyl cyclase seemed to be inactivated irreversibly by dephosphorylation. They were still responsive to ARIS but no longer to asterosap. However, in the presence of IBMX or zaprinast, inhibitors against phosphodiesterases (PDEs), they retained their capacity to undergo the acrosome reaction in response to the egg jelly or ARIS alone. IBMX and zaprinast suppressed the intracellular catabolism of cGMP, but not of cAMP. These results suggest that guanylyl cyclase and cGMP-specific, IBMX- and zaprinast-susceptible PDEs are involved in the regulation of the acrosome reaction.     

Pretreatment effects of jelly components on the sperm acrosome reaction and histone degradation in the starfish, Asterina pectinifera.

Acrosome reaction (AR) and histone degradation (HD) of Asterina pectinifera sperm are induced by co-operation of ARIS and a diffusible fraction (M8) of egg jelly. Once sperm are treated with ARIS or M8 separately for several minutes, they do not undergo the AR in response to the egg jelly. Preincubation of sperm with M8 at 0 degrees C is not effective to block the jelly-induced AR whereas inhibitory effects of ARIS remain at 0 degrees C. Jelly-induced HD is inhibited by pretreatment of sperm with ARIS but is not affected by the incubation with M8. The blockage of the jelly-induced reactions, both AR and HD, by ARIS- or M8-pretreatment can be bypassed by ionophores, A23187 and monensin.     

Regulatory mechanisms of the acrosome reaction revealed by multiview microscopy of single starfish sperm

The acrosome reaction in many animals is a coupled reaction involving an exocytotic step and a dramatic change in cell shape. It has been proposed that these morphological changes are regulated by intracellular ions such as Ca2+ and H+. We report here simultaneous visualization, under a multiview microscope, of intracellular free Ca2+ concentration ([Ca2+]i), intracellular pH (pHi), and morphological changes in a single starfish sperm (Asterina pectinifera). [Ca2+]i and pHi were monitored with the fluorescent probes indo-1 and SNARF-1, respectively. The acrosome reaction was induced with ionomycin. After the introduction of ionomycin in the medium, [Ca2+]i increased gradually and reached a plateau in approximately 30 s. The fusion of the acrosomal vacuole took place abruptly before the plateau, during the rising phase. Although the speed of the [Ca2+]i increase varied among the many sperm tested, exocytosis in all cases occurred at the same [Ca2+]i of approximately 2 microM (estimated using the dissociation constant of indo-1 for Ca2+ of 1.1 microM). This result suggests that the exocytotic mechanism in starfish sperm responds to [Ca2+]i rapidly, with a reaction time of the order of one second or less. Unlike the change in [Ca2+]i, an abrupt increase in pHi was observed immediately after exocytosis, suggesting the presence of a proton mobilizing system that is triggered by exocytosis. The rapid increase in pHi coincided with the formation of the acrosomal rod and the beginning of vigorous movement of the flagellum, both of which have been proposed to be pHi dependent. The exocytotic event itself was visualized with the fluorescent membrane probe RH292. The membrane of the acrosomal vacuole, concealed from the external medium in an unreacted sperm, was seen to fuse with the plasma membrane.     

Egg jelly components responsible for histone degradation and acrosome reaction in the starfish, Asterina pectinifera.

In the starfish, Asterina pectinifera, egg jelly induces the degradation of sperm histones as well as the acrosome reaction. We have isolated histone degradation-inducing components from the egg jelly. The histone degradation and the acrosome reaction are induced by a co-operative action of ARIS, which is an extremely large, sulfated glycoprotein with diffusible substance(s) in the jelly. Co-ARIS I, a steroidal saponin of the jelly, is effective to induce both reactions in the presence of ARIS.     

Characterization of the sperm receptor for acrosome reaction-inducing substance of the starfish,Asterias amurensis

Acrosome reaction-inducing substance (ARIS) in the jelly coat of starfish eggs is a highly sulfated proteoglycan-like molecule of an apparent molecular size over 10(4) kDa and plays a pivotal role in the induction of acrosome reaction in homologous spermatozoa. It is known in Asterias amurensis that ARIS binds to a restricted area of the anterior portion of sperm head, and that a glycan fragment of ARIS, named Fragment 1, consisting of 10 repeats or so of a pentasaccharide unit retains the biological activity of ARIS to an appreciable extent. In this report, we have shown the binding of Fragment 1, a relatively small pure glycan fragment of ARIS, to the putative ARIS receptor on the sperm surface by three independent methods. First, the specific binding of P-ARIS to isolated sperm membranes was monitored in real-time by using a surface plasmon resonance detector, namely a Biacore sensor system. The specific and quantitative binding of Fragment 1 to the intact sperm and to isolated sperm membranes was similarly monitored. Secondly, the binding of 125I-labeled Fragment 1 to the intact sperm was stoichiometrically measured, for which we had developed a unique procedure for radioiodination of saccharide chains. It is found that Fragment 1 competes with P-ARIS for the binding to ARIS-receptor, suggesting that Fragment 1 is a useful ligand in the search for ARIS receptor protein(s). Thirdly, the putative receptor molecules were specifically labeled by using Fragment 1 as a ligand for photoaffinity crosslink technique. Taking these results into account, we conclude that starfish sperm have the ARIS receptor, which consists most probably of 50 to 60 kDa proteins, of reasonably high affinity (for Fragment 1, Kd = 15 microM, Bmax = 8.4 x 10(4) per cell).     

PKA activation in concert with ARIS and asterosap induces the acrosome reaction in starfish

The acrosome reaction (AR) is a fundamental event for fertilization, which is induced in concert with acrosome reaction-inducing substance (ARIS) and asterosap, both of which are components of starfish egg jelly (EJ). During the AR, a spermatozoon undergoes a series of physiological changes, such as in intracellular cGMP concentration ([cGMP]i), pHi and intracellular Ca2+ concentration ([Ca2+]i). Affinity purification of cGMP-binding protein resulted in the isolation of a regulatory subunit of the cAMP-dependent protein kinase A (PKA), suggesting the involvement of a cAMP-dependent pathway in the AR. By using a cAMP enzyme immunoassay, [cAMP]i was found to increase in starfish spermatozoa when stimulated with ARIS and asterosap. ARIS could also increase the [cAMP]i in the presence of high pH seawater. Pretreatment of spermatozoa with two specific and cell-permeable PKA inhibitors, H89 and KT5720, prevented the induction of the AR in a concentration-dependent manner. These results suggest that PKA activity participates in the induction of the AR with ARIS and asterosap. To investigate this, we have cloned a gene that encodes a regulatory subunit of PKA that had been identified in starfish spermatozoa.     

Studies on the differentiation of egg envelopes. I. The starfish, Astropecten aurantiacus

We have studied the differentiation of the oocyte vitelline coat (VC) and jelly coat (JC) of the starfish, Astropecten aurantiacus. The precursor material of both envelopes is secreted by the oocyte while the follicle cells do not appear to participate in the secretory process. The first indication of differentiation of the VC is the deposition of a fine fibrillar material between the microvilli which emerge from the oocyte surface. External to this, a more loosely organized material becomes the precursor of the JC. At this time both layers are periodic acid-Schiff (PAS)-positive. In a later stage, the material between the microvilli acquires a more compact organization, looses its PAS-positivity while acquiring fucose binding protein (FBP) affinity. On the contrary, the JC remains PAS-positive and FBP-negative. In the full grown oocytes the VC is made up of densely packed fibrils oriented tangentially to the oocyte surface and is tightly bound to the microvilli. The observations are discussed in connection with the problem of the role of the egg envelopes in sperm-egg recognition and in the induction of the acrosome reaction.     

Structure of acrosome reaction-inducing substance in the jelly coat of starfish eggs: A mini review

Our knowledge at present on the structure of acrosome-reaction inducing substance (ARIS) in the jelly coat of starfish eggs is summarized. ARIS ia a proteoglycan-like molecule consisting of very long, linear, and highly sulfated glycans and three ARIS proteins, ARIS1-3. Detailed structures of the major glycan of ARIS and of ARIS1-3 are discussed. 3D-models of ARIS glycans are also presented. Phylogenetic distribution of ARIS proteins and/or genes indicates that ARIS genes are well preserved from the Ctenophore to Cephalochordata. In the Echinodermata, ARIS1-3 and ARIS genes were detected in all classes except for sea urchins.     

Fine structural study of the acrosome formation in the starfish Marthasterias glacialis (Echinodermata, Asteroidea)

The fine structure of the spermatogenic cells in the starfish Marthasterias glacialis was studied regarding acrosome formation. The main finding in the spermatogenesis of M. glacialis is that the formation of the pro-acrosomal vesicles seems to be initiated in late spermatogonia. Small dense bodies resulting from the division of large granulofibrillar masses were also observed in the cytoplasm of late spermatogonia. During spermiogenesis the inner acrosomal vesicle membrane becomes coated first with dense materials originated from the cytoplasmic dense bodies and then with cisternae of endoplasmic reticulum. Both coating materials are incorporated in the periacrosomal space of the mature acrosome. Besides being involved in the genesis of the periacrosomal material, cytoplasmic dense bodies were also seen in close relationship with intercellular bridges and midpiece structures of spermatids. These findings are discussed in comparison with other echinoderm spermatogenesis.     

Regulation of the starfish sperm acrosome reaction by cGMP, pH, cAMP and Ca2+

In the starfish, Asterias amurensis, three components in the jelly coat of eggs, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to induce the acrosome reaction (AR). Molecular recognition between the sperm surface molecules and the egg jelly molecules must underlie signal transduction events triggering the AR. Asterosap is a sperm-activating molecule, which stimulates rapid synthesis of intracellular cGMP, pH and Ca(2+). This transient elevation of Ca(2+) level is caused by a K(+)-dependent Na(+)/Ca(2+) exchanger, and the increase of intracellular pH is sufficient for ARIS to induce the AR. The concerted action of ARIS and asterosap could induce elevate intracellular cAMP levels in starfish sperm and the sustained increase in [Ca(2+)], which is essential for the AR. The signaling pathway induced by these factors seems to be synergistically regulated to trigger the AR in starfish sperm.