Ion channel activity of membrane vesicles released from sea urchin sperm during the acrosome reaction

The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca2+ and Na+ influx and K+ and H+ efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome reaction vesicles (ARVs), using synaptosome-associated protein of 25 kDa (SNAP-25) as a marker. Isolated ARVs have a unique protein composition. The exocytosis regulatory proteins vesicle-associated membrane protein and SNAP-25 are inside ARVs, as judged by protease protection experiments, and membrane associated based on Triton X-114 partitioning. ARVs fused with planar bilayers display three main types of single channel activity. The most frequently recorded channel is cationic, weakly voltage dependent and has a low open probability that increases with negative potentials. This channel is activated by cAMP, blocked by Ba2+, and has a PK+/PNa+ selectivity of 4.5. ARVs represent a novel membrane preparation suitable to deepen our understanding of ion channel activity in the AR and during fertilization.     

Structural requirements for species-specific induction of the sperm acrosome reaction by sea urchin egg sulfated fucan

The sulfated fucan (SF) of egg jelly induces the acrosome reaction (AR) of sea urchin sperm. Strongylocentrotus franciscanus (Sf) SF is sulfated only at the 2-position. Strongylocentrotus purpuratus (Sp) has two SF isotypes, each one being female specific. One is rich in sulfate at both the 2- and 4-positionS (SF-1), and the other is rich in sulfate at the 4-position, but not the 2-position (SF-2). Sf SF is poor at inducing the AR of Sp sperm, presumably due to lack of 4-sulfation. Sp SF-1 is better at inducing the AR of Sf sperm than Sp SF-2, hypothetically due to increased 2-sulfation. Chemical oversulfation of Sf SF increases the percentage of AR of Sp sperm, showing that 4-sulfation is important for recognition of SF by Sp sperm. Chemically oversulfated Sp SF-2 is better at inducing the Sf sperm AR, presumably because of increased 2-sulfation. The species, Strongylocentrotus drobachiensis (Sd), has an SF-2 that is exclusively 2-sulfated (like Sf), except the glycosidic linkage in Sd is alpha(1-->4), whereas in Sf it is alpha(1-->3). Sd SF-2 does not induce the AR of Sf sperm, showing the strict requirement for the alpha(1-->3) linkage in recognition between Sf sperm and SF. Egg jelly from Echinometra lucunter (El) contains sulfated galactan (SG) which differs from Sf SF only in that the monosaccharide is L-galactose, not L-fucose. This SG and Sf SF are equally potent in inducing the AR of Sf sperm, showing that modification at C6 of L-fucose is not important for proper recognition between SF and Sf sperm receptors. This system permits study of the structural basis for recognition between sulfated polysaccharide and receptors controlling signal transduction pathways in animal cells.     

Egg fucose sulfate polymer,sialoglycan, and speract all trigger the sea urchin sperm acrosome reaction

Macromolecules surrounding eggs induce the acrosome reaction (AR) of spermatozoa. In sea urchins, three egg jelly (EJ) molecules: a fucose sulfate polymer (FSP), a sialoglycan (SG), and speract mediate ionic fluxes triggering the AR. SG and speract are noninductive without FSP. Speract's role in AR induction is controversial. Here we show that speract potentiates the FSP-induced AR at pH 7.0, approximately 1 pH unit lower than natural seawater. At pH 7.0, a mixture of FSP, SG, and speract produces the intracellular pH increase necessary for maximum AR induction. Each EJ component may mediate a distinct intracellular pH control mechanism, and all three may function synergistically to increase the intracellular pH permitting AR induction. Speract peptides are an ancient family. Although important for activating cyclic nucleotide-mediated pathways in today's seawater of pH approximately 8, speract may have been more important in AR induction in the paleo-ocean of pH approximately 7.     

Evidence for a cannabinoid receptor in sea urchin sperm and its role in blockade of the acrosome reaction

Delta-9-tetrahydrocannabinol ((?)δ⁹ THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [ H]CP-55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [ H]CP-55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time-dependent manner. Specific binding of [ H]CP-55,940 to sperm, defined as total binding displaced by (?)δ⁹ THC, was saturable: K_D 5.16 ± 1.02 nM; Hill coefficient 0.98 ± 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 ± 122 cannabinoid receptors per cell. Binding of [ H]CP-55,940 to sperm was reduced in a dose-dependent manner by increasing concentrations of CP-55,940, (?)δ⁹ THC, and (+)δ⁹ THC. The rank order of potency to inhibit binding of [ H]CP-55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP-55,940 > (?)δ⁹THC > (+)δ⁹THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [ H]CP-55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation. © 1993 Wiley-Liss, Inc.     

Rho, Rho-kinase, and the actin cytoskeleton regulate the Na+ -H+ exchanger in sea urchin eggs

At fertilization, the sea urchin egg undergoes an internal pH (pHi) increase mediated by a Na+ -H+ exchanger. We used antibodies against the mammalian antiporters NHE1 and NHE3 to characterize this exchanger. In unfertilized eggs, only anti-NHE3 cross-reacted specifically with a protein of 81-kDa, which localized to the plasma membrane and cortical granules. Cytochalasin D, C3 exotoxin (blocker of RhoGTPase function), and Y-27632 (inhibitor of Rho-kinase) prevented the pHi change in fertilized eggs. These inhibitors blocked the first cleavage division of the embryo, but not the cortical granule exocytosis. Thus, the sea urchin egg has an epithelial NHE3-like Na+ -H+ exchanger which can be responsible for the pHi change at fertilization. Determinants of this pHi change can be: (i) the increase of exchangers in the plasma membrane (via cortical granule exocytosis) and (ii) Rho, Rho-kinase, and optimal organization of the actin cytoskeleton as regulators, among others, of the intrinsic activity of the exchanger.     

Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. II. Ultrastructural changes associated with inhibition of the acrosome reaction.

Pretreatment of Strongylocentrotus purpuratus sperm with delta 9-tetrahydrocannabinol (THC) prevents the triggering of the acrosome reaction by egg jelly. Examination of THC-treated sperm by transmission electron microscopy reveals that the membrane fusion reaction between the sperm plasma membrane and the acrosomal membrane is completely blocked. Electron-dense deposits are present in the subacrosomal fossa and in the centriolar fossa. The nuclear envelope is fragmented in close proximity to the electron-dense deposits. The electron-dense deposits are not bound by a limiting membrane, stain positively for lipid with thymol and farnesol, and disappear from THC-treated sperm that are extracted with chloroform:methanol (2:1) after glutaraldehyde fixation. The electron-dense deposits are lipid in nature and may be a hydrolytic product of the nuclear envelope. Electron-dense deposits are seen in sperm after 1-10 min treatment with 5-100 microM THC. The electron-dense deposits disappear after removal of THC from the sperm by washing, but the fragmented nuclear envelope in the subacrosomal fossa persists. Cannabidiol (CBD) and cannabinol (CBN) also inhibit the triggering of the acrosome reaction by egg jelly and produce ultrastructural changes in the sperm identical to those elicited by THC. Enhanced phospholipase activity stimulated by THC, CBD, and CBN may be the cause of the accumulation of lipid deposits in the sperm. Metabolites derived from this modification of membrane phospholipids may prevent triggering of the acrosome reaction by egg jelly and thereby inhibit fertilization.     

Immunoglobulins from human follicular fluid induce the acrosome reaction in human sperm

The ability of human follicular fluid (hFF), retrieved from women undergoing IVF to induce the acrosome reaction (AR) in human sperm has been documented by several laboratories. However, the nature of the active factors in the hFF and the physiological meaning of the AR induction are highly controversial. We performed a three step purification scheme for hFF and all the fractions were screened for the AR-inducing activity. AR activity was associated with a protein fraction of M_r > 180 kD that on further analysis under PAGE was found to be composed by subunits of apparent M_r 50,000 and 29,000. The N-terminal sequences of these bands showed a 100% homology with the heavy and light chains of human lgG. A polyclonal antibody raised against the purified protein and anti-human lgG were both able to suppress the acrosome reactioninducing activity of crude hFF. However, neither normal human serum nor a purified preparation of human lgG were able to mimic the AR-inducing activity of hFF. We concluded that the AR-inducing activity of hFF is, at least in part, due to the presence of antisperm antibodies. © 1994 Wiley-Liss, Inc.     

Effect of caulerpenyne,a toxin extracted from Caulerpa taxifolia on mechanisms regulating intracellular pH in sea urchin eggs and sea bream hepatocytes

The proliferation of the green marine alga Caulerpa taxifolia in the Mediterranean led us to investigate the toxic effects on marine organisms of caulerpenyne (Cyn), the major secondary metabolite synthesized by the alga. This study was performed on sea urchin eggs (Paracentrotus lividus) and isolated hepatocytes from the sea bream (Sparus aurata), in which accumulation of the toxins by metabolic processes may be of significance. Cyn provoked an acidification of seawater containing both unfertilized and fertilized eggs, as revealed by a titrable efflux of protons. The pHi in unfertilized eggs continuously increased in the presence of Cyn, whereas there was a biphasic response in both fertilized eggs and isolated hepatocytes, with a decrease of the pHi followed by recovery to the initial value. Cyn inhibited the accumulation of ¹⁴C-methylamine in acidic granules present in the cortical zone of sea urchin eggs. Dicyclocarbodiimide (DCCD), a well-known H⁺-ATPase inhibitor, provoked a similar inhibition. Both molecules increased pH in the acidic compartments of isolated bream hepatocytes. These results suggest that Cyn inhibits intracellular sequestration of protons and thus liberates protons into the cell cytoplasm from which they leak toward the extracellular medium. © 1997 John Wiley & Sons, Inc.     

三疣梭子蟹精子顶体反应前后胞内Ca~(2+)的变化

应用激光扫描共聚焦显微镜(LSCM)和Fluo-3/AM荧染技术对三疣梭子蟹精子顶体反应前后的胞内Ca2 变化进行了观察和检测.结果显示,在精子顶体反应过程中,胞内Ca2 主要分布在细胞核、穿孔器和胞质膜残存处,胞内Ca2 浓度([Ca2 ]I)总体上呈现先上升后下降的趋势.顶体反应前精子的平均荧光强度为35.95±5.71;穿孔器前伸、顶体囊膜翻转阶段精子的平均荧光强度为66.80±7.35;顶体囊膜脱落、顶体丝形成阶段精子的平均荧光强度为3.87±2.82;上述各阶段间精子荧光强度有极显著差异(P<0.01).顶体反应穿孔器前伸、顶体囊膜翻转阶段的精子相比顶体反应前精子,[Ca2 ]I显著提高;而在顶体囊膜脱落、顶体丝形成阶段,[Ca2 ]I则急剧下降,只在顶体丝基部胞质膜残存处有微量Ca2 存在.初步探讨了三疣梭子蟹精子顶体反应前后胞内Ca2 变化的功能.     

Xenoestrogenic compounds promote capacitation and an acrosome reaction in porcine sperm

There is growing evidence that endocrine disruptors bind to hormone receptors; since these receptors are present on the sperm membrane, sperm are potentially a useful model for examining estrogenic activities of endocrine disruptors. The objective of the present study was to compare the effects of two xenoestrogenic compounds (genistein and 4-tert-octylphenol) to those of two steroids (estrogen and progesterone) and heparin on in vitro capacitation and the acrosome reaction in a porcine sperm model. Porcine sperm were incubated with various concentrations (0.001-100 μM) of each chemical for 15 or 30 min, and then capacitation and the acrosome reaction were assessed using chlortetracycline. Estrogen and progesterone were considerably more potent than the other chemicals in stimulating capacitation. Estrogen stimulated sperm capacitation at all tested concentrations after 15 min of incubation (P < 0.05), whereas progesterone stimulated sperm capacitation at all tested concentrations after 15 and 30 min (P < 0.05). The effect of genistein on sperm capacitation was comparable with that of estrogen, and it was the most potent in stimulating the acrosome reaction. Genistein stimulated the acrosome reaction at all tested concentrations after 30 min (P < 0.05). However, 4-tert-octylphenol had the least effect on capacitation and the acrosome reaction. In summary, since all chemicals studied effectively altered capacitation and the acrosome reaction, it was concluded that porcine sperm could be a useful model for in vitro screening of potential endocrine disruptors. It was noteworthy that concurrent comparisons to steroids increased the ability to determine estrogenic characteristics of the tested chemicals.     

Egg sialoglycans increase intracellular pH and potentiate the acrosome reaction of sea urchin sperm.

Sea urchin egg jelly (EJ) triggers sperm acrosome reaction (AR), an exocytotic event required for membrane fusion of the gametes. Purified fucose sulfate polymer (FSP) in EJ is one inducer of the AR. Binding of FSP to its receptor regulates opening of two distinct calcium channels and also elevates intracellular pH (pH(i)). EJ also contains sialic acid-rich glycans (sialoglycans (SG)) that were isolated by beta-elimination followed by DEAE chromatography. In the presence of limiting amounts of FSP, the SG fraction markedly potentiates the AR; however, by itself SG has no activity. The SG fraction increases the pH(i) of sperm without increasing intracellular Ca(2+). The SG-induced increase in pH(i) is not blocked by nifedipine or high K(+), whereas the FSP-induced pH(i) increase is sensitive to both these agents. Treatment of the SG fraction with neuraminidase or mild metaperiodate that specifically cleaves the glycerol side chain of sialic acid abolishes the AR potentiation and ability of SG to elevate pH(i). These data are the first to show that there are at least two pathways to induce sperm pH(i) increase and that egg surface sialic acid plays a role in triggering the sperm AR.     

Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. I. Inhibition of the acrosome reaction induced by egg jelly.

delta 9-Tetrahydrocannabinol (THC) and two other major cannabinoids derived from marihuana--cannabidiol (CBD) and cannabinol (CBN)--inhibit fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of sperm (Schuel et al., 1987). Sperm fertility depends on their motility and on their ability to undergo the acrosome reaction upon encountering the egg's jelly coat. Pretreatment of S. purpuratus sperm with THC prevents triggering of the acrosome reaction by solubilized egg jelly in a dose (0.1-100 microM) and time (0-5 min)-dependent manner. Induction of the acrosome reaction is inhibited in 88.9 +/- 2.3% of sperm pretreated with 100 microM THC for 5 min, while motility of THC-treated sperm is not reduced compared to solvent (vehicle) and seawater-treated controls. The acrosome reaction is inhibited 50% by pretreatment with 6.6 microM THC for 5 min and with 100 microM THC after 20.8 sec. CBN and CBD at comparable concentrations inhibit the acrosome reaction by egg jelly in a manner similar to THC. THC does not inhibit the acrosome reaction artificially induced by ionomycin, which promotes Ca2+ influx, and nigericin, which promotes K+ efflux. THC partially inhibits (20-30%) the acrosome reaction induced by A23187, which promotes Ca2+ influx, and NH4OH, which raises the internal pH of the sperm. Addition of monensin, which promotes Na+ influx to egg jelly or to A23187, does not overcome the THC inhibition. Inhibition of the egg jelly-induced acrosome reaction by THC produces a corresponding reduction in the fertilizing capacity of the sperm. The adverse effects of THC on the acrosome reaction and sperm fertility are reversible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of Ca2+channels during the acrosome reaction of sea urchin sperm is inhibited by inhibitors of chymotrypsin-like proteases

Probable participation of sperm protease in the acrosome reaction was investigated using several inhibitors and substrates. Among those examined, L-l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) and chymostatin, chymotrypsin inhibitors, p-nitrophenyl-p′-guanidinobenzoate (NPGB), a serine protease inhibitor, and N-benzoyl-L-tyrosine ethyl ester (BTEE), a chymotrypsin substrate, inhibited the egg jelly-induced acrosome reaction of Strongylocentrotus intermedius. TPCK and BTEE, however, did not inhibit the reaction caused by ionophores, A23187, or nigericin. To know the mechanism of inhibition by chymotrypsin inhibitors and substrates of the egg jelly-induced acrosome reaction, intraccllular Ca²⁺ concentration ([Ca²⁺]_i) and pH (pH_i) were measured with fura-2 and 2′,7′-bis (carboxy-ethyl)carboxyfluorescein (BCECF), respectively. Egg jelly caused increase of [Ca²⁺]_i which was depressed by BTEE. Egg jelly also caused a transient rise of pH_i, which was not depressed by BTEE. In the presence of verapamil, the acrosome reaction by egg jelly was significantly inhibited concomitant with depressed increase of [Ca²⁺]_i. The rise of pHj was not depressed by verapamil. Thus, modes of action of BTEE and of verapamil are similar to each other. Bringing these findings together, the authors present a view that a chymotrypsin-like protease of sea urchin sperm activates verapamil-sensitive Ca²⁺ channels, which take part in the acrosome reaction.     

三疣梭子蟹精子顶体反应过程中的形态和结构变化

用离子载体A2 3187和卵水人工诱导三疣梭子蟹精子的顶体反应 ,分别获得 75 33%和 84 83%的顶体反应率。应用光镜和电镜技术观察了顶体反应前后精子形态和结构的变化。未处理精子呈陀螺形 ,由顶体、核杯和 5 - 10条核辐射臂组成。顶体包括顶体囊和顶体管。顶体囊的伞形头帽拥有约 70条辐射肋。连续发生的精子顶体反应过程被人为地分为四个阶段 :(1)头帽鼓起 ;(2 )顶体囊外翻 ;(3)穿孔器前伸 ,顶体囊膜翻转 ;(4 )顶体囊膜脱落 ,顶体丝形成。直到第四阶段才观察到钉状精子的辐射臂开始收缩。探讨了辐射臂和穿孔器前冲在精子入卵中的功能     

Video microscopic analysis of ionophore induced acrosome reactions of lobster (Homarus americanus) sperm

Sperm from the American lobster (Homarus americanus) are normally nonmotile. However, during fertilization, the sperm undergo a calcium-dependent acrosome reaction that propels them forward about 18 μMm. The reaction occurs in two phases, eversion and ejection, which take place too quickly to permit analysis by direct observation. The purposes of this study were to examine the structural changes occurring in sperm during the normal acrosome reaction and to determine the rate of the reaction using video microscopy. The reaction was induced in vitro by ionophore A23187 and recorded using a video system attached to a Nikon Nomarski interference microscope. Videotapes were played back frame by frame (30 frames/sec), and images of reactions from 10 sperm were analyzed. The acrosome reaction, including the eversion of the acrosomal vesicle and ejection of the subacrosomal material and nucleus, can be divided into 4 steps: (1) expansion of the apical cap followed by expansion of the remainder of the acrosomal cylinder; expansion of the cylinder begins at its apical end and proceeds toward its base, (2) eversion of the apical half of the acrosomal vesicle and initial contraction of the apical cap, (3) eversion of the basal half of the acrosomal vesicle, continued contraction of the apical cap, and ejection of the subacrosomal material and nucleus, and (4) final contraction of the apical cap and ejection of the acrosomal filament. During steps 2, 3, and 4, the mean forward movement of sperm is 12.7, 3.9, and 1.1 μMm, respectively. Although the time required to complete the reaction ranged from 0.66 to 5.16 s, most sperm reacted in less than 3. s, and these sperm were considered to have typical rates. For sperm that reacted in less than 3 s, both step 1 and step 4 take about 0.2 s and show little variation among sperm. the time required to complete steps 2 and 3 averaged 0.63 and 0.37 s, respectively. Forward movement of the sperm during the acrosome reaction is caused by eversion of the inner and outer acrosomal material and contraction of the apical cap. The protein(s) responsible for this contraction is not yet known. © 1993 Wiley-Liss, Inc.     

Tandem mass spectrometry identifies proteins phosphorylated by cyclic AMP-dependent protein kinase when sea urchin sperm undergo the acrosome reaction

The exocytotic acrosome reaction (AR), which is required for fertilization, occurs when sea urchin sperm contact the egg jelly (EJ) layer. Among other physiological changes, increases in adenylyl cyclase activity, cAMP and cAMP-dependent protein kinase (PKA) activity occur coincident with the AR. By using inhibitors of PKA, a permeable analog of cAMP and the phosphodiesterase inhibitor IBMX, we show that PKA activity is required for AR induction by EJ. A minimum of six sperm proteins are phosphorylated by PKA upon exposure to EJ, as detected by a PKA substrate-specific antibody. The phosphorylation of these proteins and the percentage of acrosome reacted sperm can be regulated by PKA modulators. The fucose sulfate polymer (FSP), a major component of EJ, is the molecule that triggers sperm PKA activation. Extracellular Ca(2+) is required for PKA activation. Six sperm proteins phosphorylated by PKA were identified by tandem mass spectrometry (MS/MS) utilizing the emerging sea urchin genome. Based on their identities and localizations in sperm head and flagellum, the putative functions of these proteins in sperm physiology and AR induction are discussed.     

In vitro induction of the acrosome reaction in bull sperm and the relationship to field fertility using low-dose inseminations

The acrosome reaction (AR) is a prerequisite for normal sperm fertilizing capability and can be studied in vitro after induction by various agents. The efficacy of a sperm population to undergo the AR in vivo is expected to influence male fertilizing potential. During the past two decades, a number of attempts have been made to relate the in vitro-induced AR to field fertility in several species. However, to our knowledge, no studies have combined in vitro induction of the AR with the simultaneous detection of sperm viability and acrosomal status using a high-precision flow cytometric technique. Furthermore, large-scale fertility trials using low-dose inseminations are pending. In the current study, the relationship between field fertility and the in vitro-induced AR was investigated using three ejaculates from each of 195 bulls, 156 Holstein and 39 Jersey bulls (Bos taurus), participating in a progeny test program including low-dose inseminations. A range of insemination doses, varying from 2.0 × 10⁶ to 15 × 10⁶ sperm/dose, was obtained by a controlled dilution process applied to each ejaculate. Different insemination doses were distributed at random among 75,610 experimental first inseminations in 4721 herds and 208 artificial insemination (AI) technicians. Simultaneous detection of sperm viability and acrosomal status was achieved using a triple color flow cytometric technique. Sperm samples from the bulls displayed a wide range of ability to acrosome react in response to calcium ionophore A23187. Both reproducibility of the AR response after induction and relationship between ability to acrosome react and field fertility was highly dependent on the definition of AR inducibility. Six basic and six combined AR indices were assessed. The AR index expressing the fraction of acrosome reacted sperm in the live sperm population after induction by ionophore had the highest repeatability, best described the biological variation in the studied population, and yielded the best significant predictive values on field fertility among the 12 indices considered. Moreover, the ability of sperm to acrosome react appeared to be a noncompensable trait that affects fertility regardless of the number of sperm per insemination dose. The current results therefore indicate that this sperm parameter is important in the field and also may play a role in the IVF laboratory.