Distribution of Intramembrane Particles and Its Changes during the Acrosome Reaction in Spermatozoa of the Japanese Abalone

The distribution of intramembrane particles in the plasma and acrosomal membranes of sperm of the Japanese abalone, Haliotis discus , and its changes during the acrosome reaction were studied by the freeze-fracture replica technique. The P face of the plasma membrane covering the acrosome has sparse membrane particles except in the apical region, which includes the trigger and 'truncated cone' regions. Large particles with an average diameter of 10 nm are located in this apical region. The E face of the plasma membrane has only a few particles. On the outer acrosomal membrane, many particles are randomly distributed throughout the P face, but only a small number of particles are found on the E face. Numerous particles on the P face of the inner acrosomal membrane show a regular arrangement as a dense lattice or with a concentric circular pattern. The initial change in the acrosome reaction is clearance of membrane particles from both the P and E faces of the plasma and outer acrosomal membranes around the apical region, where fusion of the two membranes occurs. As the acrosomal process elongates, the dense arrangement of particles on the inner acrosomal membrane changes via a loose lattice arrangement to a patchy distribution with particle-free areas. Then the arrangement is further disorganized becoming a sparse, random distribution.     

Maitotoxin, A Presumed Calcium Channel Activator, Induces the Acrosome Reaction in Mussel Spermatozoa

Maitotoxin, a presumed activator of the voltage-sensitive calcium channel, induced the acrosome reaction in the mussel, Mytilus edulis at physiological pH and in the starfish, Asterias amurensis at pH 9.5. The induction of acrosome reaction by maitotoxin depended upon external Ca²⁺ and was inhibited by two types of calcium channel blockers; verapamil and diltiazem. These results suggest that the activation of the voltage-sensitive calcium channel takes an important part in the initiation of acrosome reaction in Mytilus and other animals.     

Does Phospholipase A2 Participate in the Acrosome Reaction of Sea Urchin Sperm?: A Pharmacological Study

We examined whether phospholipase A₂ (PLA₂) is involved in the initiation of the acrosome reaction of sperm of the sea urchin, Strongylocentrotus intermedius , using inhibitors and an activator of this enzyme. Quinacrine and p-bromophenacyl bromide (PBPB) inhibited the egg jelly-induced acrosome reaction at 100 μM, but not the ionomycin-induced one. Depression of egg jelly-induced increase of intracellular free Ca²⁺concentration ([Ca²⁺]_i) by these reagents was expected and examined using fura 2. Quinacrine interfered with the flourescence of fura 2, but PBPB was found to depress at concentrations which could inhibit the acrosome reaction. Furthermore, melittin, which is known to stimulate PLA², caused a [Ca²⁺]_i increase and a formation of acrosomal process-like structure on sperm head. These results suggest that PLA₂ participates in the early step(s) of the acrosome reaction of sea urchin sperm.     

Induction of the Acrosome Reaction of Sea Urchin Spermatozoa by Means of Urea

Urea is an effective reagent for inducing the acrosome reaction of spermatozoa in sea urchins. Urea-treated spermatozoa are capable of fertilizing eggs in Ca-deficient sea water. The pH of the urea solution is an important factor affecting the induction of the acrosome reaction. The reaction occurs at a high percentage in urea Solution at pH's higher than 7.8, while the reaction is almost completely suppressed at pH 7.2. Ca⁺⁺ is also an important factor for the induction of the reaction, although the minimum concentration required is very low.
The acrosomal filament formed in urea solution is similar in shape to that formed in egg-water, when fixed after 10 seconds' urea-treatment. The acrosome granule material is found around the basal portion of the acrosomal filament.     

Extraneuronal Monoamine Transporter Mediates the Permissive Action of Cortisol in the Guinea Pig Trachea: Possible Involvement of Tracheal Chondrocytes

Cortisol, a member of glucocorticoids, could potentiate the action of catecholamine by a non-genomic mechanism. Although this permissive effect has been well appreciated in the anti-asthmatic medication, the underlying signaling pathway has remained mysterious. Here, we show that extraneuronal monoamine transporter (EMT), a membraneous reuptake transporter for circulating catecholamine clearance, is the direct target of cortisol in its permissive effect. We found that BSA-conjugated cortisol, which functions as a cortisol but cannot penetrate cell membrane, enhanced the spasmolytic effect of β-adrenoceptor agonist (isoprenaline) in histamine-sensitized tracheal spirals of guinea pigs, and pharmacological inhibition of EMT with famotidine was powerful enough to imitate the permissive action of cortisol. To our surprise, EMT protein expression was high in the chondrocytes of tracheal cartilage, but was undetectable in tracheal smooth muscle cells. The functionality of EMT was further confirmed with measurement of catecholamine uptake by tracheal chondrocytes. Moreover, cortisol-initiated membrane signaling could activate protein kinase C (PKC), which phosphorylates EMT and induces its internalization via a lipid raft-dependent pathway. Both of the mechanisms slow down the reuptake process by chondrocytes, leading to extracellular catecholamine accumulation and results in a more profound adrenergic signaling activation in tracheal smooth muscle cells. Thus, an EMT-centered pathway was proposed to explain the permissive action of cortisol. Collectively, our results highlight the role of EMT in the crosstalk between glucocorticoid and catecholamine. EMT may represent a promising target for adrenergic signaling modulation.     

A Simple Staining Procedure for Detecting the true Acrosome Reaction in Buffalo (Bubalus bubalis) Spermatozoa

A simple dual staining procedure for detecting the true acrosome reaction in dried smears of buffalo spermatozoa is described. Trypan blue is used first to differentiate live from dead spermatozoa and the dried smears which have been prepared are stained with Giemsa for acrosome evaluation. Four categories of spermatozoa were recognized: A) live, intact acrosome (acrosome pink, postnuclear cap clear); B) dead, intact acrosome (acrosome pink, postnuclear cap blue); C) live, detached acrosome (acrosome clear, postnuclear cap clear); and D) dead, detached acrosome (acrosome clear, postnuclear cap blue). The procedure is simple, rapid and convenient for assessing true acrosome reaction in buffalo spermatozoa. Simultaneous assessment of sperm viability and its acrosomal status in dried smears makes this procedure attractive because the true acrosome reaction can be studied thoroughly at a later state after the incubation period.     

Effects of Cations and Other Medium Components on the Zona-induced Acrosome Reaction of Hamster Spermatozoa

Although the acrosome reaction in lively motile hamster spermatozoa can occur independently of the egg or its investments ("spontaneous" acrosome reaction), it appears to be the egg investments, particularly the zona pellucida, that induces the acrosome reaction in fertilizing spermatozoa of many mammalian species. The latter is referred to as "zona-induced" acrosome reaction. Experiments were conducted to determine if the zona-induced acrosome reaction has different ion requirements from the spontaneous reaction. Like the spontaneous acrosome reaction, the zona-induced acrosome reaction required extracellular Na⁺, K⁺ and Ca²⁺. The absence of Cl⁻ and albumin in the medium inhibited the reaction. The zona-induced acrosome reaction could occur in a HCO₃⁻-free medium, but far less efficiently than in medium containing this ion. Proteinase inhibitors, benzamidine and TLCK, inhibited the zona-induced acrosome reaction. These results suggest that the chemical reactions involved in the spontaneous and zona-induced acrosome reactions are similar although the reaction-triggering mechanism is probably different.     

Ultrastructural Changes of the "Truncated Cone" during the Acrosome Reaction in Japanese Abalone Spermatozoa

An electron-dense structure termed the "truncated cone" covers the apical surface of the acrosomal contents except for the trigger region in Haliotis discus spermatozoa. The truncated cone, having a slant height of 0.3 μm and diameters of circular top and base of 0.3 and 0.6 μm, shows striations with a periodicity of 6.6 to 8.0 nm. During the acrosome reaction, the truncated cone elongates simultaneously with the protrusion of the acrosomal process through the truncated cone. As the growth of the acrosomal process further proceeds, the truncated cone transforms into a cylindrical shape and eventually reaches 1 μm in length and 0.2 μm in diameter. The elongated truncated cone is characterized by regularlly helical striations with a periodicity of 19 to 21 nm with an inclination of 40° to 46°. These results may suggest that the truncated cone is composed of coiled filaments, which coil up further during the acrosome reaction causing the truncated cone to slenderize and elongate. The elongation is also achieved by stretching of the coil. In H. discus hannai Ino, structural changes in the truncated cone show close homology to those in H. discus. No such morphologically unique organelle has been found in other species thus far.     

Preliminary Characterization of Phospholipase A2 in Lagenidium giganteum

MacKichan, J. K., Tuininga, A. R., and Kerwin, J. L. 1994. Preliminary characterization of phospholipase A₂ in Lagenidium giganteum. Experimental Mycology 18, 180-192. Phospholipase A₂ (PLA₂) hydrolyses the fatty acyl ester bond at the sn-2 position in glycerophospholipids. To better understand its regulatory roles, factors affecting PLA₂ activity in Lagenidium giganteum were investigated: divalent ions; chelators: inhibitors; pH; and substrate concentration. PLA₂ activity of L. giganteum whole cell homogenates was determined using 1-stearoyl-2-[1-¹⁴C]arachidonoyl phosphatidylcholine as substrate. The divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ all enhanced PLA² activity, while Co²⁺, Fe²⁺, and Zn²⁺ were either slightly inhibitory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while high concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca²⁺ and Mn²⁺ than Mg²⁺, reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both acid (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors, nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenacylbromide, reduced PLA₂ activity by about 80% at 5 mM concentration, 50% with 1 mM inhibitor, and had no effect at 100 μM. The relatively high levels of these compounds needed to inhibit PLA₂ hydrolysis may have been due to the presence of a cocktail of enzymes, some of which were not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without adverse effects to the mycelia. PLA₂ activity was highest in the late oospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA₂ activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes, but there was significant activity in cytosolic fractions. Separation of cell homogenates by column chromatography revealed that there were at least nine enzymes capable of cleaving fatty acids in the sn -2 position of phospholipids.     

A Naphthol Yellow S and Erythrosin B Staining Procedure for Use in Studies of the Acrosome Reaction of Rabbit Spermatozoa

Rabbit spermatozoa suspended in Krebs-Ringer-phosphate containing 0.25% glucose were smeared on polylysine-coated slides and dried in air at room temperature for 2 hr to overnight. Smears were stained in 0.1% naphthol yellow S in 1.0% acetic acid for 30 min at room temperature, blotted, rinsed in 1.0% aqueous acetic acid for 10-15 sec, drained and stained for 7 min in a mixture of equal parts of aqueous naphthol yellow S and erythrosin B (final concentration of each dye 0.1% w/v) at pH 4.6-5.0 (pH adjusted with acetic acid). Stained slides were well rinsed in distilled water adjusted to pH 4.65.0 with acetic acid, blotted, allowed to dry completely, rinsed in xylene and mounted in synthetic resin. Acrosomal caps were stained cherry-red (apical ridge) to pink (dorsal and ventral aspects); postnuclear caps stained pale pink; nuclei were either unstained or stained a very faint yellowish-pink. The mid-piece and flagellum were stained different shades of pink. The procedure is simple, rapid, and gives highly reproducible results. When present, acrosomes are easily detected regardless of the density of the smear.     

Guinea Pig Brain Histamine N-Methyltransferase: Purification and Partial Characterization

Abstract: Histamine N-methyltransferase (EC 2.1.1.8) was purified 4400–fold in 12% yield from guinea pig brain. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE-cel-lulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous by gel electrophoresis and was stable for at least 3 months at 80°C. It had an apparent molecular weight of 29 ,000 ± 1000 as determined by both gel filtration through Sephadex G-100 and by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The isoelectric point of the protein was found to be 5.3. The pH optima for methylation of histamine were determined to be 7.5 and 9.0; the K_ms for histamine and S-adenosyl-l-methionine were 13.57 ± 0.74 μM and 6.1 ± 0.12 μM, respectively; the K_i for S-adenosyl-l-homocysteine was 24.5 ± 1.45 μM.     

A Model for the Acrosome Reaction in Mammalian Sperm

The acrosome reaction is a complex, calcium-dependent reaction that results in an exocytotic event required for successful fertilization of the egg. It has long been thought that the acrosome reaction occurs upon sperm binding to the zona pellucida, a viscoelastic layer surrounding the oocyte. Recent studies have suggested that the reaction may even occur before the sperm encounters the zona, perhaps mediated by progesterone or some other agonist. It has been particularly difficult to understand differences between progesterone-induced and zona-induced reactions experimentally and whether one substance is the more biologically relevant trigger. Until this present work, there has been little effort to mathematically model the acrosome reaction in sperm as a whole. Instead, attention has been paid to modeling portions of the pathways involved in other cell types. Here we present a base model for the acrosome reaction which characterizes the known biochemical reactions and behaviors of the system. Our model allows us to analyze several pathways that may act as a stabilizing mechanism for avoiding sustained oscillatory calcium responses often observed in other cell types. Such an oscillatory regime might otherwise prevent acrosomal exocytosis and therefore inhibit fertilization. Results indicate that the acrosome reaction may rely upon multiple redundant mechanisms to avoid entering an oscillatory state and instead maintain a high resting level of calcium, known to be required for successful acrosomal exocytosis and, ultimately, fertilization of the oocyte.     

三个品种豚鼠血液蛋白多态性的比较分析

目的比较分析白毛黑眼（WHBE）豚鼠和DHP豚鼠、花色豚鼠三个品种豚鼠在13个血液蛋白位点上的多态性。方法采用垂直板浓度和pH均不连续的聚丙烯酰胺凝胶电泳法对WHBE豚鼠、DHP豚鼠和花色豚鼠的66只个体的后白蛋白（Po）、前转铁蛋白1（Prt1）、前转铁蛋白2（Prt2）、转铁蛋白1（Tf1）、转铁蛋白2（Tf2）、后转铁蛋白（Ptf）、慢α球蛋白（Sag）、红细胞酯酶（Es）、血清酯酶1（Est1）、血清酯酶3（Est3）、血红蛋白α（Hbα）、血红蛋白β（Hbβ）和白蛋白（Alb）共13个蛋白位点进行了电泳及染色,再利用电泳图谱对各蛋白位点基因频率、平均杂合度和遗传距离进行计算,然后结合聚类分析。结果 Tf1、Tf2、Ptf、Est1和Es在三个豚鼠品种中表现为多态,其中Tf1可作为识别WHBE豚鼠的遗传标记。Po、Prt1、Prt2、Sag、Est3、Hbα、Hbβ和Alb等位点在三个豚鼠品种中的表型一致。Hardy-Weinberg平衡状态分析表明,Es为DHP豚鼠的高度不平衡位点。Ptf为花色豚鼠的高度不平衡位点。在WHBE豚鼠中,Tf1为高度不平衡位点,Est1为不平衡位点。在三个豚鼠品种中,所检测的13个蛋白位点的平均杂合度的排列顺序为：花色豚鼠（0.350 1）〉WHBE豚鼠（0.339 0）〉DHP豚鼠（0.313 5）。聚类分析结果表明,花色豚鼠和WHBE豚鼠的遗传遗传距离最近（0.064 3）,DHP豚鼠与花色豚鼠的遗传距离最远（0.179 2）。结论利用这些蛋白位点可以有效鉴别WHBE豚鼠、DHP豚鼠和花色豚鼠血液蛋白的遗传多态性。     

Biochemical Studies on the Acrosome Reaction of the Starfish, Asterias Amurensis II. Purification and Characterization of Acrosome Reaction-Inducing Substance

The acrosome reaction-inducing substance (ARIS) was purified from egg jelly of the starfish, Asterias amurensis. The purification procedure included elimination of neutral glycoproteins from the ARIS fraction by isoelectric pointprecipitation and subsequent gel filtrations on Sephadex G–50 and Bio-Gel A-50m columns. The final preparation of ARIS was homogeneous as judged by cellulose acetate electrophoresis of ARIS and by ion-exchange chromatography on DEAE-Sephadex A–25 of S-carboxymethylated ARIS. ARIS is a very large, sulfated glycoprotein containing fucose, galactose, galactosamine and glucosamine as sugar components. It requires diffusible cofactor (Co-ARIS) for full biological activity. A Pronase digest of ARIS retained its capacity to induce the acrosome reaction when Co-ARIS was added to the bioassay system. The physiological significance of the carbohydrate moiety of ARIS is discussed.     

酪氨酸磷酸化蛋白在体外获能豚鼠精子上的分布与表达

为研究豚鼠精子获能过程中蛋白酪氨酸磷酸化的变化规律,将豚鼠精子悬浮于改良的TALP获能培养基中,在5% CO2 孵箱37 ℃培养,以精子与金霉素(CTC) 荧光结合类型为指标评价精子获能状态,用免疫荧光技术和Western blot方法检测酪氨酸磷酸化的蛋白在精子上的分布以及酪氨酸磷酸化水平的变化。结果显示,随着获能的进行,发生蛋白酪氨酸磷酸化的精子占总精子的百分比增加,由未获能前的36％增至获能7h时的92％。酪氨酸磷酸化的蛋白分布变广,由精子头部扩增至精子头部、鞭毛主段和中段。另外,有80,45,40kDa的三种蛋白发生酪氨酸磷酸化,其中40kDa的蛋白酪氨酸磷酸化水平自精子体外培养后呈递增趋势,45kDa的蛋白酪氨酸磷酸化自培养3h后发现并呈递增趋势,而80kDa的蛋白酪氨酸磷酸化水平在精子培养3h时最高,后呈递减趋势。     

Induction of the Acrosome Reaction in Starfish

In the starfish, Asterias amurensis , at least two distinct components of the egg jelly are required for inducing the acrosome reaction: a sulfated glycoprotein named acrosome reaction-inducing substance (ARIS) and a diffusible organic substance(s) named Co-ARIS. The following evidence suggested that ARIS and Co-ARIS cooperatively activate CA-channels of the sperm plasma membrane and eventually induce dramatic changes in sperm morphology, the acrosome reaction. 1) Pronase digest of ARIS (P-ARIS) and Co-ARIS, either as a pure or a crude preparation (Fraction M₈), were fully effective in combination for induction of the acrosome reaction in normal sea water, although they were not effective individually. P- ARIS alone induced the acrosome reaction fully in high Ca²⁺ sea water and markedly at high pHs, whereas Fraction M₈ alone did not induce the reaction even in these conditions. The reaction was not induced by increase in either the Ca²⁺ concentration or the pH of sea water, but was markedly induced in the absence of jelly components by raising both the pH and Ca²⁺ concentration together. 2) The ionophore A23187 induced the acrosome reaction appreciably when present alone and fully in the presence of monensin or Fraction M₈. Monesin alone was ineffective. 3) The jelly or a combination of ARIS and Fraction M₈ caused abrupt Ca²⁺ -uptake by the sperm. The Ca-channel blockers verapamil and diltiazem inhibited the jelly-induced acrosome reaction.     

Mechanism of the Schultz-Dale Reaction in the Denervated Diaphragmatic Muscle of the Guinea Pig

The mechanism of the contractions elicited by specific antigens in immunologically sensitized muscle tissue (Schultz-Dale responses) has been investigated on single fibers of denervated guinea pig hemidiaphragms. This preparation can be either actively or passively allergized, showing Schultz-Dale responses similar to those of visceral muscle. Specific antigens were applied with an electrically operated microtap to discrete areas of the cell surface while recording the electrical activity with intracellular microelectrodes. In this manner, a depolarizing action of the antigens on the muscle membrane was demonstrated. Brief applications of antigen gave rise to phasic potential changes (antigen potentials) similar to those elicited in the same fibers with acetylcholine-filled microtaps. However, antigen potentials occur only in denervated fibers sensitized to the specific antigen or closely related proteins; they are not seen in either innervated fibers of allergized animals or in denervated, nonallergized fibers. Repeated antigen application to the same area of the fiber causes a local irreversible desensitization. The antigen potentials are associated with a reduction in the resistance of the muscle membrane, similar to that caused by acetylcholine. It is concluded that besides causing the liberation of biogenic amines from the mast cells, antigens exert a direct action on the permeability of the muscle membrane; the molecules of antibody adsorbed to the cells appear to act as specific chemoreceptors for the antigen.