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.     

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.     

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.     

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.     

Effects of Supplementation of Various Medium Components on Chinese Hamster Ovary Cell Cultures Producing Recombinant Antibody

Thirteen vitamins, twenty amino acids, hormones, inorganic salts, and other chemical agents, which constitute typical serum-free media, were evaluated for the development of fortified medium to enhance cell growth and productivity of recombinant antibody in the cultures of the recombinant Chinese hamster ovary (rCHO) cells. Two different rCHO cell lines, rCHO-A producing recombinant antibodies against the human platelet and rCHO-B secreting recombinant antibodies against the S surface antigen of Hepatitis B, respectively, were cultivated in batch suspension mode. Concentration of interested component in the tested medium was doubled to examine the fortification effect. Growth of rCHO-A cell and its antibody production were slightly improved with addition of either choline chloride, folic acid, thiamine⋅HCl, or Long^TMR³IGF-I. On the other hand, in the cultivation of rCHO-B cell which was more sensitive to its environmental changes, hormones such as Long^TMR³IGF-I and triiodothyronine (T₃) as well as various vitamins involving choline chloride, i-inositol, niacinamide, pyridoxine HCl, and thiamine⋅HCl enhanced the cell growth and antibody production. Particularly, when concentration of consuming amino acid was doubled, remarkable increase in specific productivity was served, resulting in high final antibody concentration. These results were believed to provide a fundamental strategy of medium fortification useful for improvement of recombinant antibody production in serum-free medium. These authors contributed equally to this work     

Phospholipase A of Guinea Pig Spermatozoa: Its Preliminary Characterization and Possible Involvement in the Acrosome Reaction

Phospholipase A activity was demonstrated in guinea pig spermatozoa using [U-¹⁴C] phosphatidyl choline as a substrate. The activity had a neutral pH optimum, was stimulated by Ca²⁺ and low concentrations of detergent, and. was inhibited by EDTA, mepacrine and p-bromophenacyl bromide. Appropriate concentrations of mepacrine and p-bromophenacyl bromide inhibited the acrosome reactions of capacitated spermatozoa without interfering with their motility. These results support the notion that phospholipase A is involved in the acrosome reaction of mammalian spermatozoa.     

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.     

Effect of Phosphatidylcholine on Acrosome Breakdown and Fertilizing Capacity of Amphibian Spermatozoa

The effect of vesicles of purified egg yolk phosphatidylcholine on the fertilizing capacity and acrosome breakdown of amphibian spermatozoa was studied. When Bufo arenarum spermatozoa were incubated with either small unilamellar vesicles (prepared by sonication) or with large unilamellar vesicles (prepared by reverse-phase evaporation) a decrease in the fertilizing capacity of spermatozoa was found. At the same phosphatidylcholine concentration, large unilamellar vesicles were more inhibitory than small unilamellar vesicles. The inhibition was dependent upon the phospholipid concentration and the length of the incubation period. Small unilamellar vesicles did not modify the time course of acrosome breakdown in Leptodactylus chaquensis , while large unilamellar vesicles markedly accelerated the rate of acrosome breakdown. In both biossays, the charge of the vesicles (made either positive or negative by the addition of 5% stearylamine or 5% phosphatidic acid) did not influence their biological effect. Multilamellar vesicles did not alter the fertilizing capacity nor the acrosome breakdown. We conclude that the size and the structure of the vesicles are important parameters in determining the inhibitory capacity of phosphatidyl choline on amphibian fertilization.     

Biochemical Studies on the Acrosome Reaction of the Starfish, Asterias Amurensis I. Factors Participating in the Acrosome Reaction

In contrast with the case in sea urchin sperm, in starfish the acrosome reaction is not spontaneously induced by simply increasing the extracellular Ca²⁺ concentration or pH. At higher pHs, starfish sperm undergo morphological changes accompanied by exocytosis of the acrosomal vacuole, but they do not form acrosomal filaments. Nomarski-microscopic observation confirmed that spermatozoa undergo the acrosome reaction within the jelly coat. Acrosome reaction-inducing substance, a glycoprotein from the egg jelly, required a diffusible cofactor(s) present in the egg jelly for full activity. Several lines of evidence showed that this diffusible factor(s) is not merely Ca²⁺.     

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.     

Induction of the Acrosome Reaction of Hemicentrotus pulcherrimus Spermatozoa by the Egg Jelly Molecules, Fucose-Rich Glycoconjugate and Spem-Activating Peptide I

A fucose-rich glycoconjugate (FRG) was isolated from egg jelly of the sea urchin Hemicentrotus pulcherrimus by gel filtration. FRG induced the acrosome reaction in H. pulcherrimus spermatozoa in a concentration-dependent manner, although it showed about half the activity of the original unfractionated jelly. Synthetic sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) increased the rate of the acrosome reaction induced by FRG; the maximal rate of the acrosome reaction with FRG and SAP-I being that of the unfractionated jelly. The half-maximal increase in induction of the acrosome reaction by SAP-I with FRG occurred at 4 × 10⁻¹⁰ M SAP-I, which was almost the same concentration inducing half-maximal stimulation of sperm respiration. Pronase digestion of FRG resulted in an 50% decrease in induction of the acrosome reaction and also in the elevation of cAMP in sperm. Some reagents (monensin and 3-isobutyl-1-methylxanthine) which increase intracellular pH, Ca²⁺ and cyclic nucleotides also increased the rates of the acrosome reaction induced by FRG or pronase-digested FRG. However, the rates did not reach those with FRG or pronase-digested FRG with SAP-I. These results indicate that SAP-I promotes induction of the acrosome reaction by acting as a specific co-factor of FRG.     

Viability and Acrosome Staining of Bull, Boar and Rabbit Spermatozoa

A practical and reliable staining procedure was developed to distinguish the viability and acrosomal status of bull, boar and rabbit spermatozoa. The first stain with trypan blue or Congo red is rapid and avoids artifacts. This stain is precipitated by neutral red during the 2 min required for fixation. The precipitate gives a high contrast black color, resistant to the subsequent rinsings and persists during the time required for staining the acrosome with Giemsa. Ten classes of spermatozoa are distinguished (live or dead with intact acrosomes, loose acrosomes, damaged acrosomes, no acrosome, or with no acrosome and no postacrosomal ring). The intact acrosomes are purple, the loose acrosomes are dark lavender and the damaged acrosomes are pale lavender. The anterior part of the head of live spermatozoa with no acrosome is white or light pink and the same area of dead spermatozoa is white or pale gray. The postacrosomal ring is red. The postacrosomal area of the head of live spermatozoa is white or light pink and the same part of dead spermatozoa is black, dark violet or gray. The procedure did not give satisfactory results for stallion spermatozoa.     

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.     

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.     

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.     

培养基的组成对黄原胶发酵产量与质量的影响研究

本文着重研究了培养基各组分对胶产量与质量的影响。结果表明:氮源是极显著因素,碳源是显著因素,无机盐与柠檬酸对质量与产量的影响各有主次;以蔗糖代替葡萄糖可使产量与质量有较大提高。此外还分析了培养基中CaCO_3的加入、消泡剂的选择、H_2O_2水的添加等各因素对产胶的影响。     

培养基组成对树状多节孢(Nodulisporium sylviforme)紫杉醇产量的影响

采用正交试验法 ,研究了醋酸钠、苯丙氨酸、酪氨酸、亮氨酸对紫杉醇产生菌HQD3 3 产生紫杉醇的影响。结果表明 ,它们之间的协同作用对提高紫杉醇产量有显著影响。在改良的S - 7培养基基础上 ,再加入醋酸钠 1 0g/L、苯丙氨酸 5 0mg/L、酪氨酸 1 5mg/L、亮氨酸 6 0mg/L ,可以使紫杉醇产生菌HQD3 3 产生紫杉醇产量提高到 2 0 3 6 5 6 μg/L。