Prakash N. Srivastava

Acrosome reaction in human spermatozoa

Human spermatozoa were allowed to undergo the acrosome reaction in vitro by incubation in media with or without reagents known to accelerate the onset of the acrosome reaction. The first observable change before the acrosome reaction was a partial decondensation of the acrosomal matrix. This was followed by invaginations of the outer acrosomal membrane alone or of both the plasma and outer acrosomal membranes, which resulted in formation of many vesicles within the acrosomal cap. Subsequently, the plasma and outer acrosomal membranes fused, but the fusion was seldom seen in the acrosomal cap region. On the other hand, fusion of the two membranes was observed consistently at the anterior end of the equatorial segment of the acrosome. Soon after the membranes over the acrosomal cap disappeared, many vesicles, which were originally within the cap, were seen on or in the vicinity of the inner acrosomal membrane. These vesicles dispersed eventually, leaving the inner acrosomal membrane completely exposed. Thus the acrosome reaction in human spermatozoa seems to proceed in a way somewhat different from that in spermatozoa of most other species, although the end result of the reaction is the same.

Biochemical changes in the zona pellucida of rabbit ova induced by fertilization and sperm enzymes.

The presence of protein in the zona pellucida of the mouse and rabbit ovum has been established by removal of the zona with Pronase (1) and trypsin (2, 3). The zona pellucida of the mouse (4-6), rat and rabbit was shown to be more resistant to digestion by trypsin after fertilization than before (2). Sialic acid has been demonstrated in the zona pellucida of the rabbit ovum (7) and treatment of rabbit ova with bacterial neuraminidase reduced the number of penetrating sperm (8). The vitelline coat of invertebrate ova can be dissolved with cysteine or mercaptoethanol (9, 10). The zona pellucida is the analogous layer in mammalian ova and is also soluble in dilute mercaptoethanol solutions. The sperm acrosome contains various enzymes, such as a trypsin-like enzyme (TLE) (11, 12), a neuraminidase (13), an enzyme that disperses the corona radiata (CPE) (14), and hyaluronidase. The present study concerns biochemical changes in the zona pellucida at the time of fertilization as indicated by a change in the solubility of the zona pellucida in mercaptoethanol and by a change in susceptibility to digestion by trypsin. These changes occur at the time of fertilization and are induced in unfertilized ova by treatment with preparations of certain sperm acrosomal enzymes. Materials and Methods. Preparation of zona pellucida solutions for protein estimation and electrophoresis. Mature New Zealand white rabbits were superovulated by four subcutaneous injections of 0.25 ml of FSH (Armour) at 12-hr intervals, followed by an intravenous injection of 125 units of HCG 12 hr after the last injection of FSH. Ova were flushed from the oviducts 12 hr after administration of HCG and suspended in sterile physiological saline. The cumulus and corona layers were removed by agitation, and the ova were washed with 1 to 2 ml of either physiological saline or glass distilled water.

Mammalian sperm acrosomal neuraminidases.

Abstract A neuraminidase and a neuraminidase-like factor (NLF) were demonstrated for the first time in acrosomes of rabbit, bull, hamster, ram and human spermatozoa. The neuraminidase is similar to the known mammalian neuraminidases, but the NLF is unique in that it renders bound sialic acid reactive in Warrens colorimetric assay but does not release it from the macro-molecular complex. Partially purified decapacitation factor preparations that inhibit fertilization also inhibit NLF activity. The true neuraminidase activity appears to arise from NLF during purification. Cowpers gland mucin of the boar was used as substrate, and the effect of neuraminidase and NLF on this substrate was not duplicated by trypsin, α-chymotrypsin, pronase, α-amylase, lysozyme, hyaluronidase or detergents.

α-L-fucosidase from bull seminal plasma: Its purification and acrosome reaction promoting property

Alpha-L-fucosidase was purified from the bull seminal plasma by chromatography on DEAE-disk, octyl sepharose hydrophobic column and HPLC. The enzyme appeared to be pure as judged by the polyacrylamide gel electrophoresis both under the nondenaturing and denaturing conditions. The pure enzyme promoted the acrosome reaction of guinea pig spermatozoa in vitro. This is the first report showing that an acrosomal enzyme induces acrosome reaction which is an essential pre-requisite for the gamete interaction and fertilization.

Isolation of rabbit testicular cathepsin D and its role in the activation of proacrosin.

Abstract Cathepsin D was purified 900-fold with 30% recovery from rabbit testes using pepstatin bound Sepharose affinity chromatography. The enzyme is homogeneous as observed by acrylamide gel electrophoresis. The heat stable enzyme exhibits an apparent molecular weight of 42,000 with identical subunits of 20,000. Purified cathepsin D catalyses the conversion of proacrosin to acrosin.

Hydrolysis of p-nitrophenylphosphorylcholine by alkaline phosphatase and phospholipase C from rabbit sperm-acrosome.

Abstract p-Nitrophenylphosphorylcholine, used as an artificial substrate for phospholipase C, is readily hydrolyzed by alkaline phosphatases also, despite the reported requirement of alkaline phosphatases for a terminal phosphate for activity. p-Nitrophenylphosphorylcholine hydrolyzing activity in rabbit semen is concentrated in Hyamine-Triton extracts of sperm acrosomes or cytoplasmic droplets rather than seminal plasma. Disc electrophoresis of these extracts shows well separated zones of phosphatase and phospholipase C-like activity. Only phosphatase activity is observed in seminal plasma. It is suggested that phospholipase C is located in the sperm acrosome.

Extraction and quantification of acrosin, β‐N‐acetylglucosaminidase, and arylsulfatase‐A from equine ejaculated spermatozoa

Acrosin, Arysulfatase A, and beta-N-acetylglucosaminidase are three key enzymes localized within the mammalian acrosome that play a pivotal role in the penetration of the oocyte. The objectives of this study were to compare two methods of enzyme extraction based on the activities of these enzymes from equine spermatozoa. Method A utilized a 0.5 M Tris-maleate buffer containing 0.1% Triton X-100 and Hyamine 2389. Method B used 0.05 M Tris-HCl, 0.05 M MgCl2 in 0.05 M Tris-maleate, followed by 0.05 M Tris-maleate containing 0.1% Triton X-100. Results indicated that acrosin was initially bound in an acrosin-acrosin inhibitor complex; this complex was dissociated after incubating the extract in 2 mM HCl. Significant (P < 0.001) increases in acrosin activity were found after-acid extraction from 0.076 U/mg after Method B to 0.327 U/mg after Method A. Arylsulfatase A activity was found to have a higher mean activity (P < 0.03) after Method A (0.012 U/mg) as opposed to Method B (0.007 U/mg). Similarly, beta-N-acetylglucosaminidase was found to have a higher mean specific activity (P < 0.001) after Method A (0.037 U/mg) as compared to Method B (0.008 U/mg). This is the first report of the quantification of these enzymes from equine spermatozoa which can ultimately be used as an index of acrosomal damage in cryopreserved semen, and provide additional insight into biochemical alterations between normal vs. abnormal semen.

Purification and Properties of Aryl Sulfatases from Rabbit Sperm Acrosomes

Summary The extraction of rabbit spermatozoa with the MgCl2 treatment, followed by chromatography of extracts on a DEAE-cellulose column, yield highly purified aryl sulfatases. The enzyme preparation showed one major band with the possibility of a minor contaminant by acrylamide gel electrophoresis. The single band had two activities, that of aryl sulfatases A and B. Aryl sulfatase C was not detectable. The purified aryl sulfatases had optima at pHs 4.8, 5.6, and 6.0. The enzymes exhibited no activity at 4° but exhibited. higher activity at 50° than at 37°. The pure aryl sulfatases had higher affinity for p-nitrocatechol sulfate than for p-nitrophenol sulfate. Phosphate and sulfite ions inhibited the enzymes, but NaCl, KCl, and sulfate did not. Rabbit, ram, bull, and boar sperm acrosomal extracts showed high aryl sulfatase activities. The authors thank Mrs. Diane Fitzgerald for her skillful technical assistance.

Inhibition by Seminal Plasma of Acrosomal Enzymes in Intact Sperm

Summary Epididymal sperm have high trypsin-like enzyme (TLE) activity, CPE activity and neuraminidase activity whereas these enzyme activities are much lower in ejaculated sperm. Incubation of epididymal sperm with seminal plasma caused a decrease in each of these enzyme activities, indicating that seminal plasma inhibitors may transfer to the acrosomal enzymes during ejaculation. Boiled and centrifuged seminal plasma did not inhibit the TLE activity of epididymal sperm but did decrease the CPE and neuraminidase activity.

Purification of bull sperm hyaluronidase by concanavalin-A affinity chromatography

A new method for obtaining highly purified hyaluronidase (hyaluronate glycanohydrolase EC 3.2.1.25) in high yield is described. Bull seminal plasma was fractionated with (NH4)2 SO4 and the 30 to 65% saturation fractions were applied to a DEAE-cellulose column. The first protein peak contained hyaluronidase, beta-N-acetylglucosaminidase and beta-glucuronidase. The latter two enzymes were separated by gel filtration on Sephadex G-200. The hyaluronidase was further purified by a Concanavalin-A Sepharose 4B affinity column. By gradient elution with alpha-methyl-D-glucoside a fraction which had a specific activity of 1998 units/mg protein (57 942 National Formulary Standard units/mg protein) was obtained. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 4.3. The purified hyaluronidase did not show any beta-glucuronidase or beta-N-acetylglucosaminidase activities. The percent yield of purified hyaluronidase calculated on the basis of total activity was ten times higher than by any pervious method [Yang, C.H. and Srivastava, P.N. (1975) J. Biol. Chem. 250, 79-83].