Zona pellucida-mediated acrosomal exocytosis in mouse spermatozoa: characterization of an intermediate stage prior to the completion of the acrosome reaction

The zona pellucida (ZP)-induced acrosome reaction in mouse sperm proceeds in two steps, identified by three sperm fluorescence patterns observed sequentially with the fluorescent probe chlortetracycline. Capacitated, acrosome-intact sperm displaying a B pattern proceed to an intermediate S pattern, and then progress from the S pattern to the fully acrosome-reacted AR pattern. Previously, it was not feasible to characterize the nature of the transient intermediate S pattern. Recently, it was demonstrated that sperm bind to the ZP of eggs treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and undergo a B to S transition, but do not complete the acrosome reaction. These cells accumulate in the S pattern and fail to undergo the S to AR transition (Endo, Y., Schultz, R. M., and Kopf, G. S. 1987a. Dev. Biol. 119, 119-209). The present study utilized ZP from TPA-treated eggs to assess the state of S pattern sperm. The kinetics of the B to S transition of sperm incubated with either structurally intact or solubilized ZP from untreated or TPA-treated eggs are identical. Addition of either solubilized ZP from untreated eggs or A-23187 to S pattern sperm bound to intact or solubilized ZP from TPA-treated eggs induces the S to AR transition, while ZP from TPA-treated or fertilized eggs does not. Loss of the transmembrane pH gradient in the anterior portion of the sperm head, monitored by the fluorescent pH probe 9-N-dodecyl aminoacridine, follows the B to S transition in sperm incubated with ZP from unfertilized eggs, but no loss is observed when the B to S transition is induced using ZP from TPA-treated eggs. Subsequent addition of solubilized ZP from untreated eggs or A-23187 results in the loss of the transmembrane pH gradient of these S pattern sperm. Addition of nigericin to S pattern sperm bound to ZP from TPA-treated eggs discharges the transmembrane pH gradient and causes the S to AR transition. In contrast, nigericin added to B pattern sperm discharges the pH gradient but does not induce a B to S transition. Electron microscopic evaluation of S pattern-arrested sperm using ZP from TPA-treated eggs reveals intact plasma and outer acrosomal membranes. These results suggest that ZP from TPA-treated and fertilized eggs are modified such that the ZP ligands inducing the S to AR transition are lost or are inactivated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of a Gi protein in mouse sperm membranes by solubilized proteins of the zona pellucida, the egg's extracellular matrix.

Zona pellucida (ZP)-induced acrosomal exocytosis in mammalian spermatozoa is thought to be mediated by signal transduction cascades similar to those found in hormonally responsive cells. In order to characterize this process further, we have examined the role of GTP-binding regulatory proteins (G proteins) in coupling sperm-ZP interaction to intracellular second messenger systems in mouse sperm. An in vitro signal transduction assay was developed to assess ZP-G protein dynamics in sperm membrane preparations. Guanosine 5'-3-O-(thio)triphosphate (GTP gamma S), a poorly hydrolyzable analogue of GTP, bound to these membranes in a specific and concentration-dependent fashion which reached saturation at 100 nM. Incubation of the membrane preparations with heat-solubilized ZP resulted in a significant increase in specific GTP gamma S binding in a concentration-dependent fashion with a half-maximal response at 1.25-2 ZP/microliters. Solubilized ZP also caused a significant increase in high affinity GTPase activity in the membranes over basal levels. Mastoparan increased specific GTP gamma S binding to the sperm membranes and stimulated high-affinity membrane GTPase activity to levels consistently greater than that seen with the solubilized ZP. Mastoparan, together with solubilized ZP, gave the same level of stimulation of GTP gamma S binding as mastoparan alone. Pertussis toxin completely inhibited the ZP-stimulated GTP gamma S binding, but only decreased mastoparan-stimulated GTP gamma S binding by 70-80%. Purified ZP3, the ZP component which possesses quantitatively all of the acrosomal exocytosis-inducing activity of the intact ZP, stimulated GTP gamma S binding to the same level as solubilized ZP; ZP1 and ZP2 did not stimulate GTP gamma S binding. ZP from fertilized eggs (ZPf), which does not possess acrosome reaction-inducing activity, also failed to stimulate GTP gamma S binding to sperm membranes. These data demonstrate the direct activation of a Gi protein in sperm membrane preparations in response to the ZP glycoprotein, ZP3, that induces the acrosome reaction. These data imply that Gi protein activation is an early event in the signal sequence leading to sperm acrosomal exocytosis.     

Evidence that plasma membrane electrical potential is required for vesicular stomatitis virus infection of MDCK cells: A study using fluorescence measurements through polycarbonate supports

Summary We used fluorescence microscopy of Madin-Darby Canine Kidney (MDCK) cells grown on polycarbonate filters to study a possible link between plasma membrane electrical potential (_pm) and infectivity of vesicular stomatitis virus (VSV). Complete substitution of K⁺ for extracellular Na⁺blocks VSV infection of MDCK cells as well as baby hamster kidney (BHK) cells. When we independently perfused the apical and basal-lateral surfaces of high resistance monolayers, high K⁺ inhibited VSV infection of MDCK cells only when applied to the basal-lateral side; high K⁺ applied apically had no effect on VSV infection. This morphological specificity correlates with a large decrease in _pm of MDCK cells when high K⁺ buffer is perfused across the basal-lateral surface. Depolarization of the plasma membrane by 130 mm basal K⁺ causes a sustained increase of cytosol pH in MDCK cells from 7.3 to 7.5 as reported by the fluorescent dye BCECF. Depolarization also causes a transient increase of cytosol Ca²⁺ from 70 to 300 nm as reported by the dye Fura-2. Neither increase could explain the block of VSV infectivity by plasma membrane depolarization. One alternative hypothesis is that _pm facilitates membrane translocation of viral macromolecules as previously described for colicins, mitochondrial import proteins, and proteins secreted by Escherichia coli.We thank Kenneth Spring for many helpful discussions concerning fluorescence digitized imaging systems, James Russell for his collaboration in the design of our imaging system, Herbert Chase for suggestions on dye loading into MDCK cells, and Manfred Schubert and George Harmison for providing expertise on VSV.     

The role of protein kinases in anoxia tolerance in facultative anaerobes: purification and characterization of a protein kinase that phosphorylates pyruvate kinase

A protein kinase which phosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia-tolerant gastropod mollusc Busycon canaliculatum. Purification involved four steps: poly(ethylene glycol) fractionation, affinity chromatography on Blue agarose, ion-exchange chromatography on phosphocellulose and preparative isoelectric focusing (pI = 5.5). The activity was monitored by following changes in pyruvate kinase I50 values for L-alanine which have previously been linked to changes in the degree of enzyme phosphorylation. The correlation between enzyme phosphorylation and changes in the L-alanine inhibition constant was also directly demonstrated in the present paper by radioactively labelling PK with [tau-32P]ATP. The final purified protein kinase solution gave a single band on SDS-gel electrophoresis with a molecular weight of 37,000 +/- 2000. Kinetic analysis of the purified protein kinase (PK-kinase) showed a pH optimum of 7.0, an absolute requirement for magnesium ions (Km = 1.29 mM), a relatively high affinity for MgATP (Km = 57 microM), and inhibition by increasing salt concentrations (I50 = 55 mM KCl). The protein kinase activity was not affected by either spermine, heparin, cAMP, cGMP or concentrations of CaCl2 less than 10 mM. The enzyme did not phosphorylate either phosphofructokinase or glycogen phosphorylase, two enzymes that are also phosphorylated during anoxia in whelks. The purified enzyme is different from the catalytic subunit of cAMP-dependent protein kinase as shown by the inability of cAMP to stimulate the protein kinase at all stages of the preparation; cAMP did not activate either crude enzyme, the 7% poly(ethylene glycol) supernatant, or any of the column eluant peak fractions when measured by changes in pyruvate kinase kinetic parameters.     

Zooplankton impacts on chlorophyll and transparency in Onondaga Lake,New York,USA

The transparency of polluted, hypereutrophic Onondaga Lake, New York, USA has improved substantially in the late 1980's as a result of reductions in phytoplankton biomass, in the absence of significant reductions in external phosphorus loading. Much of this improvement has been due to the occurrence of clearing events, e.g. sudden and dramatic increases in transparency. Field measurements, laboratory experiments, and modelling analyses were utilized to identify processes regulating phytoplankton standing crop during the spring to fall interval of 1987. Changes in the zooplankton community documented over the past decade support the conclusion that increased zooplankton grazing has contributed to improvements in transparency. Herbivores now represent a greater fraction of the zooplankton population and more efficient cladocerans are present in greater numbers. Biomanipulation practices, e.g. reestablishment of piscivorous species, designed to reduce the abundance of planktivorous fish species in Onondaga Lake, may serve to reduce pressure on the grazing community and thus result in further improvements in transparency.     

Properties of pyruvate kinase and flagellar ATPase in rabbit spermatozoa: relation to metabolic strategy of the sperm cell

Rabbit sperm pyruvate kinase remains bound to the cell structure of hypotonically treated mature rabbit epididymal spermatozoa (HTRES). It displays kinetic behavior very similar to that of rabbit muscle pyruvate kinase with regard to KM values for substrates, activation by monovalent and divalent cations, inhibition by phenylalanine which is reversed by alanine, and lack of activation by fructose-1,6-biphosphate. The flagellar ATPase also remains bound to the cell structure of HTRES, whose motility may be reactivated by a source of ATP. It requires Mg+2 for activity; the KM for both ATP and MG+2 is 0.2 mM, implying that MgATP is the substrate. The ATPase activity is not inhibited by ouabain, oligomycin, or vanadate, which also do not affect reconstituted motility, and is not affected by cyclic AMP in the presence of an inhibitor of phosphodiesterase. The activities of pyruvate kinase and the flagellar ATPase in a given preparation of HTRES are comparable. Rabbit spermatozoa have a metabolic strategy which is very similar to muscle cells. This suggests that the major use of the sperm cell's metabolic machinery is maintenance of energy for the contractile work of motility and that only minor amounts of metabolic energy appear to be consumed in other reactions, including those involved in fertilization.     

Energy metabolism of spermatozoa. IV. Effect of calcium on respiration of mature epididymal sperm of the rabbit.

The effect of calcium (Ca+2) on the respiration rate of mature rab bit epididymal sperm was studied. The addition of Ca+2 did not further stimulate the respiration rate of sperm already stimulated by glucose or pyruvate. Oligomycin, which inhibits mitochondrial ATP synthesis and slows respiration, did not inhibit the uptake of mitochond rial Ca+2. The addition of the ionophore A23187, which promotes selective permeability of cell membranes to Ca+2, caused a marked stimulation of respiration when Ca+2 was added, indicating that the sperm cell membrane is not permeable to Ca+2. The stimulation of the respiration rate by pyruvate, but not glucose, was enhanced by the addition of 45 mM HCO3, which did not affect the response to added Ca+2. With or without Ca+2, cyclic AMP and dibutyl cyclic AMP did not stimulate respiration in the presence of pyruvate or glucose. The results suggest that mature rabbit sperm from the cauda epididymis are intrinsically motile, and not dependent on Ca+2.