Sara Rubinstein

Remodeling of the Actin Cytoskeleton During Mammalian Sperm Capacitation and Acrosome Reaction

Abstract The sperm acrosome reaction and penetration of the egg follow zona pellucida binding only if the sperm has previously undergone the poorly understood maturation process known as capacitation. We demonstrate here that in vitro capacitation of bull, ram, mouse, and human sperm was accompanied by a time-dependent increase in actin polymerization. Induction of the acrosome reaction in capacitated cells initiated fast F-actin breakdown. Incubation of sperm in media lacking BSA or methyl-β-cyclodextrin, Ca2+, or NaHCO3, components that are all required for capacitation, prevented actin polymerization as well as capacitation, as assessed by the ability of the cells to undergo the acrosome reaction. Inhibition of F-actin formation by cytochalasin D blocked sperm capacitation and reduced the in vitro fertilization rate of metaphase II-arrested mouse eggs. It has been suggested that protein tyrosine phosphorylation may represent an important regulatory pathway that is associated with sperm capacitation. We show here that factors known to stimulate sperm protein tyrosine phosphorylation (i.e., NaHCO3, cAMP, epidermal growth factor, H2O2, and sodium vanadate) were able to enhance actin polymerization, whereas inhibition of tyrosine kinases prevented F-actin formation. These data suggest that actin polymerization may represent an important regulatory pathway in with sperm capacitation, whereas F-actin breakdown occurs before the acrosome reaction.

Role of Hydrogen Peroxide in Sperm Capacitation and Acrosome Reaction

Abstract The generation of reactive oxygen species (ROS) has been implicated in the regulation of sperm capacitation and acrosome reaction; however, the mechanisms underlying this regulation remain unclear. To examine the cellular processes involved, we studied the effect of different concentrations of hydrogen peroxide (H2O2) on protein tyrosine phosphorylation under various conditions. Treatment of spermatozoa with H2O2 in medium without heparin caused a time- and dose-dependent increase in protein tyrosine phosphorylation of at least six proteins in which maximal effect was seen after 2 h of incubation with 50 μM H2O2. At much higher concentrations of H2O2 (0.5 mM), there is significant reduction in the phosphorylation level, and no protein tyrosine phosphorylation is observed at 5 mM H2O2 after 4 h of incubation. Exogenous NADPH enhanced protein tyrosine phosphorylation similarly to H2O2. These two agents, but not heparin, induced Ca2+-dependent tyrosine phosphorylation of an 80-kDa protein. Treatment with H2O2 (50 μM) caused approximately a twofold increase in cAMP, which is comparable to the effect of bicarbonate, a known activator of soluble adenylyl cyclase in sperm. This report suggests that relatively low concentrations of H2O2 are beneficial for sperm capacitation, but that too high a concentration inhibits this process. We also conclude that H2O2 activates adenylyl cyclase to produce cAMP, leading to protein kinase A-dependent protein tyrosine phosphorylation.

Differential Localization of Conventional Protein Kinase C Isoforms During Mouse Oocyte Development

Abstract Protein kinase C (PKC), the major cell target for tumor-promoting phorbol esters, plays a central role in signal transduction pathways. In many biological systems where Ca2+ serves as a second messenger, regulatory control is mediated by PKC. The activation of PKC depends on its binding to RACK1 receptor, which is an intracellular protein anchor for activated PKC. We demonstrate that the conventional PKC (cPKC) isoforms, PKC-α, PKC-βI, and PKC-βII, as well as RACK1, are expressed in mouse oocytes (germinal vesicle [GV]) and mature eggs (metaphase II [MII]). In GV oocytes, PKC-α, PKC-βII, and RACK1 were uniformly distributed in the cytoplasm, while PKC-βI was localized in the cytoplasm and in the plasma membrane as well. Treatment of GV oocytes with the biologically active phorbol ester, 12-o-tetradecanoyl phorbol-13-acetate (TPA), resulted in a rapid translocation of the cytosolic PKC-α, but not PKC-βI, PKC-βII, or RACK1, to the plasma membrane. This was associated with inhibition of GV breakdown. In MII eggs (17 h post-hCG), PKC-α was uniformly distributed in the cytoplasm while PKC-βI and -βII were distributed in the cytoplasm and in the plasma membrane as well. Treatment with TPA resulted in a rapid translocation of PKC-α from the cytoplasm to the plasma membrane and a significant decrease of PKC-βI throughout the cytoplasm, while it also remained in the cell periphery. No change in the distribution of PKC-βII or RACK1 was observed. TPA also induced pronucleus formation. Physiological activation of MII eggs by sperm induced cortical granule exocytosis associated with significant translocation of PKC-α and -βI, but not -βII, to the plasma membrane. Overall, these results suggest a possible involvement of cPKC isoforms in the mechanism of mouse oocyte maturation and egg activation.

Epidermal growth factor induces acrosomal exocytosis in bovine sperm

At the time of fertilization mammalian spermatozoa undergo a Ca2+‐dependent exocytotic event, which is known as the acrosome reaction (AR). We describe here that EGF‐receptor (EGFR) is localized in the head of bull spermatozoa and that epidermal growth factor (EGF) can induce the occurrence of the AR in its typical dose‐dependent manner. Previously we showed that protein kinase C (PKC) is involved in the cascade leading to AR in bull spermatozoa. Here, we show that PKC is involved in the mechanism in which EGF exerts its effect on AR. These findings together with our results which show inhibition of AR by tyrosine‐phosphorylation inhibitors, indicate that ejaculated bull sperm contain a typical 170‐kDa EGFR which is active in the mechanism leading to AR.

Involvement of MEK-Mitogen-Activated Protein Kinase Pathway in Follicle-Stimulating Hormone-Induced but Not Spontaneous Meiotic Resumption of Mouse Oocytes

Abstract Mitogen-activated protein (MAP) kinase has been reported to be activated during oocyte meiotic maturation in a variety of mammalian species. However, the mechanism(s) responsible for MAP kinase activation and the consequence of its premature activation during gonadotropin-induced oocyte meiotic resumption have not been examined. The present experiments were conducted to investigate the possible role of MAP kinase in FSH-induced and spontaneous oocyte meiotic resumption in the mouse. MAP kinase kinase (MAPKK, MEK) inhibitor, PD98059 or U0126, produced a dose-dependent inhibitory effect on both FSH-induced oocyte meiotic resumption and MAP kinase activation in the oocytes. However, the same inhibitor did not block spontaneous meiotic resumption of either denuded or cumulus cell-enclosed mouse oocytes, despite the activity of MAP kinase being totally inhibited. Immunoblotting the oocytes and the cumulus cells with the anti-active MAP kinase antibody showed that MAP kinase activity in the oocytes was detected at 8 h of FSH treatment, prior to germinal vesicle breakdown and increased as maturation progressed in the following culture period. In the cumulus cells, MAP kinase was activated even faster, its activity was detected at 1 h of FSH stimulation and increased gradually until 8 h of FSH treatment, then decreased and diminished after 12 h of FSH action. These data demonstrated that the MEK-MAP kinase pathway is implicated in FSH-induced but not spontaneous oocyte meiotic resumption.

Calcium transport and Ca2+-ATPase activity in ram spermatozoa plasma membrane vesicles.

Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca2+-activated Mg2+-ATPase and Ca2+ transport activities. Membrane vesicles that were exposed to oxalate as a Ca2+-trapping agent accumulated Ca2+ in the presence of Mg2+ and ATP. The Vmax for Ca2+ uptake was 33 nmol/mg protein per h, and the Km values for Ca2+ and ATP were 2.5 microM and 45 microM, respectively. 1 microM of the Ca2+ ionophore A23187, added initially, completely inhibited net Ca2+ uptake and, if added later, caused the release of Ca2+ previously accumulated. A Ca2+-activated ATPase was present in the same membrane vesicles which had a Vmax of 1.5 mumol/mg protein per h at free Ca2+ concentration of 10 microM. This Ca2+-ATPase had Km values of 4.5 microM and 110 microM for Ca2+ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca2+ by the vesicles. The Ca2+-ATPase activity was insensitive to ouabain. Both Ca2+ transport and Ca2+-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca2+ transport activity and a Ca2+-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin.

Bovine sperm acrosome reaction induced by G-protein-coupled receptor agonists is mediated by epidermal growth factor receptor transactivation.

We have previously demonstrated the presence of active epidermal growth factor receptor (EGFR) and its involvement in sperm capacitation and the acrosome reaction; however, the mechanism of EGFR activation was not clear. We show here that the sperm EGFR can be transactivated by angiotensin II or by lysophosphatydic acid, two ligands which activate specific G-protein-coupled receptors (GPCR), or by directly activating protein kinase A using 8Br-cAMP. This transactivation occurs in noncapacitated sperm and is mediated by PKA, SRC and a metalloproteinase. We also show that the EGFR is activated in sperm incubated under in vitro capacitation conditions, without any added ligand, but not in bicarbonate-deficient medium or when PKA is blocked. Despite the fact that EGFR is activated in capacitated sperm, this state is not sufficient to induce the acrosome reaction. We conclude that the EGFR is stimulated during capacitation via PKA activation, while further activation of the EGFR in capacitated sperm is required in order to induce the acrosome reaction. The acrosome reaction can be induced by GPCR via the transactivation of the EGFR by a signaling pathway involving PKA, SRC and metalloproteinase and the EGFR down-stream effectors PI3K, PLC and PKC.

Role of PI3-Kinase and PI4-Kinase in Actin Polymerization During Bovine Sperm Capacitation

Abstract We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian sperm capacitation. In the present study, we investigated the involvement of phosphatidylinositol 3- and 4-kinases (PI3K and PI4K) in actin polymerization, as well as the production of PIP2(4,5), which is a known cofactor for PLD activation, during bovine sperm capacitation. PIK3R1 (p85 α regulatory subunit of PI3K) and PIKCB (PI4K β) in bovine sperm were detected by Western blotting and immunocytochemistry. Wortmannin (WT) inhibited PI3K and PI4K type III at concentrations of 10 nM and 10 μM, respectively. PI4K activity and PIP2(4,5) production were blocked by 10 μM WT but not by 10 nM WT, whereas PI3K activity and PIP3(3,4,5) production were blocked by 10 nM WT. Moreover, spermine, which is a known PI4K activator and a component of semen, activated sperm PI4K, resulting in increased cellular PIP2(4,5) and F-actin formation. The increases in PIP2(4,5) and F-actin intracellular levels during sperm capacitation were mediated by PI4K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP enhanced PIP2(4,5), PIP3(3,4,5), and F-actin formation, and these effects were mediated through PI3K. On the other hand, activation of PKC by phorbol myristate acetate enhanced PIP2(4,5) and F-actin formation mediated by PI4K activity, while the PI3K activity and intracellular PIP3(3,4,5) levels were reduced. These results suggest that two alternative pathways lead to PI4K activation: indirect activation by PKA, which is mediated by PI3K; and activation by PKC, which is independent of PI3K activity. Our results also suggest that spermine, which is present in the ejaculate, regulates PI4K activity during the capacitation process in vivo.

Modified PVA–Fe3O4 Nanoparticles as Protein Carriers into Sperm Cells

Magnetite nanoparticles conjugated to protein are developed in order to potentially serve as protein carriers into bovine sperm cells. The conjugate comprises iron oxide nanoparticles that are covalently bound to an anti-protein kinase C (PKC)alpha antibody. This conjugate can serve for cellular PKC localization and the inhibition of its function. The surface of the nanoparticle is first modified with (3-aminopropyl) thrimethoxysilane to form a self-assembled monolayer, and subsequently conjugated with the antibody through amidation between the carboxylic acid end groups on the antibody and the amine groups on the surface of the nanoparticles. The anti-PKCalpha localization is proven by fluorescent microscopy and iron staining. The activity of the anti-PKCalpha conjugated with the nanoparticle is tested by recognizing PKCalpha using the Western blot method.

MAP kinase activity is downregulated by phorbol ester during mouse oocyte maturation and egg activation in vitro

The effects of protein kinase C (PKC) stimulator, phorbol 12‐myriatate 13‐acetate (PMA), on meiotic cell cycle regulation and mitogen‐activated protein (MAP) kinase changes have been studied in mouse oocytes and eggs. The results showed that MAP kinase activation itself was not necessary for germinal vesicle breakdown (GVBD), but the ability of the ooplasm to phosphorylate MAP kinase was a prerequisite for this event. At concentrations of 1.6 nM, PMA effectively inhibited GVBD and MAP kinase activation, suggesting that PMA inhibits GVBD by inhibiting molecule(s) upstream to MAP kinase. At concentrations of 16.2 nM, PMA induced metaphase‐interphase transition more effectively in eggs collected 19 hr after human chorionic gonadotropin (hCG) administration than in those collected 15 hr after hCG administration. The degree of MAP kinase activity decrease was well correlated with the time course and proportion of pronuclear formation. On the other hand, when the effect of PMA on cell cycle progression was abolished by protein phosphatase inhibitor, okadaic acid, MAP kinase was superactivated. The biologically inactive 4 α‐phorbol 12,13‐didecanoate (4 α‐PDD) had no evident effects on either GVBD and interphase transition or on MAP kinase activity. Furthermore, the effects of PMA on oocyte GVBD, egg activation, and MAP kinase activity could be overcome by the specific PKC inhibitor, calphostin C, suggesting the possible involvement of this enzyme in the regulation of MAP kinase activity. The results suggest that activation of PKC by PMA entrains a cascade of events that ultimately inhibits MAP kinase activation and GVBD in mouse oocytes and induces MAP kinase inactivation and metaphase‐interphase transition in mouse eggs. Mol. Reprod. Dev. 52:310–318, 1999.