Mammalian sperm must undergo a series of biochemical and physiological modifications, collectively called capacitation, in the female reproductive tract prior to the acrosome reaction (AR). The mechanisms of these modifications are not well characterized though protein kinases were shown to be involved in the regulation of intracellular Ca 21 during both capacitation and the AR. In the present review, we summarize some of the signaling events that are involved in capacitation. During the capacitation process, phosphatidyl-inositol-3-kinase (PI3K) is phosphorylated/activated via a protein kinase A (PKA)-dependent cascade, and downregulated by protein kinase C a (PKCa). PKCa is active at the beginning of capacitation, resulting in PI3K inactivation. During capacitation, PKCa as well as PP1c2 is degraded by a PKA-dependent mechanism, allowing the activation of PI3K. The activation of PKA during capacitation depends mainly on cyclic adenosine monophosphate (cAMP) produced by the bicarbonate-dependent soluble adenylyl cyclase. This activation of PKA leads to an increase in actin polymerization, an essential process for the development of hyperactivated motility, which is necessary for successful fertilization. Actin polymerization is mediated by PIP 2 in two ways: first, PIP 2 acts as a cofactor for phospholipase D (PLD) activation, and second, as a molecule that binds and inhibits actin-severing proteins such as gelsolin. Tyrosine phosphorylation of gelsolin during capacitation by Src family kinase (SFK) is also important for its inactivation. Prior to the AR, gelsolin is released from PIP 2 and undergoes dephosphorylation/activation, resulting in fast F-actin depolymerization, leading to the AR.

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Parkinson's disease (PD), one of the most common neurodegenerative diseases, is a multifactorial disease caused by both genetic and environmental factors. Although most patients suffering from PD have a sporadic disease, several genetic causes have been identified in recent years, including alpha-synuclein, parkin, PINK1, dardarin (LRRK2), and DJ-1. DJ-1 deletions and point mutations have been found worldwide, and loss of functional protein was shown to cause autosomal recessive PD. Moreover, DJ-1 immunoreactive inclusions are found in other alpha-synucleopathies and tauopathies, indicating that different neurodegenerative diseases might share a common mechanism in which DJ-1 might play a key role. The function of DJ-1 is still unknown; however, it is associated with various cellular processes, including response to oxidative stress, cellular transformation, RNAbinding, androgen-receptor signaling, spermatogenesis, and fertilization. This article reviews the current knowledge on DJ-1, focusing on its importance in the pathogenesis of PD.

Parkinson's disease (PD) is a slowly progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons. Dopamine is a highly toxic compound leading to generation of reactive oxygen species (ROS). DJ-1 mutations lead to early-onset inherited PD. Here, we show that DJ-1 protects against dopamine toxicity. Dopamine-exposure led to upregulation of DJ-1. Overexpression of DJ-1 increased cell resistance to dopamine toxicity and reduced intracellular ROS. Contrary effects were achieved when DJ-1 levels were reduced by siRNA. Similarly, in vivo striatal administration of 6-hydroxydopamine led to upregulation of DJ-1. Upregulation of DJ-1 was mediated by the MAP kinases pathway through activation of ERK 1, 2 in vitro and in vivo. Hence, oxidative stress, generated by free cytoplasmic dopamine, leads to upregulation of DJ-1 through the MAP kinases pathway. This mechanism elucidates how mutations in DJ-1 prompt PD and imply that modulation of DJ-1 may serve as a novel neuroprotective modality.

Actin polymerization and development of hyperactivated (HA) motility are two processes that take place during sperm capacitation. In previous studies, we demonstrated that the increase in F-actin during capacitation depends upon inactivation of the actin severing protein, gelsolin, by its binding to phosphatydilinositol-4, 5-bisphosphate (PIP2). Here, we showed for the first time the involvement of PIP2/gelsolin in human sperm motility before and during capacitation. Activation of gelsolin by causing its release from PIP2 inhibited sperm motility, which could be restored by adding PIP2 to the cells. Reduction of PIP2 synthesis inhibited actin polymerization and motility, and increasing PIP2 synthesis enhanced these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP2 and F-actin. During capacitation there was an increase in PIP2 and F-actin levels in the sperm head and a decrease in the tail. In sperm with high motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail before capacitation and translocated to the head during capacitation. We also showed that phosphorylation of gelsolin on tyrosine-438 depends on its binding to PIP2. Activation of phospholipase C by Ca(2+)-ionophore or by activating the epidermal-growth-factor-receptor inhibits tyrosine phosphorylation of gelsolin. In conclusion, the data indicate that the increase of PIP2 and/or F-actin in the head during capacitation enhances gelsolin translocation to the head. As a result the decrease of gelsolin in the tail allows keeping high level of F-actin in the tail, which is essential for the development of HA motility.

The sperm acrosome reaction occurs after the binding of the capacitated sperm to the egg zona pellucida. This study describes a novel mode of regulation of the sperm epidermal growth factor receptor (EGFR) under physiological conditions and its relevance to the acrosome reaction. Ouabain, a known Na/K ATPase blocker is present in the blood and in the female reproductive tract. We show here that physiological concentrations (nM) of ouabain enhance phosphorylation of EGFR on tyr-845, stimulate Ca(2+) influx and induce the acrosome reaction in sperm. These effects could be seen only in the presence of very low concentrations of EGF (0.1 ng/ml or 0.016 nM) added together with nano-molar ouabain. Phosphorylation, Ca(2+) influx, and the acrosome reaction are inhibited by an EGFR blocker, suggesting that trans-activation of the EGFR is involved. Moreover, our data revealed that protein kinase A and the family of tyrosine kinase, SRC, shown before to be involved in EGFR activation in sperm, mediate the acrosome reaction induced by ouabain. Ouabain alone (without EGF) at relatively high concentration (10microM) could enhance EGFR phosphorylation, Ca(2+) influx and acrosome reaction, and these processes were inhibited by EGFR blockers. Moreover, we show here that PKA and SRC family are involved in the activation of EGFR by 10 microM ouabain, further demonstrating that ouabain induces the acrosome reaction by a mechanism mediated by the trans-activation of EGFR. In conclusion, this study describes an interesting regulatory path of EGFR by physiological concentrations of ouabain and EGF found in the female reproductive tract. Neither of these compounds can activate the EGFR alone at such low physiological levels; however, when both are present, the interaction of ouabain with the Na/K ATPase leads to the priming of the EGFR, which undergoes its full activation by EGF.

Ejaculated spermatozoa must undergo a series of biochemical modifications called capacitation, prior to fertilization. Protein-kinase A (PKA) mediates sperm capacitation, although its regulation is not fully understood. Sperm contain several A-kinase anchoring proteins (AKAPs), which are scaffold proteins that anchor PKA. In this study, we show that AKAP3 is degraded in bovine sperm incubated under capacitation conditions. The degradation rate is variable in sperm from different bulls and is correlated with the capacitation ability. The degradation of AKAP3 was significantly inhibited by MG-132, a proteasome inhibitor, indicating that AKAP3 degradation occurs via the proteasomal machinery. Treatment with Ca2+-ionophore induced further degradation of AKAP3; however, this effect was found to be enhanced in the absence of Ca2+ in the medium or when intracellular Ca2+ was chelated the degradation rate of AKAP3 was significantly enhanced when intracellular space was alkalized using NH4Cl, or when sperm were treated with Ht31, a peptide that contains the PKA-binding domain of AKAPs. Moreover, inhibition of PKA activity by H89, or its activation using 8Br-cAMP, increased AKAP3 degradation rate. This apparent contradiction could be explained by assuming that binding of PKA to AKAP3 protects AKAP3 from degradation. We conclude that AKAP3 degradation is regulated by intracellular alkalization and PKARII anchoring during sperm capacitation.

Parkinson's disease (PD) is a common age-related neurodegenerative disorder. Dopamine neurotoxicity, mediated through oxidative stress, is implicated in disease pathogenesis. The vesicular monoamine transporter-2 (VMAT2) transfers dopamine into synaptic vesicles preparing it for exocytotic release and preventing its cytoplasmic oxidation. DJ-1 mutations cause early-onset familial PD. Here, we show that DJ-1 protects dopaminergic neurons and controls the vesicular sequestration of dopamine by upregulating VMAT2. Overexpression of DJ-1 protected cells against dopamine toxicity, reduced oxidative stress, and increased VMAT2 expression and function. Reduced DJ-1 levels resulted in opposite effects. Dopamine vesicular sequestration and its release upon depolarization were dependent on DJ-1 levels. Transcriptional regulation of VMAT2 expression by DJ-1 was confirmed by chromatin immunoprecipitation assay. The results were corroborated in vivo using 6-hydroxydopamine hemiparkinsonian mouse model and transgenic DJ-1 knockout mice. Our experimental data point to a novel potential protective function of DJ-1, which could be used as a therapeutic tool.