Haim Breitbart

Intracellular calcium regulation in sperm capacitation and acrosomal reaction.

Binding to the eggs zona pellucida stimulates the spermatozoon to undergo acrosome reaction, a process which enables the sperm to penetrate the egg. Prior to this binding, the spermatozoa undergo in the female reproductive tract a series of biochemical transformations, collectively called capacitation. The first event in capacitation is the elevation of intracellular calcium and bicarbonate to activate adenylyl cyclase (AC) to produce cyclic-AMP, which activates protein kinase A (PKA) to phosphorylate certain proteins. During capacitation, there is also an increase in actin polymerization and in the membrane-bound phospholipase C (PLC). Sperm binding to zona-pellucida causes further activation of cAMP/PKA and protein kinase C (PKC), respectively. PKC opens a calcium channel in the plasma membrane. PKA together with inositol-trisphosphate activate calcium channels in the outer acrosomal membrane, which leads to an increase in cytosolic calcium. The depletion of calcium in the acrosome will activate a store-operated calcium entry mechanism in the plasma membrane, leading to a higher increase in cytosolic calcium, resulting in membrane fusion and acrosome reaction.

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.

Thermotaxis of mammalian sperm cells: A potential navigation mechanism in the female genital tract

Thermotaxis of mammalian sperm cells: A potential navigation mechanism in the female genital tract

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.

Changes in calcium transport in mammalian sperm mitochondria and plasma membrane irradiated at 633 nm (HeNe laser)

The effect of light on calcium transport in mammalian sperm mitochondria and plasma membrane was studied. Digitonine-treated spermatozoa and plasma membrane vesicles were irradiated with an HeNe laser at various powers and energy doses and Ca2+ uptake was measured by the filtration method. It was found that there is an accelerated Ca2+ uptake by the mitochondria after low power HeNe irradiation and inhibition after high power. The flux of Ca2+ from the mitochondria was also examined and was found to be unaffected by the HeNe light. The ATP-dependent Ca2+ uptake by the bovine plasma membrane vesicles was not changed by the HeNe irradiation.

Effect of light on calcium transport in bull sperm cells

The effect of light on calcium transport was studied. Bull sperm cells were irradiated with an He-Ne (630 mm) laser and a 780 nm diode laser at various energy doses, and 45Ca2+ uptake was measured by the filtration technique. It was found that there is an accelerated Ca2+ transport in the irradiated cells, which means that laser light can stimulate Ca2+ exchange through the cell membrane. This may cause transient changes in the cytoplasmic Ca2+ concentration which, in spermatozoa, has a regulatory role in control of motility and acrosome reaction, and in other cells can trigger mitosis.

Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases

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(2+) 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 α (PKCα). PKCα is active at the beginning of capacitation, resulting in PI3K inactivation. During capacitation, PKCα as well as PP1γ2 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.

Changes in calcium transport in mammalian sperm mitochondria and plasma membranes caused by 780 nm irradiation

Regulation of intracellular Ca2+ concentrations are very important in control of sperm motility and acrosome reaction. It was shown previously that low‐power lasers in the visible and near‐infrared range alter Ca2+ uptake by sperm cells. In the present work the effect of a 780 nm diode laser on Ca2+ uptake by sperm mitochondria and isolated plasma membrane vesicles is investigated.

Protein synthesis in sperm : Dialog between mitochondria and cytoplasm

Ejaculated sperm are capable of using mRNAs transcripts for protein translation during the final maturation steps before fertilization. In a capacitation-dependent process, nuclear-encoded mRNAs are translated by mitochondrial-type ribosomes while the cytoplasmic translation machinery is not involved. Our findings suggest that new proteins are synthesized to replace degraded proteins while swimming and waiting in the female reproductive tract before fertilization, or produced due to the specific needs of the capacitating spermatozoa. In addition, a growing number of articles have reported evidence for the correlation of nuclear-encoded mRNA and protein synthesis in somatic mitochondria. It is known that all of the proteins necessary for the replication, transcription and translation of the genes encoded in mtDNA are now encoded in the nuclear genome. This genetic investment is far out of proportion to the number of proteins involved, as there have been multiple movements and duplications of genes. However, the evolutionary retention (or secondary uptake) of the mitochondrial machinery for translation of nuclear-encoded mRNAs may shed light on this paradox.

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.