ABSTRACT
The mechanisms involved in the regulation of mammalian sperm motility are not well understood. Calcium ions (Ca(2+)) have been suggested to play a key role in the maintenance of motility; nevertheless, how Ca(2+) modulates this process has not yet been completely characterized. Ca(2+) can bind to calmodulin and this complex regulates the activity of multiple enzymes, including Ca(2+)/calmodulin-dependent protein kinases (CaM kinases). Results from this study confirmed that the presence of Ca(2+) in the incubation medium is essential for maintaining human sperm motility. The involvement of CaM kinases in Ca(2+) regulation of human sperm motility was evaluated using specific inhibitors (KN62 and KN93) or their inactive analogues (KN04 and KN92 respectively). Sperm incubation in the presence of KN62 or KN93 led to a progressive decrease in the percentage of motile cells; in particular, incubation with KN62 also reduced sperm motility parameters. These inhibitors did not alter sperm viability, protein tyrosine phosphorylation or the follicular fluid-induced acrosome reaction; however, KN62 decreased the total amount of ATP in human sperm. Immunological studies showed that Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) is present and localizes to the human sperm flagellum. Moreover, CaMKIV activity increases during capacitation and is inhibited in the presence of KN62. This report is the first to demonstrate the presence of CaMKIV in mammalian sperm and suggests the involvement of this kinase in the regulation of human sperm motility.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Sperm Motility , Spermatozoa/metabolism , Spermatozoa/physiology , Acrosome Reaction , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Blotting, Western , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 4 , Culture Media , Dose-Response Relationship, Drug , Electrophoresis , Enzyme Inhibitors/pharmacology , Humans , Ions , Male , Microscopy, Fluorescence , Sperm Capacitation , Spermatozoa/pathology , Time FactorsABSTRACT
The parasitic protozoon Leishmania mexicana undergoes two major developmental stages in its life cycle exhibiting profound physiological and morphological differences, the promastigotes in the insect vector and the amastigotes in mammalian macrophages. A deletion mutant, Deltalmsap1/2, for the secreted acid phosphatase (SAP) gene locus, comprising the two SAP genes separated by an intergenic region of approximately 11.5 kb, lost its ability to cause a progressive disease in Balb/c mice. While in vitro growth of promastigotes, invasion of host cells and differentiation from promastigotes to amastigotes was indistinguishable from the wild-type, the mutant parasites ceased to proliferate when transformed to amastigotes in infected macrophages or in a macrophage-free in vitro differentiation system, suggesting a stage-specific growth arrest. This phenotype could be reverted by complementation with 6 kb of the intergenic region of the SAP gene locus. Sequence analysis identified two open reading frames, both encoding single copy genes; one gene product shows high homology to mitogen-activated protein (MAP) kinases. Complementation experiments revealed that the MAP kinase homologue, designated LMPK, is required and is sufficient to restore the infectivity of the Deltalmsap1/2 mutant. Therefore, LMPK is a kinase that is essential for the survival of L.mexicana in the infected host by affecting the cell division of the amastigotes.
Subject(s)
Acid Phosphatase/chemistry , Acid Phosphatase/genetics , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Genes, Protozoan , Leishmania mexicana/physiology , Leishmaniasis, Cutaneous/physiopathology , Protozoan Proteins , Acid Phosphatase/metabolism , Amino Acid Sequence , Animals , Base Sequence , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA Primers , Genetic Complementation Test , Introns , Leishmania mexicana/genetics , Leishmania mexicana/pathogenicity , Life Cycle Stages , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mutagenesis, Site-Directed , Open Reading Frames , Polymerase Chain Reaction , Rats , Restriction Mapping , Sequence Alignment , Sequence Deletion , Sequence Homology, Amino AcidABSTRACT
Trypanosoma cruzi epimastigote forms showed a tightly bound Ca(2+)-calmodulin-dependent protein kinase activity, which could be partially extracted from membranes and axonemes. The enzyme is constituted by subunits which were autophosphorylated in the absence of exogenous substrates. An antibody against CaM kinase II recognized a Ca(2+)- or Ca(2+)-CaM-dependent conformational epitope in these fractions. The detected bands were of molecular weights similar to the alpha and beta subunits of the corresponding bovine brain enzyme (60 and 50 kDa). Studies using [125I]CaM revealed the presence of a CaM-binding domain. These experiments confirm that the parasite possesses a particulate CaM kinase with characteristics similar to the bovine brain enzyme.