Capacitative Ca2+ entry is a process whereby the activation of Ca 2+ in£ux through the plasma membrane is triggered by depletion of intracellular Ca 2+ stores. Some transient receptor potential (TRPC) proteins have been proposed as candidates for capacitative Ca 2+ channels. Recent evidence indicates that capacitative Ca 2+ entry participates in the sperm acrosome reaction (AR), an exocytotic process necessary for fertilization. In addition, several TRPCs have been detected heterogeneously distributed in mouse sperm, suggesting that they may participate in other functions such as motility. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, RNA messengers for TRPC1, 3, 6 and 7 were found in human spermatogenic cells. Confocal indirect immuno£uores-cence revealed the presence of TRPC1, 3, 4 and 6 di¡erentially localized in the human sperm, and immunogold transmission electron microscopy indicated that TRPC epitopes are mostly associated to the surface of the cells. Because all of them were detected in the £agellum, TRPC channel antagonists were tested in sperm motility using a computer-assisted assay. Our results provide what is to our knowledge the ¢rst evidence that these channels may in£uence human sperm motility. ß

Numerous sperm functions including the acrosome reaction (AR) are associated with Ca 2+ in£ux through voltage-gated Ca 2+ (Ca V ) channels. Although the electrophysiological characterization of Ca 2+ currents in mature sperm has proven di⁄cult, functional studies have revealed the presence of lowthreshold (Ca V 3) channels in spermatogenic cells. However, the molecular identity of these proteins remains unde¢ned. Here, we identi¢ed by reverse transcription polymerase chain reaction the expression of Ca V 3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three Ca V 3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only Ca V 3.1 and Ca V 3.2 were detected in the head, suggesting its participation in the AR. Ca V 3.1 and Ca V 3.3 were found in the principal and the midpiece of the £agella. All Ca V 3 channels are also present in human sperm, but only to a minor extent in the head. These ¢ndings were corroborated by immunogold transmission electron microscopy. Tail localization of Ca V 3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit Ca V 3 channels did not signi¢cantly a¡ect human sperm motility. Only higher mibefradil doses that can block high-threshold (HVA) Ca V channels caused small but signi¢cant motility alterations. Antibodies to HVA channels detected Ca V 1.3 and Ca V 2.3 in human sperm £agella. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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We have recently proposed revival of the name Entamoeba nuttalli Castellani, 1908 for a virulent amoeba (P19-061405 strain) isolated from a rhesus monkey (Macaca mulatta) and located phylogenetically between E. histolytica and E. dispar. In this study, E. nuttalli was isolated from feces of captive Japanese macaques (M. fuscata) in an open-air corral in Japan. The sequence of the 18S rRNA gene in the isolates differed from the P19-061405 strain in 2 nucleotide positions, but was identical to the EHMfas1 strain isolated previously from a cynomolgus monkey (M. fascicularis). One of the E. nuttalli isolates from Japanese macaques, named the NASA6 strain, was axenized and cloned. In isoenzyme analysis, the mobilities of hexokinase and phosphate glucose isomerase in the NASA6 strain were identical to those in the P19-061405 and EHMfas1 strains, but the mobility of phosphoglucomutase was different. These results were supported by gene analyses of these enzymes. Inoculation of NASA6 strain trophozoites into the liver of hamsters led to formation of an amoebic liver abscess. The liver lesions were characterized by extensive necrosis associated with inflammatory reactions. These results demonstrate that the NASA6 strain is potentially virulent and that E. nuttalli should be recognized as a common parasite in macaques.