Spatiotemporal characterization of intracellular Ca2+ rise during the acrosome reaction of mammalian spermatozoa induced by zona pellucida.
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The mammalian sperm acrosome reaction (AR) is an essential event prior to sperm-egg fusion at fertilization, and it is primarily dependent on an increase in intracellular Ca2+ concentration ([Ca2+]i). Spatiotemporal aspects of the [Ca2+]i increase during the AR induced by solubilized zona pellucida (ZP) in hamster spermatozoa were precisely investigated with a Ca2+ imaging technique using confocal laser scanning microscopy with two fluorescent Ca2+ indicators. A rapid rise in [Ca2+]i occurred immediately after the application of ZP solution through a micropipette. The rise was always initiated in the sperm head, even when the application was directed toward the tail. The elevated [Ca2+]i was little attenuated during measurement for 30-40 s. Acrosomal exocytosis was detected as a sudden decrease of fluorescence in the acrosomal vesicle approximately 20 s after the onset of the [Ca2+]i rise. High-resolution imaging revealed that the [Ca2+]i rise in the sperm head began at the region around the equatorial segment and spread over the posterior region of the head within 0.6 s, whereas Ca2+ concentration in the acrosomal vesicle appeared to be unaltered. The [Ca2+]i rise was completely abolished under Ca2+-free extracellular conditions, indicating that it is totally attributable to Ca2+ influx. Nifedipine, an inhibitor of L-type Ca2+ channels, did not affect the rising phase of the ZP-induced Ca2+ response, but accelerated the decline of the [Ca2+]i rise and inhibited acrosomal exocytosis. The present study provides implicative information about the spatial organization of functional molecules involved in the signal transduction in mammalian AR. (+info)
Flow cytometric method to isolate round spermatids from mouse testis.
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The purpose of this study was to isolate pure populations of round spermatids from mouse testis by flow cytometry followed by cell sorting. Cell suspensions from mouse testis were enriched in germ cells by centrifugation on a discontinuous Percoll gradient, then analysed using a FACScalibur flow cytometer measuring the cell size and density. A large and well-delimited population of cells (R1) expected to contain round spermatids was observed on the dot plot diagram. Sorted R1 cells were very homogeneous in size (approximately 11 microns) and displayed the characteristic cytological aspect of round spermatids. Spermatid-specific gene expression was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of R1 cells using primers for protamine 2 gene (PRM2) and SP-10. A positive signal for SP-10 was obtained with a single cell using nested primers. The 5.5 kb transcript of c-kit, which is not expressed in spermatids, was not detected by nested RT-PCR, excluding a contamination with spermatogonia. Our results clearly established that flow cytometry followed by cell sorting allows the isolation of a highly homogeneous population of round spermatids from the testis. (+info)
Immunolocalization of CRES (Cystatin-related epididymal spermatogenic) protein in the acrosomes of mouse spermatozoa.
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The CRES (cystatin-related epididymal spermatogenic) protein is a member of the cystatin superfamily of cysteine protease inhibitors and exhibits highly restricted expression in the reproductive tract. We have previously shown that CRES protein is present in elongating spermatids in the testis and is synthesized and secreted by the proximal caput epididymal epithelium. The presence of CRES protein in developing germ cells and in the luminal fluid surrounding maturing spermatozoa prompted us to examine whether CRES protein is associated with spermatozoa. In the studies presented, indirect immunofluorescence, immunogold electron microscopy, and Western blot analysis demonstrated that CRES protein is localized in sperm acrosomes and is released during the acrosome reaction. Interestingly, while the 19- and 14-kDa CRES proteins were present in testicular and proximal caput epididymal spermatozoa, the 14-kDa CRES protein was the predominant form present in mid-caput to cauda epididymal spermatozoa. Furthermore, following the ionophore-induced acrosome reaction, CRES protein localization was similar to that of proacrosin/acrosin in that it was detected in the soluble fraction as well as associated with the acrosome-reacted spermatozoa. The presence of CRES protein in the sperm acrosome, a site of high hydrolytic and proteolytic activity, suggests that CRES may play a role in the regulation of intraacrosomal protein processing or may be involved in fertilization. (+info)
Actin filament-membrane attachment: are membrane particles involved?
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The association of actin filaments with membranes is an important feature in the motility of nonmuscle cells. We investigated the role of membrane particles in the attachment of actin filaments to membranes in those systems in which the attachment site can be identified. Freeze fractures through the end-on attachment site of the acrosomal filament bundles in Mytilus (mussel) and Limulus (horseshoe crab) sperm and the attachment site of the microvillar filament bundles in the brush border of intestinal epithelial cells were examined. There are no particles on the P face of the membrane at these sites in the sperm systems and generally none at these sites in microvilli. In microvilli, the actin filaments are also attached along their lengths to the membrane by bridges. When the isolated brush border is incubated in high concentrations of Mg++ (15 mM), the actin filaments form paracrystals and, as a result, the bridges are in register (330 A period). Under these conditions, alignment of the particles on the P face of the membrane into circumferential bands also occurs. However, these bands are generally separated by 800-900 A, indicating that all the bridges cannot be directly attached to membrane particles. Thus membrane particles are not directly involved in the attachment of actin filaments to membranes. (+info)
Acrosome formation during sperm transit through the epididymis in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula).
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In certain Australian marsupials including the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula), formation of the acrosome is not completed in the testis but during a complex differentiation process as spermatozoa pass through the epididymis. Using transmission and scanning electron microscopy this paper defined the process of acrosome formation in the epididymis, providing temporal and spatial information on the striking reorganisation of the acrosomal membranes and matrix and of the overlying sperm surface involved. On leaving the testis wallaby and possum spermatozoa had elongated 'scoop'-shaped acrosomes projecting from the dorsal surface of the head. During passage down the epididymis, this structure condensed into the compact button-like organelle found on ejaculated spermatozoa. This condensation was achieved by a complex process of infolding and fusion of the lateral projections of the 'scoop'. In the head of the epididymis the rims of the lateral scoop projections became shorter and thickened and folded inwards, to eventually meet midway along the longitudinal axis of the acrosome. As spermatozoa passed through the body of the epididymis the lateral projections fused together. Evidence of this fusion of the immature outer acrosomal membrane is the presence of vesicles within the acrosomal matrix which persist even in ejaculated spermatozoa. When spermatozoa have reached the tail of the epididymis the acrosome condenses into its mature form, as a small button-like structure contained within the depression on the anterior end of the nucleus. During the infolding process, the membranes associated with the immature acrosome are either engulfed into the acrosomal matrix (outer acrosomal membrane), or eliminated from the sperm head as tubular membrane elements (cytoplasmic membrane). Thus the surface and organelles of the testicular sperm head are transient structures in those marsupials with posttesticular acrosome formation and this must be taken into consideration in attempts to dissect the cell and molecular biology of fertilisation. (+info)
Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein.
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We produced transgenic mouse lines that accumulate mutated green fluorescent protein (EGFP) in sperm acrosome, a membrane limited organelle overlying the nucleus. The sperm showed normal fertilizing ability and the integrity of their acrosome was easily examined in a non-invasive manner by tracing the GFP in individual 'live' sperm with fluorescent microscopy. The time required for the dispersal of acrosomal contents was demonstrated to be approximately 3 s after the onset of acrosome reaction. (+info)
Scanning electron-microscopical and other observations of sperm fertilization reactions in Limulus polyphemus L. (Merostomata: Xiphosura).
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Sperm fertilization reactions of Limulus polyphemus were examined by scanning electron and/or light microscopy. The following were considered: sperm motility, attachment of sperm to egg, acrosome reaction, and penetration of the acrosomal filament. The spermatozoa after semination are non-motile and become active only in close proximity to a defined region surrounding the egg. Egg materials diffusing into this region induce sperm motility and stimulate large numbers of spermatozoa to move towards the egg surface. Each sperm initially attaches by the apical tip and undergoes the acrosome reaction which causes a more permanent secondary attachment by the adhesion of acrosomal contents to the egg surface. The acrosome reaction also initiates the penetration of the acrosomal filament through the egg envelope, an event occurring in 70-80% of the attached spermatozoa (about 10(6). Shortly after this penetration, a secondary reaction occurs which involves a spiralling of the flagellum and an incorporation into the sperm body of the flagellar fibrous components, which then become closely apposed to the sperm nucleus. These sperm fertilization reactions were performed or initiated with 0-34 M CaCl2 in whole eggs, egg sections, excised egg envelopes and/or the outer basement lamina of the egg envelope. The Limulus fertilization system is very valuable since sperm reactions can be examined biochemically, which may lead to a better understanding of the chemical mechanisms involved in sperm-egg interactions in all animal species. (+info)
Identification of Rab3A GTPase as an acrosome-associated small GTP-binding protein in rat sperm.
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The acrosome reaction is a membrane fusion event that is prerequisite for sperm penetration through the zona pellucida. To elucidate the molecular mechanisms involved in membrane fusion, the expression and localization of Rab proteins, a subfamily of small GTPases that have been shown to play key roles in regulation of intracellular membrane traffic and exocytosis, were examined in rat testis and sperm. Reverse transcription polymerase chain reaction, immunoblot analysis, and immunofluorescence microscopy revealed that Rab3A protein, which is thought to be involved in regulation of exocytosis in neurons and endocrine cells, is associated with the sperm acrosome. The protein was undetectable in acrosome-free heads prepared by sucrose density gradient centrifugation. Immunogold electron microscopy performed on ultrathin cryosections provided further evidence that Rab3A protein is associated with the acrosomal membrane. Acrosome reaction assays revealed that synthetic peptide of the Rab3 effector domain inhibited acrosomal exocytosis triggered by calcium ionophore A23187 in a concentration-dependent fashion, suggesting that Rab3A acts as an inhibitory regulator in the acrosome reaction. In view of the putative role of Rab3A protein in membrane fusion systems, these results suggest that Rab3A could be involved in regulating the mammalian acrosome reaction by controlling the membrane fusion system in sperm. (+info)