Characterization of membrane-associated actin in boar spermatozoa

Biochemical, immunological, and electron microscopic methods have been used to provide semi-quantitative estimates and to localize actin in membranes of boar spermatozoa. Immunoblots, using a monoclonal antibody raised against actin from chicken gizzard, detected the protein in caput and cauda sperm plasma membranes. Immunoassay indicated that approximately 1% of the total plasma membrane protein was actin. Monomeric actin accounted for more than one-half of the membrane actin. Approximately 30-40% of plasma membrane actin was insoluble in Triton X-100, and approximately 10% of the total actin remained insoluble after treatment with guanidine hydrochloride. The presence of F-actin in sperm plasma membranes and in plasma membrane detergent-insoluble proteins was detected by fluorescence microscopy using the specific probe NBD phallacidin. When S1 myosin subfragments attached to colloidal gold were used to localize F-actin by electron microscopy, the label was restricted to the outer acrosomal membrane of intact epididymal and ejaculated sperm. Filaments appeared in short arrays along the anterior region of the membrane. S1/gold labeled detergent-insoluble plasma membrane fractions but did not label the plasma membrane in intact sperm. Filaments were least prominent in intact caput spermatozoa and most prominent in ejaculated spermatozoa. We conclude that most actin associated with sperm membranes is in monomeric form in boar spermatozoa, but that actin filaments or protofilaments are components of the outer acrosomal membrane. These filaments may also associate with the plasma membrane overlying the acrosome.     

Identification of capacitation in boar spermatozoa by chlortetracycline staining

The functional status of boar spermatozoa undergoing capacitation in vitro was investigated. Two fluorescent stains were used: chlortetracycline (CTC) and a FITC-conjugated lectin (FITC-PSA). The first has been used for the direct identification of the capacitated boar spermatozoa, while the second, based on the identification of capacitated spermatozoa by their ability to undergo zona-induced acrosome reaction (AR), was used to confirm and validate the CTC assay in this species. Spermatozoa obtained from 5 different boars was washed and incubated under capacitating conditions. Aliquots of spermatozoa were collected at 0, 90 and 180 min of incubation and then stained with CTC or FITC-PSA. After CTC staining, 3 different fluorescent patterns were observed: Pattern A with the fluorescence uniformly distributed on the sperm head, Pattern B with the fluorescence concentrated in the post-acrosomial region, and Pattern C with the fluorescence concentrated in the acrosomial region. The percentage of spermatozoa displaying fluorescent Pattern A decreased throughout the incubation while that of spermatozoa with Pattern C showed a concomitant progressive increase. Pattern B fluorescence remained unchanged throughout the maturation period. Exposure to zonae pellucidae (ZP) brought back the levels of Pattern C fluorescence to basal values. Since only the capacitated spermatozoa are believed to react to ZP, this observation together with the rising incidence of Pattern C throughout maturation suggests that fluorescence in the acrosomial region identifies capacitated spermatozoa. The analysis of acrosome integrity carried out with FITC-PSA showed that the proportion of zona-induced AR was nearly the same as that of spermatozoa displaying Pattern C, thus confirming that CTC staining is suitable for the detection of boar sperm capacitation. In the second part of this study, CTC was used to investigate the effects of sperm origin and storage on the capacitation process. Our finding demonstrates that capacitation kinetics show wide variations in sperm samples derived from different boars; moreover, capacitation is also affected by sperm storage. While fresh semen showed a progressive increase in capacitated spermatozoa, ranging from low levels at the beginning of the culture to 46% at the end of incubation, the refrigerated semen had a relatively high percentage of capacitated spermatozoa at the beginning of culture, but this proportion increased only slightly during the following 90 to 180 min of treatment. These data indicate that CTC can be used to identify capacitated boar spermatozoa, and, because of its rapid and easy execution, it can be used routinely to identify the optimal capacitation time for different sperm samples.     

Changes in chromatin structure of boar late spermatids to mature spermatozoa by using modification with dansyl chloride

Changes in the chromatin structure of boar late spermatids maturing to spermatozoa were studied by chemical modification of their nuclei with dansyl (Dns) chloride. Protamine was isolated from the dansylated boar spermatid and sperm nuclei, and its dansylated sites and degrees of dansylation were determined by sequence analysis. The N-terminal Ala-1, Tyr-3 and Tyr-42 of the protamine molecule in cauda epididymal sperm nuclei were dansylated 27%, 22% and 40%, respectively, whereas the respective residues in late spermatid nuclei were about 1.5-times as reactive as those in cauda epididymal sperm nuclei. However, the dansyl ratio of Tyr-3 to Tyr-42 remained unchanged from the late spermatid to mature sperm nuclei. SDS treatment did not affect the reactivity of cauda epididymal protamine and that of Ala-1 of caput epididymal protamine, but raised that of Tyr-3 and Tyr-42 of caput epididymal protamine by a factor of about 1.5. As a result of the SDS treatment, caput epididymal protamine came to have almost the same reactivity as late spermatid protamine. These facts suggest that the fundamental structure, in terms of DNA-protamine interaction, of sperm chromatin was already formed at the stage of the late spermatid, and then during epididymal transit the sperm chromatin was more tightly condensed, with increasing disulfide cross-links, thereby acquiring insensitivity towards the SDS-treatment.     

Identification of anti-agglutinin for spermatozoa in epididymal boar plasma

The present report identifies epididymal boar anti-agglutinin and examines its effect on sperm motility. Boar spermatozoa from the cauda epididymidis were washed and incubated in modified Krebs–Ringer bicarbonate at 37°C (5% CO₂ in air). In the samples washed three or five times and then incubated for 3–5 h, higher rates (72–79%) of spermatozoa were associated with one another at the acrosomal region, mainly in groups of 2–5 cells (head-to-head agglutination), and many cells exhibited intensively flagellant and/or circular types of movement but rarely progressive motility. The addition of epididymal plasma or 25 kDa protein purified from it markedly inhibited the occurrence of head-to-head agglutination in washed spermatozoa, whereas heat treatment and subsequent removal of insoluble materials reduced the anti-agglutination activity of epididymal plasma. The percentages of progressively motile cells in the samples incubated with epididymal plasma or 25 kDa epididymal protein rose coincident with the reduction of sperm agglutination. These findings demonstrate that the 25 kDa epididymal protein is an anti-agglutinin for the cauda spermatozoa and that it effectively functions to maintain progressive motility of the cells in vitro. © 1994 Wiley-Liss, Inc.     

Distribution of filamentous actin in and around spermatids and in spermatozoa of Australian conilurine rodents.

The distribution of filamentous actin around the maturing sperm head and in spermatozoa of four species of Australian conilurine rodents was investigated at the light and electron microscopic levels. Similar results were obtained for all the species studied. Mechanically isolated spermatids had NBD-phallacidin-positive longitudinal bands of fluorescence over the dorsolateral surface and, in late spermatids, bands of bright fluorescence passed perpendicularly from the dorsal convex to ventral concave surface. TEM observations indicated that these regions corresponded to filaments of ectoplasmic specializations and granular filamentous material around the tubulobulbar complexes, respectively. In testicular and cauda spermatozoa NBD-phallacidin fluorescent material was present in the two ventral processes that extended from the upper concave surface of the sperm head; also fainter material occurred along the concave border and as a dorsocaudal spur. Its distribution was identical for testicular and cauda spermatozoa. TEM of late spermatids showed that in the ventral process closest to the apical hook there were between 170 and 245 filaments, which attached to the inner surface of the postacrosomal dense lamina; in the more caudal ventral process about 70 filaments occurred. No filaments were, however, visible in the mature spermatozoon but, after immunocytochemical labelling for actin, deposition of gold particles was evident over ventral processes of both late spermatids and cauda spermatozoa. Within the female tract these ventral processes made contact with the zona matrix and were taken into the egg cytoplasm unchanged in morphology. The possible functional significance of the filamentous actin in these structures is discussed.     

Immunoelectron microscopic distribution of actin in hamster spermatids and epididymal, capacitated and acrosome-reacted spermatozoa.

The distribution of actin in hamster sperm cells was studied during spermiogenesis, epididymal transit, in vitro capacitation and acrosome reaction by immunogold procedures using a polyclonal and two monoclonal antiactin antibodies. A predominant actin labeling (F-actin) was detected in the subacrosomal space of spermatids. Actin labeling was also observed under the plasma membrane of intercellular bridges and along the outer acrosomal membrane. In late spermatids there was both F-actin depolymerization and a loss of actin immunolabeling, thus suggesting a dispersion of G-actin monomers. No obvious labeling was evidenced in residual bodies. This pattern was observed with the three antiactin probes. In contrast, an actin labeling reappeared over the fibrous sheath of the flagellum in epididymal spermatozoa but only when the polyclonal antibody was used. Only one single actin reactive band was detected by immunoblotting of sperm extracts. Since the sperm tails were NBD phallacidin negative they were considered to contain either G-actin or actin oligomers rather than bundles of actin filaments. It is suggested that G-actin originating in the head of late spermatids was redistributed to the flagellum of epidymal spermatozoa. No further changes were noted after capacitation and acrosome reaction thus indicating no apparent effect on actin polymerization and distribution.     

Identification of a zona-binding protein from boar spermatozoa as proacrosin

The initial stages of fertilization in vertebrates and invertebrates are thought to involve complementary recognition molecules on spermatozoa and eggs. In a previous work (C. R. Brown and R. Jones, 1987, Development) we described one such putative molecule (a protein of approximate molecular weight 53 kDa) in detergent extracts of boar spermatozoa that has affinity for glycoproteins from the zona pellucida of pig eggs. This molecule has now been identified as proacrosin, the zymogen form of the acrosomal protease acrosin, on the basis of its electrophoretic behavior, the ability of zona glycoproteins to recognize and bind to proacrosin on Western blots, and the cross-reactivity of specific antisera to the 53-kDa molecule and proacrosin. A role is proposed for this enzyme in binding the sperm head to the zona pellucida during the initial stages of sperm-egg interaction.     

Chromatin structure visualization by immunoelectron microscopy

Antibodies elicited in rabbits by chromatin and by purified histone H2B have been used to study the structure of chromatin by immunoelectron microscopy. Chromatin spread on grids reveals a structure of closely packed spherical particles with an average diameter of 104 Å, arranged either in clusters or in linear arrays of beads, some of which have a supercoil-like arrangement. No DNA strings connecting the beads could be observed. Upon antibody binding, the diameter of the particles increases up to 300 Å. This size is compatible with a model where one layer of gamma globulin molecules 110 Å long encircles a sphere of chromatin 100 Å in diameter. The presence of rabbit gamma globulins on the enlarged beads has been verified by the addition of ferritin-labeled goat anti-rabbit gamma globulins. Anti-chromatin sera which react with nonhistone proteins but not with free histones or DNA react with more than 95% of the beads; this suggests that most of the beads contain nonhistone proteins. Since the number of nonhistone proteins is large, it is improbable that each sphere contains a full complement of these proteins. We therefore suggest that the various chromatin spheres contain different types of nonhistone proteins. About 90% of the chromatin spheres reacted with antibodies to histone H2B, suggesting that most of the chromatin beads contain this type of histone.     

Identification of Naemacyclus minor hyphae within needle tissues of Pinus sylvestris by immunoelectron microscopy

Cultural investigations revealed that Naemacyclus minor, Lophodermium seditiosum and Cenangium ferruginosum were the most frequent colonizers of asymptomatic and symptomatic Pinus sylvestris needles. Since ultrastructural observations showed that morphological features were not suitable to differentiate hyphae of N. minor from hyphae of other isolates, the on-section immunogold labelling technique was applied in combination with an anti-N. minor specific immunoserum. The specificity of this serum was tested against culture hyphae of all isolates. Anti-N. minor specific immunoserum was then used to identify N. minor hyphae in thin sections of green P. sylvestris needles. The infection loci identified were restricted to small tissue areas located in the vicinity of stomata. In the hypodermis, hyphae and endocell-containing hyphae were located within the lumina of host cells but outside the protoplast. The growth of hyphae from cell to cell occurred through pits. The hyphae spreat into the mesophyll intercellularly and continued with the intracellular colonization of moribund and dead mesophyll cells in a later stage of infection. The observed host-parasite interactions at cellular and ultrastructural level are discussed in connection with the still controversial interpretation of the pathogenicity of N. minor.     

Adsorption of seminal plasma proteins by boar spermatozoa

Seminal plasma protein adsorption by boar spermatozoa was examined using ejaculated sperm from vesiculectomized boars and seminal plasma from vasectomized boars. Sperm adsorbed 14 pg protein/sperm in 10 min. When seminal plasma proteins were radiolabeled, most of the adsorbed radiolabel was present in low M(r) proteins, particularly a 12700 M(r) protein. A 349300 M(r) seminal plasma protein was also readily adsorbed. Three radiolabeled seminal plasma proteins (307600, 165400 and 7400 M(r)) were not detected on the sperm; either they are not adsorbed by the sperm or the sperm were previously exposed to these proteins in other accessory sex gland fluids and had already adsorbed them. A 29100 M(r) sperm protein was also radiolabeled (4.9% of the adsorbed radiolabel), although there was no corresponding seminal plasma protein. Large quantities of seminal plasma protein (particularly low M(r) proteins) are adsorbed by sperm not previously exposed to seminal vesicle secretion. The functions of these proteins are yet to be determined.     

Metabolism of glycerol by mature boar spermatozoa.

Mature boar spermatozoa oxidized glycerol to carbon dioxide in the absence of any detectable activity of glycerol kinase. With triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase inhibited by the presence of 3-chloro-1-hydroxypropanone (CHOP), dihydroxyacetone phosphate accumulated in incubates when glycerol-3-phosphate was the substrate, but not when it was glycerol. Both dihydroxyacetone and glyceraldehyde could be used as substrates; in the presence of CHOP, dihydroxyacetone phosphate and fructose-1,6-bisphosphate accumulated when dihydroxyacetone was the substrate, but not when it was glyceraldehyde. The metabolic pathways glycerol----glyceraldehyde----glyceraldehyde 3-phosphate and dihydroxyacetone----dihydroxyacetone phosphate have been shown to operate in these cells.     

Effect of sex sorting on CTC staining, actin cytoskeleton and tyrosine phosphorylation in bull and boar spermatozoa

Sperm sorting is a useful technology that permits sex preselection. It presents some troubles because of low fertility after the process. The main aim of this work was to analyze the putative existence of capacitation-like changes in both boar and bull sperm subjected to sex sorting that could lead to a detriment of semen quality. The parameters used were CTC staining patterns, actin cytoskeleton organization and tyrosine phosphorylation patterns; the last two were determined by both western blotting and immunofluorescence. Sex sorted spermatozoa were compared with fresh, in vitro capacitated and in vitro acrosome reacted sperm. In both species, sex sorted sperm showed a CTC staining pattern similar to that observed after in vitro capacitation. The actin pattern distribution after sperm sorting also tended to be similar to that observed after in vitro capacitation, but this effect was more pronounced in bull than in boar spermatozoa. However, actin expression analysis through western blot did not show any change in either species. The tyrosine phosphorylation pattern in boar sperm was practically unaltered after the sex sorting process, but in bulls about 40% of spermatozoa had a staining pattern indicative of capacitation. Additionally, western blotting analysis evidenced some differences in the expression of protein tyrosine phosphorylation among fresh, capacitated, acrosome reacted and sex sorted sperm cells in both species. Our results indicate that not all the sex-sorted-related modifications of the studied parameters were similar to those occurring after “in vitro” capacitation, thus suggesting that sex sorting-induced alterations of sperm function and structure do not necessarily indicate the achievement of the capacitated status of sorted sperm.     

The crater defect in boar spermatozoa: A correlative study with transmission electron microscopy,scanning electron microscopy,and light microscopy

Nuclear vacuoles resembling the “crater defect” described in bull spermatozoa were observed in 14 boars. Both the incidence of the defect and semen quality were monitored with phase contrast microscopy over a three-month period. The percentages of cratered spermatozoa varied widely both among boars and in ejaculates from the same boar taken on different days. The presence of cratered spermatozoa at a level of 5% or more appeared to be associated with low semen quality. The defect was studied with scanning and transmission electron microscopy and was found to consist of nuclear invaginations, about 0.5 μm in diameter, containing some scanty amorphous electron-dense material. In boars showing a high incidence of spermatozoa with crater defects, abnormalities of the acrosome and perforatorium were common.     

Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit

Using a monoclonal antibody as a highly specific probe and a seminal particle-free fraction of rabbit ejaculated spermatozoa, actin has been localized in the postacrosomal region of mature rabbit spermatozoa. The sperm actin has been extracted and identified on two-dimensional PAGE immunoblots as a single spot of pI = 5.45 and Mr = 43,000. Rabbit sperm actin is present in a nonfilamentous form and is not removed by removing the plasma membrane. Unlike mature spermatozoa, however, filamentous actin is present in spermatogenic cells, as determined by rhodamine phalloidin staining. Starting as diffusely distributed in spermatocytes, actin accumulates in the subacrosomal space and appears as a band in conjunction with the developing acrosome. This band lengthens throughout the spermatid stage and becomes continuous with the postacrosomal region staining in testicular spermatozoa. Actin may therefore function during spermatogenesis to both shape the acrosome to the nucleus and to anchor inner acrosomal membrane proteins.     

Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy

In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurrs in cells growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a broad spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolase and proteins with the C-terminal SKL tripeptide. Received: 4 March 1998 / Accepted: 2 July 1998