A dendrobranchiate, Peisos petrunkevitchi (Decapoda, Sergestidae), with reptant-like sperm: a spermiocladistic assessment

The spermatozoon of Peisos petrunkevitchi differs significantly from those of any of the investigated dendrobranchiates in the anterior fusiform acrosome, lacking a spike, and embedded in (instead of capping) the nuclear region. In contrast, the position of the acrosome and the internal arrangement of its contents, as well as the apomorphic presence of a pair of centrioles (absent in all known dendrobranchiate spermatozoa) at the base of the acrosomal perforatorium, indicate a close affinity between this sperm plan and that found in reptants, especially anomurans and brachyurans. Based on the present and previous observations on decapod spermatozoal ultrastructure, we review the phylogeny of dendrobranchiate spermatozoa in the perspective of most recent phylogenetic analyses of malacostracan crustaceans.     

Spermatozoal ultrastructure in three Atlantic solenocerid shrimps (Decapoda, Dendrobranchiata)

The spermatozoal ultrastructure in three solenocerid shrimps (Solenocera membranacea, S. africana, and Pleoticus muelleri) from different Atlantic locations was examined with the aim of increasing understanding of the phylogenetic relationships in the Dendrobranchiata. A considerable structural similarity between the sperm of these species and those of penaeid shrimps supports a close affinity between the Penaeidae and Solenoceridae. However, significant differences in the sperm morphology of the previously investigated sicyoniids (namely, a greater complexity of the acrosomal complex) suggest evolutionary separation of the Sicyoniidae from the assemblage Penaeidae-Solenoceridae. Two ultrastructural features distinguish the spermatozoa of the three studied solenocerids from penaeid sperm: 1) separation of the plasma and acrosome membranes at the base of the spike and anterior region of the cap, and 2) asymmetry of the acrosomal cap, which appears to be a synapomorphy of the group. No striking ultrastructural differences were found between the spermatozoa of the closely related species S. membranacea and S. africana, whereas a great number of morphological differences separate the spermatozoa of Pleoticus from those of Solenocera (e.g., shape of the acrosomal cap, structural arrangement of the contents of the whole acrosome vesicle, thickness and distribution of the cytoplasm, and external shape of the spike).     

Protein tyrosine phosphorylation of a heparin-binding sperm membrane mitogen (HBSM) is associated with capacitation and acrosome reaction

Protein tyrosine phosphorylation in spermatozoa is associated with epididymal maturation and though to be central for attainment of a capacitated state and expression of hyperactivated motility. Heparin, the most highly sulfated glycosaminoglycans, was also the most potent at stimulating the acrosomal reaction in bovine epididymal spermatozoa. Studies using radiolabeled inorganic phosphate showed 11-fold increase (32)Pi incorporation in heparin-binding sperm membrane protein (HBSM) during spermatozoal capacitation, and the phosphorylation occurs at the tyrosine residue. Epididymal spermatozoa were induced to undergo capacitation and acrosome reaction by 70% when the cells were incubated in BWW medium supplemented with heparin. The spermatozoa pre-treated with anti-HBSM antibody showed 46% reduction in the hyperactivated motility and lowers the acrosome reaction. This was confirms by measuring the hydrolysis of benzoyl-l-arginine ethyl ether (BAEE) by the acrosomal enzyme; acrosin. The preliminary finding suggests that HBSM may play an important role in the sperm capacitation and acrosome reaction.     

Spermatozoa ultrastructure of the pink shrimp Farfantepenaeus paulensis (Decapoda: Dendrobranchiata)

Braga, A.L., Nakayama, C.L., Suita de Castro, L.A. and Wasielesky, W. 2011. Spermatozoa ultrastructure of the pink shrimp Farfantepenaeus paulensis (Decapoda: Dendrobranchiata). —Acta Zoologica (Stockholm) 00 : 1–6. The spermatozoa ultrastructure of the pink shrimp Farfantepenaeus paulensis was investigated in this morphological study. Spermatophores and spermatozoa were analyzed by electron microscopy. The pink shrimp spermatophore is divided into two regions: the appendage and the spermatophore main body, where spermatozoa are grouped in a spermatic mass. Pink shrimp spermatozoa are unistellate and are composed of main body and single spike. The spermatozoa body comprises a perinuclear cytoplasmic band, nucleus, acrosomal cap, and subacrosomal region. The spermatozoa cell mean total length was 10.71 μm, the mean body diameter was 5.56 μm, and the mean spike length and diameter were 5.15 μm and 0.85 μm, respectively.     

Ultrastructural demonstration of the model of Litopenaeus vannamei (Crustacea,Penaeidae) male sexual maturation and spermatozoal capacitation

The present study demonstrates ultrastructurally the model of Litopenaeus vannamei male sexual maturation and spermatozoal capacitation. The results show that phase 1 of the model occurred in the seminiferous tubules and includes spermatogenesis. In this phase, throughout differentiation of spermatogonia into late spermatids the following processes were observed: (1) decondensation of chromatin; (2) rupture of the nuclear envelope; (3) reduction of the cytoplasm and degeneration of organelles; (4) formation of the acrosome via fusion of cytoplasmic vesicles. Phase 2 comprised of spermatozoal maturation, a process that started with the transfer of late spermatids into the seminiferous ducts and ended with the formation of the acrosomal spike in the terminal ampoules. During this phase, development of the subacrosomal region and lateral electron-dense particles occurred in the seminiferous ducts, which is a novel finding of this species. Phase 3 was observed after spermatophore placement on the female thelycum and was mainly characterized by ultrastructural changes in the nucleus and the subacrosomal region. These results are in agreement with the model of male sexual maturation and spermatozoal capacitation proposed for L. vannamei.     

Release of acrosomal enzymes following in vitro capacitation of ejaculated rabbit spermatozoa

Significant release of the acrosomal enzymes arylsulfatase, β-N-acetylhexosaminidase and hyaluronidase was observed following the treatment of ejaculated rabbit spermatozoa for 12 hours in 20% rabbit serum for inducing in vitro capacitation, and these sperm were capable of in vivo fertilization; however, the treatment of sperm for 15 minutes in high ionic strength (380 mOsm/kg) or low ionic strength medium (305 mOsm/kg) for in vitro capacitation did not result in any significant release of the above enzymes nor were the sperm capable of in vivo fertilization. Serum-treated spermatozoa remained significantly motile following the 12 hour treatment, 51% underwent the acrosome reaction and were capable of fertilizing 66% of the ova in vivo. Identical serum treatment of lysosomes from rabbit liver resulted in a comparable release of the lysosomal enzymes. Serum treatment for in vitro capacitation resulted in vesiculation of the anterior margin of half the spermatozoa, but left their inner acrosomal membranes and equatorial segments intact. A biochemical relationship between the release of acrosomal enzymes and capacitation is suggested.     

Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.     

Ultrastructure of spermiogenesis and spermatozoa in Diurodrilus subterraneus (Polychaeta,Diurodrilidae)

Spermiogenesis in the polychaete species Diurodrilus subterraneus may be divided into six stages. These stages, as well as the ultrastructure of the mature spermatozoa, are described based on TEM studies. The spermatozoa are unusual in having a very large acrosome followed by a region containing the nucleus and several ovoid mitochondria. A secondary acrosomal membrane forms a manchette around the nucleus and mitochondria. In this region, the plasma membrane is modified, with many small, mushroom-shaped cytoplasmic processes, each including filaments. The flagellum may be divided into three sequential regions; the longest, middle one is covered by a helically arranged mucous coat. Spermatozoa of the type described here are unknown among polychaetes but show certain superficial resemblances to those in oligochaetes. The resemblance of the mushroom-shaped bodies to spermatozoal microvilli in certain gnathostomulids is discussed. The phylogenetic relationships of Diurodrilidae are considered on the basis of this new information.     

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.     

Evidence for the capacitation-associated membrane priming of mouse spermatozoa

An important feature of male fertility is the physiological priming of mammalian spermatozoa by a multifaceted process referred to as capacitation. It is a prerequisite event before spermatozoa can bind to the egg's extracellular coat, the zona pellucida, and undergo a signal transduction cascade. The net result is the fusion of the plasma membrane (PM) and underlying outer acrosomal membrane at multiple sites and the release of acrosomal contents (i.e., glycohydrolases, proteinases, etc.) at the site of sperm-zona binding. In this study, we have used an indirect immunofluorescence (IIF) assay and other staining approaches to examine capacitation-associated membrane priming of mouse spermatozoa. For IIF studies, we used affinity-purified antibodies against two glycohydrolases that cross-reacted with the acrosomal enzymes only when the uncapacitated spermatozoa were permeabilized. Incubation of spermatozoa in a medium that favors in vitro capacitation induced membrane priming that allowed the antibodies to cross-react with the acrosomal enzymes in capacitating acrosome-intact spermatozoa without permeabilization, as revealed by the appearance of several distinct fluorescent patterns, including an initial immunopositive lining over the acrosome cap to an intense immunopositive reaction throughout the acrosome. These early immunopositive patterns were followed by the appearance of intense fluorescent spots (droplets) that seem to establish contact with the PM in a time-dependent manner. Inclusion of calmodulin, a 17-kDa Ca(2+)-binding protein which promotes capacitation, in the incubation medium did not alter the overall rate of capacitation; however, its presence accelerated the initial stages of membrane priming. The potential similarities between sperm capacitation and early events of Ca(2+)-triggered membrane fusion among eukaryotes and among various stations of the secretory and endocytotic pathways are discussed.     

Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status

Living spermatozoa of seven mammalian species were treated with the thiol-alkylating fluorescent labelling compound, monobromobimane (MBBR). MB-labelling alone had no effect on sperm motility, nor on the time course or ability of golden hamster spermatozoa to undergo the acrosome reaction when capacitated in vitro. Exposure of MB-labelled spermatozoa to ultraviolet (UV) light and excitation of the MB fluorochrome resulted in virtually immediate immobilization of the spermatozoa without affecting acrosomal status. UV exposure of unlabelled spermatozoa for up to 30 sec had no effect upon motility. Immobilization of MB-labelled spermatozoa depended on the midpiece being irradiated, as irradiation of the head alone, or of the more distal parts of the principal piece, had little or no effect upon motility. Labelling with MB followed by immobilization of individually selected spermatozoa was most useful for detailing the course and site of occurrence of the acrosome reaction during penetration of the cumulus oophorus by golden hamster spermatozoa in vitro. In these often hyperactivated spermatozoa, precise determination of the acrosomal status could not often otherwise be made due to the difficulty in visualizing the acrosomal region of a vigorously thrashing, hyperactivated spermatozoon. This technique should prove valuable in a variety of studies on sperm motility, capacitation and fertilization, and could also be extended to other cell systems.     

Sperm ultrastructure of an oviparous and an ovoviviparous onychophoran species (Peripatopsidae) with some phylogenetic considerations

The spermatozoa of the Australian oviparous Ooperipatellus insignis and the South African ovoviviparous Opisthopatus cinctipes (both: Onychophora, Peripatopsidae) were studied and compared with the spermatozoal patterns already described in the taxon. The spermatozoa of both species conform with the general plan described for the Onychophora: they are filiform cells formed, in sequence, by an elongated, fully condensed nucleus capped by an acrosome and surrounded by several spiral ridges; by a mitochondrial midpiece characteristically interpolated between the nucleus and a characteristic flagellum. Major differences between the spermatozoa of both species concern their acrosome organization. The correlation between the acrosomal pattern and the size and structure of the ovarial eggs (oocytes) in onychophorans has been investigated. A parsimony analysis was performed on 21 spermatozoal characters of the species considered. Its results are congruent with those of the traditional systematics. A new set of autapomorphies characterising onychophoran sperm is suggested and some of the spermatological homologies proposed between Onychophora and Euclitellata spermatozoa are critically discussed. Our analysis suggests that spermatozoal characters are good phylogenetic markers among onychophorans, also at low taxonomic level.     

Changes of Con A Receptor Sites on Mammalian Sperms during Capacitation and Acrosome Reaction

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Surface mapping of binding of oviductin to the plasma membrane of golden hamster spermatozoa during in vitro capacitation and acrosome reaction

Oviductins are high-molecular-weight glycoproteins synthesized and secreted by nonciliated oviductal epithelial cells and have been shown to play a role in fertilization and early embryo development. The present study was carried out to examine the in vitro binding capacity of hamster oviductin to homologous sperm and to determine the sites of its localization in untreated, capacitated, and acrosome-reacted spermatozoa. Freshly prepared epididymal and capacitated sperm as well as acrosome-reacted sperm were incubated with oviductal fluid prepared from isolated hamster oviducts, fixed and then probed with a monoclonal antibody against hamster oviductin. Results obtained with pre-embedding immunolabeling experiments revealed binding of oviductin to the acrosomal cap and the apical aspect of the postacrosomal region. Immunolabeling of both regions appeared to be more intense in capacitated spermatozoa. Acrosome-reacted sperm showed an immunoreaction of moderate intensity over the postacrosomal region. The plasma membrane overlying the equatorial segment also exhibited a weak labeling. Quantitative analysis obtained with the surface replica technique indicated that oviductin had a higher binding affinity for the acrosomal cap than the postacrosomal region and that the binding of oviductin to the latter plasma membrane domain was enhanced during capacitation. Binding of oviductin to the postacrosomal region, however, was attenuated after acrosome reaction. Immunolabeling for oviductin was found to be the weakest over the equatorial segment regardless of the experimental conditions. The binding of hamster oviductin to specific membrane domains of the homologous sperm and the changes in its distribution during capacitation and acrosome reaction may be important for the function of hamster oviductin preceding and during fertilization.     

Sperm morphology of salamandrids (Amphibia, Urodela): implications for phylogeny and fertilization biology

Mature spermatozoa belonging to four salamander species, Salamandrina terdigitata, Triturus alpestris, Triturus carnifex and Triturus vulgaris, have been investigated by electron microscopy. The sperm ultrastructure of these species was compared with that of previously examined urodeles (36 species and 20 genera) and with that of anurans and caecilians. Many phylogenetic considerations may be inferred as a consequence of comparative spermatology. Urodela appears to be a monophyletic order characterized by three sperm synapomorphies: the acrosomal barb, nuclear ridge and marginal filament. Cryptobranchoidea are confirmed to form a monophyletic suborder having two synapomorphic characters: absence of mitochondria in the tail, and cylindrical shape of the tail axial rod. Within the family Salamandridae, sperm morphology confirms the phylogenetic distance between Salamandrina and Triturus, as already pointed out on the basis of molecular and morphological characters. The very complex ultrastructure of spermatozoa confirms a previous opinion that internal fertilization is the ancestral condition of the Amphibia.     

Influence of a capacitation period on human sperm acrosome loss.

The present study investigates whether a 5 hour capacitation period modifies the ability of human spermatozoa to undergo induced acrosomal loss. Human sperm acrosomal loss was induced by treatment with either the calcium ionophore A23187, low concentrations of the phospholipid dilauroylphosphatidylcholine (PC12), or 2 hours incubation in conditioned medium prepared from human cumulus cells (CM/CC). The use of a dual staining method (FITC-ConA and Hoechst 33258) for simultaneous assessment of acrosomal status and viability demonstrated that induction of acrosomal loss with calcium ionophore was not dependent on a capacitation period. A short (5 hour) incubation period was not sufficient to induce acrosomal loss with CM/CC above spontaneous acrosome reaction rates in medium alone. A significant capacitation-dependent increase (P < 0.05) in acrosomal loss was observed when human spermatozoa were incubated with PC12. Induction of acrosomal loss of capacitated human spermatozoa with PC12 therefore provides a simple assay for the simultaneous assessment of human sperm capacitation and the acrosome reaction in vitro.     

Spermatozoal ultrastructure of Penaeus kerathurus and Penaeus japonicus (Crustacea,Dendrobranchiata)

Summary An ultrastructural comparison between the unistellate spermatozoa of the shrimps Penaeus kerathurus and P. japonicus reveals a number of similarities that are common among dendrobranchiates, but also some marked differences which would confirm the validity of a potential use of sperm structure in systematic and phylogenetic studies. Typical morphological features shared by the spermatozoa of P. kerathurus and P. japonicus are: a membrane-bound acrosomal vesicle consisting of a cap and spike; non-membrane-bound filamentous chromatin; a perinuclear cytoplasmic band including degenerative membranous organelles (mostly mitochondria), small vesicles with a dense core and parallel membrane lamellae. Discordant spermatozoal characteristics between both species involve a significantly different size (ca. 5 m in length by ca. 3 m in diameter in P. kerathurus; ca. 8 m in length by ca. 4.7 m in diameter in P. japonicus), the occurrence of intranuclear lipid droplets only in P. kerathurus and the presence of a deeper subacrosomal space in this species as compared to P. japonicus. It is very likely that the most significant difference between both species is, however, the appearance of microtubule bundles in the spermatozoon of P. japonicus. So far, the occurrence of spermatozoal microtubules in decapod crustaceans appears to be restricted to reptantian species, whereby the finding of such elements in sperm of a dendrobranchiate shrimp could be of phylogenetic interest.     

Fertility differences among male rabbits determined by heterospermic insemination of fluorochrome-labeled spermatozoa

Spermatozoa from different bucks were stained with different fluorochromes, mixed, and inseminated heterospermically. By altering the interval between insemination and luteinizing hormone injection, spermatozoa were allowed to reside in the female tract approximately 5, 10, or 15 h prior to ovulation. The number of functional spermatozoa, from each male of a pair used, that was transported to the site of fertilization was estimated by counting total number of differently stained spermatozoa that surrounded or fertilized each oocyte. Spermatozoa from split ejaculates within a male competed against each other equally, indicating that the staining procedure did not affect fertilization or functional spermatozoal transport rates. Three pairs of males with high initial semen quality (greater than 80% motility) differed in fertility primarily due to functional spermatozoal transport. Spermatozoal survival in the female tract and capacitation time played a role in differences in male fertility when heterospermic insemination occurred at variable times relative to ovulation. Differences in fertilization not accounted for by spermatozoal transport ratio raised the possibility that rate of egg penetration due to acrosomal enzyme differences may be important in determining male fertility. Therefore, total acrosin, hyaluronidase, and arylsulfatase activity in spermatozoa from specific bucks used in fertilization experiments were determined. Although there were trends favoring high fertility when enzyme content was higher, the difference was significant only for arylsulfatase in one buck.     

Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon

Experiments to bind fluorescein-conjugated Arachis hypogea (peanut) agglutinin (FITC-PNA) to washed human spermatozoa demonstrated that this lectin binds to the acrosome region in air-dried preparations. Since there was no binding when labelling was performed in suspension, and comparable labelling to that seen in air-dried preparations was seen when spermatozoa treated with saponin (to lyse the plasma membrane) were labelled in suspension, the lectin must bind to an intracellular structure, probably the outer acrosomal membrane. This was confirmed by ultrastructural localization of colloidal gold-conjugated lectin in saponin-treated spermatozoa. Treatment of spermatozoa with the detergent Nonidet P-40 caused a marked change in the binding pattern: more spermatozoa showed binding in the equatorial segment of the acrosome with no binding in the anterior cap region. A comparable, less marked, change was seen when spermatozoa were incubated overnight under conditions known to support the capacitation and spontaneous acrosome reactions. Treatment with the calcium ionophore A23187 for 1 h to induce acrosome reactions artificially in uncapacitated spermatozoa resulted in the appearance of patchy acrosome fluorescence. From these experiments it is concluded that PNA binds specifically to the outer acrosomal membrane, and that FITC-PNA-labelling may be used to monitor the human sperm acrosome reaction.