Ultrastructural aspects of spermiogenesis and synspermia in the brown spider Loxosceles intermedia (Araneae: Sicariidae)

This study reports ultrastructural and cytochemical aspects of spermiogenesis and synspermia in the brown spider Loxosceles intermedia. The roundish early spermatids are initially interconnected by cytoplasmic bridges, forming groups of four cells. During spermiogenesis, these cells pass through a series of modifications: (1) progressive nuclear condensation brings chromatin into a fibrillar arrangement; (2) the nucleus becomes long and asymmetric, with a short post-centriolar elongation; (3) formation of the long, cone-shaped acrosome and the F-actin acrosomal filament; (4) establishment of the implantation fossa and the 9x2+3 pattern flagellum, which extends away from the sperm cell body. Eventually, the entire cell undergoes twisting and folding resulting in a synspermium, containing four sperm cells in which the flagellum and nucleus are delimitated by the plasma membrane, as individualized structures, but remain involved by the fused remaining cytoplasm and plasma membrane. Reaching the vas deferens, the synspermia are surrounded by a basic glycoproteic secretion. Synspermia are considered a derivative character, probably developed in this Sicariidae species, as well as in other Haplogynae, as an adaptation to improve the reproductive strategy.     

Ultrastructural investigations of testes and spermiogenesis in two species of halacarid mites (Halacaridae, Actinedida, Actinotrichida): Thalassarachna basteri from the Baltic Sea and Halacarellus thomasi from McMurdo Sound (Antarctica)

Testes and sperm cells of two species of halacarid mites, Thalassarachna basteri from the Baltic Sea and Halacarellus thomasi from McMurdo Sound (Antarctica), were investigated. Testes are paired structures, composed of a glandular and a germinal part. The testicular lumen is filled with a very complex secretion that contributes to sperm cell aggregates. Early spermatids of T. basteri display unusual chromatin condensation within the nucleus, but the formation of an acrosomal complex with a small acrosomal vesicle and a long acrosomal filament can be regarded as typical for the group. Tubular invaginations of the plasmalemma occur at the cell periphery. A deep, ring-like infold divides the cell into one part containing the chromatin body and another containing mainly the invaginations and the acrosomal complex. The mature sperm cell is ovoid, aflagellate and surrounded by a distinct secretion sheath. In H. thomasi only a limited number of spermiogenesis stages were observable. Chromatin condensation was rather similar and peripheral invaginations also occurred. However, no acrosomal complex was observed in the early stages. The division of the mature sperm cells into two halves was even more pronounced in H. thomasi, since one half of the cell contained masses of convoluted structures. The same half also contained a structure that remotely resembled an acrosomal complex. The observed differences between T. basteri and H. thomasi sperms support the placing of the two halacarids in separate genera.     

Formation of primary sperm conjugates in a haplogyne spider (Caponiidae,Araneae) with remarks on the evolution of sperm conjugation in spiders

Sperm conjugation, where two or more sperm are physically united, is a rare but widespread pheno-menon across the animal kingdom. One group well known for its different types of sperm conjugation are spiders. Particularly, haplogyne spiders show a high diversity of sperm traits. Besides individual cleistospermia, primary (synspermia) and secondary (coenospermia, “spermatophore”) sperm conjugation occurs. However, the evolution of sperm conjugates and sperm is not understood in this group. Here, we look at how sperm are transferred in Caponiidae (Haplogynae) in pursuit of additional information about the evolution of sperm transfer forms in spiders. Additionally, we investigated the male reproductive system and spermatozoa using light- and transmission electron-microscopy and provide a 3D reconstruction of individual as of well as conjugated spermatozoa. Mature spermatozoa are characterized by an extremely elongated, helical nucleus resulting in the longest spider sperm known to date. At the end of spermiogenesis, synspermia are formed by complete fusion of four spermatids. Thus, synspermia might have evolved early within ecribellate Haplogynae. The fused sperm cells are surrounded by a prominent vesicular area. The function of the vesicular area remains still unknown but might be correlated with the capacitation process inside the female. Further phylogenetic and functional implications of the spermatozoa and sperm conjugation are discussed.     

Spermatozoa and spermiogenesis of Liphistius cf. phuketensis (Mesothelae, Araneae, Arachnida) with notes on phylogenetic implications

The present study deals with the spermatozoa and spermiogenesis of Liphistius cf. phuketensis, a representative of the most primitive and enigmatic spider group Mesothelae. The general organization of the spermatozoa is very similar to the condition known from Amblypygi supporting a sister-group relationship between Araneae and Amblypygi. Besides plesiomorphic characters such as, e.g., an elongated and corkscrew shaped nucleus, the sperm cells are characterized by several apomorphic characters, e.g., the giant body and conspicuous membranous areas which are formed at the end of spermiogenesis. As the transfer form, coenospermia are formed at the end of spermiogenesis, which strongly supports the idea that this type of sperm aggregation is the primitive transfer form within spiders. A very remarkable character of the spermatozoa of some groups of arachnids is the coiling of the main cell organelles at the end of spermiogenesis. Previously, the Mesothelae were believed to be the only spider group which does not show a complete coiling of the main cell organelles. With the present study the first evidence of a complete coiling of spermatozoa within this primitive spider group could be documented, indicating that this character is part of the ground pattern of spider spermatozoa. Consequently, the incomplete coiling seems to be a synapomorphy of certain species of Mesothelae, which sheds new light on the discussion of the phylogenetic relationships of this group.     

Protein kinase C-mediated phosphorylation of the two polybasic regions of synaptotagmin VI regulates their function in acrosomal exocytosis

We have previously reported that synaptotagmin VI is present in human sperm cells and that a recombinant protein containing the C2A and C2B domains abrogates acrosomal exocytosis in permeabilized spermatozoa, an effect that was regulated by phosphorylation. In this report, we show that each individual C2 domain blocks acrosomal exocytosis. The inhibitory effect was completely abrogated by phosphorylation of the domains with purified PKCbetaII. We found by site-directed mutagenesis that Thr418 and/or Thr419 in the polybasic region (KKKTTIK) of the C2B domain--a key region for the function of synaptotagmins--are the PKC target that regulates its inhibitory effect on acrosomal exocytosis. Similarly, we showed that Thr284 in the polybasic region of C2A (KCKLQTR) is the target for PKC-mediated phosphorylation in this domain. An antibody that specifically binds to the phosphorylated polybasic region of the C2B domain recognized endogenous phosphorylated synaptotagmin in the sperm acrosomal region. The antibody was inhibitory only at early stages of exocytosis in sperm acrosome reaction assays, and the immunolabeling decreased upon sperm stimulation, indicating that the protein is dephosphorylated during acrosomal exocytosis. Our results indicate that acrosomal exocytosis is regulated through the PKC-mediated phosphorylation of conserved threonines in the polybasic regions of synaptotagmin VI.     

Fine structure of spermiogenesis, spermatozoa and spermatophore of Saxidromus delamarei (Saxidromidae, Actinotrichida, Acari)

Ultrastructural details of spermiogenesis, spermatozoa and the spermatophore of the early derived actinedid mite Saxidromus delamarei are described. Spermatids and mature sperm cells are provided with up to four acrosomal complexes and nuclei derivatives (chromatin bodies). Due to this reason, the sperm cells may be classified as synspermia, a sperm type found only in some spiders until now. The acrosomal complex is composed of a remarkably complicated vacuole and filament. Other peculiarities of sperm structure correspond to those found in prostigmatic mites, i.e. penetration of the chromatin body by the acrosomal filament and the presence of peripheral invaginations of the plasmalemma. The sperm cells are covered by a thin secretion layer of probably proteinaceous material. Stalked spermatophores are rather large, but simply structured and contain relatively few sperm cells. The results are discussed taking systematical and behavioural aspects into account. In particular, it is suggested that the peculiar mating behaviour of these mites secures both sperm transfer and first male's sperm priority and that this allowed reduction of sperm numbers.     

The ultrastructure of the peculiar synspermia of some Dysderidae (Araneae, Arachnida)

The present study reports on the ultrastructure features of spermatozoa and spermatogenesis of several species of Dysderidae (Dysdera crocata, Dysdera erythrina, Dysdera ninnii, Harpactea arguta, Harpactea piligera, Dasumia taeniifera). Dysderid spiders are known to possess a peculiar sperm transfer form known as synspermia, characterized by fused spermatozoa surrounded by a secreted sheath. Until now the exact mode of formation of the synspermia is unknown. The present study demonstrates that the spermatids are connected via narrow cell bridges during the entire spermiogenesis as is usual, although in Dysderidae they do not separate at end of the spermiogenesis. Instead, they fuse completely within the testes shortly after the spermatid has coiled to get a spherical shape. The number of fusing sperm cells is different in the different observed species. The species of the genus Harpactea thus have synspermia consisting of two fused spermatozoa; whereas in the species of the genus Dysdera four sperm cells are fused and in D. taeniifera at least three spermatozoa are fused. In contrast with other known families with this peculiar form transfer of sperm, the synspermia in Dysderidae are mainly characterized by a conspicuous vesicular area which extends through the entire synspermium surrounding the cell organelles. Thus, all main cell components (e.g., nucleus, acrosomal vacuole, and axoneme) are covered by the vesicular membrane. The vesicular area seems to be functional and probably it is important during sperm activation in female genital system. Simultaneously to the extension of the vesicular area, the synspermium accumulates large amounts of glycogen. The glycogen is mainly located around the centriolar adjunct and along the axoneme accompanying the postcentriolar elongation of the nucleus. A further peculiar feature is the extremely elongated acrosomal vacuole, which seems to be synapomorphic trait for sperm cells of dysderids. Interestingly, spermatogenesis, including the fusion, exclusively occurs within the testes (in contrast to the formation of coenospermia). In the vas deferens only synspermia were found. The secreted sheath surrounding the spermatozoa is finally synthesized in the parts of the vasa deferentia, which are close to the genital opening where numerous vacuoles and microvilli are seen in the epithelial cells.     

A reevaluation of the fertilizin hypothesis of sperm agglutination and the description of a novel form of sperm adhesion

The classical isoagglutination of sea urchin sperm by egg jelly is not an agglutination of cells, as proposed by the fertilizin-antifertilizin hypothesis. Sperm motility is required to obtain the isoagglutination of Strongylocentrotus purpuratus sperm, and the sperm do not adhere to each other in the isoagglutination clusters, which cannot be fixed for microscopy and which disperse rapidly into individual cells when sperm motility is inhibited. These observations suggest that isoagglutination is the swarming of freely moving sperm to a common focus and is quite distinct from the agglutination of sperm by known crosslinking agents (antibodies or lectins).A previously unrecognized form of sperm agglutination is described which follows induction of an acrosome reaction by egg jelly, ammonia, or the ionophore A23187 in a suspension of sea urchin or sand dollar sperm. The sperm form rosettes of up to 100 cells in which the newly extended acrosomal processes adhere to each other. Rosettes can form containing sperm of different species, in which the acrosomal processes adhere without species preference.As observed by transmission electron microscopy, the acrosomal process of Lytechinus pictus sperm consists of an acrosomal tubule covered by a sheath of extracellular material. Rosette formation results from attachment between the extracellular materials of adjacent sperm.Less frequently, the acrosomal process of one sperm adheres to the midpiece of another by fusion of the acrosomal tubule and midpiece plasma membranes.     

Double spermatogenesis in Chelicerata

Sperm dimorphism is a rare phenomenon in Chelicerata. Until now, it was known only from three species of the opilionid genus Siro (Sironidae, Cyphophthalmi). Fertilizing (eusperm) and nonfertilizing spermatozoa (parasperm) develop in the same cyst and are thus sister cells. The fine structure of the spermatozoa of two species has been examined and is compared here. In contrast to Siro rubens, S. duricorius spermatozoa lack an acrosomal complex. Both sperm types produce a transitional process, a more or less modified flagellum, which is later retracted. Hence, the spermatozoa are aflagellate. Eusperm and parasperm of all three species form highly ordered sperm balls that are stored in the deferent duct. Reviewing and adding new results about the sperm dimorphism in this arachnid taxon provides the basis for some considerations of another enigmatic morphological character found in Uropygi and Amblypygi, i.e., the tubular accessory genital glands that show holocrine extrusion. These glands are suggested to represent modified, infertile derivatives of the testis anlage. Their secretion is produced in a way reminiscent of a strongly degenerated spermatogenesis. Consequently, these products may be regarded as strongly degenerated germ cells representing a line of germ cell development, which has been separated very early in spermatogenesis from the usual line leading to fertilizing sperm cells. This further, although less evident, case of probable dichotomous germ cell development is discussed with respect to the controversial phylogenetic-systematic relationships between Uropygi (Thelyphonida and Schizomida), Amblypygi, and Araneae.     

COMPARATIVE SPERMATOLOGY OF FOUR SYMPATRIC SPECIES OF SIPHONARIA (PULMONATA: BASOMMATOPHORA)

The fine structure of the modified sperm and spermatogenesisof four sympatric species of Siphonaria is described. The morphologyof the sperm of all species is very similar. The head, whichis about 6 µm long, is composed of a nucleus with fibrouschromatin capped by an acrosome (about 1 µm long) comprisedof an acrosomal pedestal and apical vesicle. The midpiece hasa mitochondrial derivative which surrounds a single glycogenhelix, posterior to which is a glycogen piece. Although differencesbetween each species exist, the value of sperm morphology forpurposes of taxonomy in this genus is questioned. Comparisonwith other basommatophorans however suggests that sperm morphologymay be of value at a higher taxo-nomic level. The morphologicalchanges that occur during spermatogenesis are similar to thosedescribed for other molluscs with modified sperm, except thatduring early spermiogenesis the Golgi body and smooth endoplasmicreticulum become highly developed. This proliferation of theSER and Golgi occurs at the same time as elongation of the spermatid.Throughout spermatogenesis, the germ cells are closely associatedwith a somatic cell which, because of structural similaritieswith the somatic cell of mammalian seminiferous epithelium,has been termed a Sertoli cell. After the spermatids have beenreleased from the Sertoli cells of the testis, maturation continuesin the hermaphrodite duct where the acrosome reaches its finalsize and glycogen accumulates in the glycogen compartment ofthe mid-piece. (Received 25 April 1990; accepted 1 September 1990)     

The Multi-PDZ domain protein MUPP1 as a lipid raft-associated scaffolding protein controlling the acrosome reaction in mammalian spermatozoa

The success of acrosomal exocytosis, a complex process with a variety of interrelated steps, relies on the coordinated interaction of participating signaling molecules. Since scaffolding proteins are known to spatially organize sequential signaling pathways, we examined whether the Multi-PDZ domain protein MUPP1, recently identified in mammalian spermatozoa, is functionally active in controlling acrosomal secretion in mammalian sperm cells. To address this question, permeabilized mouse sperm were loaded with inhibitory antibodies against MUPP1 as well as with a photosensitive Ca(2+) chelator which allows a controlled release of acrosomal Ca(2+). The results revealed that MUPP1 controls initial tethering and docking of the acrosomal vesicle, whereas syntaxin 2, a t-SNARE protein also expressed in the acrosomal cap of mammalian spermatozoa, appears to take part in the final process of acrosomal fusion. Interestingly, using immunogold electron microscopy, it was found that MUPP1 is detectable in the region of the periacrosomal membrane. Furthermore, in isolated detergent-insoluble glycolipid-enriched membrane domains from epididymal spermatozoa, MUPP1 was found to show a striking association with the Triton X-100 insoluble membrane fraction, which did not change significantly upon sperm capacitation or partial chemical extraction of cholesterol. This evidence points to a role of MUPP1 as a membrane raft-associated molecular organizer, and suggests that mammalian spermatozoa may use a scaffolding protein and distinct membrane subdomains to spatially organize components involved in the process of acrosomal exocytosis.     

Calcineurin-mediated Dephosphorylation of Synaptotagmin VI Is Necessary for Acrosomal Exocytosis

Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. In general, exocytosis is initiated by a cytosolic calcium increase. In this report we show that calcium affects several factors during human sperm acrosomal exocytosis. By using an antibody that specifically recognizes synaptotagmin VI phosphorylated at the polybasic region of the C2B domain, we showed that a calcium-dependent dephosphorylation of this protein occurred at early stages of the acrosomal exocytosis in streptolysin O-permeabilized sperm. We identified the phosphatase as calcineurin and showed that the activity of this enzyme is absolutely required during the early steps of the secretory process. When added to sperm, an inhibitor-insensitive, catalytically active domain of calcineurin was able to rescue the effect of the specific calcineurin inhibitor cyclosporin A. This same domain dephosphorylated recombinant synaptotagmin VI C2B domain, validating this protein as a new substrate for calcineurin. When sperm were treated with catalytically active calcineurin before stimulation, exocytosis was inhibited, an effect that was rescued by the phosphomimetic synaptotagmin VI C2B-T418E,T419E mutant domain. These observations indicate that synaptotagmin must be dephosphorylated at a specific window of time and suggest that phosphorylated synaptotagmin has an active role at early stages of the acrosomal exocytosis.     

Pisum sativum agglutinin used as an acrosomal stain of porcine and caprine sperm

Fluorescein isothiocyanate-labelled pisum sativum agglutinin (FITC-PSA) was evaluated as an acrosomal stain for porcine and caprine sperm that had previously been stained with Hoechst 33258 to assess cell viability. The FITC-PSA procedure was as accurate as other procedures in assessing acrosomal presence or absence on either fresh or liquid-stored porcine sperm. Approximately half of the incubated porcine sperm with acrosomal loss maintained membrane impermeability to the Hoechst 33258; these were potentially viable acrosome-reacted sperm. The FITC-PSA procedure was significantly correlated with the assessment of acrosomal status of cryopreserved caprine sperm by transmission electron microscopy (TEM). However, TEM results indicated a higher percentage of caprine sperm with acrosomal loss. Ability to penetrate zona-free hamster ova was not highly correlated with the percentage of viable acrosome-reacted porcine or caprine sperm. The FITC-PSA procedure provides an estimate of viable acrosome reactions and may be a useful tool in the evaluation of sperm fertility.     

Acrosome reaction in spermatozoa from hagfish (Agnatha) Eptatretus burgeri and Eptatretus stouti: acrosomal exocytosis and identification of filamentous actin

Spermatozoa of the hagfishes Eptatretus burgeri and Eptatretus stouti, caught in the sea near Japan and North America, respectively, were found to undergo the acrosome reaction, which resulted in the formation of an acrosomal process with a filamentous core. The acrosomal region of spermatozoa of E. stouti exhibited immunofluorescent labeling using an actin antibody. The midpiece also labeled with the antibody. The acrosomal region showed a similar labeling pattern when sperm were probed with tetramethylrhodamine isothyocyanate (TRITC)-phalloidin; the midpiece did not label. Following induction of the acrosome reaction with the calcium (Ca2+) ionophore ionomycin, TRITC-phalloidin labeling was more intense in the acrosomal region, suggesting that the polymerization of actin occurs during formation of the acrosomal process, as seen in many invertebrates. The potential for sperm to undergo acrosomal exocytosis was already acquired by late spermatids. During acrosomal exocytosis, the outer acrosomal membrane and the overlying plasma membrane disappeared and were replaced by an array of vesicles; these resembled an early stage of the acrosome reaction in spermatozoa of higher vertebrates in which no formation of an acrosomal process occurs. It is phylogenetically interesting that such phenomena occur in spermatozoa of hagfish, a primitive vertebrate positioning between invertebrates and high vertebrates.     

Presence of a trypsin-like protease in starfish sperm acrosome.

Marthasterias glacialis sperm cells were treated with ionophore A23187, centrifuged, and the supernatants were assayed for esterase activity. With N-benzoyl-L-arginine ethyl ester-HCl (BAEE) as substrate, a net activity was determined which was not detectable when N-acetyl-L-tyrosine ethyl ester (ATEE) was used. The BAEE trypsin-like activity was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-L-lysine chloromethyl ketone-HCl (TLCK), and phenyl methyl sulfonyl fluoride (PMSF), but not by L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK). The presence of proteolytic activity in acrosomal exudates was further demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic zymography (gelatin-SDS-PAGE). The presence of several bands of low proteolytic activity and of one band of high proteolytic activity, which also has the lower molecular weight, together with the fact that all are inhibited by benzamidine, suggests the existence of a trypsin-like proteinase system. The effect of the acrosomal exudate on the oocyte jelly coat was investigated by SDS-PAGE analysis. All jelly proteins appeared to be digested by the acrosomal enzymes. Furthermore, if SBTI is added shortly after insemination, the sperm fail to fertilize the oocytes. These results indicate that the starfish sperm acrosomal vesicle contains a trypsin-like protease which may be involved in sperm penetration through the oocyte jelly coat.     

Equatorial segment protein defines a discrete acrosomal subcompartment persisting throughout acrosomal biogenesis

The equatorial segment of the acrosome underlies the domain of the sperm that fuses with the egg membrane during fertilization. Equatorial segment protein (ESP), a novel 349-amino acid concanavalin-A-binding protein encoded by a two-exon gene (SP-ESP) located on chromosome 15 at q22, has been localized to the equatorial segment of ejaculated human sperm. Light microscopic immunofluorescent observations revealed that during acrosome biogenesis ESP first appears in the nascent acrosomal vesicle in early round spermatids and subsequently segregates to the periphery of the expanding acrosomal vesicle, thereby defining a peripheral equatorial segment compartment within flattened acrosomal vesicles and in the acrosomes of early and late cap phase, elongating, and mature spermatids. Electron microscopic examination revealed that ESP segregates to an electron-lucent subdomain of the condensing acrosomal matrix in Golgi phase round spermatids and persists in a similar electron-lucent subdomain within cap phase spermatids. Subsequently, ESP was localized to electron-dense regions of the equatorial segment and the expanded equatorial bulb in elongating spermatids and mature sperm. ESP is the earliest known protein to be recognized as a marker for the specification of the equatorial segment, and it allows this region to be traced through all phases of acrosomal biogenesis. Based on these observations, we propose a new model of acrosome biogenesis in which the equatorial segment is defined as a discrete domain within the acrosomal vesicle as early as the Golgi phase of acrosome biogenesis.     

Identification of actin in boar spermatids and spermatozoa by immunoelectron microscopy

Actin was localized in testicular spermatids and in ionophore-treated ejaculated sperm of boar by use of a monoclonal anti-actin antibody labeled with colloidal gold. With the on-grid postembedding immunostaining of Lowicryl K4M sections, actin was identified in the subacrosomal region of differentiating spermatids, in the microfilaments of the surrounding Sertoli cells, and in the myoid cells of the tubular wall. Ejaculated sperm, labeled with the preembedding method, showed actin between the plasma membrane and the outer acrosomal membrane of the equatorial segment. Indirect immunofluorescence was positive in the equatorial segment and in the acrosomal cap of intact sperm, whereas reacted sperm at the anterior head region retained fluorescence only in the inner acrosomal membrane. Rhodamine-phalloidin failed to stain intact and reacted sperm. The distribution of actin in sperm head membranes (inner acrosomal membrane, membranes of the equatorial segment), which are retained after the acrosome reaction, is discussed.     

Effect of increasing trypsin concentrations on seminal coagulum dissolution and sperm parameters in spider monkeys (Ateles geoffroyi)

Seminal coagulum formation in spider monkeys (Ateles geoffroyi) interferes with the efficient recovery and evaluation of spermatozoa. The main objective was to assess the effect of increasing concentrations of trypsin on dissolution of seminal coagulum and spermatic parameters. Seminal coagulum was incubated at 37 °C without trypsin or in the presence of increasing trypsin concentrations (0.1%, 1.0%, and 5.0%). For each sample, coagulum dissolution time was measured, and sperm concentration, viability, motility, and morphology were evaluated using light microscopy and/or transmission electronic microscopy (TEM). Trypsin concentrations of 1.0% and 5.0% more rapidly liquefied seminal coagulum, averaging 32 and 21 min, respectively, compared with nontrypsinized controls, with maintenance of greater sperm viability (70.8% and 72.5%, respectively). Coagulum treated with 1.0% trypsin and the liquid ejaculate fraction averaged higher sperm motility (40.1% and 55.6%, respectively) than control samples, and both 1.0% and 5.0% trypsin treatment allowed recovery of increased numbers of motile spermatozoa. There was greater sperm fragmentation at the head and midpiece level after treatment with 1.0% and 5.0% trypsin (55.8% and 55.9%); however, the percentage of normal morphology in structurally intact spermatozoa did not differ relative to controls. With transmission electronic microscopy imaging, there were similar percentages of spermatozoa with plasma membrane swelling in the midpiece and acrosomal regions in trypsin-treated samples and controls. In conclusion, trypsin treatment of spider monkey seminal coagulum exerted a concentration-dependent effect on dissolution time and various spermatic parameters. Higher trypsin concentrations caused more rapid liquefaction of coagulum and recovery of greater numbers of motile spermatozoa, but may adversely affect fragmentation of spermatozoa and could compromise sperm function and cryopreservation potential.     

Acrosomal status in fresh and capacitated human ejaculated sperm

The acrosomal status of human sperm was evaluated by immunofluorescence utilizing a specific monoclonal antibody that recognizes target antigen(s) localized in the acrosomal cap region. Spontaneous acrosomal loss was first examined in sperm preparations used for successful in vitro fertilization of human eggs. In these sperm populations, less than 20% of the sperm underwent degenerative or spontaneous acrosomal loss following 24 h of incubation. The correlation of acrosomal loss with changes in motility and viability suggested that sperm senescence was not necessarily coupled to acrosomal loss. Chemical induction of acrosomal loss by calcium ionophore A23187 and lysophosphatidylcholine (LPC) was characterized. Maximal ionophore induction (10 microM A23187 in media containing calcium) was observed in cells exposed to capacitating conditions in vitro; sperm exposed to noncapacitating conditions did not readily acquire the ability to respond to ionophore. The reaction induced by ionophore was slow (60 min), and at least 30% of the cells were always resistant to induction. In contrast, LPC induced rapid, synchronous acrosomal loss in either freshly ejaculated or capacitated sperm in the presence or absence of extracellular calcium, suggesting that this loss was not a physiologic reaction. These studies may provide a basis for evaluating capacitation and ultimately fertility potential in the human male.