Acrosomal Cap Research Articles

FPP modulates mammalian sperm function via TCP-11 and the adenylyl cyclase/cAMP pathway.

Fertilization promoting peptide (FPP; pGlu-Glu-ProNH2), which is found in seminal plasma, promotes capacitation but inhibits spontaneous acrosome loss in mammalian spermatozoa in vitro. Adenosine, known to modulate the adenylyl cyclase (AC)/cAMP pathway, elicits these same responses whereas FPP + adenosine produces an enhanced response, leading to the hypothesis that FPP and adenosine modulate the same signal transduction pathway but act via different receptors. TCP-11, the product of a t-complex gene, is the putative receptor for FPP: Fab fragments of anti-TCP-11 antibodies have the same effect as FPP on mouse spermatozoa and Gln-FPP, a competitive inhibitor of FPP, also competitively inhibits responses to the Fab fragments. In the present study, specific binding of 3H-FPP to sperm membranes was significantly inhibited by 200 nM Gln-FPP and anti-TCP-11 Fab fragments (1/25 dilution), thus confirming that FPP, Gln-FPP, and Fab fragments compete for the same binding site. In addition, spermatozoa treated with A23187 to induce the acrosome reaction bound significantly less 3H-FPP than untreated cells, suggesting that a large proportion of the FPP binding sites are associated with the acrosomal cap region; TCP-11 is located in this region. In other experiments, 100 nM FPP significantly stimulated cAMP production in mouse sperm membranes, permeabilized cells and intact cells. Furthermore, Gln-FPP inhibited production of cAMP in response to FPP but not to adenosine (10 microM) or its analogue NECA (100 nM), supporting the involvement of two different receptors. Finally, anti-TCP-11 Fab fragments (1/25 dilution) significantly stimulated cAMP production, whereas low Fab (1/200; nonstimulatory when used alone) plus adenosine (10 microM) significantly enhanced the stimulation of capacitation by adenosine. These results support the hypotheses that TCP-11 is the receptor for FPP and that FPP<-->TCP-11 interactions modulate AC/cAMP.

Localisation of low molecular weight sperm antigens during capacitation and acrosome reaction.

FITC-labelled sperm-specific antibodies against hamster spermatozoa were utilized as probes in acrosome reaction assays. An indirect immunofluorescence test demonstrated the localisation of two sperm proteins of 19 kDa and 23 kDa on the anterior acrosomal cap region of washed cauda epididymal sperm. These proteins were not detected in reacted acrosome or on immature or immotile sperm. Antisperm agglutinating antibodies specific to these two low molecular weight sperm antigens could be useful probes for evaluating the acrosomal status of mammalian spermatozoa.

Human sperm non-nuclear progesterone receptor expression is a novel marker for fertilization outcome.

In a prospective, blind study, we have examined the relationship among the expression of human sperm surface progesterone receptors, the ability to undergo a mannose-stimulated acrosome reaction and the rate of fertilization in vitro. Individual aliquots of motile spermatozoa were surface-labelled with progesterone and/or mannose-fluoresceinated ligands. Spontaneous acrosome loss and the increase in acrosome reactions following exposure of spermatozoa to mannose ligands were assessed using rhodaminated Pisum sativum agglutinin. Progesterone fluoresceinated ligand binding was observed to occur in two patterns: (i) a uniform distribution of labelling over the acrosome cap (pattern II), and (ii) labelling limited to the equatorial and postacrosomal regions of the human sperm head (pattern III). A conversion of pattern II to pattern III binding was observed and was associated with the acrosome reaction. Pattern III binding was highly correlated with both fertilization potential and the ability to undergo a mannose-stimulated acrosome reaction (P < 0.001). In contrast, normal sperm mannose receptor expression was seen in five men whose abnormal progesterone receptor expression/function and inability to acrosome react after mannose treatment were correlated with their reduced fertility in vitro. In conclusion, surface progesterone receptor aggregation enhances the mannose ligand-stimulated acrosome reaction. Such detection of defective sperm surface progesterone receptor expression/function may be useful in the evaluation and management of male infertility.

Ultrastructure of gametes and intracytoplasmic sperm injection: the significance of sperm morphology

The aim of this study was to determine characteristic malformations of sperm ultrastructure in patients with severe subfertility undergoing intracytoplasmic sperm injection (ICSI). Although light microscopy (LM) can reveal major abnormalities of the three parts of the spermatozoon (head, mid-piece and flagellum), the various cell organelles of the spermatozoon and their fine structure remain unevaluated by LM. Insight into the submicroscopic organization of the spermatozoon and its complex organellar system may contribute to a better understanding of the preconditions for success or failure of fertilization. An in-depth evaluation of semen quality by transmission electron microscopy (TEM) can improve the diagnosis of male subfertility and can give substantial information about the fertilizing competence of spermatozoa. Thus, in this study 56 ejaculated sperm samples from patients with severe male subfertility or previous failed attempts at in-vitro fertilization were assessed by LM and TEM prior to ICSI to evaluate the most important sperm defects causing extreme subfertility. LM analysis was performed according to World Health Organization criteria. It could be confirmed that severe head defects are mostly involved in long-term infertility and fertilizing failure in classical IVF treatments. The most frequent head defects are disorders of the nuclear membranes and the acrosomal cap and disorganization of the chromatin structure. These defects of sperm fine structure seem to be associated with dysfunctional sperm-oocyte recognition, binding and fusion with the oolemma. Chromatin alterations and signs of decondensation or karyolysis are frequently associated with a deterioration of the nuclear membranes and may be due to impaired spermiogenesis. However, our results and the success of ICSI proved that severe sperm defects have no predictive value and do not impair the fertilization process, and also that the maturity of spermatozoa does not play an important role. Fine structure analysis revealed the pleiomorphology and heterogeneity of human spermatozoa.

Molecular cloning and characterization of P47, a novel boar sperm-associated zona pellucida-binding protein homologous to a family of mammalian secretory proteins.

P47, a peripherally associated 47-kDa protein of porcine spermatozoa, was identified by affinity chromatography in the fraction of solubilized plasma membrane proteins bound to immobilized porcine zona pellucida glycoproteins. N-terminal and internal amino acid sequences revealed structural similarity between P47 and rat O-acetyl ganglioside synthase, bovine mammary gland protein (MGP)57/53 and mouse milk fat globule protein E8-polypeptides of unknown function secreted by mammary gland epithelial cells in both species. A polyclonal antibody directed against bovine MGP57/53 displayed cross-reactivity with P47. Indirect immunofluorescence analysis located porcine P47 on the acrosomal cap of testicular sperm and on sperm recovered along different sections of the ductus epididymidis, as well as on swim-up and in vitro-capacitated sperm. Porcine P47 was demonstrated on sperm bound to the zona pellucida of a homologous oocyte. Western blot analysis identified P47 (or MGP57/53) homologous proteins in porcine and human milk. Like the sperm-associated protein, porcine milk P47 possesses affinity for isolated, biotinylated sow oocyte zona pellucida glycoproteins. Reverse transcription-polymerase chain reaction was used to isolate P47 homologous cDNAs from porcine testis and mammary gland tissues as well as from bovine, mouse, and human testis. P47 proteins deduced from these cDNA sequences showed 60-100% amino acid sequence identity. These proteins display a mosaic structure organized into two N-terminal, tandemly arranged epidermal growth factor (EGF)-like domains followed by a region with similarity to C1 and C2 domains found in blood clotting factors V and VII. The second EGF-like domain contains an arginine-glycine-aspartic acid sequence, a motif often found in integrin receptor ligands. P47-like proteins are not expressed solely in testicular and mammary gland tissues. Northern blot analysis showed that P47 mRNA is transcribed in several porcine and bovine tissues. These data indicate a potential role for boar sperm-associated P47 in membrane remodeling and/or as a zona pellucida binding protein.

Binding of a bovine oviductal fluid catalase to mammalian spermatozoa.

Hydrogen peroxide (H2O2) is a reactive oxygen species that at low concentration is toxic to sperm. H2O2 inhibits not only sperm viability but also the acrosome reaction, sperm-egg binding, and oocyte penetration. Catalase activates the decomposition of H2O2 into water and oxygen, thus removing an initiator of free radical chain reactions leading to lipid peroxidation. Since the oviduct is known to enhance sperm survival, we hypothesized that it might secrete catalase. We found that oviductal fluid, harvested from washed cells collected at the slaughterhouse, possessed catalase-specific activity that varied during the estrous cycle. Catalase activity increased during the cycle and reached its maximal level just before ovulation (Days 18-20). No significant difference in activity was seen between fluid from the isthmus and that from the ampulla. Indirect immuno-staining of spermatozoa incubated in the oviductal fluid revealed the association of catalase in the region of the acrosomal cap. Addition of a commercial antibody directed against bovine liver catalase completely inhibited catalase activities from the oviductal fluid. Catalase activity was also detected in porcine oviductal fluid, human oviductal fluid, and cervical mucus. Western blots of oviductal fluid probed with the anti-catalase antibody revealed two major bands at 60 and 40 kDa. An immunoaffinity column was used to purify oviductal catalase, showing a unique band at about 60 kDa when analyzed by SDS-PAGE. The purified protein was incubated with bovine, boar, and human sperm, and Western blots of these sperm after several washes detected a band at 60 kDa, indicating that the protein was bound to sperm membranes. However, bovine liver catalase did not bind to sperm. Since H2O2 is one of the key reactants in the chain reaction of free radical production, this enzyme may play an important role in sperm survival within the female tract.

Protein C inhibitor may modulate human sperm-oocyte interactions.

Protein C inhibitor (PCI) is a heparin-binding plasma serine protease inhibitor that was originally identified as an inhibitor of activated protein C. PCI has a broad protease specificity, inhibiting several proteases in hemostasis and fibrinolysis by acting as a suicide substrate. Recently it has been reported that proteases of the reproductive system, such as acrosin, prostate-specific antigen, and tissue kallikrein, can also be effectively inhibited by PCI. However, a direct relation between PCI and physiological events during fertilization has not yet been established. An attempt was made to monitor and localize the inhibition of the sperm protease acrosin by PCI. Localization experiments for PCI on epididymal spermatozoa showed that PCI is present on the acrosomal cap of human spermatozoa, which demonstrates the early presence of PCI in the male reproductive tract. Induction of the acrosome reaction in ejaculated human spermatozoa resulted in the disappearance of PCI from the plasma membrane overlying the acrosomal head and the appearance of a strict distribution at the equatorial segment of human spermatozoa. The activity of acrosin in sperm extracts could be effectively inhibited by PCI. Zona-binding assays showed that active PCI is able to block sperm-egg binding in a concentration-dependent manner. The combination of the potent inhibition of acrosin and sperm-egg binding by PCI and the localization studies suggested that PCI may protect spermatozoa against premature acrosome reaction and degradation, thereby modulating the acrosin activity so that it can coincide with binding to the oocyte.

TCP-11, the product of a mouset-complex gene, plays a role in stimulation of capacitation and inhibition of the spontaneous acrosome reaction

Tcp-11 is a candidate for a distorter gene within the t-complex on mouse chromosome 17; although t-complex genes appear to affect sperm function, relatively little is known about mechanisms whereby these genes might play a specific physiological role. We present evidence that the protein TCP-11 is found on the surface of mature epididymal spermatozoa. Although detected on both the acrosomal cap region of the head and the flagellum of acrosome-intact cells, it is absent from the heads of acrosome-reacted cells. When epididymal spermatozoa were incubated in the presence of anti-TCP-11 IgG Fab fragments for a total of 120 min and assessed using chlortetracycline fluorescence, we observed a stimulation of capacitation and an inhibition of spontaneous acrosome loss, suggestive of enhanced fertility compared with untreated suspensions. In vitro fertilization experiments confirmed that Fab-treated suspensions became fertile more quickly and then maintained high fertility. Because these responses were remarkably similar to those obtained using the TRH-related peptide FPP (fertilization promoting peptide; pGlu-Glu-ProNH2) and adenosine, we investigated responses to Fab fragments, FPP, and adenosine. Results indicated that the Fab fragments appear to work at the same extracellular site as FPP, one that is distinct from the adenosine site of action. Further evidence for this conclusion was obtained using pGlu-Gln-ProNH2, an FPP-related tripeptide known to competitively inhibit responses to FPP; as with FPP, pGlu-Glu-ProNH2 inhibited the stimulatory effect of Fab fragments in a concentration-dependent manner. From these results we suggest that TCP-11 may be the receptor for FPP and that the adenylate clyclase/cyclic AMP pathway may be the signal transduction pathway activated by interactions between extracellular effector molecules (e.g., Fab fragments or FPP acting as an agonist) and TCP-11. A mechanism such as this that promotes capacitation but inhibits spontaneous acrosome loss in vivo would play a very important role by helping to maximize the fertilizing potential of the few spermatozoa that reach the site of fertilization. The fact that there is a human homolog of Tcp-11 suggests that this gene could play an important role in regulation of human, as well as mouse, sperm function.

Pentoxifylline induces hyperactivation and acrosome reaction in spermatozoa of golden hamsters: changes in motility kinematics.

Pentoxifylline (PF) is used to enhance motility of spermatozoa from infertile human subjects. We have previously shown that 0.45 mM PF improved capacitation of spermatozoa and fertilization of oocytes in vitro in hamsters. The present study was carried out to assess PF-induced changes in motility kinematics of hamster spermatozoa by a computer-aided sperm analyser (CASA) and determine the timing of onset of hyperactivation (HA) and acrosome reaction (AR) in PF-treated spermatozoa. Motility kinematics were analysed by CASA for 0-8 h in the absence or presence of 0.45 mM PF in Tyrode's medium supplemented with lactate, pyruvate and polyvinyl alcohol (TLP-PVA) or in TLP-PVA with bovine serum albumin (TALP-PVA). Conventional assessment was also made on the percentage of motility and quality of motility of spermatozoa; values were expressed as sperm motility index (SMI). Both in TALP-PVA and TLP-PVA, PF markedly increased SMI, especially the quality of motility (P < 0.02) by 2-3 h which was sustained up to 6 h. The motility kinematic data of PF-treated spermatozoa in TALP-PVA showed that average path velocity, curvilinear velocity and amplitude of lateral head displacement significantly (P < 0.05) increased as early as 2 h, with the expected decrease in straightness (STR) and linearity (LIN). Similar changes were also observed with PF-treated spermatozoa in TLP-PVA. Moreover, the percentage of hyperactivated spermatozoa in PF-treated samples was significantly (P < 0.001) higher than the untreated control at 2 h. To determine whether PF could induce AR, independent of bovine serum albumin, quantitative AR was assessed by observing the presence or absence of acrosomal cap on viable spermatozoa. PF significantly (P < 0.001) increased the percentage of AR as early as 2 h, reaching maximum at 4 h both in TALP-PVA (P < 0.05) and in TLP-PVA (P < 0.001). These results show that, in hamsters, PF induces early onset (by 2 h) of HA and AR and increases the proportion of spermatozoa undergoing physiological maturation.

Cloning and sequence analysis of rat fertilin alpha and beta--developmental expression, processing and immunolocalization.

Fertilin alpha and beta are members of the MDC (metalloproteinase-like, disintegrin-like, cysteine-rich) protein family and are expressed on the sperm surface where they have been proposed to play a role in mammalian fertilization. Inhibition of sperm-oocyte binding and sperm-oocyte fusion make fertilin an attractive target for the development of an immunocontraceptive vaccine. Full-length cDNAs encoding alpha and beta fertilin subunits were isolated from a rat testis cDNA library and sequenced. Using reverse transcription-polymerase chain reaction (RT-PCR), the developmental expression of fertilin alpha and beta was determined in pre-pubertal and mature rat testes. Fertilin alpha mRNA was present at all stages of development, suggesting that it is not exclusively expressed in post-meiotic germ cells. In contrast, fertilin beta mRNA was first identified in day 19 testes, coincident with the presence of pachytene spermatocytes. Polyclonal antisera raised against a 28-residue peptide (corresponding to part of the disintegrin domain) and two recombinant fusion proteins identified a 90 kDa protein in testicular sperm extracts and a 60 kDa protein in caput and cauda epididymidal sperm extracts, the predicted sizes for rat fertilin beta precursor and mature protein respectively. Indirect immunofluorescence using the anti-peptide antisera stained the acrosomal cap of permeabilized testicular, caput and caudal spermatozoa and elongating spermatids in testicular sections.

Sperm-egg interaction in the painted frog (Discoglossus pictus): an ultrastructural study.

The ultrastructure of sperm changes and penetration in the egg was studied in the anuran Discoglossus pictus, whose sperm have an acrosome cap with a typical tip, the apical rod. The first stage of the sperm apical rod and acrosome reaction (AR) consists in vesiculation between the plasma membrane and the outer acrosome membrane. The two components of the acrosome cap are released in sequence. The innermost component (component B) is dispersed first. The next acrosome change is the dispersal of the outermost acrosome content (component A). At 30 sec postinsemination, when the loss of component B is first observed, holes are seen in the innermost jelly coat (J1), surrounding the penetrating sperm. Therefore, this acrosome constituent might be related to penetration through the innermost egg investments. At 1 min postinsemination, during sperm penetration into the egg, a halo of finely granular material is observed around the inner acrosome membrane of the spermatozoon, suggesting a role for component A at this stage of penetration. Gamete-binding and fusion take place between D1 (the egg-specific site for sperm interaction) and the perpendicularly oriented sperm. Spermatozoa visualized at their initial interaction (15 sec postinsemination) with the oolemma are undergoing vesiculation. The first interaction is likely to occur between the D1 glycocalyx and the plasma membrane of the hybrid vesicles surrounding the apical rod. As fusion is observed between the internal acrosome membrane and the oolemma, it can be postulated that gametic interaction might be followed by fusion of the latter with the apical rod internal membrane that extends posteriorly into the inner acrosome membrane. Insemination of the outermost jelly layer (J3) dissected out of the egg, and observations of the ultrastructural changes of spermatozoa in this coat, indicate that J3 rather than the vitelline coat (VC) induces the AR. Interestingly, at the late postinsemination stage, VC fibrils are seen crosslinking the inner acrosome membrane. The role of this binding is here discussed.

Sperm‐egg interaction in the painted frog (Discoglossus pictus): An ultrastructural study

The ultrastructure of sperm changes and penetration in the egg was studied in the anuran Discoglossus pictus, whose sperm have an acrosome cap with a typical tip, the apical rod. The first stage of the sperm apical rod and acrosome reaction (AR) consists in vesiculation between the plasma membrane and the outer acrosome membrane. The two components of the acrosome cap are released in sequence. The innermost component (component B) is dispersed first. The next acrosome change is the dispersal of the outermost acrosome content (component A). At 30 sec postinsemination, when the loss of component B is first observed, holes are seen in the innermost jelly coat (J1), surrounding the penetrating sperm. Therefore, this acrosome constituent might be related to penetration through the innermost egg investments. At 1 min postinsemination, during sperm penetration into the egg, a halo of finely granular material is observed around the inner acrosome membrane of the spermatozoon, suggesting a role for component A at this stage of penetration. Gamete-binding and fusion take place between D1 (the egg-specific site for sperm interaction) and the perpendicularly oriented sperm. Spermatozoa visualized at their initial interaction (15 sec postinsemination) with the oolemma are undergoing vesiculation. The first interaction is likely to occur between the D1 glycocalyx and the plasma membrane of the hybrid vesicles surrounding the apical rod. As fusion is observed between the internal acrosome membrane and the oolemma, it can be postulated that gametic interaction might be followed by fusion of the latter with the apical rod internal membrane that extends posteriorly into the inner acrosome membrane. Insemination of the outermost jelly layer (J3) dissected out of the egg, and observations of the ultrastructural changes of spermatozoa in this coat, indicate that J3 rather than the vitelline coat (VC) induces the AR. Interestingly, at the late postinsemination stage, VC fibrils are seen crosslinking the inner acrosome membrane. The role of this binding is here discussed. Mol. Reprod. Dev. 47:323–333, 1997. © 1997 Wiley-Liss, Inc.

Interaction of acrosome-reacted macaque sperm with the macaque zona pellucida.

In this study we investigated the ultrastructure of macaque sperm induced to acrosome-react with calcium ionophore A23187, and the interaction between these acrosome-reacted sperm and the macaque zona pellucida. Transmission electron microscopy revealed that the majority of ionophore-treated sperm retained the vesiculated acrosomal cap or "shroud." Untreated, acrosome-reacted sperm on the zona had a similar ultrastructural appearance. In sperm-zona binding experiments, a mean of 4.5 ionophore-treated sperm were bound per zona after 1 min of coincubation compared with 41 sperm per zona in the solvent control. Vigorous pipetting was used to remove the acrosomal shrouds from approximately 50% of acrosome-reacted sperm before incubation with oocytes. Significantly more of these mechanically treated sperm were bound to the zona after a 4-min coincubation compared with acrosome-reacted sperm that were not pipetted. The number of mechanically treated sperm bound to the zona was the same whether the sperm and oocytes were coincubated in calcium-free medium or in control medium. The percentage of mechanically treated sperm that were acrosome-reacted on the zona also was not different in the two media. We conclude that macaque sperm that undergo the acrosome reaction on the zona surface are bound by the acrosomal shroud before zona penetration. When sperm acrosome-react before interaction with the oocyte, their zona binding capacity is significantly reduced. Removal of the acrosomal shroud and exposure of the inner acrosomal membrane increases the affinity of sperm for the zona. This sequence occurs naturally during the transition from primary binding to secondary binding on the zona surface.

Evolution of the Spermatozoon in Australasian Rodents

The head of the spermatozoon in eutherian mammals contains a nucleus, acrosomal cap and cytoskeleton. It is generally spatulate, paddle-shaped or pear-shaped, but in most murid rodents it is hook-shaped with the anterior region of the nucleus surrounded by an elaborate acrosome and an extension of the subacrosomal cytoskeleton as a perforatorium. This type of spermatozoon is present in Australasian Rattus, together with several other New Guinean genera. However, in most Australasian hydromyine rodents a far greater complexity of structural organisation of the sperm head has evolved in which two further elaborate processes extend from its upper concave surface. These processes contain a huge extension of the cytoskeleton within which filamentous actin is present. By contrast, the form of the sperm head in a few species of Pseudomys, Notomys and Solomys is highly divergent and is either truncated, spatulate or pear- shaped. The evolutionary trends of change in sperm head shape are discussed and it is suggested that the falciform sperm head with the two extra processes in most of the hydromyine rodents is one of the most morphologically complex sperm head types to have evolved in eutherian mammals; it contains a far more extensive development of the cytoskeleton than that of any other mammalian spermatozoon.

Identification of hydrolase binding activities of the acrosomal matrix of hamster spermatozoa.

The interior of the mammalian sperm acrosome contains a structural framework, the acrosomal matrix, that may regulate both the distribution of hydrolases within the acrosome and their release during the acrosome reaction. To define the biochemical basis of this interaction, we examined the binding of two acrosomal hydrolase, proacrosin and N-acetylglucosaminidase (NAGA), to a purified acrosomal matrix fraction of hamster spermatozoa. Proacrosin-acrosin was chromatographically purified from acid extracts of hamster spermatozoa and consisted of four size variants of 50 kDa, 49 kDa, 45 kDa, and 43 kDa. Each of the four isoforms exhibited the same N-terminal amino acid sequence through 16 residues, suggesting that they may be modified by cleavage at the C-terminus. Polyclonal antiserum against the proacrosin isoforms specifically binds the acrosomal cap as shown by immunofluorescence microscopy. Neither proacrosin nor NAGA were solubilized when sperm were permeabilized with Triton X-100 under low ionic strength conditions; however, both hydrolases were releases by extraction with Triton X-100 containing 0.5 M NaCl. An acrosomal matrix fraction isolated under low ionic strength conditions retained bound proacrosin-acrosin and NAGA, and both hydrolases were released from the matrix by subsequent high-salt extraction. After high-salt treatment, the acrosomal matrix retained specific binding sites for both proacrosin and NAGA. In a blot overlay assay, a set of acrosomal matrix polypeptides between 29 kDa and 24 kDa specifically bound proacrosin. These data suggest that specific interactions between acrosomal matrix polypeptides and hydrolases represent a mechanism to sequester hydrolases within the acrosome and to regulate their release during the acrosome reaction.

Localization of seminal plasma transforming growth factor-β1 on human spermatozoa: an immunocytochemical study

To localize immunocytochemically transforming growth factor beta1 (TGF-beta1) in human spermatozoa. Incubation of spermatozoa with anti-TGF-beta1 antiserum at various pH. Human volunteers in an academic research institute. Young healthy fertile men. Semen specimens were collected. At neutral pH and at physiological pH of seminal plasma, TGF-beta1 immunostaining was detected predominantly at the postacrosomal region of the head, at the neck, and at the middle piece of the tail. Transforming growth factor-beta1 also was found occasionally at the axial filament complex of the connecting piece and the ring. The acrosomal cap section of the head, the principal piece, or the end piece of the tail were immunocytochemically negative for TGF-beta1. The TGF-beta1 immunostaining pattern at acidic pH was similar to that at neutral pH and at physiological pH of seminal plasma, but a greater intensity of immunostaining was found at acidic pH than that at neutral pH. These results suggest that an in vivo activation of seminal plasma latent TGF-beta1 may take place in the acidic environment of vagina, which results in a greater amount of activated TGF-beta1 and, in turn, with an enhanced "coating" of TGF-beta1 to spermatozoa.

Surface localization of P34H an epididymal protein, during maturation, capacitation, and acrosome reaction of human spermatozoa.

During epididymal transit, spermatozoa acquire new surface antigens that are involved in the acquisition of their fertilizing ability. We have previously described a 34-kDa (P34H) human epididymal sperm protein that shows antigenic and functional homologies with the hamster P26h. P34H is localized on the acrosomal cap of human spermatozoa and has been proposed to be involved in the interaction with the zona pellucida. The aim of this study was to document the expression of P34H on the sperm surface during transit along the male and female genital tracts. Immunohistochemical techniques were performed on human testes and epididymides by means of an antiserum specific for P34H. No labelling was detected on those spermatozoa found within the seminiferous tubules or in the vasa efferentia. P34H first appeared in the caput epididymidis and was restricted to the acrosomal cap. Signal intensity then increased considerably from the proximal corpus to the cauda region of the epididymis. After ejaculation, the same pattern of P34H distribution was observed, but the intensity was much lower than that characterizing the cauda epididymal spermatozoa. Strong labeling was restored after incubation in B2 medium and was maximal after 5 h of capacitation. After acrosomal exocytosis induced by a Ca2+ ionophore, the percentage of P34H-labeled spermatozoa decreased proportionally to the number of acrosome-reacted spermatozoa as determined by Pisum sativum-fluorescein isothiocyanate (FITC) labeling. P34H appeared to be strongly anchored to the sperm plasma membrane during epididymal transit as indicated by the requirement for detergent to extract this surface antigen from ejaculated spermatozoa. This confirms the importance of P34H binding to the sperm plasma membrane during epididymal maturation. We have previously proposed that P34H is involved in sperm-zone pellucida interaction. The appearance and accumulation of P34H on the sperm plasma membrane during epididymal maturation, followed by its inaccessibility associated with ejaculation, its unmasking during capacitation, and finally its elimination after the acrosome reaction, are in agreement with te proposed function of this sperm antigen.

Purification of P26h: a hamster sperm protein.

P26h is a 26 kDa glycoprotein, located on the acrosome cap of hamster spermatozoa, involved in the species specificity of gamete interaction. We have purified this protein from hamster spermatozoa collected from the distal cauda region of the epididymis. Its purification was realized following a three-step procedure: detergent extraction, ion-exchange chromatography, and chromatofocusing. Protein partitioning using Triton X-114 (the first step) showed a ratio of 5:1 between the resulting aqueous and detergent phase. P26h was found almost exclusively in the aqueous phase where it represented about 10-12% of the total protein content. When the desalted aqueous phase was loaded on a carboxymethyl column for cation-exchange chromatography, about 80% of the proteins did not bind to the matrix and were eliminated. P26h was eluted from the column with a linear gradient of salt. At this point, P26h had a rate of purification estimated at 45-55%; three other major proteins of <21, 45, and 63 kDa remained in the sample. These undesired proteins were eliminated by submitting these samples to chromatofocusing using a PBE 94 polybuffer exchanger column. Indeed, P26h was collected almost in the dead volume of the column while the other proteins were eluted much later. Two-dimensional gel electrophoresis and Western blotting were performed to determine the purity of P26h. Only one major spot was detected, confirming the purity of P26h. Usefulness of this purified sperm antigen in the understanding of the physiology of mammalian fertilization is discussed.

Localization by indirect immunofluorescence of nitric oxide synthase in mouse and human spermatozoa.

The localization of nitric oxide synthase was studied in mouse epididymal spermatozoa and freshly ejaculated human sperm. A rabbit antiserum against the neuronal isoform of the enzyme was used, and antibody binding was detected with a fluorescein isothiocyanate-conjugated polyclonal antibody specific for rabbit IgG. In mouse spermatozoa, the percentage of cells staining specifically ranged from 88% to 98%. Samples were examined after 0-, 90- and 150-min incubations in vitro. Three different patterns of staining were observed: (a) Pattern I, intense fluorescent staining localized in the acrosome and in a segment of the tail; (b) Pattern II, fluorescent staining localized only in the tail; and (c) Pattern III, faint fluorescent staining localized in the acrosomal cap and in the tail. The potential physiological significance of these patterns is discussed. Nitric oxide synthase was also localized in the acrosome of freshly ejaculated human sperm.

Boar spermadhesins AQN-1 and AQN-3: oligosaccharide and zona pellucida binding characteristics.

AQN-1 and AQN-3 form part of the complement of surface-associated lectins which coat the plasma membrane overlying the acrosomal cap of in vitro capacitated boar spermatozoa. They belong to the spermadhesin protein family and have binding affinity for glycoconjugates of the zona pellucida, the extracellular investment surrounding mammalian eggs. The oligosaccharide and zona pellucida binding characteristics of spermadhesins AQN-1 and AQN-3 were investigated using a solid-phase assay and biotinylated glycoprotein ligands. Both sperm proteins bind glycoproteins containing Gal beta (1-4)-GlcNAc and Gal beta-(1-3)-GalNAc oligosaccharide sequences with dissociation constants (Kd) of 0.08 to 0.8 microM, and to zona pellucida glycoproteins with Kd = 0.15-0.25 microM. However, 5-N-acetyl neuraminic acid alpha (2-3/6)-linked to the galactose residue decreases the affinity of glycosylated ligands to AQN-1 three-fold, although it did not affect oligosaccharide binding to AQN-3. In addition, AQN-3 binds preferentially to glycoproteins with either a linear or tri- and tetraantennary carbohydrates than to those containing diantennary N-acetyllactosamine structures. The similar but distinct oligosaccharide recognition capabilities of spermadhesins AQN-1 and AQN-3 (this work) and AWN-1 (Dostálová, Z, Calvete, J.J., Sanz, L., and Töpfer-Petersen, E. (1995) Eur. J. Biochem. 230, 329-336) suggest that, in the pig, sperm-zona pellucida binding might be mediated by lectins displaying similar although distinct carbohydrate-recognition abilities.