Proacrosin Activation Research Articles

Proacrosin activation mechanisms in capacitated and frozen-thawed boar spermatozoa.

The main objective of the current study was to explore the different activation mechanisms of capacitation and freeze-thawed spermatozoa. Using SDS-PAGE and Western blotting, the conversion process of boar proacrosin during freeze-thawing and capacitation of spermatozoa was analyzed. The results revealed that capacitated spermatozoa exhibited a greater fluorescence area than that of the freeze-thawed spermatozoa, which were smaller than those of the fresh group. Fresh spermatozoa displayed 45- and 35- kDa protein bands, while those of freeze-thawed andcapacitated spermatozoa displayed 45-, 35- and 28-kDa bands. In summary, these data indicate that proacrosin is activated, thus becoming α- and β-acrosins and a 28-kDa protein during capacitation and freeze-thawing.

Activation of proacrosin accompanies upregulation of sp32 protein tyrosine phosphorylation in pig sperm

This study investigated the relationship between acrosin activation and pig sperm proacrosin binding protein (sp32) phosphorylation levels. Differently processed pig spermatozoa (fresh semen sperm, capacitation sperm, acrosome reaction sperm, capacitation-like sperm, and thawed sperm) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. The fresh semen and capacitation sperm groups both produced proacrosin protein bands of 55 kDa; however, the result of the fresh semen sperm group was clearer than that of the capacitation sperm group. The thawed sperm group showed a shallow strip at 55 kDa. The capacitation and acrosome reaction sperm groups produced obvious proacrosin protein bands at 35 kDa, and the strips of the capacitation sperm group were again clearer. A faint band was visible at 32 kDa in the acrosome reaction sperm group. The capacitation, thawed, and acrosome reaction sperm groups showed significant strips in sp32, and the bands of the acrosome reaction sperm group were shallower than those of the 2 other groups. The capacitation and thawed sperm groups produced significant strips at 40 kDa, and the capacitation sperm group produced an additional strip at 55 kDa. In conclusion, sp32 phosphorylation levels can promote proacrosin activation into the active acrosin.

Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo)

This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After freezing-thawing, additional bands appeared in sperm extract and seminal plasma. These bands were of different molecular weight regarding the presence or absence of NPGB. These data suggest that the mechanism of damage to the proacrosin/acrosin system is different for liquid storage and cryopreservation. Liquid storage seems to increase in the susceptibility of the proacrosin/acrosin system to be activated during extraction. Kazal inhibitors of turkey seminal plasma are involved in the control of proacrosin activation. The disturbances of the proacrosin/acrosin system of turkey spermatozoa can be related to a disturbance in the induction of the acrosome reaction. Our results may be important for a better understanding of the proacrosin/acrosin system of turkey spermatozoa and disturbance to this system during liquid storage and cryopreservation.

Anti-human proacrosin antibody inhibits the zona pellucida (ZP)–induced acrosome reaction of ZP-bound spermatozoa

The anti-acrosin monoclonal antibody AcrC5F10 inhibited proacrosin activation, proacrosin-human zona pellucida glycoprotein A (ZPA) binding, and the zona pellucida (ZP)-induced acrosome reaction of the ZP-bound spermatozoa but had no significant effect on sperm-ZP binding. These results suggest that proacrosin-acrosin may play an important role in the ZP-induced acrosome reaction of spermatozoa after primary binding to the ZP.

Cell biology of acrosomal proteins: Zellbiologie akrosomaler Proteine

Acrosin is a multifunctional enzyme combining several functional properties within a single molecule: the catalytic triad of the proteinase, hydrophobic domains responsible for the special membrane-associating character of the enzyme and the carbohydrate binding sites by which the molecule can bind to the zona pellucida. Acrosin occurs in the sperm acrosome as an inactive precursor, proacrosin, with a molecular mass of 53-55 kDa. Proacrosin is activated by a single proteolytic clip between Arg23 and Val24 generating the high molecular mass acrosin. The activation of proacrosin to the biologically active enzyme which occurs concomitantly with the acrosome reaction appears to be regulated on and by the zona pellucida. It is hypothesized that alternating cycles of binding to the zona, digestion of the zona and release from the zona together with the forward motility of the spermatozoon would be required to achieve penetration.

Bovine epididymal sperm proacrosin-acrosin system: quantification and partial characterization

Several studies suggest that acrosin, an acrosomal trypsin-like serine proteinase, plays a role in fertilization. The enzyme is present in an enzymatically inactive precursor form, called proacrosin and is believed to be converted to the enzymatically active form(s) through one/multiple physiological event(s) prior to the sperm penetration of the zona pellucida. Although, the proacrosin-acrosin system of several species has been well documented, the study of the enzyme system in bovine caput and cauda epididymis (where the maturation of spermatozoa occurs) has not been characterized. The present study demonstrates the quantification and partial characterization of the proacrosin-acrosin proteinase system in unpurified acrosomal extracts of bovine caput and cauda epididymal sperm. Proacrosin activation followed the sigmoidal type of activation curve. Activation experiments demonstrate that almost 80-90% of this protein exists in zymogen (proacrosin) form either in ejaculated or caput and cauda epididymal spermatozoa. Time-course activation studies showed that the zymogen in isolated spermatozoa was completely converted to active non-zymogen form in 3 and 5 h after removal from the cauda and caput regions, respectively, at pH 8.0 at 25 degrees C. This conversion was markedly inhibited by calcium in a dose dependent manner and the inhibition was reversible. On the other hand, calcium has a stimulatory effect on the hydrolytic activity of acrosin. These studies reveal that the proacrosin-acrosin system can be identified in crude extracts of bull epididymal and ejaculated sperm.

Inhibition of the vacuolar H(+)-pump with bafilomycin A1 does not induce acrosome reaction or activate proacrosin in mouse spermatozoa

Acrosomal protease activation is regarded as an important event triggered by acrosomal reaction and leading to sperm passage through zona pellucida. Mammalian acrosome has an internal acid pH that probably helps to maintain inactive proenzymes that otherwise could be precociously activated and prevent normal fertilization. In this work, we have studied the effect of bafilomycin A1, a potent and specific inhibitor of vacuolar H(+)-pump (V-ATPase), on acrosome reaction and proacrosin activation. We used the pH-sensitive probe Lysotracker Green DND-26 to monitor qualitatively intra-acrosomal pH in cauda epididymal mouse spermatozoa. Our results showed that loss of Lysotracker label induced by bafilomycin A1 (acrosome alkalinization) did not induce acrosome reaction or proacrosin activation. We also developed a new technique for imaging the acrosome, and for evaluating the acrosome reaction, in live mouse spermatozoa using Lysotracker DND-26. These results showed that the V-ATPase is a key regulator of mammalian acrosome pH, and that acrosome alkalinization is not the only prerequisite to activate proacrosin under in vivo conditions. Our results suggest that acrosome alkalinization and acrosome reaction are two processes that could be independently regulated during mammalian sperm capacitation.

Biochemical and molecular studies of the proacrosin/acrosin system in patients with unexplained infertility

Biochemical and molecular studies of the proacrosin/acrosin system in patients with unexplained infertility

Evaluation of the proacrosin/acrosin system and its mechanism of activation in human sperm extracts

Acrosin is an acrosomal protease synthesized as a proenzyme and activated into β-acrosin during the acrosome reaction. In the present study, a set of sensitive assays was developed to identify the proacrosin/acrosin system and to evaluate its activation pattern in human sperm extracts. Immunocytochemical analysis with monoclonal antibody (Mab) AcrC5F10 showed specific staining on the acrosome of permeabilized ejaculated and capacitated spermatozoa. Acrosome reaction was associated with a decrease in staining. AcrC5F10 specifically recognized a 55-kDa band (proacrosin) in Western immunoblots. Activation studies showed enzymatically active intermediates of 39 and 35 kDa after zymography. Immunoreactive bands of 52, 43, 34, 21–26 and 16 kDa were identified in the activation patterns developed with AcrC5F10. Activation was completely inhibited in the presence of 9 mM CaCl 2 or 100 mM benzamidine. A multiple sequence alignment revealed partial conservation of putative cleavage sites in the proacrosin sequence. The tests described allow the detection of human proacrosin in spermatozoa and sperm protein extracts, as well as the evaluation of the proenzyme activation pattern. They can be used to study the effect of inhibitors upon proenzyme activation. In addition, alterations in proacrosin activation in semen samples with abnormal acrosin enzymatic activity can be analyzed using these assays.

Acrosin activity in turkey spermatozoa: assay by clinical method and effect of zinc and benzamidine on the activity

We optimized a clinical assay developed for measuring total acrosin activity for mammalian and fish semen for use in turkey spermatozoa. The main modifications included dilution of semen to a final concentration of 25 to 1000 × 10 3 spermatozoa, an increase of Triton X-100 concentration to 0.05% and 1 hr preincubation without substrate. Acrosin activity in turkey spermatozoa was much higher than in human spermatozoa (about 100-times) but similar to that of boar sperm. To optimize this assay for turkey spermatozoa, it was necessary to use higher Triton X-100 concentrations in the reaction mixture. There was a better catalytic efficiency at higher temperatures and a special requirement for a preincubation period for proacrosin activation. We observed high inhibition of acrosin activity by zinc added during preincubation (90% at 0.01 mM of zinc chloride). Benzamidine also inhibited turkey acrosin, and the extent of inhibition was similar for the incubation or preincubation period. When zinc ions were added during incubation, this inhibition was lower (24%). The results suggest that zinc influences proacrosin activation of turkey spermatozoa. This influence may be important for successful long-term storage of spermatozoa in the hen's oviduct.

CDNA Cloning and Functional Analysis of Ascidian Sperm Proacrosin

cDNA cloning and functional analysis of proacrosin from the ascidian Halocynthia roretzi were undertaken. The isolated cDNA of the ascidian preproacrosin consists of 2367 nucleotides, and an open reading frame encodes 505 amino acids, which corresponds to the molecular mass of 55,003 Da. The mRNA of proacrosin was found to be specifically expressed in the gonad by Northern blotting and in the spermatocytes or spermatids by in situ hybridization. The amino acid sequences around His(76), Asp(132), and Ser(227), which make up a catalytic triad, showed high homology to those of the trypsin family. Ascidian acrosin has paired basic residues (Lys(56)-His(57)) in the N-terminal region, which is one of the most characteristic features of mammalian acrosin. This region seems to play a key role in the binding of (pro)acrosin to the vitelline coat, because the peptide containing the paired basic residues, but not the peptide substituted with Ala, was capable of binding to the vitelline coat. Unlike mammalian proacrosin, ascidian proacrosin contains two CUB domains in the C-terminal region, in which CUB domain 1 seems to be involved in its binding to the vitelline coat. Four components of the vitelline coat that are capable of binding to CUB domain 1 in proacrosin were identified. In response to sperm activation, acrosin was released from sperm into the surrounding seawater, suggesting that ascidian acrosin plays a key role in sperm penetration through the coat. These results indicate that ascidian sperm contains a mammalian acrosin homologue, a multi-functional protein working in fertilization.

A basic 18-amino acid peptide contains the polysulfate-binding domain responsible for activation of the boar proacrosin/acrosin system.

Proacrosin is the zymogen of acrosin, a serine protease localized in the acrosomal matrix of mammalian sperm. Proacrosin/acrosin binds to solubilized zona pellucida glycoproteins (ZPGs) and various polysulfates in a non-enzymatic mechanism. In addition, both polysulfates and ZPGs induce proacrosin activation once they bind to the polysulfate-binding domain (PSBD) of the enzyme. We show here that the peptide (43)IFMYHNNRRYHTCGGILL(60) inhibited the proacrosin activation induced by either fucoidan or ZPGs. In addition, the peptide was recognized by the monoclonal antibody C5F10, which is directed against the PSBD region. Our data suggest that the PSBD is composed of many "subsites" that may or may not interact with each other.

Functional interactions between sulphated polysaccharides and proacrosin: implications in sperm binding and digestion of zona pellucida.

Acrosin is a serine protease located within mammalian acrosome as inactive proacrosin. Sulphated polymers bind to proacrosin and acrosin, to a domain different from the active site. Upon binding, these polymers induce proacrosin activation and some of them, such as fucoidan, inhibit sperm binding to the zona pellucida. In this work we have studied the interaction of solubilised zona pellucida glycoproteins (ZPGs), heparin and ARIS (Acrosome Reaction Inducing Substance of Starfish) with boar and human acrosin. We have found that ARIS, solubilised ZPGs and fucoidan, but not heparin, inhibit the binding of the monoclonal antibody against human acrosin C5F10 to boar or human proacrosin. These results suggest that fucoidan, solubilised ZPGs and ARIS bind to a related domain on the proacrosin surface. Moreover, ARIS was able to induce human proacrosin activation. On the other hand, neither ARIS nor heparin from porcine intestinal mucosa or bovine lung induced hamster sperm acrosome reaction or sperm motility. Recent data showed that acrosin is involved in dispersal of the acrosomal matrix after acrosome reaction. Thus, the control of the ZPG glycan chains over proacrosin activation may regulate both sperm penetration rate and limited proteolysis of zona pellucida proteins.

The polysulphate binding domain of human proacrosin/acrosin is involved in both the enzyme activation and spermatozoa-zona pellucida interaction.

Mammalian acrosin is a protease present as a zymogen in the acrosome of a non-reacted mammalian sperm, and in vitro is able to carry out limited hydrolysis of homologous and heterologous zonae pellucidae. On the other hand, sulphated polymers and zona pellucida glycoproteins bind to acrosin on a domain different from the active site, named the polysulphate binding domain (PSBD). Thus it is believed that acrosome-reacted spermatozoa bind to glycan chains of the zona pellucida through PSBD participating as secondary binding receptor. The aim of the present work was to study the role of PSBD during both human gamete interaction and acrosin activation. In this work we present evidence that the anti-human acrosin monoclonal antibody C5F10 is directed to an epitope located on or near the PSBD on human proacrosin/acrosin. Moreover, we show that this antibody is able to inhibit both proacrosin activation induced by fucoidan and the sperm binding to the zona pellucida. Our results suggest that the same PSBD is involved in both sperm secondary binding, during zona pellucida penetration, and proacrosin activation.

Relationship between molecular conversions of acrosin and the progression of exocytosis in the calcium ionophore-induced acrosome reaction

In this study we used a previously characterized monoclonal antibody to analyze the molecular conversions of acrosin during the acrosomal exocytosis induced by ionophore A23187. Before sperm exposure to the ionophore, most of the sperm acrosin was in the form of proacrosin (55-kDa and 53-kDa forms). Upon exposure to the ionophore, the concentration of proacrosin in sperm samples decreased rapidly and was negatively correlated with the progression of exocytosis. After 1 h of ionophore treatment, proacrosin was quantitatively converted into the two active acrosin forms, α-acrosin (49 kDa) and β-acrosin (36 kDa). However, products of further acrosin conversions were not found after this treatment. As compared with the speed of acrosin activation during sperm contact with the ionophore, the ionophore-induced release of acrosin from the sperm cells into the soluble fraction was apparently delayed, and only the active acrosin forms (49 kDa and 36 kDa) were found in sperm incubation media. External Ca 2+ influenced the speed of proacrosin conversion in a concentration-dependent manner. The ionophore-induced activation of proacrosin and acrosome reaction were partially inhibited by trypsin inhibitors. The results suggest that proacrosin activation is an essential step in the mechanism of the acrosomal exocytosis.

Activation and subsequent degradation of proacrosin is mediated by zona pellucida glycoproteins, negatively charged polysaccharides, and DNA

Boar proacrosin (E.C. 3.4.21.10, Mw 53 kD) was isolated by a modified method and subjected to autoactivation. Previously described molecular intermediates of 49 and 43 kD and a stable form (beta-acrosin, 35 kD) were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoactivation was expedited in the presence of either zona pellucida glycoproteins, fucoidan, or DNA. The end point of this accelerated conversion was the complete degradation of otherwise stable beta-acrosin via the formation of a characteristic active intermediate protein of 30 kD. All intermediate molecular forms observed during proacrosin activation/conversion exhibited the N-terminal sequence of the boar acrosin heavy chain, indicating a C-terminal processing mechanism. Hence zona pellucida glycoproteins stimulate proacrosin activation as well as acrosin degradation. Such a mechanism of proenzyme activation and degradation is to our knowledge described here for the first time and points to a previously unrecognized role of zona pellucida during gamete interaction.

Determination of active, non-zymogen acrosin, proacrosin and total acrosin in different andrological patients

A spectrophotometric assay is described which, due to improved extraction conditions, allows quantitative determination of enzymatically active, non-zymogen acrosin, proacrosin and total acrosin activity from human sperm acrosomes. Acrosomal proteinase activity is assessed by acid extraction of the sperm pellet and the suspension medium before and after snap-freezing, followed by zymogen autoactivation. Release of acrosin from the acrosome can be used as a sensitive biochemical marker to characterize acrosomal membrane stability, severe disturbance of which may be the cause of impaired male fertility. Acrosin activities in different populations of semen specimens are reported and compared to data available in the literature. Different degrees of acrosomal membrane alterations are observed in men with oligozoospermia, tetratozoospermia and polyzoospermia. Particularly in oligozoospermia, a significant increase of active, non-zymogen acrosin points to severe acrosomal membrane alterations and, in addition, to a premature activation of proacrosin, which may impair fertilization in certain individuals. Finally, acrosin activity is shown to be significantly influenced by the time of sexual abstinence. It is concluded that determination of acrosin may be a useful indicator of the fertility potential in men.

In vitro interactions of calmodulin with the ovine proacrosin-acrosin system.

The authors studied the interaction of calmodulin (CaM) with proacrosin and acrosin from ram spermatozoa. CaM binding evaluated by the [125I]-CaM overlay procedure was shown to occur preferentially with both proacrosin and acrosin in the presence of EGTA; in the presence of Ca2+, the interaction was less intense. Further studies with native proenzyme preparations showed that proacrosin activation at pH 7.1 or 8.0 was significantly accelerated in the presence of CaM and EGTA (t1/2 = 23 min vs. 55 min for EGTA alone at pH 7.1), but not in the presence of Ca2+ (t1/2 = 73 min). The enzymatic activity of acrosin towards benzoyl arginine paranitroanilide, however, was not significantly affected by CaM whether Ca2+ was absent or present. Finally, the authors demonstrated that acrosin hydrolyzed CaM rapidly and extensively in the presence of EGTA. These results indicate that CaM interacts in vitro with proacrosin and acrosin, and that acrosin can attenuate CaM activity through proteolysis. Whether these interactions also occur in vivo and are involved in some aspects of spermatozoa function remains to be determined.

Proacrosin activation and acrosin release during the guinea pig acrosome reaction

The kinetics of proacrosin activation and release from guinea pig spermatozoa during the nonsynchronous acrosome reaction were studied. Epididymal spermatozoa were incubated at 37 degrees C in a defined medium (pH 7.8) containing 1.7 mM Ca2+. After 195 min, 78% of the motile spermatozoa had undergone the acrosome reaction as determined by light microscopy. Acrosin and proacrosin levels in the spermatozoa and medium were measured at the beginning of the incubation period. Most of the total acrosin activity (78%) was associated with the spermatozoa, of which greater than 90% was in the form of proacrosin. Proacrosin represented a small, stable fraction (23%) of the total acrosin in the medium; it did not activate to acrosin while in the medium. After 195 min, a decrease in sperm-associated total acrosin (42%; p less than 0.05) was accompanied by an increase in the total acrosin level in the medium (115%; P less than 0.05). No change in the relative proacrosin content (percent of total acrosin) was evident in either medium or spermatozoa. Additional experiments quantified acrosin and proacrosin during the progression of the acrosome reaction. Both the loss of sperm-associated total acrosin and the increase in total acrosin levels in the medium were highly correlated with the fraction of acrosome-reacted spermatozoa (r = 0.954 and 0.922, respectively; P less than 0.001). However, the rate of acrosin appearance in the medium was only 60% (P less than 0.001) of the rate of acrosin loss from the spermatozoa. The fractional proacrosin content of spermatozoa (94%) and medium (31%) remained unchanged during the acrosome reaction (r = 0.15 and 0.30, respectively; P greater than 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the human sperm acrosome reaction by proteinase inhibitors

The analogue of the second messenger cAMP, dibutyryl cAMP (dbcAMP), was shown to induce the human sperm acrosome reaction to the same extent as calcium ionophore A23187, providing preliminary evidence for the involvement of the adenylate cyclase system in the acrosome reaction (AR) of human spermatozoa. Using the human synchronous acrosome reaction system, proteinase inhibitors were tested for their effect on the dbcAMP-induced human sperm acrosome reaction. The proteinase inhibitor 4'-acetamidophenyl 4-guanidinobenzoate (AGB), an inhibitor of proacrosin activation and of acrosin, when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, significantly (P less than 0.01) inhibited the acrosome reaction at final concentrations of 1 x 10(-4) M to 1 x 10(-6) M in comparison to dbcAMP treatment alone. At concentrations less than 1 x 10(-6) M, no significant inhibitory effect was seen. Similarly, para-aminobenzamidine (pAB), also an inhibitor of proacrosin activation and of acrosin, significantly (P less than 0.01) inhibited the dbcAMP-induced acrosome reaction at final concentrations of 1 x 10(-4) M to 1 x 10(-6) M when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, in comparison to stimulation by dbcAMP alone. However, at concentrations less than 1 x 10(-6) M, no significant (P greater than 0.05) inhibitory effect was seen. These results indicate that a serine proteinase, most likely acrosin, has a role in the human sperm acrosome reaction and suggest that the enzyme functions after the involvement of the adenylate cyclase system.