Maturation of guinea pig sperm in the epididymis involves the modification of proacrosin oligosaccharide side chains

Proacrosin from guinea pig cauda epididymal sperm has a lower molecular weight compared with the testicular zymogen. In this study, we have examined the structural basis of this change and where the conversion in proacrosin molecular weight occurs during sperm maturation. Immunoblotting of trifluoromethanesulfonic acid-deglycosylated testicular and cauda epididymal sperm extracts with antibody to guinea pig testicular proacrosin demonstrated that the polypeptide backbones of proacrosins from the testis and cauda epididymal sperm had the same molecular weights (approximately 44,000). Keratanase, an endo-beta-galactosidase specific for lactosaminoglycans, partially digested testicular proacrosin but had no effect on proacrosin from cauda epididymal sperm. In extracts of testis, caput epididymis, and corpus epididymis analyzed by immunoblotting, anti-proacrosin recognized a major antigen with an apparent molecular weight (Mr) of 55,000, although a 50,000-Mr minor antigen began to appear in the corpus epididymis. By contrast, extracts of cauda epididymis, vas deferens, and cauda epididymal sperm had the 50,000 Mr protein as the only immunoreactive antigen. By enzymography following electrophoresis, the major bands of proteolytic activity in extracts of testis, caput epididymis, and corpus epididymis had 55,000 Mr. A band of protease activity with 55,000 Mr also appeared in extracts of the corpus epididymis. However, the most prominent bands of proteolytic activity in cauda epididymis, vas deferens, and cauda epididymal sperm had 50,000 Mr. In addition, two other major protease activities were detected with 32,000 and 34,000 Mr; the relationships of these proteases to proacrosin are unclear. From these results, we conclude that the oligosaccharides of proacrosin are altered during epididymal transit and that this modification occurs in the corpus epididymis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of three major proteases associated with guinea pig sperm acrosomes

The major proteases associated with guinea pig sperm were investigated by using immunological and electrophoretic techniques. Three major proteases were detected following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis in gels containing 0.1% gelatin. These enzymes had molecular weights of 47,000, 34,000, and 32,000 relative to reduced protein standards and 58,500, 40,000, and 37,500 relative to unreduced standards. All three protease activities were present in acid extracts of sperm, detergent extracts of sperm, and the soluble acrosomal components of sperm released following induction of the acrosome reaction with the Ca2+-ionophore A23187. As determined by indirect immunofluorescence, an antibody to purified boar acrosin specifically cross-reacted with the acrosomes of guinea pig sperm. Decreased fluorescence was associated with sperm that had lost their acrosomes. Immunoblot analysis demonstrated that this antibody reacted with the 47,000 Mr protease but not the 32,000 and 34,000 Mr proteases. All three proteases were maximally active in the pH 7.0-8.5 region and were inhibited by classical inhibitors of acrosin activity. During a 3-hour incubation period, MgCl2 (10 mM) inhibited the activities of the 32,000 and 34,000 Mr proteases while the 47,000 Mr protease was stimulated. Although these proteases shared properties that would classify them as trypsin-like proteases, only the 47,000 Mr protease could be definitely classified as a member of the proacrosin-acrosin family based upon cross-reaction with an antibody to purified boar acrosin.     

Guinea pig proacrosin is synthesized principally by round spermatids and contains O-linked as well as N-linked oligosaccharide side chains

Proacrosin is the zymogen precursor of acrosin, a sperm protease believed to play an essential role in fertilization. In this study, we used primary cultures of guinea pig spermatogenic cells to examine the temporal appearance and mechanisms of synthesis and processing of proacrosin during acrosome development. Following [35S]methionine incorporation and immunoprecipitation, cultured spermatogenic cells were found to synthesize two forms of proacrosin (Mr 54,000 and 57,000). Proacrosin was synthesized mainly by round spermatids. By immunoblotting, proacrosin became very prominent in round spermatids and persisted throughout spermiogenesis. Pulse-chase experiments demonstrated that the Mr 54,000 form of proacrosin was converted to the Mr 57,000 form, presumably reflecting posttranslational processing of carbohydrate side chains. When spermatogenic cells were cultured in the presence of tunicamycin, the synthesized proacrosin had an Mr of 54,000. However, in vitro translation of mRNA extracted from guinea pig testis followed by immunoprecipitation indicated that the core polypeptide of proacrosin has an Mr of 44,000. Guinea pig spermatogenic cells incorporated glucosamine and fucose into the oligosaccharides of proacrosin. Treatment of guinea pig testis proacrosin with N-glycosidase or O-glycosidase reduced the Mr by 3-7%. These results indicate that proacrosin is synthesized by postmeiotic cells and the enzyme contains N- and O-linked oligosaccharides.     

Regulation of proacrosin conversion in isolated guinea pig sperm acrosomal apical segments

Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proacrosin has been identified in extracts of intact guinea spermatozoa as a major silver staining band which reacted immunologically with antibodies made against purified proacrosin from guinea pig testis. Proacrosin exhibited an approximate Mr of 50,000 and was rapidly converted to an Mr 45,000 protein following induction of the acrosome reaction with 2.0 mM CaCl2 and 1 micrograms/ml A23187. Apical segments isolated at pH 6.0 from guinea pig spermatozoa also contained a major silver staining band of Mr 50,000 which cross-reacted with antibodies to guinea pig testis proacrosin. Subcellular fractionation of spermatozoa indicated that proacrosin remained in the particulate fraction of homogenized spermatozoa and was enriched within the isolated acrosomal apical segment. When apical segments isolated at pH 6.0 were incubated at pH 7.5, proacrosin was rapidly converted to the Mr 45,000 form observed in spermatozoa undergoing the acrosome reaction. The conversion process in isolated apical segments was inhibited by leupeptin and was accelerated in the presence of calcium, magnesium, and manganese. Zinc completely inhibited the conversion of proacrosin to the Mr 45,000 protein. Neither proacrosin nor the Mr 45,000 protein were released into the supernatant fluid during the incubation of apical segments at pH 7.5. Furthermore, the proteins were resistant to solubilization by 150 mM NaCl and 1% Triton X-100 but were solubilized by treatment of apical segments with 1 M NaCl. These results provide evidence as to the identity and subcellular distribution of proacrosin in intact guinea pig sperm prior to zymogen conversion and suggest that isolated apical segments exhibit a subset of the exocytotic reactions leading to completion of the acrosome reaction.     

Comparative activation studies with extracted and purified human proacrosin

Proacrosin was purified from acid extracts of human spermatozoa by concanavalin A precipitation and Bio-Gel P-100 chromatography. Two molecular weight forms of proacrosin were obtained, a major one with a Mr of 70,000-71,000 and a minor one with a Mr of 47,000-53,000. In contrast to sperm extracts, the purified forms of proacrosin were free of acrosin inhibitor(s) and nonzymogen acrosin. By modulating pH, ionic strength and temperature, the activation of proacrosin in sperm extracts was compared to only the major form of purified proacrosin, since it seemed to be the source of the lower molecular weight form of proacrosin. In both preparations, proacrosin activation occurred maximally over a broad pH range (7.6-8.8 for purified proacrosin and 7.6-9.6 for extract). Additionally, an ionic strength of 0.1 and above caused a decrease in proacrosin activation in both preparations. Similarly, proacrosin was sensitive to short incubation periods at 45 degrees C and above which caused a decrease in the amount of proacrosin found in both preparations.     

Studies on the rabbit proacrosin-acrosin system

A single molecular form (Mr = 68,000 approx) of a homogeneous preparation of rabbit testis proacrosin (S. K. Mukerji and S. Meizel (1979) J. Biol. Chem. 254, 117;21-11728) was initially converted by autoactivation into an acrosin (Mr = 68,000); both gave a single activity and protein bands with similar electrophoretic mobilities (Rm = 0.25) when subjected to polyacrylamide disc gel electrophoresis on 7.5% gel at pH 4.5. Two additional bands (Rm values of 0.395-0.412 and 0.497-0.519, respectively) were noticeable only when proacrosin was activated further after attaining maximum activity. The slowest- and the fastest-moving bands were separated into two acrosin activity peaks by Sephadex G-100 gel-filtration chromatography on a calibrated column. The molecular weights of the two proteins, determined by rechromatography on the same column, was estimated to be 68,000 and 34,000, respectively. Also, sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of three acrosins gave protein bands which corresponded to molecular weights of approximately 68,000, 52,000, and 34,000, respectively. Electrophoresis data suggest that the loss of acrosin activity generally observed following prolonged activation of proacrosin is caused by self-aggregation of the Mr 34,000 form of acrosin. This property was not shown by Mr 68,000 acrosin. Initial acrosin (Mr = 68,000) was activated by divalent cations such as Ca2+ and Mg2+. The enzyme was inhibited by Zn2+, Fe2+, Hg2+, and sulfhydryl blockers such as 5,5'-dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and iodoacetate, apparently due to their reaction with one out of six titratable sulfhydryl groups per mole of acrosin. Probably Zn2+ is involved in acrosomal stabilization. The initial rabbit acrosin (Mr = 68,000) appears to be the major and most stable form, and is generated from proacrosin with little structural alteration. This may be the functionally active form which plays an essential role in mammalian fertilization.     

Evaluation of the human sperm proacrosin-acrosin system using gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophesis

Proteolytic enzymes in extracts of human sperm have been identified and partially characterized using a technique which incorporates gelatin into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) system. Initially, semen characteristics from four donors were evaluated. Following this, washed sperm were acid extracted and proacrosin and acrosin activities determined spectrophotometrically. Proteinase activity in unactivated sperm extracts was then extracts was then demonstrated using the gelatin-SDS-PAGE system. Three major (Mr approximately equal to 47,000-54,000) and four faint (Mr approximately equal to 34,000-38,000) bands of digestion were observed. Upon activation of sperm extracts it was observed that maximum esterase activity occurred within 7 min of activation while maximum proteinase activity required approximately 15 min. When gels were washed and incubated in the presence of 50 mM benzamidine, no digestion bands were observed. This indicates that all of the digestion bands were due to trypsin-like proteinases. Finally, upon serial dilution of sperm extracts it was found that this SDS-PAGE system is sensitive enough to detect proteinase activity from as few as 30,000 sperm.     

Purification and initial characterization of proacrosins from guinea pig testes and epididymal spermatozoa

Previous studies showed that interspecies differences in proacrosin size may exist. We purified guinea pig proacrosins, one from testes and two from epididymal spermatozoa, by gel filtration and cation exchange at acidic pH. Final purification was by cation exchange at pH 8.0 in 6 M urea. Testis proacrosin migrated with 62,000 Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). One sperm proacrosin migrated with 43,000 Mr, and the other as a 56,000/54,000 Mr doublet in SDS-PAGE. These results represent the first purification of three forms of proacrosin from one species, and the first purification to homogeneity of a 43,000 Mr proacrosin. The proacrosins autoactivate at pH 8.0 with similar kinetics, copurify until the last purification step, and share antigenic determinants. It is possible that the sperm proacrosins are derived from the testis proacrosin, perhaps by proteolysis. The sizes of the three guinea pig proacrosins reported in this study are similar to those reported for proacrosins from other species. Apparent interspecies differences in proacrosin size may be primarily a question of which of at least three possible forms of the zymogen predominates in a species.     

Quantification and partial characterization of the hamster sperm proacrosin-acrosin system

The proacrosin-acrosin proteinase system was measured and partially characterized in unpurified extracts of washed hamster epididymal sperm. Autoactivation experiments demonstrated that proacrosin accounted for greater than 98% of the acrosin activity in the sperm extracts from individual animals. Several bands of proteinase activity were observed on gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic (gelatin-SDS-PAGE) zymography. The major proteinase activities in the nonactivated extracts corresponded to relative molecular masses (Mr) of 51,000 to 56,000, while less distinct digestion occurred with relative molecular masses of 37,000 to 49,000. It was demonstrated that after a serial dilution of the sperm extract, the proteinase activity in as few as 6,000 sperm could readily be detected by the gelatin-SDS-PAGE methods. Time-course activation studies showed that the zymogen was completely converted to active proteinase in 45-60 min at pH 8.0 and 25 degrees C. This autoconversion process was markedly inhibited by calcium, sodium, and heparin. However, each of these compounds stimulated the proteolytic activity of acrosin. These studies demonstrate that the proacrosin-acrosin system can be investigated in extracts of nonpurified hamster epididymal sperm.     

Rabbit testis proacrosin: Immunological similarities between testis,sperm proacrosins and the initially formed testis acrosin

Anti-rabbit proacrosin IgG was prepared from goat serum following immunization with a homogeneous preparation of rabbit testis proacrosin. The “auto-activation” products of purified testis proacrosin were separated into 68,000 and 34,000 molecular weight (mol wt) acrosins by Sephadex G-100 column chromatography. Immunodiffusion analysis of testis and epididymal sperm proacrosins and acrosins on agarose gel against goat anti-rabbit testis proacrosin showed immunological identity between rabbit testis and sperm proacrosins and the initial testis acrosin (mol wt 68,000). However, the 34,000 mol wt form of testis acrosin showed weaker reaction with the antibody and only partial identity with the proacrosin and the 68,000 mol wt form of acrosin. These results suggest that there is no major structural difference between testis and sperm proacrosins and between proacrosin and the 68,000 mol wt acrosin, but such a structual change occurs when the 34,000 mol wt acrosin is formed.     

Differential release of guinea pig sperm acrosomal components during exocytosis

The contents of the sperm acrosome are compartmentalized at the biochemical and morphological levels. Biochemically, the acrosome can be considered to be comprised of two compartments: one consisting of readily soluble proteins and one containing a particulate acrosomal matrix. To test the hypothesis that compartmentalization affects the release of acrosomal components during the course of secretion in guinea pig sperm, we examined the relationship between the presence of specific proteins and acrosomal status and monitored the recovery of acrosomal constituents in the medium surrounding sperm induced to undergo exocytosis with the ionophore A23187. Cysteine-rich secretory protein 2 (CRISP-2), a soluble component of the acrosome, was rapidly lost from the acrosome soon after ionophore treatment. However, acrosomal matrix components remained associated with the sperm for longer periods. AM67, a matrix component and the guinea pig orthologue of the mouse sperm zona pellucida-binding protein sp56, was released at a slower rate than was CRISP-2 but at a faster rate than were two other matrix proteins, AM50 and proacrosin. Coincident with their release from the sperm, AM50 and proacrosin were posttranslationally modified, probably by proteolysis. The release of proacrosin from the matrix appears associated with the conversion of this protein to the enzymatically active acrosin protease. These results provide strong support for the hypothesis that compartmentalization plays a significant role in regulating the release of proteins during the course of acrosomal exocytosis. Acrosomal matrix proteins remain associated with the sperm for prolonged periods of time following the induction of acrosomal exocytosis, suggesting that transitional acrosomal intermediates may have significant functions in the fertilization process.     

Formation of the perinuclear theca in spermatozoa of diverse mammalian species: relationship of the manchette and multiple band polypeptides

The perinuclear theca is a novel cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. Immunoreactive proteins less than Mr 55,000 were prominent in rat sperm heads and mouse sperm: guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately Mr 50,000. Different immunoreactive proteins were present in isolated sperm tails. The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. Immunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub- and post-acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus.     

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.     

Difference of acrosomal serine protease system between mouse and other rodent sperm

A fraction of acrosomal proteins dispersed during calcium ionophore A23187‐induced acrosome reaction was prepared from cauda epididymal sperm of wild‐type and acrosin‐deficient mice, rat, and hamster. The acrosome‐reacted sperm were further extracted by Nonidet P‐40 to obtain the detergent‐soluble protein fraction. Activities of serine proteases in the two protein fractions were examined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the presence of gelatin. A mixture of 42‐ and 41‐kDa gelatin‐hydrolyzing proteases was found in both fractions of the wild‐type mouse sperm, whereas the acrosin‐deficient mouse sperm contained the active 42‐kDa protease and apparently lacked the activity of the 41‐kDa protease. However, exogenous bovine pancreatic trypsin compensated for the absence of acrosin in the protein fractions of the mutant mouse sperm; the gelatin‐hydrolyzing activity of the 41‐kDa protease appeared when the sperm proteins of the mutant mice were treated with pancreatic trypsin. Two‐dimensional polyacrylamide gel electrophoresis revealed that the 42‐ and 41‐kDa proteases were distinguished from acrosin by the isoelectric point and immunoreactivity with affinity‐purified antibody against an oligopeptide corresponding to the N‐terminal amino acid sequence of mouse proacrosin. Moreover, the gelatin‐hydrolyzing proteins corresponding to these two proteases were not detected in rat and hamster sperm, in spite of the treatment of the sperm extracts with pancreatic trypsin, and the total amount of gelatin‐hydrolyzing activities in mouse was much smaller than those in rat and hamster. These results may reflect the difference of the serine protease system for the sperm penetration through the egg zona pellucida between mouse and other rodent animals, possibly explaining why the acrosin‐deficient mouse sperm are capable of penetrating the zona pellucida. Dev. Genet. 25:115–122, 1999. © 1999 Wiley‐Liss, Inc.     

The zymogen form of acrosin in testicular,epididymal, and ejaculated spermatozoa from ram

The sperm-specific proteinase acrosin (EC 3.4.21.10) is found in spermatozoa as a zymogen. We have looked for different forms of this zymogen in testicular, epididymal, and ejaculated spermatozoa from ram and have compared total sperm extracts made immediately after cell disruption with extracts made later from isolated sperm heads. We have concluded that the autoactivatable zymogen form, known generally as proacrosin, is the only form of acrosin within intact mature ram spermatozoa; no other zymogen form was detected, although lower levels of proacrosin were found in some samples of testicular spermatozoa. From studies of the activation process, it appears that ram proacrosin is truly autoactivatable; no evidence could be found for the involvement of any auxiliary enzyme. Estimations of the molecular weight of proacrosin using gel chromatography (60,000) and SDS-polyacrylamide gel electrophoresis (51,300) indicated that the zymogen is monomeric. Comparison with the molecular weight of ram acrosin (44,000 or 40,000, using the two respective methods) indicated that a single acrosin molecule is derived from each zymogen molecule. The sperm acrosin inhibitor (molecular weight 11,000 or 8,000) was present in testicular spermatozoa as well as in ejaculated spermatozoa; there was no evidence that it was produced as a result of zymogen activation.     

The interaction of boar sperm proacrosin with its natural substrate, the zona pellucida, and with polysulfated polysaccharides

Boar sperm acrosin is an acrosomal protease with trypsin-like specificity, and it functions in fertilization by assisting sperm passage through the zona pellucida by limited hydrolysis of this extracellular matrix. In addition to a proteolytic active site domain, acrosin binds the zona pellucida at a separate binding domain that is lost during proacrosin autolysis. In this study, we quantitate the binding of proacrosin to the physiological substrate for acrosin, the zona pellucida, and to a non-substrate, the polysulfated polysaccharide fucoidan. Binding was analogous to sea urchin sperm bindin that binds egg jelly fucan and the vitelline envelope of sea urchin eggs. Proacrosin was found to bind to fucoidan and to the zona pellucida with binding affinities similar to bindin interaction with egg jelly fucan. These interactions were competitively inhibited by similar relative molecular mass polysulfated polymers. Since bindin and proacrosin have distinctly different amino acid sequences, their interaction with acidic sulfate esters demonstrates an example of convergent evolution wherein different macromolecules localized in analogous sperm compartments have the same biological function. From cDNA sequence analysis of proacrosin, this binding may be mediated through a consensus sequence for binding sulfated glycoconjugates. Proacrosin binding to the zona pellucida may serve as both a recognition or primary sperm receptor, as well as maintaining the sperm on the zona pellucida once the acrosome reaction has occurred.     

Effects of various conditions of semen storage on the acrosin system of human spermatozoa

Stability of the human sperm acrosin system (major components: non-zymogen acrosin, proacrosin and acrosin inhibitor) was studied under various conditions of semen storage used clinically or in the laboratory. Freezing at -196 degrees C caused a profound decrease in total acrosin content and in the amount of this enzyme present in zymogen form (proacrosin), but resulted in some increase in non-zymogen acrosin. Acrosin inhibitor did not appear to be significantly affected by this treatment. No relationship was present between the decreases in sperm motility induced by freezing to -196 degrees C and the alterations in total acrosin, proacrosin and non-zymogen acrosin. Storage of whole semen at -20 degrees C had deleterious effects on all the components of the acrosin system measured except for non-zymogen acrosin. Major decreases in the total acrosin, proacrosin and acrosin inhibitor occurred after only 1 day at -20 degrees C and continued slowly thereafter. Whole semen kept at room temperature for up to 24 h after ejaculation did not show any significant changes in the sperm acrosin system. Seminal plasma did not have a detrimental or stabilizing effect of acrosin and proacrosin when spermatozoa were kept at room temperature. However, removal of seminal plasma and re-suspension of spermatozoa in 0.9% NaCl resulted n the liberation of a significant amount of the acrosin inhibitor from the spermatozoa and the apparent activation of some of the proacrosin to acrosin.     

Adenosine 3':5'-monophosphate receptor proteins in mammalian brain.

cAMP receptor proteins present in mammalian brain were identified and characterized with the use of the photoaffinity label 8-azido[32P]cAMP. Cytosol and membrane fractions from various regions of rat, guinea pig, and bovine brain contained two specific cAMP receptor proteins with apparent molecular weights of 47,000 and 52,000 to 55,000. Subcellular fractionation studies showed the highest amounts of these cAMP receptor proteins associated with cytosol fractions and synaptic membrane fractions. For both the cytosol and membrane fractions, the two cAMP receptor proteins represented almost all of the proteins specifically labeled by 8-azidol[32P]cAMP and appeared to be the regulatory subunits of cAMP-dependent protein kinases.     

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.