Fibrinogen-Related Protein, Fgl2, of Hamster Cauda Epididymal Fluid: Enzymatic Characterization, and Identification of Fgl2-Binding Proteins and Ligand of Defective Hamster Sperm Organelles.

The luminal environment of the mammalian epididymidis performs a dual function; sperm maturation and maintaining sperm viability. We previously identified a secretory protein (260/280KDa oligomers) of hamster cauda epididymal principal cells that binds to nonviable sperm. The 260/280KDa oligomers are composed of 64kDa FGL2 (fibrinogen-like protein-2) and 33kDa FGL1) (fibrinogen-like protein-1). The potential mechanism by which FGL2 binds to degenerative sperm is not clearly demonstrated. In this study, we report the downstream sequence of prothrombinase activity of FGL2, the identification of organelles, and characterize candidate proteins that bind FGL2. The following reaction sequence confirms that FGL2 is a phospholipid-activated serine protease; the conversion of prothrombin to thrombin by FGL2, followed by the conversion of soluble fibrinogen to insoluble fibrin polymers by thrombin. FGL2 binds intensely to tails than heads of de-membranated sperm. A spectrum of polypeptides of cauda sperm tails binds to FGL2. Proteomic analyses of 65KDa, 16kDa, and 13kDa polypeptides of tails correspond to a-Kinase anchor protein 4, glutathione peroxidase 4, and cytochrome c oxidase subunit 4, respectively. Annexin V, a calcium-dependent phosphatidylserine-binding protein localized to the flagellum and co-precipitated with FGL2. We have demonstrated a novel protective mechanism for recognizing and eliminating defective spermatozoa from viable sperm population.

Fibrinogen in Hamster Cauda Sperm.

Epididymis plays a vital role in promoting sperm maturation and maintaining sperm viability. It has been shown the presence of nonviable sperm in cauda epididymis. We previously identified a secretory protein (260/280KDa oligomers) of hamster cauda epididymal principal cells that binds to nonviable sperm. The 260/280KDa oligomers are composed of 64kDa FGL2 (fibrinogen-like protein-2) and 33kDa FGL1) (fibrinogen-like protein-1). In addition, we have demonstrated that FGL2 is a phospholipid-activated serine protease; the conversion of prothrombin to thrombin by FGL2 followed by the conversion of soluble fibrinogen to insoluble fibrin polymers by thrombin. In the present study, we have shown the presence of a 56kDa fibrinogen ß in hamster cauda sperm. The potential role of fibrinogen in hamster physiology is being discussed.

Biochemical alterations in the proacrosin-acrosin system during epididymal maturation of the rat spermatozoa.

Acrosin, an acrosomal serine protease, is believed to have a role in fertilization. The enzyme is synthesized in an enzymatically inactive precursor form, proacrosin, and is processed to enzymatically active form(s). In the studies presented here, maturation-associated changes in the proacrosin-acrosin system of rat spermatozoa are reported. Acid-solubilized components of spermatozoa from caput, corpus, and cauda epididymidis were resolved on gelatin-sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and the proteolytic bands visualized by enzymography. These studies reveal the presence of one form (52 kDa), two forms (52 and 41 kDa), and four forms (52, 41, 34, and 31 kDa) in the spermatozoa from caput, corpus, and cauda, respectively. The findings suggest that the enzymatically inactive high molecular weight component (proacrosin) present in the caput spermatozoa is partially converted to the low molecular weight components (acrosin) during epididymal transit. The sensitivity of these molecular forms to an inhibitor of acrosin, p-nitrophenyl p'-guanidino benzoate (NPGB), and the fact that all four forms cross-reacted with the antibody against guinea pig testis proacrosin, suggest that these molecular forms are proacrosin-acrosin components. To understand the mechanism of the changes in molecular forms, spermatozoa from caput, corpus, and cauda regions were subjected to in vitro activation, and the acid-solubilized components resolved on gelatin-SDS-polyacrylamide gel. A smaller component of 34 kDa was generated from both the caput and corpus spermatozoa. No changes in the molecular form(s) of cauda spermatozoa were observed, even after in vitro activation for 4 hours. Inclusion of NPGB during in vitro activation blocked generation of the new molecular form from the caput spermatozoa. These studies indicate that intra-acrosomal events during epididymal transit may be important in the production of functionally mature spermatozoa.

Bovine proacrosin from cauda epididymal sperm: purification, characterization and partial sequencing at N-terminus.

1. In the present study, we isolated the two forms of proacrosin from acid extracts (pH 3.0) of cauda epididymal bovine spermatozoa by ammonium sulfate fractionation, gel filtration on Sephadex G-150 and affinity chromatography on Concanavalin A Sepharose 4B. The overall purification was 13-fold with respect to crude acid acrosomal extract. 2. The apparent molecular weight of the proacrosins determined by SDS-PAGE were 44,000 and 38,000. Both forms have proteinase activity on gelatin-SDS-polyacrylamide gel electrophoretic zymography. 3. The M(r) = 38,000 component was isolated by reverse phase HPLC. Thirty-nine amino acid residues at the N-terminus have about 72 and 77% sequence similarity with boar and human proacrosin, respectively. 4. The amino acid sequence of 14 amino acids at the N-terminus of the high molecular weight component (M(r) = 44,000) was determined after electroblotting on a polyvinylidene difluoride membrane. This portion of the molecule is identical with that of the low molecular weight component. 5. Proacrosin autoactivation followed the sigmoidal activation curve.

Effect of chlorpromazine on bovine sperm respiration.

Chlorpromazine (CPZ), an antipsychosis drug, causes the in vitro inhibition of sperm motility in various species, but the effect of this drug on sperm respiration is not well studied. This study was undertaken to evaluate the effect of this drug on ejaculated bovine sperm endogenous respiration. CPZ acted as a stimulator and an inhibitor of respiration at 0.1 and 0.75 mM CPZ, in the respiration medium, respectively. CPZ caused the release of sperm protein in the supernatant in a dose-dependent manner and the lactate dehydrogenase activity has also been demonstrated in the supernatant. Succinate and glycerol 3-phosphate supported the respiration among the respiratory substrates used in the present study, even after inhibition of endogenous respiration by 0.75 mM CPZ. Chlorpromazine has biphasic actions on bovine sperm respiration. The mechanism of action of the drug is discussed.

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.

The kinetics of inhibition of human seminal plasma acid phosphatase by sodium fluoride.

Purified human seminal plasma acid phosphatase is inhibited by sodium fluoride and L(+) tartrate and 50% inhibitions (I50) have been obtained at 7.3 X 10(-5) and 3.0 X 10(-4) M concentrations, respectively. By Lineweaver-Burk plot, sodium fluoride shows non-competitive type of inhibition. Ackermann plot indicates that sodium fluoride is a reversible inhibitor of seminal plasma acid phosphatase. On polyacrylamide activity gel electrophoresis and by densitometric scan, a single molecular form of acid phosphatase has been identified in human seminal plasma. Inhibition patterns by sodium fluoride and L(+) tartrate have also been performed by polyacrylamide activity gel electrophoresis and almost complete inhibition of hydrolysis of alpha-napthol phosphate is observed at 20 mM and 100 mM of sodium fluoride and L(+) tartrate, respectively.

Proacrosin-acrosomal matrix binding interactions in ejaculated bovine spermatozoa.

The mechanisms regulating hydrolase release during the mammalian sperm acrosome reaction are poorly understood. The present study demonstrates that specific domains of the acrosomal matrix of bovine spermatozoa function to maintain a particulate proacrosin pool and to regulate proacrosin/acrosin release. In sonicated sperm suspensions, 50-60% of the total proacrosin activity was sedimentable, and the amount of sedimentable proacrosin activity remained unchanged over time. Serial centrifugation and resuspension experiments demonstrated that the particulate proacrosin fraction resulted from an equilibrium binding of proacrosin to a stable sperm structure. To identify the proacrosin-binding structure of the acrosome, a purified sperm head fraction was isolated on sucrose density gradients. The sperm heads were extracted with Triton X-100, and a homogeneous acrosomal subfraction, the matrix complex associated with the outer acrosomal membrane (OMC), was isolated on Percoll density gradients. A centrifugation assay was then used to demonstrate that the OMC specifically binds proacrosin in a dose-dependent manner. These data demonstrate that the OMC represents a stable structural component of the acrosome that maintains a particulate proacrosin pool. We propose that the OMC regulates proacrosin release during the acrosome reaction and maintains elevated acrosin concentrations at the site of sperm-egg interaction.

Temporal expression and localization of protein farnesyltransferase during spermiogenesis and posttesticular sperm maturation in the hamster.

Spermiogenesis and posttesticular sperm maturation in the epididymis are distinct developmental processes that result in a polarized spermatozoon possessing a plasma membrane partitioned into segment-specific domains of distinct composition and function. The mechanisms that specify the distribution of intracellular organelles and target proteins to restricted membrane domains are not well understood. In this study we examined the expression pattern and distribution of protein farnesyltransferase (FTase) in hamster spermatids and epididymal spermatozoa to determine if protein lipidation may represent a potential mechanism to regulate protein association with specific organelles or the plasma membrane. Round spermatids exhibited only weak immunostaining with antibody against the beta-subunit of FTase, whereas elongating spermatids exhibited a high level of FTase expression that was segregated to the cytoplasmic lobe surrounding the anterior flagellum. Although FTase was released with the residual body, mature spermatids retained FTase within the midpiece and cytoplasmic droplet. In epididymal spermatozoa, FTase remained associated with the cytoplasmic droplet during its migration to the midpiece-principal piece junction; following release of the cytoplasmic droplet, no immunodetectable FTase was noted in the midpiece segment. Immunoblotting demonstrated the presence of both the alpha and beta subunits of FTase in sperm lysates. The temporal expression pattern and restricted distribution of FTase in spermatids and epididymal spermatozoa suggest a potential role in regulating protein association with specific organelles and/or membrane domains of the mature spermatozoon.

Acrosome biogenesis in the hamster: ultrastructurally distinct matrix regions are assembled from a common precursor polypeptide.

The hamster sperm acrosome contains a stable matrix complex that binds specific hydrolases and appears to regulate their release during the acrosome reaction. This complex comprises two contiguous but ultrastructurally distinct regions that are segregated to specific sites within the acrosome. In this study, we define the temporal expression, processing, and localization of major matrix proteins of 29 kDa (AM29) and 22 kDa (AM22) during spermiogenesis and post-testicular sperm maturation in the epididymis. Peptide mapping, N-terminal microsequence analysis, immunoblotting, and immunocytochemistry were used to demonstrate that AM29 and AM22 of mature spermatozoa are structurally related and appear to arise from a common 40-kDa precursor protein expressed in round spermatids. A monoclonal antibody that recognized only the mature forms of the matrix proteins and a polyclonal antibody that recognized both the precursor and fully processed matrix proteins were prepared and used to demonstrate that the precursor protein is present in the acrosome of round spermatids and that it undergoes size processing during the terminal stages of spermiogenesis so that the mature matrix polypeptides are evident in epididymal spermatozoa. Finally, using light and electron microscopic immunocytochemistry, we demonstrated that the matrix polypeptides are excluded from the equatorial segment and are localized to both structurally distinct matrix domains of the mature acrosome. These data show that processing of the major proteins of the acrosomal matrix occurs in a temporally regulated fashion after their transport to the acrosome and that the processed products can assemble into ultrastructurally distinct matrix elements.

Identification of a hamster epididymal region-specific secretory glycoprotein that binds nonviable spermatozoa.

Even though the epididymis produces an environment promoting sperm maturation and viability, some sperm do not survive transit through the epididymal tubule. Mechanisms that segregate the epididymal epithelium and/or the viable sperm population from degenerating spermatozoa are poorly understood. We report here the identification and characterization of HEP64, a 64-kDa glycoprotein secreted by principal cells of the corpus and proximal cauda epididymidis of the hamster that specifically binds to and coats dead/dying spermatozoa. The HEP64 monomer contains approximately 12 kDa carbohydrate and, following chemical deglycosylation, migrates as a approximately 52-kDa polypeptide. Both soluble (luminal fluid) and sperm-associated HEP64 are assembled into disulfide-linked high molecular weight oligomers that migrate as a doublet band of 260/280 kDa by nonreducing SDS-PAGE. In the epididymal lumen, HEP64 is concentrated into focal accumulations containing aggregates of structurally abnormal or degenerating spermatozoa, and examination of sperm suspensions reveals that HEP64 forms a shroudlike coating surrounding abnormal spermatozoa. The HEP64 glycoprotein firmly binds degenerating spermatozoa and is not released by either nonionic detergent or high salt extraction. Electron microscopic immunocytochemistry demonstrates that HEP64 localized to an amorphous coating surrounding the abnormal spermatozoa. The potential mechanisms by which this epididymal secretory protein binds dead spermatozoa as well as its possible functions in the sperm storage function of the cauda epididymidis are discussed.

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.

Rat sperm plasma membrane mannosidase: localization and evidence for proteolytic processing during epididymal maturation.

Previous studies from this laboratory have identified a novel alpha-D-mannosidase on the sperm plasma membranes of several species, including man, which may have a role in fertilization. The polyclonal antibody raised against an isoform of the enzyme purified from rat epididymal fluid was found to cross-react with the alpha-D-mannosidase activity present in the detergent-solubilized spermatozoa and sperm plasma membranes. In the present study, we have used affinity-purified as well as monospecific anti-mannosidase IgG to demonstrate that the sperm mannosidase is an integral plasma membrane component of the rat sperm and is localized on the periacrosomal region of the sperm head. In addition, we demonstrate proteolytic processing of the membrane-bound alpha-D-mannosidase during maturation of spermatozoa. The membrane fractions prepared from testis, and spermatozoa from the caput, corpus, and cauda regions of the epididymis, were solubilized in SDS and resolved by SDS-PAGE. The resolved polypeptides, when subjected to Western blot analysis using affinity-purified anti-mannosidase IgG as the primary antibody, revealed the presence of three specific immunoreactive bands (apparent M(r), 135, 125, and 115 kDa) in the membranes from testis, caput, and corpus spermatozoa. However, the cauda sperm plasma membranes showed only one immunoreactive band of apparent M(r) 115 kDa. The disappearance of the 135-and 125-kDa forms and the appearance of a sharp 115-kDa band on cauda spermatozoa suggests a precursor-product relationship between various molecular forms of the enzyme. Trypsin treatment of testicular and caput sperm membranes largely converted the precursor forms to the mature (115-kDa) form. The in vitro proteolysis resulted in an elevated level of the alpha-D-mannosidase activity in the caput (but not cauda) sperm plasma membrane. Inclusion of trypsin inhibitors (benzamidine and aprotinin) largely prevented the conversion of precursor form to the mature form. These data are consistent with the observed increase in the levels of sperm enzyme activity as spermatozoa move from the caput to the cauda region and suggest that the increase is due to the conversion of enzymatically inactive/less active high molecular weight precursor forms (135 and 125 kDa) into enzymatically active mature form (115 kDa) during sperm maturation.

Identification and characterization of a Mg2+-dependent and an independent Ca+2-ATPase in microsomal membranes of rat testis.

Rat testicular microsomal membrane fraction contains both Mg+2-dependent and Mg+2-independent Ca+2-ATPase activity. The latter activity is about two times higher than the former. Calcium ion required for maximum activation of Mg+2-independent Ca+2-ATPase in 3.0 mM, whereas for the dependent one it is 2.5 mM. Both the enzymes are resistant to cold shock upto seven days. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities, respectively. The pH optima for dependent one is 7.5, whereas for the independent one it is 8.5. Temperature optima for the former is 37 degrees C and for latter one it is 40 degrees C. Among all the nucleotides tested, ATP is found to be the best substrate for both the enzymes. The optimum concentration of ATP for dependent and independent enzyme activities are 3.0 mM and 1.5 mM respectively. Divalent metal ions like Zn+2, Ba+2 and Mn+2 have been found to inhibit Mg+2-dependent Ca+2-ATPase activity whereas Mg+2-independent Ca+2-ATPase activity is inhibited by the divalent ions except zinc which is found to stimulate the enzyme activity. Both the enzymes are inhibited by vanadate, EDTA and EGTA. I50, for vanadate is 0.05 and 0.125 mM for dependent and independent activities, respectively. Sulfhydryl groups modifying agents e.g., NEM, DTNB and chlorpromazine are found to affect the enzyme activities in different ways. Thus NEM and chlorpromazine are found to inhibit and DTNB stimulate the enzyme activities in both the cases.

The interaction of chloroquine with transport ATPase and acetylcholine esterase in microsomal membranes of rat in vitro and in vivo.

Chloroquine, an antimalarial drug has been found to inhibit Na+, K+-ATPase activity in vitro in the microsomal membranes of rat brain on time, temperature and concentration dependent manner. There have been stimulation of Na+,K+-ATPase, Ca+2-ATPase and acetylcholine esterase activities in vivo studies at lower concentration of drug or shorter period of treatment with the drug, whereas higher concentrations or longer periods of treatment lead to inhibition in the microsomal membranes of different organs.

Purification and characterization of rat epididymal-fluid alpha-D-mannosidase: similarities to sperm plasma-membrane alpha-D-mannosidase.

We have previously reported the occurrence and partial characterization of a novel alpha-D-mannosidase activity on rat sperm plasma membranes [Tulsiani, Skudlarek and Orgebin-Crist (1989) J. Cell Biol. 109, 1257-1267]. Here, we report the presence of a similar alpha-D-mannosidase activity in a soluble form in rat epididymal fluid. The soluble enzyme was purified nearly 500-fold with 9-12% recovery to a state approaching homogeneity using: (1) (NH4)2SO4 precipitation; (2) affinity chromatography on immobilized mannan and D-mannosamine; (3) ion-exchange (DE-52) column chromatography; (4) molecular-sieve chromatography. The enzyme was eluted from the final column (Sephacryl S-400) at an apparent molecular mass of 460 kDa. When resolved by SDS/PAGE (under denaturing conditions), the enzyme showed a major protein band (115 kDa) and few very minor bands. The polyclonal antibody raised against the major protein band was found to cross-react with the alpha-D-mannosidase activity present in epididymal fluid (soluble) and detergent-solubilized spermatozoa from the rat and mouse. This result suggested that the soluble and membrane-bound enzyme activities shared a common antigenic site(s). The antibody was used to characterize further the alpha-D-mannosidase activity(ies) present in the rat epididymal fluid and rat sperm plasma membranes. Data from these studies show that the two forms are similar in (a) subunit molecular mass, (b) substrate specificity and (c) inhibitory effect of several sugars. These similarities suggest that the soluble and membrane-bound alpha-D-mannosidase activities are isoforms. Immunoprecipitation studies after solubilization of the testis and epididymal particulate fraction from sexually immature rats show that the testis (but not the epididymis) contains the immunoreactive alpha-D-mannosidase activity. This result and the fact that spermatozoa from the rat rete testis show alpha-D-mannosidase activity indicate that the sperm enzyme is synthesized in the testis during spermatogenesis.

Beta-D-galactosidase of rat spermatozoa: subcellular distribution, substrate specificity, and molecular changes during epididymal maturation.

In previous studies, we reported that rat epididymal fluid acid beta-D-galactosidase, which optimally cleaves a synthetic substrate (PNP beta-D-galactoside) at pH 3.5, shows maximum activity at pH 6.8 when a glycoprotein is used as a substrate [Skudlarek MD, Tulsiani DRP, Orgebin-Crist M-C. Biochem J 1992; 286: 907-914]. We now describe a similar pH-dependent substrate preference for rat sperm beta-D-galactosidase. We found that only 10-14% of total beta-D-galactosidase (and other glycosidase) activity was associated with spermatozoa. The remaining enzyme activities were present in soluble form in the luminal fluid. When the glycosidase levels were expressed per 10(6) sperm, all enzymes showed a progressive increase in spermatozoa from the caput to the corpus or proximal cauda followed by a sharp decline in spermatozoa from the distal cauda epididymidis. The observed decrease in beta-D-galactosidase activity could not be explained by the loss of cytoplasmic droplets (which have a low enzyme activity relative to spermatozoa) or the presence of inhibitors/activators of the enzyme activity in spermatozoa from the proximal or distal epididymis. However, we found that the changes in beta-D-galactosidase activity during sperm maturation in the epididymis were accompanied by changes in the molecular form(s) of the enzyme. Western blot analysis using an antibody to beta-D-galactosidase showed a progressive processing of the 82-kDa immunoreactive band in caput spermatozoa to an 80-kDa diffuse band in cauda spermatozoa. The sperm-associated beta-D-galactosidase form(s) does not appear to be due to adsorption and/or binding of the luminal fluid beta-D-galactosidase, which contained a 97-kDa form in fluid from the caput and two forms, of 97 kDa and 84 kDa, in corpus and cauda fluids. The observed difference in the molecular forms of the sperm and luminal fluid was found to be due to differential glycosylation, since de-N-glycosylation of various forms of beta-D-galactosidase generated a single immunoreactive form of 70 kDa. Subcellular localization studies and assay for the beta-D-galactosidase activity in the enriched plasma membrane and acrosomal membrane fractions suggested the likelihood that the activity of beta-D-galactosidase and other glycosidases is present in the acrosome and is readily released during sperm disruption. The evidence suggests that sperm beta-D-galactosidase may be functional within the acidic environment of the acrosome during sperm maturation as well as in the neutral environment of the oviduct after the zona-induced acrosome reaction.

Evidence for the presence of high-mannose/hybrid oligosaccharide chain(s) on the mouse ZP2 and ZP3.

Previous studies from this laboratory have identified a novel alpha-D-mannosidase on plasma membranes of rat, mouse, hamster, and human spermatozoa [Tulsiani et al. J Cell Biol 1989; 109:1257; Biol Reprod 1990; 42:843]. Inhibition of the mouse sperm surface alpha-D-mannosidase inhibits sperm-egg binding in vitro, suggesting that the sperm enzyme may have a receptor-like role in binding to the complementary molecules (presumably mannose-containing oligosaccharide [OS] chains) on the mouse zona pellucida (ZP) glycoconjugates [Cornwall et al. Biol Reprod 1991; 44:913]. In the studies reported here, we demonstrate the presence of high-mannose/hybrid-type OS on mouse zona components. Zona-intact eggs, prepared from superovulated mice, were radioiodinated, and the individual zona components (ZP1, ZP2, and ZP3) were isolated by electrophoresis followed by electroelution. The purified ZP components, when resolved by immobilized concanavalin A column chromatography, showed the following results: 1) Nearly all of the ZP1 applied to the immobilized lectin eluted in the column flow-through (effluent) fractions, and no radioactivity eluted with alpha-methyl mannoside, suggesting that ZP1 may not contain high-mannose/hybrid OS. 2) A significant amount of both ZP2 and ZP3 bound to the immobilized lectin, and nearly 16% and 8% of the two components, respectively, were repeatedly eluted with alpha-methyl mannoside.(ABSTRACT TRUNCATED AT 250 WORDS)

Reproductive features of the eastern mole (Scalopus aquaticus) and star-nose mole (Condylura cristata).

Since moles are closely related to shrews, the gametes and reproductive tracts of the star-nose mole (Condylura cristata) and the eastern mole (Scalopus aquaticus) were examined to gain further insight into unusual reproductive traits of the Soricidae. Moles display many of these soricid traits, but with some important differences. The cumulus oophorus of Scalopus, ovulated about 16 h after hCG injection, was largely dispersed by hyaluronidase and, though quite dense, was nevertheless more similar to that of higher mammals than to the compact 'ball of the soricid cumulus. Within the female tract in these moles, approximately 85% of the length of the oviduct comprises a narrow ampulla with numerous differentiated crypts that, in shrews, house spermatozoa. However, in contrast to shrews, moles produce considerably larger numbers of spermatozoa, which challenges the proposal that, in shrews, oviductal sperm crypts specifically permit lower sperm production by the males. In the sperm head of these two moles, the acrosome displays the long rostrum that is typical of other Insectivora, and the perforatorium has the barbs by which soricid spermatozoa probably bind to the zona pellucida. Perhaps allied to this, immunoblots indicated that the immunoreactive acrosomal matrix of Scalopus spermatozoa is simpler than the polypeptide complex of the bovine and hamster acrosomal matrix.

Reproductive characteristics of the african pygmy hedgehog, atelerix albiventris.

To obtain further perspective on reproduction and particularly gamete function among so-called primitive mammals presently grouped in the Order Insectivora, we have examined the African hedgehog, Atelerix albiventris, in light of unusual features reported in shrews and moles. Atelerix proves to share many but not all of the characteristics seen in these other insectivores. The penis of Atelerix has a 'snail-like' form, but lacks the surface spines common in insectivores and a number of other mammals. Hedgehog spermatozoa display an eccentric insertion of the tail on the sperm head, and they manifest the barbs on the perforatorium that, in shrews, probably effect the initial binding of the sperm head to the zona pellucida. As a possible correlate, the structural matrix of the hedgehog acrosome comprises only two main components, as judged by immunoblotting, rather than the complex of peptides seen in the matrix of some higher mammals. The Fallopian tube of Atelerix is relatively simple; it displays only minor differences in width and in the arborized epithelium between the isthmus and ampulla, and shows no evidence of the unusual sperm crypts that characterize the isthmus or ampulla, depending on the species, in shrews and moles. In common with other insectivores, Atelerix appears to be an induced ovulator, as judged by the ovulation of some 6-8 eggs by about 23 h after injection of hCG. The dense cumulus oophorus appeared to have little matrix, in keeping with the modest dimensions of the tubal ampulla and, while it was not quite as discrete as that of soricids, it did show the same insensitivity to 0.5% (w/v) ovine or bovine hyaluronidase.