Biochemical characterization and epididymal localization of the maturation-dependent ram sperm surface antigen ESA152

We examine here the biochemical properties and epididymal localization of a maturation dependent ram sperm surface antigen. A monoclonal antibody, ESA152, identifies an antigen that is present on the surface of ejaculated sperm, but is absent from testicular sperm. Crosslinking of the ESA152 antigen with bivalent antibodies induces the acrosome reaction, redistributing the antigen into the anterior region of the sperm head where it associates with the fusion product of the plasma membrane and the outer acrosomal membrane. The ESA152 antigen appears as a polypeptide of 18 kDa on immunoblots of SDS-polyacrylamide gels. The ESA152 epitope includes the sialic acid termini of N-linked oligosaccharides, as shown by its sensitivity to neuraminidase and endoglycosidase F. The ESA152 antigen is a highly hydrophobic integral membrane protein that resists aqueous extraction, partitions into the detergent phase of Triton-X-114, and solubilizes in chloroform-methanol mixtures. The anchoring of ESA152 is unaffected by phosphtidylinositol specific phospholipase C. The antigen is absent from extracts of caput and corpus epididymidis but appears abruptly in the first segment of the cauda. Immunofluorescence reveals that the ESA152 epitope first appears in clusters of cells in the luminal epithelium of the proximal cauda, prior to or concurrent with its appearance on sperm. © 1993 Wiley-Liss, Inc.     

Purification and partial characterization of a sperm motility-inhibitory protein of ram cauda epididymal plasma

Mammalian sperm remain quiescent but fertile for several weeks in cauda epididymis. Although several sperm quiescent factors of epididymal plasma have been identified in goat, pig and cattle; however, little is known in sheep. In the present study, purification and characterization of a novel sperm quiescent protein of ovine cauda epididymal plasma (CEP) was carried out. The sperm quiescent protein was partially purified by hydroxyapatite gel adsorption chromatography followed by DEAE-sepharose® anion exchange chromatography. In the latter, the sperm quiescent activity was eluted both in 0.05 and 0.2 M potassium phosphate buffer (pH 7.5) fractions having a predominant protein of about 80 and 70 kDa with 87% and 63% homogeneity, respectively. The proteins were designated as motility-inhibitory factor of sheep I and II (MIFS-I and II), respectively. Significant (about 60%) inhibition of sperm motility was observed following treatment of cauda epididymal sperm with 6 and 12 µg/mL of partially purified MIFS-I and II, respectively. Specific activities of the partially purified MIFS-I and II were 563 and 261 U/mg of protein, while the fold-purification of the activity were 5119 and 2373, respectively. Both the proteins were heat-labile and the activity was completely lost following incubation at 100°C for 5 min. Further, the partially purified MIFS-I (5 µg/mL) caused significant reduction in in vitro sperm capacitation and slight decline in tyrosine phosphorylated p72 and p52 proteins; however the protein was nontoxic to sperm. Mass spectrometric analysis of MIFS-I revealed significant identity with human semaphorin 3D. Both dot blot and western blot analysis demonstrated cross-reactivity of MIFS-I with polyclonal anti-human SEMA3D antibody. It was concluded that the MIFS-I of ovine CEP was putative ovine semaphorin 3D protein having potent sperm quiescent and decapacitating activities and it possibly acts through inhibition of protein tyrosine phosphorylation.     

Cross-linking a maturation-dependent ram sperm plasma membrane antigen induces the acrosome reaction

ESA152 is a highly hydrophobic 18 kDa sialoglycoprotein, which becomes expressed on ram sperm in the proximal cauda epididymis. ESA 152 is expressed on all regions of the sperm surface, most strongly on the posterior region of the head, most weakly on the anterior region of the head. In this paper, we show that induction of the acrosome reaction with Ca2+ ionophore causes ESA152 to be redistributed from the posterior to the anterior region of the head plasma membrane. Cross-linking ESA152 with bivalent antibody causes similar redistribution and induces the acrosome reaction. Induction of the acrosome reaction with ESA152 antibody requires Ca2+ but is insensitive to (10 ng/ml) pertussis toxin.     

Effect of transportation temperature on the quality of cauda epididymal spermatozoa of ram

The objective of this study was to develop a protocol for ram epididymal sperm preservation that could be applied to wild ruminants for collection and preservation of spermatozoa from dead or hunted animals. Ram testicles collected from abattoirs were used to study the effect of two transportation temperatures viz. ambient temperature (AT) and refrigeration temperature (RT) on the cauda epididymal sperm quality at recovery and during preservation up to 72h at 4°C. For AT the testicles were transported in normal saline in a container (17.9-21.5°C) where as for RT the testicles were transported in an ice-chest (4.9-6°C). The results of the current study revealed that intact acrosome was significantly higher (P<0.01) and other quality parameters like sperm motility, live sperm count, sperm concentration and major sperm abnormalities were also higher (P>0.05) for RT than AT. The mean percent sperm motility for RT and AT was 81.67% and 78.33%, respectively. The corresponding figures were 92.08% and 90.46% for mean live sperm, 98.33% and 90.50% for intact acrosome, 0.50% and 0.33% for major sperm defects. The percent minor abnormality was 79.50% for RT and 77.67% for AT. The most prevalent minor defect was distal cytoplasmic droplet (70-80%). The mean sperm motility for RT and AT at 0h was 82.50% and 75.00%, respectively and the corresponding values at 72h of preservation were 60.00% and 45.83%. The mean live sperm at 0h for RT and AT were 92.92% and 88.92%, respectively and the corresponding figures at 72h were 81.50% and 73.17%. The mean intact acrosome at 0h for RT and AT was 98.58% and 90.58%, respectively and at 72h the corresponding values were 91.66% and 82.25%. The sperm motility, live sperm count and intact acrosome decreased significantly (P<0.05) from 0h to 72h of preservation for both transportation temperatures. The sperm motility, live sperm count and intact acrosome also varied significantly between the transportation temperatures. The major sperm abnormality for both RT and AT at each hour of preservation up to 72h was less than 0.5%. The study concluded that epididymides or testicles should be transported to the laboratory at RT (4.9-6°C) either in an ice-chest or portable refrigerator for their processing, evaluation and storage.     

Lateral regionalization and diffusion of a maturation-dependent antigen in the ram sperm plasma membrane

We have used a monoclonal antibody ESA 152 in fluorescence recovery after photobleaching (FPR) studies of a maturation-dependent surface antigen of ram sperm. The antibody is an immunoglobulin G secreted by a hybridoma derived from NS1 mouse myeloma cells. The ESA 152 antigen is not detectable in testicular sperm. It is localized on the surface of ejaculated sperm where it is present on all regions of the surface, but tends to be concentrated on the posterior region of the head. The ESA 152 antigen can be extracted by detergents or chloroform-methanol. The extracted antigen is sensitive to proteases and migrates with an apparent Mr approximately 30,000 in SDS-containing 10-20% polyacrylamide gradient gels. FPR measurements of ESA 152 lateral mobility in the membrane yield diffusion coefficients in the range 10(-9)-10(-8) cm2/s, values typical of lipids but observed for proteins only at the fluid dynamic limit where diffusion is controlled by lipid fluidity. Immobile fractions, typical of membrane proteins, are observed on all regions. When the antigen is stained by a fluoresceinated Fab fragment of the ESA 152 antibody, the diffusibility is highly regionalized, with particularly low, but rapid, recovery on the midpiece. Cross-linking of the antigen with the intact ESA 152 antibody induces a redistribution in which the antigen is excluded from the posterior head region. This cross-linking is accompanied by increases in ESA 152 diffusibility on both the anterior head and the midpiece.     

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.     

Surface transformation of ram spermatozoa in uterine, oviduct and cauda epididymal fluids in vitro

Genital tract fluids were collected continuously from conscious ewes through catheters inserted surgically into the uterus and oviducts. Cauda epididymal spermatozoa and fluid were obtained through catheters inserted into the transected vas deferens. The washed spermatozoa were labelled using the surface-specific chloroglycoluril-Na125I procedure. High-resolution electrophoretic analysis of sperm plasma membrane preparations revealed a partial loss of a major surface component (i.e. Mr 97,000) during incubation in uterine and oviduct fluids. This specific loss resulted in a shift in radioactivity distribution toward an Mr 24,000 component which had been previously identified as a sialoglycoprotein. No significant changes in the distribution of radiolabelled surface components were detectable when the spermatozoa were incubated in synthetic medium. Incubation of unlabelled spermatozoa in 125I-labelled uterine fluid showed that adsorption of exogenous fluid components was highly selective; an Mr 16,000 polypeptide was greatly enriched on the sperm surface although it was only a minor component in the incubation fluid. Adsorption of labelled oviduct fluid components was also selective and involved predominantly high molecular weight components (i.e. Mr 140,000, 95,000, 78,000, 53,000). When spermatozoa were incubated in labelled cauda epididymal fluid after exposure to unlabelled uterine and oviduct fluids, several fluid components were incorporated by the plasma membrane, indicating that surface renovation of 'capacitated' spermatozoa may be a more general process rather than a specific event. These results suggest that capacitation of ram spermatozoa involves loss of specific surface proteins as well as selective adsorption of exogenous fluid components and point to a polypeptide in uterine fluid as an active constituent.     

The fertilizing capacity of ram testicular spermatozoa,freshly collected and after storage in cauda epididymal fluid

Epididymal fluid may contain substances which promote development of the fertilizing capacity of testicular spermatozoa under in vitro conditions, provided that the spermatozoa are exposed to such substances for long periods of time. In an attempt to resolve this question, the fertilizing capacity of testicular spermatozoa was assessed before and after storage in cauda epididymal fluid and comparisons made with ejaculated spermatozoa from the same rams. Of the 13 eggs examined from the group of ewes inseminated with ejaculated spermatozoa 61.5% were found to be in the 2-to 8-cell stage. No fertilized eggs were recovered from ewes impregnated with freshly collected testicular spermatozoa. Nor were any cleaved eggs obtained from the group of ewes inseminated with testicular spermatozoa stored in cauda epididymal fluid at 4°C for 7 to 11 days. We suggest there-fore, that in order to develop maximal fertilizing capacity, mammalian spermatozoa must be exposed to specific concentrated testicular and epididymal secretions in a sequential order and within strict time limits.     

ATP-induced reactivation of ram testicular, cauda epididymal, and ejaculated spermatozoa extracted with Triton X-100

It was possible to demembrante and reactivate not only freshly collected testicular, cauda epididymal, and ejaculated ram sperm but also sperm that had been stored for several days at 0 degrees C and for several months at -196 degrees C in rete testis fluid or egg yolk citrate media. Sperm were usually washed free of seminal plasma before demembranation, but this was not essential for reactivation. Bovine serum albumin (1.0%) in the wash medium increased the survival of sperm, but more than 0.25% in the extraction medium decreased reactivation. A macro-molecular component of cauda epididymal fluid also inhibited the reactivation of testicular sperm. Triton X-100 concentrations between 0.01% and 1.00% in the extraction medium were satisfactory for demembranating the sperm. Rapid cooling (i.e., cold shock) mimicked the effect of detergent in making the sperm responsive to added ATP and demonstrated that damage to ram sperm in cold shock does not involve the axoneme. Ejaculated and cauda sperm were reactivated immediately on addition of ATP and activity persisted for up to 10 min. Testicular sperm, on the other hand, required about 4 min to become fully reactivated. The optimal ATP concentration for activation of sperm was 0.1-1.0 mM. Magnesium ions (0.1-1.0 mM) were important for reactivation, and testicular sperm required a higher magnesium concentration than did cauda or ejaculated sperm. Manganese ions were almost as effective as magnesium for reactivating cauda epididymal and ejaculated sperm. Cobalt and cadmium ions were much less active for cauda and ejaculated sperm and none of these ions were effective for testicular sperm. Fluoride (25-50 mM) inhibited reactivation. The presence of 50 microM cAMP in the extraction medium or preincubation of testicular sperm with theophylline or caffeine increased low levels of activation, but this was not evident with ejaculated or cauda sperm. We conclude that the motor apparatus is already functionally assembled in spermatozoa on leaving the testis, but some fine adjustment must take place during maturation in the epididymis.     

Changes in lectin-binding features of ram sperm surfaces associated with epididymal maturation and ejaculation

Ram sperm, isolated from the caput, corpus, and cauda epididymidis, plus ejaculated cells were washed free of loosely bound components and tested for their ability to bind fluorescein-conjugated lectins (Con A, SBA, RCA, PNA, ECA and WGA) as assessed by epiluminescent-fluorescence light microscopy and flow cytometry. Detailed preliminary studies established an appropriate lectin-to-sperm ratio and incubation conditions for quantitative comparisons of sperm cell types and permitted a detailed analysis of both the amount of lectin bound as well as its distribution on the various aspects of the cell surface. Con A (mannose positive) bound weakly over the entire surface, with little change associated with maturation in the male tract. SBA (N-acetylgalactosamine positive) bound moderately strongly to caput sperm, with an emphasis on the apical ridge portion of the cell; during epididymal transit this binding was greatly diminished and was regained upon ejaculation. RCA, PNA, and ECA (galactose positive) gave generally equivalent results, where initially strong binding to the entire sperm surface decreased (over all parts of the surface except the anterior head) during epididymal maturation, with no change associated with ejaculation. WGA (sialic acid positive) binding initially was weak, but increased with epididymal transit and ejaculation. In vitro incubations with beta-galactosidase and neuraminidase confirmed the assignments given above. These data, when coupled with previous reports describing the heterogeneous distribution of proteins and lipids and changes in their distribution associated with epididymal maturation, serve to quantitatively describe changes in those aspects of the cell surface that are probably responsible for the acquisition of the capacity of the sperm to bind successfully to the oocyte.     

Localization of a maturation-dependent epididymal sperm surface antigen recognized by a monoclonal antibody raised against a 135-kilodalton protein in porcine epididymal fluid.

A specific 135-kDa protein was purified from porcine cauda epididymal fluid. Analysis of its N-terminal amino acid sequence revealed it to be a new protein. Stable clones of hybridomas that produced monoclonal antibodies against the purified 135-kDa protein were established. A clone, B-11, reacting both with epididymal fluid and with sperm plasma membranes was selected and used in this study. Immunoblotting analysis showed that B-11 reacted only with a 135-kDa protein among epididymal fluid proteins. In contrast, B-11 did not recognize a similar 135-kDa sperm protein but did strongly react with a 27-kDa protein among sperm membrane proteins, extracted by NP-40 in the presence of protease inhibitors. B-11 also reacted only with a 27-kDa protein fragment among trypsin digests of the 135-kDa epididymal protein. The 135-kDa protein was first detected, by ELISA or immunoblotting analysis, at the beginning of the corpus epididymis. Maximal levels were reached in the distal corpus and levels were slightly decreased in the cauda epididymis. On the other hand, the surface of caput sperm were found to contain small amounts of antigen(s), the concentration of which gradually increased during epididymal transit. In immunocytochemical studies, the antigen was detectable in the epithelial cells from the initial segment to the corpus of the epididymis but not in the caudal cells. In the lumen, the presence of the 135 kDa protein was apparent in the corpus (at a maximum in the middle and distal corpus) and to a lesser degree in the caudal lumen. The 27-kDa protein was distributed all over the equatorial region of the acrosome of less than 10% of caput epididymal sperm. As sperm passed through the corpus epididymis, the percentage of immunoreactive cells increased and the protein was restricted to specific domains of the sperm head. Thus, on the mature sperm, antigen was localized in a crescent-shaped area of the equatorial segment just behind the anterior part of the acrosome and on the apical rim of the sperm head. This is the first observation of a sperm surface antigen derived from an epididymal protein as a proteolytic fragment that interacts with specific regions of the sperm membrane during the process of spermatozoa maturation.     

Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system

Studies were carried out to analyze the cryoprotecting efficacy of several amino acids by use of a chemically defined synthetic medium (modified Ringer's solution) and goat cauda epididymal sperm as the model system. Motile goat cauda sperm dispersed in the synthetic medium were subjected to a freezing protocol in a computer-controlled bio-freezer, cooling 0.25 degrees C x min(-1) to 5 degrees C, 5 degrees C x min(-1) to -20 degrees C, and 20 degrees C x min(-1) to -100 degrees C, prior to being plunged into liquid nitrogen. In the absence of amino acids, sperm cells completely lost their flagellar motility. Of all the amino acids tested, l-alanine showed maximal cryoprotection potential. l-Alanine at 135 mM offered optimum cryoprotection potential: recovery of sperm forward motility and total motility were 14 +/- 2% and 19 +/- 2%, respectively. l-Glutamine, l-proline, and glycine at optimum concentration (100-150 mM) cryopreserved approx. 11-17% total motility of the sperm cells, whereas amino acids such as l-arginine, l-lysine, and l-histidine offered little cryoprotection (0-5%) to the cells. Increasing the amino acid concentration beyond the optimum level sharply decreased the recovery of the sperm motility, which therefore showed a biphasic cryoprotection profile. Addition of amino acids enhanced (approx. 7-10%) the cryoprotection efficacy of the well-known cryoprotectants glycerol and a combination of glycerol and dimethyl sulfoxide. The presence of glycerol caused a marked reduction (from 100-150 mM to 20-70 mM levels) in the optimal cryoprotective concentration of the amino acids. The combined cryoprotecting action of glycerol, dimethyl sulfoxide, and amino acids provided motility recovery as high as 52%. The observation that amino acids and dimethyl sulfoxide had an additive effect in augmenting the cryoprotecting potential of glycerol suggests that the mechanism of their action is different from that of glycerol. This cocktail of cryoprotectants may be useful for cryopreservation of semen of various species.     

Changes in binding of a 27-kilodalton chimpanzee cauda epididymal protein glycoprotein component to chimpanzee sperm

Motility patterns of caput epididymal chimpanzee sperm, caput epididymal chimpanzee sperm incubated in vitro with chimpanzee cauda epididymal fluid, and cauda epididymal chimpanzee sperm were assessed quantitatively. Sperm recovered from the caput epididymis showed no motility, whereas sperm recovered from cauda epididymis showed progressive forward motility. After incubation in cauda fluid, approximately 25% of caput epididymal sperm showed some motile activity. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed that the surface of caput epididymal sperm, incubated in cauda fluid, was modified by the appearance of a major protein-glycoprotein surface component with an apparent molecular weight of 27 kilodaltons (kD). THis 27-kD component was not detected on caput epididymal sperm incubated in buffer or in caput fluid. However, it was present in cauda fluid and on cauda epididymal sperm. Binding to caput epididymal sperm was cell specific in that chimpanzee erythrocytes incubated in cauda fluid did not bind this 27-kD cauda fluid component. Motility patterns of ejaculated chimpanzee sperm and of ejaculated chimpanzee sperm incubated in the uterus of adult female chimpanzees also were assessed quantitatively. Ejaculated sperm showed progressive forward motility, whereas in utero incubated ejaculated sperm showed hyperactivated motility typical of capacitated sperm. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed the loss of a 27-kD component from the surface of ejaculated sperm after in utero incubation. No significant change in the ¹²⁵I-distribution pattern was detectable when ejaculated sperm were incubated in buffer. These results suggest that the lumenal fluid component, which becomes adsorbed to the surface of chimpanzee sperm during maturation in the epididymis and which is removed from the surface of mature chimpanzee sperm in the female reproductive tract, affects sperm motility.     

Preliminary characterization of the multiple forms of ram sperm hyaluronidase.

An investigation was made of the inter-relationships and characteristics of various hyaluronidase forms isolated from ram spermatozoa. They were shown to be members of an oligomeric series, apparently formed by intermolecular disulphide cross-linking. Two monomer species were detected, alpha (Mr 89,600) and beta (Mr 81,200). Although the alpha species predominated, the two were evenly distributed throughout the oligomer population, and they shared antigenic determinants; the beta species did not arise from the alpha species as a result of catabolism following cell disruption. The oligomeric series was of the form [Hyal]n, where n = 1, 2, 4, 5, 6, 7 etc.; no trimer was detectable. Though essentially cationic, part of the hyaluronidase population also had anionic characteristics, probably due to oxidation of free thiol groups. In the anionic subpopulation tetramers and higher oligomers predominated, whereas the non-anionic subpopulation was composed of monomers, dimers and tetramers. The pH optimum of the monomer was 4.3 in 0.2 M-NaCl/0.1 M-sodium citrate, whereas that of the anionic oligomers was 4.9. Both serum albumin and polylysine stimulated enzyme activity at pH 4.0 in the absence of NaCl; polylysine was particularly effective. NaCl diminished the stimulatory effects, and essentially suppressed them above the pH optimum. The specific activities of different oligomer populations were the same as that of the monomer, and conversion of oligomers into monomer by reduction had likewise no effect upon the specific activity. Low concentrations of poly(vinyl alcohol), poly(ethylene glycol) or polyvinylpyrrolidone stabilized soluble hyaluronidase activity by preventing the enzyme's binding to surfaces; solutions of anionic oligomers were further stabilized by NaCl. Enzyme preparations were stable for several months frozen in the presence of poly(vinyl alcohol) and salt.     

Characterization of secretory proteins from cultured cauda epididymal cells that significantly sustain bovine sperm motility in vitro

Epididymis provides a safe environment in which stored-spermatozoa could survive for days before ejaculation. In vitro studies suggested that epididymal proteins seem to be implicated in sperm survival during coincubation with cultured epididymal cells. This study was basically designed to confirm if secretory proteins from bovine epididymal cell cultures provide sperm protection against rapid loss of sperm motility in vitro. Bovine spermatozoa were incubated in conditioned media (CM), which were prepared from cultured cauda epididymal cell (CEC). Motion parameters were recorded using a computer-assisted sperm analyzer. Sperm-free protein extracts from CM were fractionated by ultrafiltration through a 10-kDa cut off membrane. A significantly positive effect on sperm motility was observed when spermatozoa were incubated in CM (54 +/- 4%) and CM > 10 kDa (57 +/- 4%) compared to CM < 10-kDa fraction (30 +/- 3%) or fresh media (34 +/- 3%), after a 6-hr incubation period. This beneficial effect on sperm motility was abolished when the CM > 10-kDa fraction was heat-treated at 100 degrees C for 10 min. The CM > 10 kDa fraction provides factors that remained active even though spermatozoa were washed twice after a 2-hr preincubation period. To identify potential beneficial factors, bovine spermatozoa were incubated with radiolabeled proteins obtained using (35)S-methionine in culture medium. SDS-PAGE analysis of proteins extracted from CM-preincubated spermatozoa revealed the presence of a 42-kDa protein strongly associated to the sperm surface. This 42-kDa spot was trypsin-digested and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as a protein homologue to a 35-kDa bovine estrogen-sulfotransferase. This protein can play a role in epididymal biology and sperm function. Taken together, these results suggest that specific epididymal proteins can be implicated in the sperm protection in vitro, and can be characterized in our cell culture system.     

Characterization of cAMP-dependent protein kinase and its endogenous substrate proteins in ram testicular,cauda epididymal,and ejaculated spermatozoa

Mammalian spermatozoa have been shown to possess cAMP-dependent protein kinase (A-PK) and endogenous substrate proteins for this enzyme. A study of the kinase system was undertaken to determine changes that may be associated with sperm maturation by comparing immature testicular with mature cauda epididymal and ejaculated spermatozoa. Absolute activity levels of A-PK, stimulated over a concentration range of 10^?9 to 10^?5 M, was significantly greater in testicular than ejaculated spermatozoa. At an optimal cAMP concentration (10^?6M), testicular spermatozoa had significantly greater amounts of cAMP-dependent protein kinase activity than did cauda or ejaculated spermatozoa. Electrophoretic analysis and autoradiography of NP-40-soluble protein extracts revealed the presence of two substrate proteins (M_r = 62,000 and 44,000) in all three types of spermatozoa. In addition, a phosphoprotein (M_r = 20,000) was detected in mature cauda and ejaculated but not immature testicular spermatozoa. The phosphorylation of these substrate proteins was both dose and time dependent. Examination of cyclic AMP phosphodiesterase activity revealed significantly higher levels in testicular than ejaculated spermatozoa. These results indicate marked alterations in cAMP-modulated protein phosphorylation and dephosphorylation systems in ram spermatozoa during epididymal maturation.     

Tyrosine phosphorylation as evidence of epididymal cauda participation in the sperm maturation process of Corynorhinus mexicanus bat

The bat Corynorhinus mexicanus provides an interesting experimental model for the study of epididymal sperm maturation because after spermatogenesis and the regression of the testes, this bat stores sperm in the epididymal cauda for several months. Earlier research conducted by our group suggested that sperm maturation in this species must be completed in the caudal region of the epididymis. One of the major signal transduction events during sperm maturation is the tyrosine phosphorylation of sperm proteins. The aim of the present study was to comparatively evaluate tyrosine phosphorylation in spermatozoa obtained from the caput, corpus and cauda of the epididymis during the sperm storage period. The maturation status of the sperm was determined by the percentage of capacitation and tyrosine phosphorylation in sperm obtained from the epididymis. The highest proportion of tyrosine phosphorylation was registered after the sperm had reached the cauda epididymis during the middle of the storage period. In conclusion, in Corynorhinus mexicanus and most likely in other chiropteran species with an asynchronous male reproductive pattern, epididymal sperm maturation ends in the caudal region of the epididymis and is related to the time that the sperm remains in the epididymis before mating activity.