The development of regionalized lipid diffusibility in the germ cell plasma membrane during spermatogenesis in the mouse

The lipids and proteins of sperm cells are highly regionalized in their lateral distribution. Fluorescence recovery after photobleaching studies of sperm membrane component lateral diffusibility have shown that the sperm plasma membrane is also highly regionalized in the extents and rates of diffusion of its surface components. These studies have also shown that regionalized changes in lateral diffusibility occur during the differentiative processes of epididymal maturation and capacitation. Unlike mammalian somatic cells, sperm cells exhibit large nondiffusing lipid fractions. In this paper, we will show that both regionalized lipid diffusibility and nondiffusing lipid fractions develop with the morphogenesis of cell shape during spermatogenesis in the mouse. Pachytene spermatocytes and round spermatids show diffusion rates and the nearly complete recoveries (80-90%) typical of mammalian somatic cells. In contrast, stage 10-11 condensing spermatids, testicular spermatozoa, cauda epididymal spermatozoa, as well as the anucleate structures associated with these later stages of spermatogenesis (residual bodies and the cytoplasmic droplets of condensing spermatids and testicular spermatozoa), exhibit large nondiffusing fractions. Both the diffusion rates and diffusing fractions observed on the anterior and posterior regions of the head of stage 10-11 condensing spermatids are the same as the values obtained for these regions on testicular spermatozoa. Possible mechanisms of lipid immobilization and possible physiological implications of this nondiffusing lipid are discussed.     

ATP-binding cassette transporter G2 activity in the bovine spermatozoa is modulated along the epididymal duct and at ejaculation

During their epididymal maturation, stabilizing factors such as cholesterol sulfate are associated with the sperm plasma membrane. Cholesterol is sulfated in epididymal spermatozoa by the enzyme estrogen sulfotransferase. Because of its role in the efflux of sulfate conjugates formed intracellularly by sulfotransferases, the ATP-binding cassette membrane transporter G2 (ABCG2) might have a role in the translocation of this compound across the plasma membrane. In the present study we showed that ABCG2 is present in the plasma membrane overlaying the acrosomal region of spermatozoa recovered from testis, epididymis, and after ejaculation. Although ABCG2 is also present in epididymosomes, the transporter is not transferred to spermatozoa via this mechanism. Furthermore, although epididymal sperm ABCG2 was shown to be functional, as determined by its ability to extrude Hoechst 33342 in the presence of the specific inhibitor Fumitremorgin C, ABCG2 present in ejaculated sperm was found to be nonfunctional. Additional experiments demonstrated that phosphorylation of ABCG2 tyrosyl residues, but not its localization in lipid rafts, is the mechanism responsible for its functionality. Dephosphorylation of ABCG2 in ejaculated spermatozoa is proposed to cause a partial protein relocalization to other intracellular compartments. Prostasomes are proposed to have a role in this process because incubation with this fraction of seminal plasma induces a decrease in the amount of ABCG2 in the associated sperm membrane fraction. These results demonstrate that ABCG2 plays a role in epididymal sperm maturation, but not after ejaculation. The loss of ABCG2 function after ejaculation is proposed to be regulated by prostasomes.     

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.     

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.     

Development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane

Ram spermatozoa were obtained from different regions (caput, corpus, and cauda) of the epididymis and their plasma membrane was removed using a nitrogen cavitation treatment (750 psi, 10 min equilibration at 4 degrees C). Membrane was recovered after sucrose gradient centrifugation and identified using 125I-succinylated concanavalin A (125I-succConA) as a surface marker. Based on fluorescein isothiocyanate-succConA (FITC-succConA) labeling and electron microscopy, cavitation removed plasma membrane from the anterior sperm head in the area overlying the acrosome. Cholesterol was the major sterol in plasma membrane, with desmosterol present in sperm entering the epididymis (caput sperm) but negligible in sperm after epididymal transit (cauda sperm). Ethanolamine and choline phosphoglycerides represented 70-80% of membrane phospholipids, with the ethanolamine fraction decreasing relative to choline phosphoglycerides during epididymal transit. The molar ratio of cholesterol to phospholipid increased in the plasma membrane during maturation. The bulk phospholipid-bound fatty acids consisted primarily of palmitoyl acyl groups (16:0) in caput sperm and docosahexaenoyl acyl groups (22:6) in cauda sperm. The choline phosphoglyceride fraction was purified and analyzed. It consisted of a mixture of ether acyl glycero-3-phosphocholine and diacyl phosphoglyceride, with the dominant acyl residue, at all stages of epididymal maturation, being 22:6 throughout epididymal transit. The significance of these findings relative to acquisition of fertilization capacity by sperm during epididymal maturation is discussed.     

Lectin binding sites on the plasma membrane of epididymal and ejaculated chimpanzee sperm

Lectins have been used to analyze variations in the distribution and density of exposed saccharides of the sperm plasma membrane during physiologic maturation and after ejaculation. Studies have been conducted in a number of nonprimate species but have been conducted to only a limited extent in nonhuman primates. In this study, pure suspensions of chimpanzee sperm from the caput and cauda epididymis and from the ejaculate were labeled with lectins conjugated to fluorescein isothiocyanate in order to visualize changes in the distribution of exposed membrane glycocomponents. The lectins used were Con A, DBA, RCA-I, and WGA. Con A binding showed minimal change during epididymal transit, with an increased binding to the flagellum after ejaculation. DBA binding was relatively constant in all specimens. RCA-I showed distinct changes in binding pattern between epididymal and ejaculated sperm. On ejaculated sperm strong fluorescence was limited to the posterior head and to the midpiece. WGA binding increased during epididymal passage and decreased after ejaculation. There appears to be a wide variety of saccharide groups available for lectin binding on the surface of epididymal and ejaculated chimpanzee sperm. The general similarity in binding patterns of caput and cauda epididymal chimpanzee sperm exposed to Con A and DBA might reflect the fact that sperm morphology does not change during epididymal transit in this species, thus implying a more stable membrane structure than is present in other primates so far studied.     

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.     

Évolution de la composante lipidique de la membrane plasmique des spermatozoïdes durant la maturation épididymaire

One aspect of mammalian post-testicular sperm maturation is the progressive change in their plasma membrane lipid composition. These modifications in lipids allow sperm cells to fuse with oocytes during fertilization. A significant share of these sperm lipid changes occurs during their descent through the epididymal tubule. It then continues within the female genital tract during the capacitation process, an essential prerequisite for acrosomic reaction and hence fertilization. This review presents what is known concerning the sperm plasma membrane lipid changes during epididymal maturation in various mammalian models. In the first section, after a brief presentation of the classic eukaryotic cell plasma membrane lipid organization, the emphasis is on the particularities of sperm plasma membrane lipids. The second section presents the different changes occurring in the three major classes of lipids (i.e. phospholipids, sterols and fatty acids) during the sperm’s epididymal descent. The final section briefly describes the mechanisms by which these lipid changes might happen in the epididymal lumen environment. The role played by lipid-rich vesicles secreted by the epididymal epithelium via apocrine secretory processes is highlighted.     

Regulation of calcium content in bovine spermatozoa

Plasma membrane vesicles isolated from bovine epididymal and ejaculated spermatozoa have widely different capabilities for transporting Ca2+. Spermatozoa were ruptured by nitrogen cavitation, and the plasma membrane fraction was harvested after low speed and sucrose gradient centrifugation; purity was assessed by marker enzyme analyses, electron microscopy, and sedimentation properties. Plasma membrane vesicles isolated from epididymal sperm accumulate Ca2+ passively at a faster rate and to a greater extent than vesicles prepared from ejaculated sperm. Ca2+ transport across bovine sperm plasma membranes is an ATP-independent, Na+-dependent process that obligatorily exchanges intravesicular Na+ for external Ca2+. The rate of Na+/Ca2+ exchange is significantly lower in ejaculated sperm vesicles than in those of epididymal sperm. Bovine plasma membranes contain little or no Ca2+-dependent ATPase activity. It is suggested that, at the time of ejaculation, calcium flux into bovine sperm is prevented by the interaction of the plasma membrane with putative factors in seminal fluid that specifically interfere with Na+/Ca2+ exchange. We have isolated a protein from seminal plasma that prevents calcium accumulation by bovine epididymal sperm (Rufo, G. A., Jr., Singh, J. P., Babcock, D. F., and Lardy, H. A. (1982) J. Biol. Chem. 257, 4627-4632). A protein with properties resembling those of the seminal calcium transport inhibitor is found on the membrane vesicles from ejaculated sperm but not on membranes from epididymal sperm. We conclude that this protein binds strongly to the plasma membrane of bovine sperm and is responsible for preventing calcium uptake by ejaculated sperm.     

Changes in sperm plasma membrane lipid diffusibility after hyperactivation during in vitro capacitation in the mouse

We have used the technique of fluorescence recovery after photobleaching to measure the diffusibility of the fluorescent lipid analogue, 1,1'-dihexadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate on the morphologically distinct regions of the plasma membranes of mouse spermatozoa, and the changes in lipid diffusibility that result from in vitro hyperactivation and capacitation with bovine serum albumin. We found that, as previously observed on ram spermatozoa, lipid analogue diffusibility is regionalized on mouse spermatozoa, being fastest on the flagellum. The bovine serum albumin induced changes in diffusibility that occur with hyperactivation are also regionalized. Specifically, if we compare serum incubated in control medium, which maintains normal motility, with those hyperactivated in capacitating medium, we observe with hyperactivation an increase in lipid analogue diffusion rate in the anterior region of the head, the midpiece, and tail, and a decrease in diffusing fraction in the anterior region of the head.     

Lipid phase transition in the plasma membrane of the goat epididymal maturing spermatozoa

Microviscosity of the highly purified plasma membranes isolated from the maturing goat caput, corpus and cauda epididymal sperm, was measured using l,6-diphenyl-l,3,5-hexatriene as the lipophilic probe at varying temperatures (12–42°C). As shown by the Arrhenius plot of the data each of the maturing sperm membranes had two distinct lipid phase transitions in the temperature zones 19–25°C and 34–37°C. The low-temperature transitions for the immature caput- and mature cauda-sperm membranes were noted at 19–20°C, and 24–25°C, respectively, whereas both these membranes showed high temperature transition at 36–37°C. The maturing corpus-sperm membrane had phase transitions at 21–22°C and 35–36°C that were significantly different from those of the immature/mature sperm membranes. The data implicate significant alteration of the sperm membrane structure during epididymal maturation. The phase transition of the mature male gametes at 36–37°C may have a great impact on the subsequent events of the sperm life cycle since the mature spermatozoa that are stored in the epididymis a few degrees below the body temperature, experience higher temperature when ejaculated into the female reproductive tract.     

Lipid dynamics in the plasma membrane of ram and bull spermatozoa after washing and exposure to macromolecules BSA and PVP.

Seminal plasma proteins and macromolecules in the external medium have a major influence on the functionality of sperm plasma membranes. In this investigation we have examined their effects on lipid diffusion in the surface membrane of ram and bull spermatozoa as measured by fluorescence recovery after photobleaching (FRAP). Results show that progressive removal of seminal plasma from ram spermatozoa by repeated centrifugation and resuspension in media +/- 4% bovine serum albumin (BSA) or 0.4% polyvinlypyrrolidone (PVP) causes a reduction in lipid diffusion in all regions of the membrane. By contrast, bull sperm membranes respond with an increase in diffusion in all regions. Repeated washing of bull spermatozoa whose membranes were previously immobile (i.e., showed no recovery after FRAP) restored lipid diffusion suggesting an inhibitory effect of seminal plasma proteins. Further analysis by atomic force microscopy revealed a close association between BSA and the plasma membrane. It is concluded that diffusion of lipids in the plasma membrane of ejaculated ram and bull spermatozoa is influenced by seminal plasma proteins and the composition of the suspending medium. Mol. Reprod. Dev. 59:306-313, 2001.     

Seminal plasma proteins regulate the association of lipids and proteins within detergent-resistant membrane domains of bovine spermatozoa

Maturing spermatozoa acquire full fertilization competence by undergoing major changes in membrane fluidity and protein composition and localization. In epididymal spermatozoa, several proteins are associated with cholesterol- and sphingolipid-enriched detergent-resistant membrane (DRM) domains. These proteins dissociate from DRM in capacitated sperm cells, suggesting that DRM may play a role in the redistribution of integral and peripheral proteins in response to cholesterol removal. Since seminal plasma regulates sperm cell membrane fluidity, we hypothesized that seminal plasma factors could be involved in DRM disruption and redistribution of DRM-associated proteins. Our results indicate that: 1) the sperm-associated proteins, P25b and adenylate kinase 1, are linked to DRM of epididymal spermatozoa, but were exclusively associated with detergent-soluble material in ejaculated spermatozoa; 2) seminal plasma treatment of cauda epididymal spermatozoa significantly lowered the content of cholesterol and the ganglioside, GM1, in DRM; and 3), seminal plasma dissociates P25b from DRM in epididymal spermatozoa. We found that the seminal plasma protein, Niemann-Pick C2 protein, is involved in cholesterol and GM1 depletion within DRM, then leading to membrane redistribution of P25b that occurs in a very rapid and capacitation-independent manner. Together, these data suggest that DRM of ejaculated spermatozoa are reorganized by specific seminal plasma proteins, which induce lipid efflux as well as dissociation of DRM-anchored proteins. This process could be physiologically relevant in vivo to allow sperm survival and attachment within the female reproductive tract and to potentiate recognition, binding, and penetration of the oocyte.     

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.     

Changes in sperm surface membrane and luminal protein fluid content during epididymal transit in the boar

The surface membrane protein of boar sperm and the proteins in the fluid surrounding the gametes were analyzed during epididymal transit. The present study demonstrated that sequential dramatic changes occur in protein composition of the sperm membrane and epididymal fluid during epididymal transit. The maturation process of the boar sperm surface was characterized by a complex sequential evolution of the composition and orientation of macromolecules in the sperm membrane. Epididymal maturation resulted in the progressive disappearance of most of the surface testicular compounds, which were either renewed or masked by new permanent or transient low molecular weight polypeptides on the boar sperm surface membrane. In the fluid surrounding the spermatozoa, composition of the luminal proteins was altered throughout the epididymal transit and several new compounds were characterized. Very few proteins were correlated either with blood plasma or sperm surface compounds.     

Boar sperm plasma membrane protein profile: Correlation with the zona-free hamster ova bioassay

This study was designed to compare differences among porcine sperm plasma membrane proteins with the ability of spermatozoa to interact with zona-free hamster ova. Sperm plasma membrane vesicles were recovered from 24 ejaculates from 10 fertile boars, and from cauda epididymal spermatozoa from 3 fertile and 1 very subfertile boar. Solubilized sperm plasma membrane proteins were run on 1D SDS-PAGE gels, transferred to western blots, stained, and analyzed for quantity of protein per band by scanning laser densitometry. Variation in the quantities of individual sperm plasma membrane proteins in the 20 identified bands were statistically compared with the ability of spermatozoa from the same ejaculate to penetrate zona-free hamster ova. The percentages of plasma membrane protein present in 3 bands (90, 84 and 60 kD) were positively correlated with the ability of spermatozoa from the same ejaculate to fuse with zona-free hamster ova (P = 0.002, 0.01, 0.04; R = 0.53, 0.40, 0.38, respectively). The quantities of protein in 2 other bands (69 and 35 kD) were significantly but negatively correlated with the results of the zona-free hamster ova bioassay (P = 0.02, 0.01; R = -0.42, -0.37, respectively). The sperm plasma membrane profiles were quantitatively similar between the ejaculated samples and the fertile epididymal samples. Six epididymal sperm plasma membrane proteins were present in statistically different quantities in the subfertile boar sample and the 3 fertile controls. The 90 kD band positively correlated with the hamster ova bioassay in the ejaculated samples was not detected in the subfertile epididymal sperm plasma membrane sample. These results suggest that protein(s) in one or more of the 3 positively correlated ejaculated sperm plasma membrane protein bands may be involved in sperm-oocyte interaction.     

Epididymal SPAM1 is a marker for sperm maturation in the mouse

Sperm adhesion molecule 1 (SPAM1), is a glycosyl phoshatidylinositol-linked sperm membrane protein that is dually expressed in testis and epididymis. Epididymal SPAM1 is secreted in all three regions of the epididymis in all mammalian species studied, including humans. It shares the same molecular mass and neutral hyaluronidase activity as the testicular and sperm isoforms that are responsible for the penetration of the cumulus during fertilization. Using wild-type (W/T) sperm and those from mice homozygous for either a null (Spam1-/-) or mutant Spam1 allele, which results in decreased mRNA and protein, we demonstrate that sperm binding of epididymal SPAM1 occurs in vitro after exposure to W/T sperm-free epididymal luminal fluid (ELF). Binding or adsorption that occurred after incubation at room temperature or 32 degrees C was detected immunocytochemically and confirmed quantitatively using flow cytometry. The localization of SPAM1 on the plasma membrane of Spam1-null sperm mimicked that seen in the W/T. The remarkable increase in binding on W/T caudal sperm indicates that they are not fully saturated with SPAM1 during storage, and suggests that uptake of epididymal SPAM1 in vivo augments testicular SPAM1. Spam1-null sperm exposed to W/T ELF for 45-60 min during in vitro capacitation to allow epididymal SPAM1 binding showed a highly significant (P < 0.001) increase in cumulus penetration after 6-7 h compared to those incubated in ELF from null males. Similarly, the number of cumulus-free oocytes was also highly significantly greater (P < 0.001) than that for sperm capacitated in W/T SPAM1-antibody-inhibited ELF. Because epididymal SPAM1 uptake significantly increases cumulus penetration, we conclude that it is a marker of sperm maturation.     

Lipid diffusion in the plasma membrane of ram and boar spermatozoa during maturation in the epididymis measured by fluorescence recovery after photobleaching

Maturation of spermatozoa in the epididymis involves remodelling of many protein and lipid components of the plasma membrane. In this investigation we have examined whether (a) diffusion of lipid molecules in the surface membrane changes during epididymal maturation; (b) diffusion is spatially restricted; and (c) differences in lipid diffusion can be related to known changes in membrane composition. For this purpose we have used the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusion of the lipid reporter probe ODAF (5‐(octa‐decanoyl)aminofluorescein) in spermatozoa from two species: ram, where substantial changes in membrane lipids occur during passage through the epididymis, and boar, where there are relatively few changes. Results on ram spermatozoa show that between the testis and cauda epididymidis, diffusion coefficients values (D) for ODAF increase significantly in all the surface domains. Percentage recovery values (%R) remain constant irrespective of maturational status. In boar spermatozoa, however, D and %R values do not change significantly between epididymal regions. Cholesterol, which has widespread effects on the behaviour of lipid molecules in cell membranes, was visualized by binding of filipin. In both species filipin was concentrated over the acrosomal domain and cytoplasmic droplet of testicular spermatozoa, but in the epididymis it had a heterogenous distribution over the whole head and tail. These results are discussed in relation to the establishment and maintenance of lipid domains in spermatozoa and their influence on development of fertilizing capacity. Mol. Reprod. Dev. 52:207–215, 1999. © 1999 Wiley‐Liss, Inc.     

Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies

The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. Immunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bands of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.     

Adenine nucleotide changes at initiation of bull sperm motility.

Testicular and cauda epididymal sperm were obtained via catheters previously implanted in the rete testis and proximal vas deferens of bulls and were used to examine the relationships among sperm motility, cyclic adenosine 3':5'-monophosphate (cAMP) level, adenine nucleotide levels, and rates of glucose and oxygen consumption. Testicular, cauda epididymal, and ejaculated sperm contain cAMP-stimulated protein kinase, adenylate cyclase, and nucleotide phosphodiesterase. Treatment of the nonmotile testicular sperm with phosphodiesterase inhibitors resulted in a doubling of cellular cAMP concentration and a 25% increase in their glucose consumption. No change in motility, ATP level, or rate of oxygen consumption was observed. Sperm in neat cauda epididymal semen had flagellating tails but no progressive motility. Dilution of these sperm into glucose-containing buffer resulted in an increase in intracellular cAMP concentration and a decrease in ATP level with concomitant increases in ADP and AMP levels. These biochemical changes occurred within 30 s after dilution and apparently preceded the initiation of progressive motility by most cells. Since sperm in neat cauda epididymal semen became progressively motile when diluted with neat cauda epididymal plasma as well as accessory sex gland fluid or buffer, composition of the fluid surrounding the sperm is not responsible for the initiation of progressive motility upon dilution nor does cauda epididymal plasma contain an inhibitory factor. Perhaps release from contact immobilization provides the stimulation for the initial acquisition of progressive motility by cauda epididymal sperm. We conclude that during epididymal passage sperm develop from a cell physically unresponsive to changes in cAMP concentration to a form which initiates progressive motility upon changes in cAMP concentration.