Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis.

To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures.     

Alterations in distribution of surface and intracellular antigens during epididymal maturation of rat spermatozoa

The surface membrane of mammalian spermatozoa is known to undergo considerable conformational and organizational changes during epididymal maturation. However, much less is known about remodelling of intracellular membranes. In this communication we have used specific immunological markers to study the behavior of several antigens both on and within rat spermatozoa as they mature in the epididymis. Four monoclonal antibodies (McAbs) designated 5B1, 1B5, 2D6, and 1B6 were used to probe testicular and caput and cauda epididymal spermatozoa by indirect immunofluorescence and immunogold labeling techniques. None of the McAbs bound to testicular spermatozoa; in all cases, they became reactive only on spermatozoa which had reached the caput epididymis. McAb 5B1 was restricted to the outer acrosomal membrane (OAM) of the acrosomal cap domain. The epitope first appeared on antigen(s) with molecular mass (Mr) of approximately 200 kDa in immature spermatozoa, but later in mature spermatozoa the antigen(s) had Mr of approximately 160 kDa. The antigen(s) recognized by 1B5 McAb on the other hand was initially distributed over the OAM of the entire acrosomal domain (cap + equatorial segment), but during maturation it became progressively more restricted in area until in cauda spermatozoa only the anterior tip of the OAM bound the McAb. McAb 2D6 also bound to the entire OAM and acrosomal contents of caput spermatozoa, but, unlike 5B1 and 1B5 McAbs, reactivity was transient. That is, staining was first detected in caput spermatozoa but then disappeared in corpus and cauda spermatozoa. In contrast to all of the above, 1B6 McAb bound to the surface membrane overlying the entire head domain of caput spermatozoa, but during maturation it became restricted to the postacrosomal domain. These results indicate that, in addition to remodeling of the surface membrane during epididymal maturation, extensive processing of intracellular membrane antigens also takes place and that it is very active within the acrosome. The nature of these intracellular processing events remains to be elucidated, but they may have important consequences for membrane fusion and cell recognition phenomena during fertilization.     

Alpha-mannosidase activity in stallion epididymal fluid and spermatozoa

The expression of α-D-mannosidase activity was fluorometrically and electrophoretically assessed in spermatozoa, epididymal fluid and homogenates of stallion epididymal tissue. Enzyme activity had regional differences; it was higher (P < 0.05) in samples from the cauda epididymal region than in samples from the proximal caput region (largely composed of efferent ducts). Based on enzyme activity, as a function of pH of the assay substrate, electrophoretic analysis in native and native/SDS-PAGE conditions, and the effect of inhibitors or activators, we inferred the presence of at least two catalytically active forms of α-D-mannosidase. The neutral form of the enzyme (α-mannosidase II) was activated by Co²⁺, whereas the acid form (optimum pH 3.5 to 4.0) was sensitive to swainsonine (an inhibitor of α-mannosidase I), stabilized or stimulated by Zn²⁺, and not activated by Co²⁺ (activator of the neutral form). The activity of the acid form of the enzyme was highest in the epididymal fluid, where it seemed to be mainly in a secretory form. This form of the enzyme may have a role in plasma membrane remodeling associated with sperm maturation. In contrast, the activity of α-mannosidase II was higher in mature spermatozoa. It has been postulated that α-mannosidase II may act as a receptor in the recognition and binding of the complementary carbohydrate moieties present on the zona pellucida. With non-denaturing electrophoresis, α-D-mannosidase had an electrophoretic mobility of 0.35 and 0.24. When resolved by 1D and 2D SDS-PAGE (under denaturing conditions) the enzyme had a major protein band of molecular weight 154 kDa in spermatozoa and epididymal samples. Based on its properties under native conditions, we inferred that this enzyme might interact with other proteins and form transitory aggregates.     

Capacity of rete testicular and cauda epididymal boar spermatozoa to undergo the acrosome reaction and subsequent fusion with egg plasma membrane

The capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane was examined in rete testicular and cauda epididymal spermatozoa from boars. Sperm penetration assay using zona-free hamster eggs demonstrated that the penetration rates for rete testicular spermatozoa preincubated for induction of the acrosome reaction for 2 and 3 h were 55% and 97%, respectively. However, most of the eggs (93%) were penetrated with polyspermy by cauda epididymal cells preincubated for 2 h. Results obtained by the triple-stain technique revealed the percentages of acrosome-reacted spermatozoa in the rete testicular and cauda epididymal samples preincubated for 3 h to be 61% and 74%, respectively. These results indicate that many rete testicular spermatozoa possess the capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane in vitro, which appears to be completely established only after sperm transit through at least the proximal part of the epididymis. © 1993 Wiley-Liss, Inc.     

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.     

Reduction of intramembranous particles in the periacrosomal plasma membrane of boar spermatozoa during in vitro capacitation: a statistical study

Membrane remodeling in the periacrosomal plasma membrane (PAPM) of boar spermatozoa during incubation in capacitation medium was examined by the freeze-fracture technique. In the preservation medium (PM) group, the major small (about 8 nm) intramembranous particles (IMP) and the minor large (> 10 nm) IMP were distributed evenly in the PAPM. The IMP-free area increased during capacitation. To correct the IMP-free area, arithmetically redistributed (ARD)-IMP density was used for statistical analysis. In the PM group, the mean density +/- SD of large IMP was 379 +/- 64 and 266 +/- 58/microm2, and that of small IMP was 1450 +/- 155 and 672 +/- 252/microm2 in protoplasmic (P) and external (E) faces, respectively. During capacitation, the significant (P < 0.01) reduction of large IMP density was encountered only in the E face of a few incubation groups, while that of the small IMP density occurred in the P face by 2 h. Consequently, reduction of the total IMP density of both faces was not significant in the large IMP, but it was significant (P < 0.01) in the small IMP. One-fifth of the total small IMP density reduced by 2 h. Filipin-sterol complexes (FSC) were numerous in the PAPM, and FSC-free areas also increased during capacitation. The mechanism of IMP-free area formation and the behavior of the small IMP in the PAPM during capacitation were discussed in relation to membrane stability.     

Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa

A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2-4 h Giemsa staining at 25-40° C is recommended for stallion semen.     

Cell surface changes in the proteins of rabbit spermatozoa during epididymal passage

Differences in the exposure of spermatozoa surface components during epididymal passage have been examined using lactoperoxidase-catalyzed ¹²⁵I-iodination or labeling with ¹²⁵I-diazodiiodosulfanilic acid. Labeled surface proteins obtained from caput and cauda epididymides were solubilized in detergent, separated by sodium dodecylsulfate polyacrylamide slab gel electrophoresis, and identified by radiography. Densitometer scans of autoradiograms revealed increased amounts or exposures of surface proteins of ～35,000, ～39,000, ～50,000, and ～78,000 molecular weight on the cauda epididymal spermatozoa.     

The development of signal transduction pathways during epididymal maturation is calcium dependent

Capacitation has been correlated with the activation of a cAMP-PKA-dependent signaling pathway leading to protein tyrosine phosphorylation. The ability to exhibit this response to cAMP matures during epididymal maturation in concert with the ability of the spermatozoa to capacitate. In this study, we have addressed the mechanisms by which spermatozoa gain the potential to activate this signaling pathway during epididymal maturation. In a modified Tyrode's medium containing 1.7 mM calcium, caput spermatozoa had significantly higher [Ca2+]i than caudal cells and could not tyrosine phosphorylate in response to cAMP. However, in calcium-depleted medium both caput and caudal cells could exhibit a cAMP-dependent phosphorylation response. The inhibitory effect of calcium on tyrosine phosphorylation was also observed in caudal spermatozoa using thapsigargin, a Ca(2+)-ATPase inhibitor that increased [Ca2+]i and precipitated a corresponding decrease in phosphotyrosine expression. We also demonstrate that despite the activation of tyrosine phosphorylation in caput spermatozoa, these cells remain nonfunctional in terms of motility, sperm-egg recognition and acrosomal exocytosis. These results demonstrate that the signaling pathway leading to tyrosine phosphorylation in mouse spermatozoa is negatively regulated by [Ca2+]i, and that maturation mechanisms that control [Ca2+]i within the spermatozoon are critically important during epididymal transit.     

Heterogeneous distribution of filipin-sterol complexes in nuclear membranes

Filipin, a sterol-specific polyene antibiotic, has been shown by electron microscopy to form complexes in membranes of mouse urinary bladder cells. Following instillation of a glutaraldehyde-filipin-dimethylsulfoxide solution into the bladder lumen, filipin-cholesterol complexes appear as membrane corrugations in thin sections and as 20–25 nm protuberances and depressions on PF and EF faces in freeze-fracture replicas. The complexes are observed in plasmalemma, Golgi membrane, rough endoplasmic reticulum and nuclear membrane of five different cell types (urothelial, endothelial, mesothelial, smooth muscle and fibroblasts). In the present report, we direct particular attention to the localization of numerous filipin-cholesterol complexes present in the nuclear envelopes of these cells. Our results suggest that enrichment of cell membranes with cholesterol occurs at an earlier stage in the flow-differentiation process than previously suspected. In addition, the unequal distribution of complexes in favor of the outer nuclear membrane suggests that it has a higher cholesterol content than the inner membrane.     

Immunological approach to the characterization of the outer acrosomal membrane of boar spermatozoa

Antiserum to purified boar spermatozoan outer acrosomal membrane (OAM) was raised in rabbits and adsorbed with boar liver and serum glutaraldehyde cross-linked immunoadsorbents. The IgG fraction of the antiserum was purified by (NH₄)₂SO₄ precipitation followed by ion-exchange chromatography. Indirect immunofluorescence showed bright fluorescent staining of the acrosomal cap of boar spermatozoa and to a lesser extent of the acrosomes of bull and goat spermatozoa after incubation with anti-OAM-IgG. Immuno-electron microscopy further confirmed the specificity of the antibody for the OAM. Preincubation of the anti-OAM-IgG with isolated OAM, completely abolished its reactivity. When tested by ELISA, anti-OAM-IgG reacted with boar, bull, goat, and human spermatozoa; however, its binding activity to boar spermatozoa was significantly greater as compared to spermatozoa from the other species tested. In an effort to identify OAM antigens recognized by this antiserum, the isolated boar OAM was labeled either with ³H or with ¹²⁵I and solubilized by mild detergent treatment. The extracted components were immunoprecipitated with anti-OAM-IgG and protein A-bearing S. aureus and the thus isolated antigens were analysed on SDS-PAGE. The results suggest that anti-OAM-IgG recognized one high molecular ³H-labeled glycoprotein (270 kd), and four ¹²⁵I-labeled polypeptides of lower molecular weight of the boar OAM.     

Distribution of carnitine and acylcarnitine in the hamster epididymis and in epididymal spermatozoa during maturation

The highest levels of carnitine and acylcarnitine were found in the cauda epididymidis, and spermatozoa from the cauda contained greater amounts of total carnitine (free carnitine plus acylcarnitine) than those removed from the corpus or caput epididymidis. Spermatozoa from the distal cauda contained significantly greater amounts of both free and total carnitine than those removed from the proximal cauda epididymidis. The acylcarnitine:carnitine ratio was 1.7 and 0.37 in caput and cauda spermatozoa, respectively and 1.7 and 1.3 in caput and cauda fluid, respectively. It is suggested that the accumulation of carnitine is involved in sperm maturation and that acylcarnitine serves as an energy substrate for epididymal spermatozoa.     

Distribution of calmodulin and calmodulin-binding proteins in membranes from bovine epididymal spermatozoa

Reproducible concentrations of calmodulin representing approximately 0.1% of the membrane protein were detected in purified plasma membranes from bovine epididymal spermatozoa. When membranes were isolated in the presence of 1 mM EGTA, the amount of calmodulin associated with the plasma membranes was not reduced. Calmodulin-binding proteins were detected in both purified plasma membranes and in a mixed membrane fraction containing both plasma membranes and cytoplasmic droplet membranes. A calcium-dependent, calmodulin-binding protein of apparent molecular weight 123,000 was detected in both fractions. In the presence of 1 mM EDTA, putative calcium-independent calmodulin-binding proteins of apparent molecular weights 93,000, 32,000, 18,000, and 15,000 were detected in the plasma membrane fraction. The 15,000 Mr polypeptide was also present in the mixed membrane fraction but the three proteins of higher molecular weight were reduced or absent in this fraction.     

Changes in plasma membrane glycoproteins of rat spermatozoa during maturation in the epididymis

Glycoproteins on the plasma membrane of testicular and cauda epididymidal spermatozoa have been labeled with galactose oxidase/NaB [3H]4 and sodium metaperiodate/NaB[3H]4, followed by analysis on SDS polyacrylamide gels. The major glycoprotein labeling on testicular spermatozoa has a molecular weight 110,000 whereas on cauda epididymidal spermatozoa greater than 90% of the radio-label is incorporated into proteins of molecular weight 32,000. These 32,000-mol wt X proteins are homologous with proteins of similar molecular weight purified from the epididymal secretion and which have been shown previously to be synthesized in the caput epididymidis under hormonal control. Immunofluorescence revealed that the 32,000-mol wt proteins are present on the flagellum of mature but not immature spermatozoa and that they have a patchy distribution suggesting that they are mobile within the plane of the membrane. The membrane-bound 32,000-mol wt proteins possess hydrophobic domains as revealed by charge-shift electrophoresis and they also label with a lipophilic photoaffinity probe suggesting that they are in contact with the lipid bilayer. The evidence indicates that there is a considerable reorganization of the molecular structure of the plasma membrane of spermatozoa during maturation in the epididymis and that some of the changes are brought about by a direct interaction with epididymal secretory proteins.     

Influence of recovery method and microbial contamination on the response to freezing–thawing in ibex (Capra pyrenaica) epididymal spermatozoa

The method of sperm recovery may influence the initial quality of sperm samples and their response to freezing–thawing. The aim of the present work was to compare two methods for collecting epididymal spermatozoa in order to improve the quality of recovered sperm and reduce possible contamination. Testes were obtained from 23 legally hunted, adult ibex males. The sperm mass of the right epididymis was collected by small longitudinal and transverse cuts made in the cauda epididymidis. The sperm mass of the left epididymis was collected by retrograde flushing from the vas deferens to the cauda epididymidis (using a cannula), employing a Tris, citric acid, glucose, egg yolk-based medium. The flushing method recovered more spermatozoa (P < 0.001) than the cutting method. After freezing–thawing, greater acrosomes damage (P < 0.001) and more morphological abnormalities (P < 0.05) were seen among the sperm cells recovered by the cutting method than among those obtained by retrograde flushing. The method of sperm recovery did not, however, influence the microbial contamination rate. In frozen–thawed samples that were microbially contaminated, motility was significantly reduced (P < 0.05) and membrane integrity tended to be poorer (P = 0.06). In conclusion, retrograde flushing is recommended for ibex sperm collection since it would appear that microbial contamination is no more of a problem than that encountered with the cutting method, while a larger number of sperm cells more resistant to freezing–thawing can be obtained.     

Effects of cyclic AMP on the motility of mature and immature hamster epididymal spermatozoa studied by reactivation of the demembranated cells

Hamster spermatozoa from the caput and cauda epididymides were demembranated with 0.04% Triton X-100 and reactivated with 1 mM ATP. Motility parameters were analysed by video recording and stroboscopic photography. In the absence of added cAMP, reactivated cauda sperm showed percentage motility and forward swimming patterns similar to those of intact cells, but velocities were lower. When 2 or 20 μM cAMP was present, the velocities were increased but there was no effect on beat frequencies or percentage of forward progressing sperm. Cyclic AMP also markedly increased the percentage of cauda sperm which at first displayed nonprogressive “looping” movement. Addition of cAMP to the reactivation medium greatly improved the otherwise feeble and irregular motility of the demembranated caput sperm by increasing the percentage motility and beat frequencies of nonprogressive cells. It also induced forward motility with beat frequencies and velocities similar to cauda sperm reactivated in the absence of cAMP, but looping was never seen, indicating a change in the flagellar apparatus with maturation. The time required for the exhibition of the cAMP effects was reduced when caput sperm were reactivated in extracts of another previously maximally reactivated caput sperm preparation. The results suggest the production of some potent compound(s) by the axonemes for the manifestation of the cAMP effects.     

Distribution of plasma membrane rosettes and kinetics of cellulose formation in xylem development of higher plants

Summary Germ roots of several higher plants—maize (Zea mays), mung bean (Vigna radiata) and cress (Lepidium sativum)—were freeze-fractured without cryoprotection in order to confirm and extend the informations on frequency and distribution of plasma membrane particle complexes with respect to cellulose formation. In all three objects the PF of developing xylem elements showed rosette accumulations in the regions of wall thickenings. The rosette-distribution pattern ranges from random in a young stage, to more grouped in a probable intermediate stage to strictly localized in later stages. The frequency of rosettes increases from stage to stage.In all three objects the EF of developing xylem elements is relatively poor in particles. Observations of terminal globules were rare and undistinct. This leads to the assumption that rosettes on the PF and terminal globules on the EF are not part of the same complex.A comparison of the number and distribution of microtubules underlying the xylem wall thickenings with rosette frequency and distribution leads to the conclusion that there seem to be no direct connections between these two structures. Microtubules may be involved in grouping of rosettes, thus indirectly orienting microfibril deposition. Calculations based on the observed rosette frequencies and the amount of wall material formed indicate that in xylem development 1,000 nm elementary fibril per rosette per minute may be formed and that the active phase of one rosette may be about 10 minutes.Abbreviations EF exoplasmic fracture face - PF protoplasmic fracture face     

Changes of elemental concentrations around and on the surface of fowl sperm membrane during maturation in the male reproductive tract and after in vitro storage

X-ray microprobe analysis was performed to investigate the changes of elemental concentrations around or on the membrane of the head, midpiece, and principal piece regions of individual fowl spermatozoa during maturation in the male reproductive tract and after storage in vitro at 4°C. The pattern of change of elemental concentrations during maturation and postejaculation was, in general, similar in the three different subcellular regions; i.e., concentrations of sodium, potassium, chlorine, and calcium decreased gradually during sperm passage through the male reproductive tract and after storage. Phosphorus concentration remained almost constant in the male tract and decreased gradually after storage. In contrast, magnesium, zinc, and copper concentrations showed an interesting pattern: concentrations increased significantly during maturation to a maximum at ejaculation and decreased again after storage. The ratios of sodium to potassium in the midpiece region showed patterns similar to those of magnesium, zinc, and copper concentrations.     

Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm

Transmission electron microscopy was used to confirm that a monoclonal antibody (F79.3E2; class IgGlK) was specifically localized to an antigen in the acrosomal ground substance of stallion sperm. This antibody was used to develop and validate an indirect immunofluorescent procedure to evaluate integrity of the plasma-acrosomal membranes of stallion sperm. The concept was that primary monoclonal antibody would be “shielded” from its acrosomal antigen by an intact plasma membrane. Conversely, sperm with damaged plasma-acrosomal membranes would exhibit green acrosomal fluorescence when viewed with an epifluorescence microscope. A lipophilic counterstain (red fluorescence) was used to insure that all sperm were visualized. Sperm in fresh-extended or frozen-thawed semen were incubated with hybridoma supernatant containing monoclonal antibody for 30 min at 37°C, then a second antibody (rabbit anti-mouse IgG-FITC) was added for 30 min at 37°C. Unbound antibody was removed by dilution and centrifugation. Sperm were resuspended in phosphate-buffered saline containing Evan's blue as a counterstain. All sperm fluoresced bright red, regardless of the status of cell membranes, except that in cells with damaged plasma-acrosomal membranes, the green fluorescence associated with antibody was overriding for the rostral portion. By counting fluorescent and nonflourescent “acrosomes”, the percentage of sperm with intact plasma-acrosomal membranes was easily determined. Evaluation of five mixtures of undamaged and damaged sperm by this procedure gave a correlation of 0.91 between the percentage of damaged sperm in a mixture and the percentage of sperm with a fluorescent acrosome. Intra- and interassay coefficients of variability were < 6%.