Fine structure distribution of non-specific acid phosphatase in the head region of mouse spermatozoa from various regions of the male reproductive tract.

The fine structure distribution of non-specific acid phosphatase was determined in the head region of mouse spermatozoa from the testes, the caput, corpus and cauda epididymidis and the ductus deferens. Enzymatic localization was achieved by the Gomori technique. The postacrosomal dense lamina, the nuclear side of the inner acrosomal membrane and the space between the plasmalemma and the outer acrosomal membrane showed reaction product in spermatozoa from the testis and caput epididymidis. Spermatozoa from the cauda epididymidis exhibited reaction product only between the plasmalemma and the outer acrosomal membrane. Spermatozoa from the corpus epididymidis and from the ductus deferens showed no reaction product in the head region. The changes observed in the distribution of acid phosphatase in the sperm head during epididymal transport may reflect maturational events.     

Effect of sodium and potassium concentrations and pH on the maintenance of motility of rabbit and rat epididymal spermatozoa.

Spermatozoa were collected from the caput and cauda epididymidis of rabbits and rats and diluted in Hank's solution containing BSA, with various concentrations of Na+ and K+. Ionic strength and osmolarity were kept constant. Motility was assessed at various intervals during incubation at 25 degrees C. In the pH range 7.05--7.20, the motility of rabbit spermatozoa was not affected by changes in the ratio of K+ to Na+. Similarly, the motility of rat cauda spermatozoa was not altered, but that of caput spermatozoa was slightly depressed by a high K+/Na+ ratio. In the pH range 5.45--5.85, rabbit cauda and caput spermatozoa had much greater motility in media with a high K+/Na+ ratio. The reverse result was obtained for the rat. These findings indicate that the motility of epididymal spermatozoa is influenced by external Na+ and K+ concentrations and that this phenomenon is pH-dependent.     

Motility of undiluted bull epididymal spermatozoa collected by micropuncture

Motility of whole undiluted semen collected from different regions of the bull epididymis by micropuncture was determined by examining a droplet under paraffin oil. Bull caudal spermatozoa showed vigorous motility in undiluted semen. This motility was less in samples collected from nearer the testis: samples from the distal caput showed weak but detectable motility while those from the proximal and mid-caput were completely quiescent. Motility of spermatozoa from the distal caput and the proximal corpus was markedly increased after incubation at 34 or 37 degrees C for 1 h, but was depressed by incubation at 25 degrees C. Similar but smaller effects were observed with spermatozoa collected from the mid-corpus and the mid-cauda, except that motility of spermatozoa from the mid-corpus was reduced after incubation for 1 h at 37 degrees C. The inhibitory effect of low temperature was completely reversible. Incubation of caudal spermatozoa under anaerobic conditions produced partial and reversible inhibition of sperm motility. The results suggested that bull epididymal spermatozoa may not be completely quiescent in their native environment as previously assumed.     

Changes in the motility patterns of spermatozoa from the rabbit epididymis as assessed by computer-aided sperm motion analysis

Sperm maturation in the epididymis includes changes in their potential for motility that enables spermatozoa to reach the egg and penetrate its investments. The motility characteristics of spermatozoa from the testis, the epididymis, and vas deferens of the rabbit were investigated by computer-assisted sperm analysis (CASA). Various forms of motility were displayed by sperm from different regions of the epididymis released into incubation medium Testicular sperm were motile, although nonprogressive. The maximum percentage motility was expressed by sperm in the proximal cauda epididymidis, and forward progression was developed by spermatozoa from the distal caput. Once forward progression was established, the curvilinear velocity was about the same for sperm from all regions of the tract, whereas straight-line velocity increased between the mid-corpus and cauda and paralleled the decline in lateral displacement of the head. The maintenance of motility in vitro was best maintained by sperm from the distal regions of the tract although sperm from the distal caput maintained motility better than sperm from the proximal and midcorpus regions. Analysis of the motile sperm cells revealed several types of trajectories (“irregular,” “small circular,” “large circular and arcs,” “jagged” and “straight-line”) that were analyzed by discriminant analysis using the variables generated by CASA. Accuracy of classification varied from 70% to 96%, depending on the type of track. The classification function was then applied to the changes that occurred during incubation and showed that irregular trajectories gave way to small and then large circular tracks and progressive forms as sperm matured. © 1996 Wiley-Liss, Inc.     

The effects of carnitine,glycerylphosphorylcholine, caffeine,and egg yolk on the motility of rat epididymal spermatozoa

Experiments were performed to further the understanding of epididymal processes involved in the acquisition of sperm motility. Samples of luminal contents were collected by micropuncture from four regions of the rat epididymis. These samples were incubated in various diluents to observe the effects of the diluents on sperm motility. Consonant with previous reports, 40 mM glycerylphosphorylcholine (GPC) and 60 mM DL-carnitine reduced overall motility scores of cauda epididymidal spermatozoa but did not prevent normal initiation of motility. Additionally, control sperm cells and cells treated with carnitine could reinitiate full motility after becoming immotile. Spermatozoa treated with GPC could not reinitiate motility. The sperm cells in our system thus react to GPC and carnitine in fundamentally different ways, the exact nature of which remains to be determined. Spermatozoa from the distal caput epididymidis evidenced high motility scores when diluted in a 5% egg yolk + 10 mM caffeine diluent. It was demonstrated, however, that the subjective appearance of full motility in these immature cells was not supported by actual progressive motility as measured in an assay of linear distance traveled. It was concluded that neither 10 mM caffeine, 5% egg yolk, nor their combination was sufficient to induce progressive motility in immature rat spermatozoa.     

Sperm morphological abnormalities appearing in the male rabbit reproductive tract

The role of the excurrent duct system in producing and/or eliminating morphologically abnormal spermatozoa may modify the semen parameters and interfere with sperm fertilizing capacity. To study this process, changes in the morphology of spermatozoa during their transit through the reproductive tract in sexually mature rabbits were investigated. The incidence of head, midpiece and tail abnormalities as well as of multiple defects in a single spermatozoon, and the position of the cytoplasmic droplet along the sperm midpiece were evaluated in samples from the testis, 6 regions of the epididymis and the vas deferens. Spermatozoa were characterized by rapid migration of the cytoplasmic droplet when passing from the proximal to the distal caput of the epididymis, and spermatozoa with no droplet predominated in the distal epididymis and vas deferens. In passing from the testis to the proximal caput of the epididymis, the incidence of spermatozoa with an abnormal midpiece and those with multiple defects decreased significantly. The proportion of spermatozoa with abnormal heads was also lower in the testis, but no statistically significant differences were found, whereas there was no change in the proportion of those with abnormal tails. These results indicate that there must be a mechanism for the disposal of defective spermatozoa. No evidence of spermiophagy by luminal macrophages was observed in the extracts, although a few spermatozoa exhibited signs of degeneration, suggesting, that although intraepithelial phagocytosis has not been clearly demonstrated in the nonexperimental rabbit, sperm cells may undergo a form of autolysis within the lumen of the duct.     

Two-dimensional electrophoresis of proteins in principal cells, spermatozoa, and fluid associated with the rat epididymis

Spermatozoa, fluids, and principal cells from different regions of the epididymis were characterized by two-dimensional electrophoresis. Rete testis fluid was collected after 36-h efferent duct ligation, and cauda epididymal fluid was collected by retrograde perfusion through the vas deferens. Spermatozoa were collected after their exudation from minced caput and corpus epididymal tissue. Principal cells were recovered after enzymatic disaggregation and centrifugal elutriation of epididymides. Two-dimensional polyacrylamide gel electrophoresis was used to prepare protein profiles of all samples. Comparison of the proteins found in rete testis fluid versus those found in cauda epididymal fluid revealed a dramatic change in composition, including the loss, addition, or retention of specific proteins as well as changes in the relative concentrations of certain proteins. Prominent cauda epididymal fluid proteins, possibly contributed by the epididymal epithelium, were detected at 16, 23, and 34 kDa. After epididymal transit, a considerable decrease was observed in the number of aqueous-soluble sperm proteins. Differences in the protein composition of epididymal epithelial principal cells from the caput versus corpus epididymidis were also noted, suggesting that functional differences exist for these epididymal regions. Of particular interest was the occurrence of a prominent protein of approximately 20-23 kDa found in all sperm samples, in fluids, and in caput and corpus principal cells. However, this protein was absent in cauda epididymal sperm after 36-h efferent duct ligation. The rapid loss of this protein from sperm after efferent duct ligation suggests that this surgical intervention may affect spermatozoa residing within the epididymis.     

Induction and enhancement of progressive motility in hamster caput epididymal spermatozoa

Progressive motility was induced in hamster caput epididymal spermatozoa incubated in Tyrodes medium containing 50 mM theophylline, 1.0% Fraction V bovine serum albumin, and 15% (v/v) heat-treated human seminal plasma. Under these induction conditions, however, the maximum percent of caput spermatozoa exhibiting progressive motility (21%) and the time during which motility was sustained (120 min) were significantly less (p less than 0.05) than that of controls from the cauda epididymidis. Moreover, in contrast to caudal spermatozoa, the majority of the induced caput spermatozoa exhibited some degree of flagellar bending at the neck or midpiece. In subsequent experiments the procedure for motility induction was modified to achieve levels of motility in caput spermatozoa equivalent to those observed for caudal spermatozoa. The addition of 5 microM diamide, a sulfhydryl oxidant, to the induction medium prevented the flagellar angularity observed in induced caput sperm preparations. The percentage of caput spermatozoa induced to progressive motility was increased to levels characteristic of caudal spermatozoa (48%) by the addition of hamster caudal epididymal fluid (CEF) to the induction medium. Finally, the viability of the induced caput spermatozoa was significantly enhanced (p less than 0.05) by the removal of Fraction V albumin from the induction medium. In the presence of CEF and in the absence of albumin, 50% of the caput spermatozoa acquired progressive motility and sustained this motility for 4 h. Moreover, when fatty acid-free, charcoal-extracted albumin instead of Fraction V albumin was utilized in the induction procedure, a maximum of 43% of the caput spermatozoa acquired progressive motility and maintained this motility for 4 h, suggesting that the decreased sperm viability observed in the presence of Fraction V albumin was due to a contaminant of albumin, possibly fatty acids. The studies described herein demonstrate for the first time that immature quiescent caput epididymal spermatozoa can be induced to acquire progressive and sustained motility equivalent to that observed in mature caudal epididymal spermatozoa.     

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.     

Maturation of spermatozoa in the epididymis of the Chinese hamster

Chinese hamster spermatozoa gain their ability to move when they descend from the testis to the distal part of the caput epididymis, but it is not until they enter the corpus epididymis that they become capable of fertilizing eggs. The maturation of the spermatozoa proceeds as they further descend the tract and perhaps continues even in the vas deferens. During transit between the distal caput and proximal cauda epididymides, small membrane-limited vesicles (and tubules) appear on the plasma membrane over the acrosomes of the spermatozoa. The number of vesicles appearing on the sperm brane reaches a maximum when the spermatozoa are in the proximal cauda epididymis. It declines sharply in the distal cauda epididymis. Spermatozoa in the vas deferens are free of the vesicles. The origin, chemical nature, and functional role of the vesicles that appear on the sperm surface during epididymal transit must be the subject of further investigation.     

Immunocytochemical localization of nuclear protamine in boar spermatozoa during epididymal transit

Protamine was specifically demonstrated in boar spermatozoa collected from the rete testis, caput, corpus and cauda epididymidis and the ejaculate by immunoelectron microscopy, using anti-boar or anti-ram protamine antisera and an indirect post-embedding immunogold technique. Spermatozoa from all collection sites stained after incubation although with different degrees of labelling. Controls were negative. Labelling increased from the rete testis towards the epididymal corpus, where it was most intense, decreasing sharply thereafter. The weakest binding of the assayed antibodies was obtained in the ejaculated spermatozoa but it could be reversed by in-vitro induction of chromatin decondensation with sodium dodecyl sulphate and the metal-chelating EDTA. The finding of a significant decrease in the immunolabelling detected from the corpus epididymidis onwards indicates a critical point for the interaction between DNA and the protamines in boar spermatozoa during the epididymal maturation.     

A determinant of Mr 34,000 expressed by hamster epididymal epithelium binds specifically to spermatozoa in co-culture

A murine monoclonal antibody raised against hamster cauda epididymal spermatozoa was shown to recognize an Mr 34,000 component of epididymal epithelium. Antigen was localized by immunocytochemistry on the surface and in the apical cytoplasm of principal cells in the proximal corpus epididymidis but not in the caput or initial segment regions. Spermatozoa from the corpus epididymidis expressed antigen on their post-acrosomal plasma membrane and annulus. Epididymal principal cells from the proximal corpus region when cultured in vitro bound antibody on their apical surface for at least 5 days. Spermatozoa from the caput epididymidis co-cultured with epithelium expressed antigen after incubation for 8 and 24 h. These results suggest that a surface change to epididymal spermatozoa during maturation in vivo may also be elicited during in-vitro culture.     

Evolution of the flagellar waveform of ram spermatozoa in relation to the degree of epididymal maturation.

Motility and flagellar movement of ram spermatozoa along the epididymis were analysed in vitro. From the caput to the cauda of the epididymis, the percentage of motile and progressive spermatozoa increases. No flagellar bending was observed in spermatozoa from the testis or the epididymal anterior caput. When spermatozoa reached the distal caput of the epididymis, a static curvature, associated with an initiation of the flagellar beating, appeared on the flagella. This curvature normally disappeared during epididymal transit. Its disappearance was associated with an increase in the flagellar beat efficiency. Our results suggest that the initiation of motility is related to two mechanisms involving: (1) the presence of a transient static curvature, and (2) the establishment of a symmetric regular beating of the flagellum.     

Dynamics of the thiol status of rat spermatozoa during maturation: analysis with the fluorescent labeling agent monobromobimane

Mammalian spermatozoa undergo maturation as they pass through the epididymis. Maturation is accompanied by the oxidation of thiols to disulfides. Disulfides are probably involved in sperm chromatin condensation and tail structure stabilization. In this work, we used the fluorescent thiol-labeling agent monobromobimane to determine the changes occurring in thiols and disulfides in rat sperm heads and tails during maturation. Spermatozoa were obtained from testis, epididymis (caput, corpus, cauda, and vas deferens), and ejaculate. Intact spermatozoa were labeled with monobromobimane, with or without pretreatment with dithiothreitol. Labeling was evaluated microscopically, and quantitative analysis was carried out spectrofluorimetrically with labeled globin used as a standard. Samples were also analyzed by gel electrophoresis. The total amount of thiols and disulfides remained the same during the entire period of sperm maturation (26 +/- 0.5 nmoles thiols + disulfides/10(6) spermatozoa). However, the reactive thiols decreased markedly between the corpus and the cauda (from greater than 90% of total in testis and 75% in corpus to about 25% in cauda), with little or no further change in vas deferens and ejaculated sperm. Trypsin treatment followed by sucrose gradient was used to separate the heads from the tails. Thiols comprised 84% of the total SH + SS in the heads and 74% in the tails of caput spermatozoa, decreasing to 14% and 45%, respectively, in cauda sperm. Thus, the decrease in reactive thiols involved both heads and tails-oxidation to disulfides being very marked in the head. Electrophoresis revealed that oxidation of thiols to disulfides occurred in many protein fractions during maturation in the epididymis.     

Acquisition of sperm motility and its maintenance during storage in the lizard, Lacerta vivipara

Lizard spermatozoa, which are non-motile in the testis, develop the ability to swim as they pass along the excurrent duct. The addition of caffeine, a phosphodiesterase inhibitor, induced forward motility in spermatozoa from the caput epididymidis and increased the velocity of spermatozoa from the distal part of the epididymis. Caffeine had no effect on the motility of testicular spermatozoa. This suggests that sperm motility in this species is cyclic AMP-dependent but this factor alone is not sufficient to induce testicular sperm motility. In samples from the distal region of the epididymis, sperm motility was maximal in April just after the breeding season and then decreased significantly during the following months. A parallel can be drawn between these data and the levels of testosterone in the plasma. In the lizard, as in mammals, the epididymis may play an important role in the maturation of spermatozoa.     

The effect of sulfhydryl oxidation on the morphology of immature hamster epididymal spermatozoa induced to acquire motility in vitro

Immotile spermatozoa from the caput epididymidis become progressively motile when incubated in medium containing theophylline, seminal plasma, and albumin. We previously reported that under these incubation conditions the spermatozoa induced to acquire motility exhibited a marked flagellar angularity, with the sperm head or midpiece bent 90-180 degrees towards the tail. In addition, we demonstrated that sperm flagellar bending did not occur when the sulfhydryl oxidant diamide was added to the motility induction medium. In the present study, we examined further the effect of sulfhydryl oxidation on the morphology and sulfhydryl content of immature caput spermatozoa induced to acquire motility in vitro. We found that flagellar bending was prevented and sperm flagellar straightness was maintained in a dose-dependent manner by diamide. Moreover, flow cytometric analysis of caput sperm sulfhydryls using the sulfhydryl reagent monobromobimane (mBBr) revealed that 1) diamide oxidizes caput sperm sulfhydryls, and 2) less than 15% of the total reactive sperm sulfhydryls were oxidized at diamide concentrations capable of preventing sperm angulation. Sodium tetrathionate (NaTT), another sulfhydryl oxidant, and hamster cauda epididymal fluid (CEF) containing sulfhydryl oxidase enzyme activity also maintained flagellar straightness in induced caput spermatozoa and oxidized sperm sulfhydryls. The flagellar straightness in caput spermatozoa treated with sulfhydryl oxidants, however, was temporary; with extended incubation, diamide- or CEF-treated spermatozoa exhibited flagellar bending. Additional studies showed that the flagellar straightness observed in sulfhydryl-oxidized spermatozoa was sustained when nitrofurantoin, an inhibitor of glutathione reductase, was included in the induction medium. Flow cytometric analysis of nitrofurantoin-treated spermatozoa showed that nitrofurantoin maintained the sperm disulfides formed by diamide and prevented the reduction of sperm disulfides back to sulfhydryls. Taken together, these studies demonstrate the significance of sulfhydryl oxidation in maintaining the morphology of immature caput epididymal spermatozoa induced to acquire motility in vitro and suggest that sulfhydryl oxidation may be important in the development of motility during sperm epididymal maturation in vivo.     

Effects of osmolality, bicarbonate and buffer on the metabolism and motility of testicular, epididymal and ejaculated spermatozoa of boars.

Spermatozoa were collected from the rete testis of conscious boars, from the cauda epididymidis by retro-flushing, and by ejaculation. Testicular spermatozoa showed no progressive motility, and that of ejaculated was greater than that of epididymal spermatozoa. Glycolysis and respiration of testicular spermatozoa, while lower than that of the more mature cells, were only slightly affected by the incubation conditions. Epididymal spermatozoa converted 83% of the glucose they utilized to CO2 or lactate, but testicular cells converted only 35% to these metabolites. Synthesis of lipid was greatest by testicular spermatozoa. With the more mature cells hyperosmolar conditions depressed CO2 production, but increased lactate production, and these changes were greater for ejaculated than for epididymal spermatozoa. Glycolysis plus respiration of these cells was related to their motility. These results were interpreted as showing increasing motility, glycolysis and respiration with maturation, but also decreased synthetic capacity and increased sensitivity to the environment.     

Glycoproteins: a variable factor in surface transformation of ram spermatozoa during epididymal transit

To determine sequential surface glycoprotein changes in ram spermatozoa during epididymal maturation, labeling procedures were used that were specific for galactosyl, galactosaminyl, and sialyl residues. Spermatozoa and fluids were collected from the rete testis through surgically inserted catheters or flushed from the lumen of selected regions of the epididymis: i.e., caput, proximal and distal corpus, and cauda epididymidis. Ejaculated spermatozoa were collected by electrical stimulation. Electrophorectic analysis of galactose (GAO)-sodium boro[3H]hydride (NaB3H4)-treated spermatozoa revealed a sharp overall decrease in carbohydrate residue labeling during sperm transport through the efferent ducts and caput epididymidis, whereas several high molecular weight components in the 600K to 250K zone persisted throughout epididymal transit. Preincubation of spermatozoa with neuraminidase (NEUA) exposed galactose residues that had not been labeled with GAO alone (i.e., 97K, 43K, 24K) in both cauda epididymal and ejaculated spermatozoa. Treatment with sodium metaperiodate-NaB3H4 labeled many of the surface components displayed by NEUA-GAO-treated spermatozoa and revealed an overall shift in sialyl residue labeling from high molecular weight components in immature testicular spermatozoa to low molecular weight components in mature cells. The labeling procedures applied allowed only a qualitative interpretation of the results and they presumably represent the minimum possible changes. Nonetheless, our results demonstrate that glycoproteins are a major factor in surface transformations of ram spermatozoa in the epididymis, especially during the initial stages of maturation.     

Maturation of hamster epididymal sperm motility and influence of the thiol status of hamster and rat spermatozoa on their motility patterns

A method for objective quantification of hamster sperm movement parameters as an indicator of maturation along the epididymis was established using a computerised system. Analysis of spermatozoa released into medium from five epididymal regions showed that the most drastic increases in percentage motility and curvilinear velocity (VCL) occurred from the distal corpus to the beginning of the proximal cauda and in straight-line velocity (VSL) from the beginning to a more distal site within the proximal cauda region. Both high osmolarity (400 mOsm/kg) and the thiol-oxidising agent diamide (10 μM) increased flagellar straightness of distal corpus spermatozoa, but VSL was increased only with the latter. The thiol-reducing agent dithiothreitol (DTT, 1mM) stimulated and maintained percentage motility and velocities of spermatozoa from the caput, stimulated only percentage motility of distal corpus sperm, but decreased velocities of those from the proximal cauda in prolonged incubation. In rats, diamide increased path straightness but not velocities of caput spermatozoa and yet caused immotility within 15 min, whereas DTT prolonged the maintenance of in vitro motility. The slight increases in kinematic parameters in the presence of DTT were enhanced by a 2-min preincubation with diamide. The finding that the effects of DTT and diamide were not compensatory suggests that the influence of the SH/S-S status on sperm movement is multifaceted, with decreasing sensitivity to stimulation upon sperm maturation. © 1994 Wiley-Liss, Inc.