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.     

Preventive effect of Desferal on sperm motility and morphology

Transition metal ions, mainly iron, are involved in the generation of highly reactive hydroxyl radicals, which are the most powerful inducers of oxidative damage to all biomolecules. The lipids in sperm membranes are highly susceptible to oxidation. Sperm lipid peroxidation (LPO) leads to decrease of motility and reduction of likelihood for sperm‐oocyte fusion. The excess radical production may affect also the spermatozoa morphology. The aim of the present study was to investigate the effect of Desferal on the LPO, motility, and morphology of boar sperm subjected to oxidative stress. After collection, the ejaculates were equally separated and diluted in a commercial semen extender (experiment 1) or in physiological saline (experiment 2). The ejaculates of the 2 experiments were divided into aliquots, which were incubated with one of the following agents: FeSO₄ (0.1mM), H₂O₂ (0.5mM), or FeSO₄ + H₂O₂ (Fenton system), in the presence or absence of Desferal. The application of Desferal in the incubation medium had a protective effect against FeSO₄ + H₂O₂‐induced sperm damage, namely, decrease of LPO; decrease the quantity of immotile spermatozoa and decrease the number of morphological abnormalities, regardless of the used medium. In experiment 2, the presence of FeSO₄ in the incubation medium induced LPO in the same range as the combination FeSO₄ + H₂O₂, in which the effect was reduced by Desferal. Thus, the supplement of Desferal to media used for sperm storage and processing could be a useful tool for diminishing oxidative injury and improving the quality of the semen.     

Synthesis of highly unsaturated phosphatidylcholines in the development of sperm motility: a role for epididymal glycerol-3-phosphorylcholine

Summary Interpretation of the experimental literature on epididymal glycerophosphorylcholine metabolism according to a recently proposed de novo pathway for the synthesis of acyl-specific phosphatidylcholine suggests that epididymal glycerophosphorylcholine is an intermediate of this proposed pathway. This glycerophosphodiester is postulated to be utilized by spermatozoa to synthesize docosahexaenoic phosphatidylcholine, proposed to be required for the development of sperm motility. A defect in glycerophosphorylcholine synthesis might be responsible for some forms of asthenozoospermia.     

Hamster sperm motility transformation during development of hyperactivation in vitro and epididymal maturation

The transformation of hamster sperm motility during capacitation in vitro and during maturation in the caudal epididymis was analyzed and compared using videomicrography. Sperm recovered from the distal portion of the caudal epididymis, as well as ejaculated sperm recovered from the uterus exhibited low amplitude, planar flagellar beating. By 3 hr of incubation under capacitating conditions, the caudal epididymal sperm were swimming in helical patterns apparently produced by significantly increased acuteness of flagellar bending and by torsion seen as abrupt, periodic turning of the head. By 4 hr, most sperm were hyperactivated, swimming in circles resulting from asymmetrical, planar flagellar bending that was significantly more acute than the preceding patterns. When motility parameters of fresh sperm were compared with those of sperm swimming in the transitional helical pattern and with hyperactivated sperm, transitional sperm had significantly higher net and average path velocities than the others, indicating that they covered space at the greatest rate. This suggests that the transitional phase plays an important role in sperm transport. Sperm recovered from the proximal region of the caudal epididymis, near the corpus, swam in either the helical or hyperactivated patterns, or a mixture of the two. The means of their flagellar curvature ratios and linear indices were intermediate between helical and hyperactivated mean values. Thus, sperm undergoing final maturation in the caudal epididymis reverse the pattern of development of hyperactivation. Also, the development of hyperactivated motility must therefore entail induction of a preexisting potential for flagellar movement, rather than a maturational process.     

Effect of overdose zinc on mouse testis and its relation with sperm count and motility

The purpose of this study was to investigate the effects of excessive zinc intake on the testes and on sperm count and motility in mice. Thirty Balb c mice were divided randomly into 3 groups of 10 animals in each. Group I acted as controls; group II was supplied with drinking water containing 1.5 g/100 mL Zn, and group III was supplied with drinking water containing 2.5 g/100 mL Zn. The animals were sacrificed after 3 wk supplementation and the epididymis and testis were quickly excised. A negative correlation between Zn dose and sperm count and motility was found. The sperm count in group III was significantly lower than in groups II and I (p<0.05). The sperm motility in group III was significantly lower than in the controls (p<0.05). Degenerative changes, including spermatic arrest, degeneration of seminiferous tubules, and fibrosis in interstitial tissue, were observed in group III animals. These results show that high doses of zinc significantly alter sperm motility.     

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.     

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.     

Relationship between sperm density, spermatocrit, sperm motility and spawning date in wild and cultured haddock

Semen was collected repeatedly from captive haddock Melanogrammus aeglefinus and the effect of seasonality on various sperm parameters was investigated. No differences in sperm traits were observed for wild and cultured haddock. A highly significant positive relationship existed between spermatocrit and spermatozoa density. A significant increase in mean spermatocrit occurred throughout the spawning season but the amount of variability explained by collection date was low (35·1%) due to variability between males. Each of 10 males sampled repeatedly throughout the spawning season demonstrated an increase in spermatocrit. No relationship existed between spermatocrit and proportion of motile spermatozoa when spermatocrit was ≤70%. Motility was reduced in semen samples with spermatocrits >70%. The proportion of spermatozoa that were motile decreased with time since activation. Some motility was still observed after 60 min in sea water (0·1–15·2%) for sperm collected at all times within the spawning season. Of those spermatozoa that were motile, the proportion that exhibited forward swimming motion decreased and the proportion that had only vibratory movement increased with time post‐activation. The speed of forward swimming spermatozoa showed no significant relationship with spermatocrit at any time between 0 and 60 min after activation. Swimming speed was negatively related to time since activation, decreasing from 174–240 μm s⁻¹ at 0 min to 80–128 μm s⁻¹ at 60 min after activation.     

Effects of exposure to static magnetic fields on the morphology and morphometry of mouse epididymal sperm

Morphologic and morphometric sperm characteristics of mouse epididymal extracts from animals exposed to static magnetic fields were evaluated. For this purpose, animals were exposed for 35 days to a field of 0.7 T generated by a commercial permanent magnet for either 1 or 24 h per day. The values of morphometric parameters were obtained using the morphometric module of the Sperm Class Analyzer® computerized image analysis system, and percentages of abnormalities were calculated. The size of sperm heads was unaffected by exposure to static magnetic fields. Lack of hook was a sperm head abnormality found significantly more frequently in animals exposed continually than in nonexposed animals, showing a possible alteration to the spermatogenic process after exposure to static magnetic fields. The percentage of sperm with coiled tails or of sperm with abnormal midpiece or tail was not altered by exposure. Bioelectromagnetics 19:377–383, 1998. © 1998 Wiley-Liss, Inc.     

Diethylstilbestrol-treated adult rats with altered epididymal sperm numbers and sperm motility parameters, but without alterations in sperm production and sperm morphology

In this study, we characterized estrogenic effects of diethylstilbestrol (DES) on reproductive parameters in male rats to identify a minimal dose level that alters epididymal and sperm functions but has little or no effect on sperm production and/or spermatogenesis. Adult rats (five animals/group) received s.c. injections of 0.2 ml of corn oil containing DES at a rate of 1.0 mg, 200 microg, 40 microg, 8 microg, 1.6 microg, or 320 ng x rat(-1) x day(-1) for 12 days. The control group received corn oil only. DES effects were similar in the 8-microg group and higher dose groups and included significant (P < or = 0.05) reductions in 1) absolute and relative weights of the head and body of the epididymis (EP), tail of the EP, and seminal vesicle, 2) numbers of sperm in both regions of the EP, and 3) motility characteristics in sperm collected from the tail of the EP. Conversely, no significant changes were observed in relative testis weight, daily sperm production, spermatogenesis, seminiferous epithelial height in stage VII, and sperm morphology. All of the above parameters in the 1.6-microg group (except seminal vesicle weight) and 320-ng group were comparable to those of controls. Plasma testosterone (T) level was reduced to an almost undetectable level in the > or = 8-microg groups and to a very low level in the 1.6-microg group (0.35 vs. 2.36 ng/ml in controls or 320-ng group), but LH level was unaltered. In a parallel fertility study, males received DES at a rate of 40, 8, or 1.6 microg x rat(-1) x day(-1) for 12 days prior to and 12 days during cohabitation (1:1) with untreated females. Of the 15 females cohabited with treated males (5 females/dose), none in the 40-microg and 8-microg groups and 1 in the 1.6-microg group formed a copulatory plug and delivered 8 pups, in contrast to 5/5 copulatory plugs and 13-15 pups/litter in the controls. DES at a rate of 8 microg x rat(-1) x day(-1) for 12 days reduced EP weights, sperm numbers in the EP, and sperm motility patterns but caused minimal to no alterations in daily sperm production, spermatogenesis, or sperm morphology. Factors other than T, or in addition to lower T, may be responsible for DES-induced reproductive disorders (despite lower T, sperm contents and sperm motility patterns in the EP were normal in the 1.6-microg group). Deficits in EP sperm functions and/or sexual behavior (as evident from absence of copulatory plugs) probably accounted for reduced fertility in treated males.     

Objective analysis of stallion sperm motility

An image-analysis system utilizing a microcomputer and CellSoft computer-assisted semen analysis software package was evaluated to assess stallion sperm motility characteristics. Analyses were performed at 37°C on a 6 μl drop of diluted semen placed on a glass slide and covered with an 18 mm² coverslip. Four groups of 25 cells each per slide, four slides per ejaculate and four ejaculates from each of three stallions were analyzed in a nested model. The percentage of motile sperm cells, mean velocity (μm/sec), mean linearity, and mean angular head displacement (μm) were measured. Statistical analysis of variance components showed that within ejaculates, more variation was accounted for in the differences among groups of 25 cells than among slides. Predicted standard deviations calculated for combinations of slides and groups of cells showed that a combination of two slides from which a total of 400 cells were analyzed resulted in a mean intra-assay coefficient of variation (CV) of 5.7% for the four measured variables. The following are individual coefficients of variation: percentage of motile cells (7.8%), mean velocity (6.4%), mean linearity (1.9%) and mean angular head displacement (6.6%). When ejaculate differences were included in the model and predicted standard deviations were calculated for a single ejaculate, the mean inter-assay CV was 9.2%. Mean velocity (6.4%) and mean linearity (4.7%) were more repeatable among ejaculates than either the percentage of motile sperm (14.4%) or angular head displacement (11.2%). It was concluded that this system is precise enough to determine differences in motility characteristics of stallion semen samples.     

Duration of epididymal sperm transit in hamster: An autoradiographic study

The time required for passage of spermatozoa through the epididymis has been determined in hamster employing quantitative light microscopic autoradiography with ³H-thymidine. The time required for spermatozoa to traverse the caput is 3 days; corpus, 2 days; proximal cauda, 2 days; distal cauda, 6 days. An additional 2 days are required for passage of spermatozoa through the proximal ductus deferens. The total duration of sperm transit through the ductus epididymis in sexually rested hamster has been estimated at about 15.0 days.     

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.     

Estimation of sperm numbers in insects by fluorometry

Summary To facilitate the study of mating biology in the desert leaf-cutter antAcromyrmex versicolor, methods were developed that allowed storage and easy quantification of sperm samples collected from both male and female reproductive tracts. Sperm samples stored frozen were sonicated, stained with a fluorescent DNA stain, and the fluorescence emitted by the stained sperm heads was measured. The intensity of fluorescence was shown to be a linear function of the number of sperm in the sample as determined by counting.     

Effects of prenatal exposure to a 900 MHz electromagnetic field on 60-day-old rat testis and epididymal sperm quality

We investigated the effects of exposure in utero to a 900 megahertz (MHz) electromagnetic field (EMF) on 60-day-old rat testis and epididymis. Pregnant rats were divided into control (CG; no treatment) and EMF (EMFG) groups. The EMFG was exposed to 900 MHz EMF for 1 h each day during days 13 ? 21 of pregnancy. Newborn rats were either newborn CG (NCG) or newborn EMF groups (NEMFG). On postnatal day 60, a testis and epididymis were removed from each animal. Epididymal semen quality, and lipid and DNA oxidation levels, apoptotic index and histopathological damage to the testis were compared. We found a higher apoptotic index, greater DNA oxidation levels and lower sperm motility and vitality in the NEMFG compared to controls. Immature germ cells in the seminiferous tubule lumen, and altered seminiferous tubule epithelium and seminiferous tubule structure also were observed in hematoxylin and eosin stained sections of NEMFG testis. Nuclear changes that indicated apoptosis were identified in TUNEL stained sections and large numbers of apoptotic cells were observed in most of the seminiferous tubule epithelium in the NEMFG. Sixty-day-old rat testes exposed to 900 MHz EMF exhibited altered sperm quality and biochemical characteristics.     

Relationships between sperm morphometry and sperm motility in the Atlantic salmon

Relationships between spermatozoal design and swimming behaviour were investigated using the significant natural variance in sperm traits in Atlantic salmon Salmo salar. In vitro motility and fertilization experiments were conducted with 86 Atlantic salmon to measure sperm form and function under natural fertilization conditions. Spermatozoal traits of Atlantic salmon showed narrow variance within individuals but differed extensively between samples: mean sperm length varied from 32·3 to 39·5 μm, mean velocity ranged from 18 to 127 μm s⁻¹, and ejaculate longevity varied from 18 to 78 s. In addition to variation in sperm morphometry between fish, a negative relationship was also found between sperm head length and flagellum length. This natural variation in sperm form and function between males is counter-intuitive since measures are from a single Atlantic salmon population where all males are adapted to a common fertilization environment. No evidence was found that longer sperm, or sperm with longer flagella, achieved faster swimming velocities. Also no evidence was found for a trade-off between mean sperm velocity and ejaculate longevity. There were significant negative associations, however, between sperm total and flagellum length and ejaculate longevity, so that males with longer sperm had shorter-lived gametes. This finding has previously been reported in a study across fish species, supporting the theory that increased hydrostatic forces generated by longer flagella may trade against sperm cell longevity.     

Cholesterol and phospholipid levels of washed and percoll gradient centrifuged mouse sperm: Presence of lipids possessing inhibitory effects on sperm motility

Levels of DNA, cholesterol, and phospholipids of mouse caudal epididymal and vas deferens sperm that were processed through simple washing and Percoll gradient centrifugation were measured. The DNA and cholesterol contents of washed sperm and Percoll gradient centrifuged (PGC) sperm (DNA = 3.6 ± 0.3 pg/sperm and 3.4 ± 0.3 pg/sperm, respectively; cholesterol = 0.219 ± 0.057 nmole/μg DNA and 0.224 ± 0.030 nmole/μg DNA, respectively, for washed and PGC sperm) were not significantly different from each other; however, the phospholipid level of PGC sperm was only one half of that of washed sperm (0.315 ± 0.071 nmole/μg DNA versus 0.720 ± 0.075 nmole/μg DNA, respectively). The presence of 0.3% bovine serum albumin (BSA) in the culture medium used in sperm washing did not change the cholesterol and phospholipid contents of washed sperm. Similarly, the cholesterol and phospholipid levels of washed sperm and PGC sperm that were further incubated in BSA-containing medium for 30 min remained the same. Interestingly, substantial amounts of lipids, as determined by the cholesterol and phospholipid levels, were released into the supernatants of the sperm washes, and sperm needed to be washed at least twice to ensure their stable levels of cholesterol and phospholipids. The lipid mixture in the first sperm wash supernatant was shown to have inhibitory effects on PGC sperm motility. © 1996 Wiley-Liss, Inc.     

Boar sperm cytoplasmic droplets: Their ultrastructure,their numbers in the epididymis and at ejaculation and their removal during isolation of sperm plasma membranes

Cytoplasmic droplets of the boar are progressively lost from the flagellum of boar spermatozoa during epididymal transit, at ejaculation and during the nitrogen cavitation technique for isolation of plasma membranes. Apparently very fragile, these structures are broken up in the fluids of the reproductive tract and in the buffer used during the nitrogen cavitation procedure. The maximal potential contamination of cytoplasmic droplet internal vesicular membranes in plasma membrane fractions was determined to be 2.2% of the entire membrane surface area collected. The highly sensitive silver-stained, two-dimensional (2-D) polyacrylamide (PAGE) gels of boar sperm plasma membranes did not reveal cytoplasmic droplet, internal membrane, marker polypeptides, further demonstrating the high purity of plasma membrane preparations. In addition, freeze-fracture demonstrates that the internal membranes of the cytoplasmic droplet show few intramembranous particles and these may contribute little protein to plasma membrane preparations. The presence of two forms of vesicular elements in boar sperm Cytoplasmic droplets (typical vesicles and collapsed vesicles) is described.