Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility.

Under moderate conditions, reactive oxygen species (ROS) have been shown to inhibit sperm motility after several hours of incubation. The rapid decrease in flagellar beat frequency observed within the first hour of contact between ROS and spermatozoa was associated with a rapid loss of intracellular adenosine triphosphate (ATP). Motility of intact spermatozoa ceased when their ATP concentration was reduced by 85 +/- 5%. Axonemal damage was confirmed when ROS-treated spermatozoa could not reactivate motility after demembranation in a medium containing magnesium adenosine triphosphate (Mg.ATP). However, in conditions allowing rephosphorylation of the axonemes (addition of cyclic adenosine monophosphate, or cAMP, and protein kinase or sperm extracts to the demembranation medium), the motility could reactivate. Three lines of evidence suggested that ATP depletion induced by ROS treatment was responsible for the effects observed in spermatozoa. First, the rapid decrease in intracellular ATP observed after ROS treatment was closely followed by a decrease in beat frequency, loss of intact sperm motility, and axonemal damage due to insufficient phosphorylation. Second, incubation of spermatozoa with the combination pyruvate-lactate allowed maintenance of sperm ATP at a normal level and prevented the effects of ROS; furthermore, spermatozoa immobilized after ROS treatment, then supplemented with pyruvate-lactate, were able to reinitiate motility in parallel with an increase in their ATP level. Third, treatment of spermatozoa with rotenone, an ATP depleting agent, produced effects similar to ROS treatment and could also be reversed by the addition of pyruvate-lactate. These data are consistent with the conclusion that ROS treatment produced axonemal damage mostly as a result of ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of sperm motility patterns in the murine epididymis

The maturation of sperm motility in the epididymis of the mouse was assessed using a computer-assisted sperm analysis system. Spermatozoa were immotile in the most proximal regions of the epididymis but developed motility rapidly in the proximal caput epididymis; the percentage motility remained high thereafter. In the caput, flagellar beat was erratic with little progression, but in the proximal corpus region circular movement patterns were reflected in reduced linearity (LIN) and straightness (STR) of the sperm tracks, although velocities were little changed and wobble (WOB) increased. In the mid-corpus region, however, all velocities, LIN, STR and WOB, increased markedly. In more distal regions there was little change in these parameters. Distribution curves of the kinematic parameters of spermatozoa obtained from each region indicated that the most heterogeneous population was that from the mid-corpus epididymis; the most homogeneous was that from the mid-cauda region. Individual sperm tracks revealed slowly progressing spermatozoa in the distal caput, transforming to motion in small circles, interrupted by more linear progression. More distally, linear progression was interrupted by looping movements and a generally progressive path was observed thereafter, with less deviation from the average path as the spermatozoa matured. Spermatozoa displaying motion compatible with passing the uterotubal junction were first found in the proximal corpus epididymis, in agreement with earlier in-vivo fertilization studies on where fertilizing capacity is achieved with epididymal spermatozoa.     

冷冻复温后低温保护剂和精子激活剂对精子功能影响的研究

本文对冷冻复温后精子的功能进行了研究观察，发现精液保护剂（甘油－卵黄－枸橼酸钠）对冷冻复温后精子的存活有明显的保护作用，而单纯应用卵黄或甘油溶液却对精子功能存在不利的影响。体外处理精子时应用ＡＴＰ、肌苷对精子的功能无任何的改善。两种浓度的苯甲酸钠咖啡因（１２ｍＭ和４ｍＭ）对冷冻复温后精子在不同时间内的存活和ＣＭ穿透力均有明显的提高作用     

Control of Epididymal Sperm Motility; An Approach to Male Fertility Regulation

Thermal immobilization was designed as a simple approach to study factors controlling sperm motility. Heating dissected epididymis or isolated epididymal sperm at 48°C to 52°C for 5 min abruptly and completely immobilized the sperm. The thermally immobilized sperm showed some alteration of membrane lipids, as indicated by the response of the surface ATPase to temperature change. However, no qualitative change in the surface negative charge was detectable by the binding of the cells to DEAE-Sephadex beads. The immobilized cells can metabolize ATP into ADP and AMP similar to the control epididymal sperm. The ATP-dependent sliding or disintegration of sperm flagella was completely prevented in the immobolized cells. The findings suggested that heat-treatment disrupted the integrity of the sperm membrane and the motile apparatus, resulting in the loss of sperm motility. The development of post-testicular antifertility agents should aim at interfering specifically with these components of the sperm.     

The maturation of sperm motility in the epididymis and vas deferens of the vervet monkey, Cercopithecus aethiops

Among the diverse facets of sperm maturation, changes in motility are conspicuous and hence studies of sperm kinematics might provide good indices for sperm maturation. Accordingly, the maturation of sperm motility in the epididymis and vas deferens of the vervet monkey, Cercopithecus aethiops, was assessed using a computer-aided sperm motility analysis system. The results revealed clear trends in the development of both sperm motility per se and in the movement characteristics of motile spermatozoa from different regions of the epididymis, the vas deferens and the ejaculate, reflecting maturational changes associated with the attainment of functional motility and fertility. Motion of spermatozoa from the caput epididymis was sluggish and irregular. As the spermatozoa moved through the corpus epididymis, motility increased sharply, and continued to improve through the cauda epididymis and vas deferens. Despite the high proportion of motile cells, full maturation of motion capabilities was not completed in spermatozoa from the corpus epididymis. Only once spermatozoa had reached the cauda epididymis and vas deferens did they attain their full vigour, and swam rapidly (greater VCL, VSL and VAP) with straightline trajectories (greater LIN, WOB and STR; lower ALH, MAD and CURV). After acquiring their maximal percentage motility and progressive velocity in the cauda epididymis and vas deferens, a slight decline in motility and vigour occurred in ejaculated spermatozoa, and was possibly associated with the ageing of stored spermatozoa. The results from this investigation have revealed clear trends in the maturation of the motility of vervet monkey spermatozoa during their transit through the epididymis and vas deferens and final emergence in the ejaculate, and have provided crucial baseline information on the reproductive physiology of this potentially valuable biomedical model to serve as a reference for future studies in reproductive toxicology.     

alpha-Glucosidase, sperm ATP concentrations, and epididymal function

It has been suggested that alpha-glucosidase may be a marker of epididymal patency and function. Spermatozoal ATP concentrations decrease during passage through the epididymis, indicating efficient maturation. We correlated sperm motility with seminal plasma alpha-glucosidase activity and spermatozoal ATP. The sperm motility correlation with alpha-glucosidase activity was significantly positive, and the sperm motility correlation with spermatozoal ATP was significantly negative. It appears that high-alpha-glucosidase activity and low-spermatozoal ATP were present in semen with good sperm motility and could possibly indicate efficient epididymal function.     

Serum factors stimulate the motility of human spermatozoa

Motile human sperm were collected from a Percoll gradient and the effects on sperm motility of human serum, various serum fractions, follicular fluid and seminal plasma were assessed. In culture medium alone (RPMI-1640) sperm motility was lost after about 5 h. The addition of male blood serum both enhanced sperm motility and prolonged viability very significantly. Albumin, seminal plasma and follicular fluid all stimulated sperm motility but to a much lesser extent than did blood serum. No difference was noted between male serum or female serum which had been collected during the follicular or luteal phases of hormone-stimulated cycles and which contained high levels of oestradiol. Serum fractions obtained by separation on Sephacryl S-300 column were tested for their ability to enhance sperm motility. The most pronounced effect, much superior to that achieved by the albumin fraction, was obtained by a fraction with a molecular weight of around 200 kD. In conclusion, certain factors in human serum, which are different from albumin, strongly support sperm motility. The high serum concentrations of oestradiol resulting from hormone stimulation for in-vitro fertilization do not invalidate the use of serum from the same patient during sperm preparation, or in the medium used for ovum insemination and culture.     

Effects of Methyl Xanthines upon the Spermatozoa Adenosinetriphosphate (ATP) Concentration in Normal Semen

The aim of the present study was to ascertain the effect of methyl xanthines upon the spermatozoa ATP concentration in normal semen in vitro. 26 normal semen were studied. The specimens were diluted after liquefaction with equal volume of Lopata's buffer or Lopata's buffer plus 1 or 6 mM either of caffeine or pentoxifylline. The samples of semen were incubated at room temperature during 90, 180 and 240 minutes before motility and ATP determination by the firefly luciferin-luciferase method. Significant variation was observed in sperm motility nevertheless variations in the ATP concentration was not induced by any of the methyl xanthines we used.     

奥硝唑对大鼠精子的影响及作用机制探讨

目的: 探讨奥硝唑降低大鼠精子活动力的影响因素及作用机制。　方法: 20只SD大鼠随机分为低剂量组(n=5)、高剂量组(n=8)和正常对照组(n=7)。低剂量组和高剂量组分别给予 200、400mg/kg奥硝唑灌胃,正常对照组给予0. 5%羧甲基纤维素钠灌胃,连续 20d。末次给药 24h后戊巴比妥钠麻醉,取附睾及睾丸,观察精子密度、精子活动力;检测睾丸、附睾组织匀浆中乳酸脱氢酶、α 葡糖苷酶、丙二醛、果糖的浓度变化。观察睾丸与附睾有无形态学改变。　结果: 奥硝唑 200、400mg/kg,连续灌胃给药 20d,能显著降低精子活动力(P<0. 01),对精子密度无明显影响(P>0. 05)。与正常对照组比较,高剂量组乳酸脱氢酶水平明显降低 (P<0. 01),丙二醛含量明显升高(P<0. 05)。　结论: 奥硝唑通过增加细胞脂质过氧化产物丙二醛,抑制附睾中的能量转换酶或非蛋白类物质使精子细胞受损、活动力降低,这是奥硝唑致弱精子症动物模型的作用机制之一。     

The effect of serum on motility of human spermatozoa in culture

The motility of spermatozoa was higher at 6-22 h in Ham's F10 culture medium supplemented with 20-30% human serum than with lower proportions of serum or with 1% human serum albumin. Heat treatment (56 degrees C, 1 h), charcoal extraction and dialysis (18,000 molecular weight cut-off) of the serum did not reduce sperm motility suggesting that high molecular weight components are responsible for maintenance of motility. Renewing the medium (Ham's F10 with 30% serum) at 7 h resulted in better sperm motility and velocity at 22 h. At 22 h the pO2, pCO2, pH and sodium concentrations were not different in replenished and control cultures, but the concentration of glucose was higher and that of potassium lower if the medium was changed. These results suggest that addition of 20-30% human serum and renewal of medium at intervals is beneficial for sperm culture and may be of use in in vitro fertilization.     

Effects of propranolol and caffeine on movement characteristics of human sperm

Human sperm, separated on Percoll gradients and transferred to cell culture medium, were exposed to various concentrations of propranolol (0.8-800 microM) or caffeine (3 microM-17 mM) for 4 h. Their motility pattern was analysed after 5 min and 4 h, employing the computerized Cellsoft system. Curvilinear velocity (VCL), percentage motile sperm, linearity (LIN), mean amplitude of lateral head displacement (ALHMEAN) and beat cross-frequency (BCF) were assessed. Both drugs had a practically immediate effect on the sperm. Propranolol concentrations greater than 80 microM had a negative effect on all movement variables, except VCL and ALHMEAN, which showed a slight, non-significant, initial increase. LIN and the percentage motile sperm appeared to be somewhat negatively affected at lower concentrations (80 microM) than the other variables, and were reduced further at higher concentrations and with time. The presence of 800 microM propranolol immobilized all sperm within 4 h. Caffeine at 1.7 and 5 mM, increased VCL and ALHMEAN. In contrast, the highest caffeine concentration tested (17 mM) had a negative effect on all variables at 4 h after addition.     

Comparison between a swim-up and a Percoll gradient technique for the separation of human spermatozoa

Two methods of separating human sperm were compared using twenty-two semen samples. The sperm were separated by a swim-up technique or by self-migration on a Percoll gradient followed by medium change. After separation, the sperm obtained were assessed for progressive motility, ATP content, energy charge index ([ATP + 0.5 ADP]/[ATP + ADP + AMP]) and morphology. In general, and especially for semen samples containing less than 20 X 10(6) sperm/ml, separation by Percoll gradient selected sperm that were superior to those separated by the swim-up technique. The relatively high energy charge index (greater than 0.8) showed that the sperm tolerated the separation conditions well. It is suggested that self-migration on a Percoll gradient should prove useful for obtaining sperm of high quality.     

Role of the epididymis in sperm competition

Although it is generally understood that the testes recruited kidney ducts for reproductive function during the evolution of vertebrates, little is understood of the biological significance of the adaptation. In the context of the evolution of the mammalian epididymis, this report provides evidence that a major role of the epididymis is to enhance a male＇s chance of achieving paternity in a competitive mating system. A unique example of sperm cooperation in monotremes is used as evidence that the epididymis produces sperm competition proteins to form groups of 100 sperm into bundles that have a forward motility nearly thrice that of individual spermatozoa. As it required 3-h incubation in vitro under capacitation conditions to release motile sperm from the bundles, it is suggested that the monotremes provide an example of capacitation that is quite different from capacitation in higher mammals. It is suggested that variation between species in the intensity of sperm competition could explain the variation that occurs between species in the amount of post-testicular sperm maturation and storage in the epididymis, an explanation of why the human epididymis does not play as important a role in reproduction as the epididymis of most mammals. （Asian J Androl 2007 July; 9： 493-499）     

Sperm membrane modulation by Sapindus mukorossi during sperm maturation

Aim: To observe the alterations in the biochemical and biophysical changes in the sperm membrane during sperm maturation in male rats treated with the water extract of the fruit pericarp of S. mukorossi. Methods: Adult male Sprague-Dawley rats were gavaged the aqueous extract of the fruit pericarp of S. mukorossi at a dose of 50 mg/kg/d for 45 days. On day 46, the sperm parameters were observed in different sections of the epididymis and the sperm superoxide dismutase and the lipid peroxidation was determined and compared with the controls. The testis and epididymis were routinely prepared for histological examination under the light microscope. Results: No significant differences in the sperm number and morphology were observed between the control and treated groups. However, a significant inhibition (P<0.05-0.01) of sperm motility in the caput, corpus and cauda regions of the epididymis was seen in the treated group. No significant histopathological changes were found in the testis and epididymis. The     

Influence of complement on sperm motility

Isolated sperm from normo-, oligo- and astheno-spermic men were incubated for 20 h in medium supplemented with 8% heat-inactivated or untreated human serum, and in medium with heated or untreated serum deficient in complement factor C3. Before and after incubation, sperm motility was assessed by means of a computer-assisted semen analyser. The results did not show significant differences between the motility of sperm incubated in heated or untreated serum. It is concluded that heating of homologous serum is not necessary for preserving sperm motility and in some cases may even be disadvantageous.     

Contribution of epididymal factors to sperm maturation and storage

Summary.  In fertile men, the majority of epididymal spermatozoa acquire the potential to fertilize (assessed with sperm function assays) on passage into the corpus and cauda regions of the epididymis. Although secretions of the epididymal epithelium are clearly important for sperm maturation and survival, their role in this process has yet to be fully determined. Alterations in epididymal sperm membranes may result from the incorporation of protein, sugar and lipid determinants. Most probably, factors of epididymal origin act in concert with constitutional changes to spermatozoa, which together permit full sperm function. Epididymal spermatozoa incubated with epididymal epithelial cell cultures can undergo some maturation in vitro , which can lead to the development of sperm fertilizing capacity. Co-incubations of human sperm with epididymal epithelial cultures, at 37 °C with medium replenished every other day, led to 50% of spermatozoa retaining motility after 8 days. In one case, a few spermatozoa survived for 17 days, the inherent maximal survival time of human spermatozoa in situ. An important aspect of coculture experiments is that close interactions between spermatozoa and epithelial cells can be examined in detail. This coculture technique may yield important information related to epididymal sperm maturation and storage.     

Addition of caffeine to equine thawed sperm increases motility and decreases nitrite concentration

The aim of this study was to improve the quality of frozen-thawed equine sperm by the addition of caffeine to it. Semen from nine stallions was frozen and different concentrations of caffeine (3, 5 and 7.5 mM) were added to frozen-thawed semen. The sperm kinetic parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite and hydroperoxide concentrations of frozen-thawed semen were measured using spectrophotometry. Sperm fertility was evaluated by artificial insemination (AI) of 16 mares with thawed ejaculates (control and 5 mM caffeine-treated groups). Compared to that in the control, the addition of 5 mM caffeine induced an increase in sperm motility (38.9 ± 2.8 versus 32.6 ± 3.4%), and a decrease in nitrite concentration (11.4 ± 2.1 versus 12.8 ± 2.9 µM/µg protein, p < .05). Moreover, the pregnancy rate from AI in the caffeine group was significantly higher (62.5%) than that in the control group (12.5%). These data suggest that caffeine reduced the nitrite concentration and enhanced sperm motility in thawed equine sperm, thus increasing the fertility rate in mares inseminated with caffeine-treated equine semen.     

Successful lowering of epididymal carnitine by administration of pivalate to rats

This study was designed to lower the epididymal content of carnitine in male rats and to examine subsequent effects on fertility and sperm motility. As carnitine is transported from serum into the epididymal lumen a method to lower serum carnitine was sought. Administration of 20 mmol/L sodium pivalate in the drinking water for up to 5 weeks substantially lowered serum carnitine (to 20% of control levels within 1 week) and reduced epididymal carnitine content (to 25% of control levels in the proximal and 52% of control in distal regions) within 2 weeks. Carnitine in distal cauda epididymal fluid was also reduced (to 30% of control levels) but no changes were observed in the sperm carnitine content. The percentage motility and kinematic parameters of spermatozoa released from four epididymal regions and diluted into artificial medium were unaltered by the treatment, and all males retained their fertility in mating tests performed at weekly intervals. Increasing the dose of sodium pivalate administered to 60 mmol/L for 2 weeks lowered serum carnitine concentration more but did not further decrease epididymal carnitine content and altered neither sperm motility nor male fertility. The rat epididymis secretes an excessive amount of carnitine into its lumen so that substantially lowering the tissue content does not reduce sperm carnitine or affect their motility or fertilizing ability.     

Effects of piperine on hamster sperm capacitation and fertilization in vitro

The effect of piperine on the fertilizing ability of hamster sperm was investigated in vitro . Sperm were incubated in a capacitation medium for 3 h prior to co-incubation with hamster eggs in a fertilization medium for another 3 h. Addition of 0.18–1.05 mM piperine to the capacitation medium reduced both the percentage of eggs fertilized and the degree of polyspermia in a dose-dependent manner. When piperine was added to the fertilization medium alone, a significant reduction in fertilization was observed only at high doses (0.70–1.05 mM). The presence of piperine in the capacitation medium inhibited the acrosome reaction in a dose-dependent manner but had no effect on sperm motility, whether this was measured quantitatively or qualitatively. Piperine also inhibited the influx of calcium into sperm during capacitation. It is suggested that such an inhibition might be a major cause of a reduction of the acrosome reaction and the subsequent impairment of fertilizing ability of sperm.     

Famotidine does not affect human sperm fertility characteristics (motility,viability and DNA integrity) in vitro

Famotidine, a histamine‐2 receptor antagonist, is commonly used to relieve the acid‐related gastrointestinal diseases; however, its effect on human sperm parameters, and hence on sperm function, is still undetermined. Here, we intended to measure human sperm motility, viability, and DNA integrity of ejaculated human sperm in the presence of famotidine at 0, 0.1, 1 and 10 mM concentrations in vitro. Forty‐nine semen samples of normal count, motility, and morphology were included in this study. Sperm motility was assessed using Makler counting chamber and a phase contrast optics (200× magnification), whereas sperm viability was assessed using eosin–nigrosin staining procedure. The effect of famotidine on sperm DNA integrity was measured using flow cytometry. Famotidine at 0.1, 1 or 10 mM had insignificant effect on human sperm motility (progressive, p = .9594; and total, p = .8420), sperm viability (p = .6471), and content of DNA breaks in sperm (p > .05) compared with the control. In conclusion, famotidine at 0.1, 1 or 10 mM did not alter human sperm motility, viability or DNA integrity in vitro. Although, these findings indicate safety of famotidine in human sperm, further in vivo studies are required to establish the drug's safety.