Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligozoospermic sperm populations

Suspensions of capacitating human spermatozoa were analyzed for potential hyperactivated movements using videomicrographic methods. Analysis was carried out on aliquots of 22 sperm suspensions, which were proved fertile several hours later during human in vitro fertilization. After approximately 3 h of capacitation, 22.1% of the fertile spermatozoa displayed motility patterns designated as hyperactivated. Over 80% of these hyperactivated spermatozoa moved with a wide-amplitude, two-dimensional whiplash pattern, displaying marked lateral displacement of the head. Only 8.4% of capacitating spermatozoa from oligozoospermic patients showed these hyperactivated movements. The incidence of hyperactivated movements by fertile and oligozoospermic spermatozoa could be significantly increased after exposure to various motility stimulants. The clinical significance of hyperactivation as a functional assay of fertilizing capacity is discussed.     

Detection of progesterone receptors in human spermatozoa and their correlation with morphological and functional properties

In previous reports, it has been demonstrated that progesterone (P) stimulates capacitation, hyperactivation of human sperm motility and initiates the acrosome reaction (AR). This last effect has been related to the presence of non-genomic receptors for the steroid, localized on the sperm head plasma membrane. These receptors can be detected after treating spermatozoa with the non-permeable conjugate Progesterone - 3-(O-carboxymethyl) oxime: bovine serum albumin-fluorescein isothiocyanate (P-BSA-FITC). In the present study, the presence of progesterone receptors was determined in a selected sperm population with normal morphology and high progressive motility. In addition, other parameters such as the AR, hypo-osmotic swelling test, stability of chromatin and capacitating effect of P were evaluated. The percentage of P-BSA-FITC positive-spermatozoa present in the selected sperm population was higher than in total seminal spermatozoa. Furthermore, spermatozoa incubated with P showed a higher percentage motility and AR than did control spermatozoa. The HOS test indicated that membrane integrity of P-treated spermatozoa was not different to that found in the control sperm suspensions. Unexpectedly, the total sperm population treated with P showed a marked susceptibility to nuclear decondensation with reducing agents. According to these results, the selected sperm population of this study, able to respond to P, may be similar to that with good motility and normal morphology selected in the female reproductive tract, before fertilization.     

Protein tyrosine phosphorylation under capacitating conditions in porcine fresh spermatozoa and sperm cryopreserved with and without alpha tocopherol

The aim of this study was to evaluate the capacitation behaviour of fresh and α-tocopherol frozen spermatozoa. Spermatozoa frozen with or without α-tocopherol and fresh semen were incubated under capacitating conditions. Aliquots were collected at 0, 15, 30, 45, 60, 90, 120 and 180 min of incubation time. Parameters of semen quality were evaluated by optical microscopy and capacitation was determined by the epifluorescence chlortetracycline technique. Protein tyrosine phosphorylation was examined by Western immunoblotting. Motility, viability and intact spermatozoa were higher ( P  < 0.05) in fresh semen compared with frozen samples. These parameters significantly decreased, in every treatment, throughout the incubation time. Fresh semen showed a progressive increase in capacitated spermatozoa, reaching 25 ± 3% at 180 min. Cryopreserved semen had a fast increase at the beginning of incubation time (28 ± 5% at 45 min and 28 ± 3% at 30 min for samples with or without α-tocopherol, respectively). The amount of an MW 32 kDa tyrosine-phosphorilated protein, associated with capacitation, increased throughout incubation for fresh semen and spermatozoa cryopreserved with α-tocopherol. The supplementation with α-tocopherol preserved sperm plasma membrane, reflected not only in the acrosome integrity but also in a greater efficiency of energy production.     

A positive role for the superoxide anion in triggering hyperactivation and capacitation of human spermatozoa

Capacitation of human spermatozoa is essential for fertilization, and is characterized visually by hyperactivated motility. We have investigated whether reactive oxygen species could induce these two events in human spermatozoa. The addition of xanthine + xanthine oxidase + catalase (X+XO+CAT: generation of superoxide anion and removal of hydrogen peroxide) and foetal cord serum (FCS), a known biological inducer of capacitation and hyperactivation, to spermatozoa, induced levels of hyperactivation (15.4 ± 1.6% and 8.0 ± 1.0%, respectively) which were significantly higher than that of controls (5.4 ± 0.6%). The hyperactivation measured was part of the capacitation process. Furthermore, the addition of superoxide dismutase prevented the capacitation and hyperactivation induced by X+XO+CAT or by FCS. These results suggest that the superoxide anion may be involved in capacitation and hyperactivation of human spermatozoa.     

Evaluation of human sperm hyperactivated motility and its relationship with the zona-free hamster oocyte sperm penetration assay

The authors studied hyperactivated motility of human spermatozoa as a method of evaluating capacitation by examining its relationship to results of zona-free hamster oocyte sperm penetration assays (SPA) of semen samples from 50 men attending the infertility clinic. Hyperactivated motility was assessed in the seminal plasma and after swim-up preparation of spermatozoa at 1, 3, and 24 hours of incubation in capacitation media using a computer-assisted semen analysis system equipped with a hyperactivation module. Hyperactivated motility reached a peak at 1 hour and plateaued at 3 hours. The percentage of spermatozoa in seminal plasma with star-spin hyperactivated motility was significantly lower in the group showing no penetration in the SPA. The hyperactivated motility characteristics did not differ in the groups with positive or negative penetration. Correlation analysis failed to show any significant relationship between the hyperactivated motility parameters and SPA score. When the hyperactivated motility characteristics were compared in samples with normal and abnormal semen analyses, the total percentage of spermatozoa with hyperactivated motility and the percentage with star-spin at 3 hours were significantly lower in the group with abnormal semen analysis. The data indicate that lower hyperactivated motility of spermatozoa was found in patients with a score of zero for SPA and in patients with abnormal semen analysis. It was concluded that although no direct correlations were found between the results of SPA and hyperactivated motility, evaluating hyperactivated motility may still be useful as an early indicator of capacitation abnormalities of human spermatozoa not measured by SPA.     

Effect of freezing–thawing on the expression of mannose-ligand receptors on human spermatozoa: the impact on sperm capacitation and acrosome reaction

The aim of this study was to evaluate the change in the expression of mannose-ligand receptors following a freezing-thawing procedure, in order to assess its impact on sperm capacitation and acrosome reaction. Twenty semen samples were obtained from fertile donors. Sperm samples were divided into two equal volumes. One aliquot was cryopreserved and the other aliquot was incubated at 32 degrees C. After 2 h the frozen sample was thawed and both samples were further incubated at 32 degrees C to allow capacitation. Mannose receptors were examined following 4 and 22 h of incubation using a mannosylated-BSA-FITC probe. The expression of mannose-ligand receptors on the sperm plasma membrane was determined according to the fluorescence pattern: pattern I represents pre-capacitation, pattern II represents capacitated spermatozoa and pattern III represents acrosome-reacted spermatozoa. After 4 h incubation in capacitating medium, the percentages of patterns I, II and III were 90, 7 and 3% for fresh spermatozoa and 89, 8 and 3% for frozen-thawed spermatozoa, respectively (P > 0.05). Following 22 h of incubation, the percentages of patterns I, II and III were 84, 11 and 5 for fresh spermatozoa and 83, 11 and 6% for frozen-thawed spermatozoa, respectively (not significant at P > 0.05). The percentages of patterns II and III in fresh and frozen-thawed spermatozoa were increased by the same magnitude with longer incubation in the capacitating conditions. It was concluded that the freezing-thawing procedure for human spermatozoa does not affect the expression of mannose-ligand receptors and the dynamics of sperm pre-fertilization processes.     

In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation

The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham′s F10 medium plus bovine albumin at 37° and 5% CO₂ for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P < 0.05) increase in oxygen‐free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P < 0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity.     

Changes in membrane lipid order with capacitation in rhesus macaque (Macaca mulatta) spermatozoa

Lipophilic fluorescent dye merocyanine 540 is believed to stain cell membranes with increasing affinity as the lipid components become more disordered and has been associated with changes in membrane fluidity. The aim of this study was to determine whether membrane lipid disorder is associated with capacitation of macaque spermatozoa. To induce capacitation, spermatozoa from 5 rhesus macaques were incubated at 37 degrees C (5% CO(2) in air) for 2 hours in a modified Biggers-Whitten-Whittingham medium containing 30 mg/mL bovine serum albumin and 36 mmol/L NaHCO(3). Caffeine (1 mmol/L) and dbcAMP (1.2 mmol/L) were added to the medium, and incubation was performed for an additional 30 minutes. Sperm motility was determined by computer-assisted sperm analysis, and membrane lipid order and sperm viability was determined by flow cytometry with merocyanine (2.7 micromol/L) and Yo-Pro-1 (25 nmol/L), respectively. Tyrosine phosphorylation of proteins in sperm tail was immunohistochemically examined by means of anti-phosphotyrosine (alpha-PY; clone 4G10). Capacitation resulted in a significant increase in the amplitude of lateral head displacement and beat cross frequency (P < .005) and a significant decrease in linearity and straightness in capacitated spermatozoa (P < .005), compared with control spermatozoa, which suggests the expression of hyperactivated motility. Animals in which capacitation was induced had a significant increase in the number of spermatozoa showing tyrosine phosphorylation of tail proteins (P < .0001) and a significant increase in the intensity of merocyanine fluorescence (P < .0001), compared with control animals. The observed decrease in membrane lipid order after capacitation was induced was not associated with surface exposure of phosphatidylserine, as determined by flow cytometry with annexin V-Alexa Fluor 488. Merocyanine may be a useful tool for investigating the role of the plasma membrane on capacitation and other cytotoxic events in macaque spermatozoa.     

Speciesspezifische Motilitätsmuster hyperaktivierter Säugetierspermatozoen und die quantitative Auswertung der Hyperaktivierung von Bullenspermatozoen/Species Specific Motility Patterns of Hyperactivated Mammalian Spermatozoa and Quantitative Analysis of the Hyperactivation of Bull Spermatozoa

Movement tracks of spermatozoa of human, lion, tiger, cow, pig and sheep are recorded by dark field photography (fluid layer thickness 16.7 microns, exposure time 1 s). Comparison before and after capacitation by an incubation of 2 h in modified tissue culture medium TCM 199 with 10% fetal calf serum resulted in two quite different patterns of hyperactivated spermatozoa: 1) Tracks are broadened due to enlarged lateral head displacement or radius of rotating head movements respectively (tiger, lion) and show beside that a markedly increase in erratic motility (human). 2) In the studied species of artiodactyla, cow, pig and sheep, a qualitative new, panicle-like pattern arised as a result of superposition of spermatozoa head pendular movements around the axis of forward motility and the other one around the head axis. This new type of tracks allows a simple quantitative analysis of hyperactivation of bull spermatozoa, first described in this report. The method is applied to investigations on efficacy of capacitation media and provides evidence for high individual differences of semen donors in capacitation success.     

Hyperactivated motility is coupled with interdependent modifications at axonemal and cytosolic levels in human spermatozoa.

Whether the motility characteristics of hyperactivated spermatozoa were determined by stable changes at the axonemal level and whether the presence of cytosolic factors was required for the expression of these changes was investigated. Different degrees of sperm hyperactivation were produced in Percoll-washed spermatozoa after incubation for 1 hour to 3 hours at 37 degrees C in Ham's F-10 supplemented with human blood plasma or fetal cord serum. Decomplemented fetal cord serum induced the highest percentage of hyperactivation (19 +/- 3%), followed by human plasma (13 +/- 2%). Fetal cord serum that was not decomplemented did not induce a level of hyperactivation (1.7 +/- 0.2%) significantly different from control levels (0.9 +/- 0.2%). Dialyzed fetal cord serum induced intermediate levels of hyperactivation (6 +/- 1%). The motility characteristics of demembranated sperm models of hyperactivated spermatozoa induced by decomplemented fetal cord serum and nonhyperactivated spermatozoa were compared by videomicroscopy and computer-assisted digital image analysis. After demembranation with Triton X-100 and reactivation of motility by Mg. adenosine triphosphate (Mg.ATP), hyperactivated and nonhyperactivated spermatozoa showed similar motility characteristics. However, hyperactivated spermatozoa that were demembranated and reactivated in cytosolic extracts from hyperactivated spermatozoa had significantly higher (P less than 0.05) linear velocity (33 +/- 4 mu/sec) and lower linearity (0.23 +/- 0.04) than control spermatozoa that were demembranated and reactivated in control cytosolic extracts (velocity = 24 +/- 1 mu/sec; linearity = 0.32 +/- 0.02). The data suggest that the expression of hyperactivated motility requires interdependent changes at the axonemal and cytosolic levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperactivation of Human Spermatozoa: Measurement of Motility before and after Incubation

The motility rate of sperm during capacitation process was determined by calculating a sperm motile efficiency index (SMEI) using a hemocytometer. The SMEI values for the sperm from 100% of normal fertile men and 64% of infertile patients increased significantly by the fourth hour of incubation, while for 36% of infertile patients there was no increase. A correlation between the increase of SMEI and original motility, original SMEI and original semen amount was not possible. In addition, a decrease of the SMEI value was observed after sperm incubated for 3 hours were added to the seminal plasma of the donor. It is concluded that in human spermatozoa "hyperactivation and dehyperactivation" can be measured by SMEI.     

Loss of acid phosphatase from rat spermatozoa as a method for assessing the acrosome reaction

A method is presented for evaluating the extent of the acrosome reaction by measuring the release of acrosomal acid phosphatase from rat spermatozoa during incubation under capacitating conditions. Treatment of spermatozoa with lysophosphatidylcholine or Triton X-100 released the acid phosphatase from the sperm cell. Using this enzymatic method we could not detect an alteration in enzyme activity following 5 h incubation under capacitating conditions. The effect of in vitro capacitation for 5 h in the absence or presence of heparin or ionophore A23187 was studied. Incubation in the presence of heparin (10 micrograms ml-1) caused a 32% increase in enzyme activity. After exposure of the spermatozoa to ionophore A23187 (0.5 microM) 16% increase of enzyme activity could be detected.     

Effect of natural antioxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen-thawed bull spermatozoa

Summary.  Bovine spermatozoa from frozenthawed semen are sensitive to lipid peroxidation. Vitamin E protects sperm membrane against oxidative damage. Sperm capacitation produces structural changes on the plasma membrane. Reactive oxygen species could be involved in the capacitation process. The aim of this work was to study the influence of natural antioxidants on the plasma membrane and the influence of reactive oxygen species during bovine sperm capacitation. Sperm samples were frozen in a standard diluent, with and without vitamin E (1 mg ml^-1). Heparin (60 μg ml^-1) was used as a sperm capacitation inductor. Sperm capacitation was evaluated by chlorotetracycline assay. Lipid peroxidation was determined by the 2-thiobarbituric acid assay. A diminution of thiobarbituric acid reactive substances was observed in sperm samples frozen with vitamin E ( P < 0.05). The addition of vitamin E to the freezing diluent had no effect on the capacitated pattern ( P > 0.05).
When vitamin E and vitamin E + vitamin C were added to the capacitation medium, a significant decrease in the percentage of capacitated spermatozoa ( P < 0.05) was observed in both cases. The addition of superoxide dismutase (0.1 mg ml^-1) or H₂O₂ (50 μM) in the incubation medium, decreased the percentage of capacitated spermatozoa ( P < 0.05). Vitamin E protects the plasma membrane against lipid peroxidation during sperm capacitation, and the presence of superoxide anion would be necessary for frozen-thawed bull sperm capacitation.     

The influence of homogenous zona pellucida on human spermatozoa hyperactivation, acrosome reaction and zona binding

The study aimed to evaluate the changes in sperm motion characteristics and the occurrence of hyperactivation among sperm populations after exposure to human zona pellucida. Motile spermatozoa samples were used to evaluate the sperm-zona binding capacity, zona-induced acrosome reaction and changes in sperm motion characteristics. Sperm motion characteristic changes studied included straight line velocity, curvilinear velocity, amplitude of lateral head displacement, straightness and beat cross frequency. Recordings were performed on semen immediately after liquefaction, 3 h capacitation and after exposure to solubilised human zona pellucida. The semen samples were divided into morphology categories, namely six (16 +/- 1.4% normal forms, normal patterns), 31 (8 +/- 1.7% normal forms, G-pattern) and 27 (3 +/- 1.3% normal forms, P-pattern). The Hemizona Indices for the three morphology groups namely normal, G-patterns and P-patterns, were 77 +/- 6%, 61 +/- 5% and 41 +/- 5% respectively (P 相似文献   

Participation of superoxide anion in the capacitation of cryopreserved bovine sperm

Mammalian spermatozoa must undergo a preparation period known as capacitation to become capable of fertilizing oocytes. Controlled amounts of reactive oxygen species (ROS), such as superoxide anion (O2.-) and hydrogen peroxide (H2O2) have been shown essential for capacitation and acrosome reaction. The presence of an oxidase in the sperm plasma membrane has been suggested. The objective of the present study was to provide evidence for the production of O2.- by capacitating cryopreserved bovine spermatozoa. Percentages of capacitation and acrosome reaction were determined by the chlortetracycline assay. The effect of several nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors on capacitation was also studied. H2O2 production was determined by the fluorometric assay using the p-hydroxyphenylacetic acid-horseradish peroxidase system. Superoxide dismutase (SOD) activity was determined spectrophotometrically at 480 nm. Heparin-dependent capacitation was inhibited by all NADPH oxidase inhibitors tested (p < 0.05). Significant levels of H2O2 were produced during capacitation with heparin; such production was inhibited by diphenyleneiodonium, one of the NADPH oxidase inhibitors. The addition of catalase to the incubation medium failed to modify the capacitation rate; inhibition was only observed when SOD was present (p < 0.05). Endogenous SOD activity was diminished during heparin-dependent capacitation (p < 0.05). Similar levels of acrosome reaction induced by lysophosphatidylcholine were obtained in both heparin and O2.--dependent capacitation. Overall results suggest the participation of a sperm oxidase in bovine sperm capacitation. H2O2, generated by O2.- dismutation, failed to participate in capacitation, although this ROS may have been able to decrease endogenous SOD activity. Exogenous O2.- promotes physiological capacitation in cryopreserved bovine sperm, thus allowing the acquisition of fertilizing capacity.     

Analysis of the lipid content and the motility of human sperm after follicular fluid treatment*

Summary The effect of human follicular fluid (hFF) on the cholesterol and phospholipid content and the movement characteristics of human spermatozoa were studied. Semen was selected by a discontinuous Percoll gradient and incubated during in vitro capacitating conditions with B2 medium supplemented with hFF 20%. Percoll pelleted spermatozoa were incubated in either B2 (B2-Percoll) or B2 supplemented with hFF (hFF-Percoll). In hFF-Percoll, we observed a time-dependent (24 h) decrease in both the cholesterol and phospholipid contents (cholesterol: 10.1 vs. 8.7 nmol 10^-7 spermatozoa; phospholipids: 17.5 vs. 15.7 nmol 10^-7 spermatozoa, P < 0.05). This decrease in cholesterol and phospholipids in human spermatozoa was concomitant with a high straight line velocity, a high progressive motility percentage and an increased value of lateral head displacement without any significant alteration of the spermatozoal membrane. No modification of the cholesterol: phospholipid ratio after 2 and 24 h of incubation in either B2-Percoll (0.61, 0.54) in hFF-Percoll (0.59, 0.63) was observed when compared with original control semen. It is suggested that the decrease in cholesterol and phospholipids in hFF-Percoll may be taken into account for the changes of membrane modification as part of the capacitation process.     

Effect of bafilomycin A1, a specific inhibitor of vacuolar (V-type) proton ATPases, on the capacitation of rabbit spermatozoa

Mammalian spermatozoa maintain precisely regulated ionic gradients that must be modified during capacitation and the acrosome reaction. In other cell types, ionic gradients are mainly regulated by the presence in plasma membranes of three metabolically different types of ATPases. The modifications induced during in vitro capacitation of rabbit spermatozoa by the specific inhibition of V-type H+-ATPases with bafilomycin A were studied. We used chlortetracycline binding to rabbit spermatozoa to monitor capacitation, and the coomassie brilliant blue method to identify acrosome-reacted sperm cells. There was a significant difference between the percentage of epididymal (66 +/- 7%) and ejaculated (43 +/- 11%) spermatozoa capacitated in vitro, after a 6-h incubation period in the presence of Ca2+ without ATPase inhibitor. The presence of bafilomycin significantly reduced these numbers (25 +/- 11 and 16+/- 8%, epididymal and ejaculated spermatozoa, respectively) and eliminated the difference. Ejaculated spermatozoa capacitated in the absence of bafilomycin showed a linear increase in the percentage of acrosome reactions induced by the addition of A23187 (12 +/- 5, 23+/- 6 and 31 +/- 5 after 15, 30 and 45 min). The presence of 0.2 micromol l-1 bafilomycin during the capacitation incubation induced a significant decrease in the acrosome reaction percentages (4 +/- 2, 8 +/- 3 and 14 +/- 4 after 15, 30 and 45 min). The addition of bafilomycin after the capacitating period had no effect upon the induction of the acrosome reaction by A23187. These results indicate that vacuolar ATPases play an important role during rabbit sperm capacitation. However, once the spermatozoa have been capacitated, V-type ATPases do not have a significant participation during the acrosome reaction.     

Kinematics of human spermatozoa incubated under capacitating conditions

Suspensions of seminal plasma-free human spermatozoa were prepared by swim-up from semen and studied using high magnification videomicrography after incubation under capacitating conditions for 1.5-2 h. Three subpopulations of capacitating spermatozoa showing different patterns of motility could be distinguished visually: forward progressive, transition phase, and hyperactivated motility. The purpose of this study was not to determine the relative proportions of spermatozoa in these three categories but to describe their movement characteristics. Manual track plotting and analysis allowed value derivation for the curvilinear, average path and straight-line velocities (VCL, VAP, and VSL respectively); for the three progression ratios of linearity (LIN = VSL divided by VCL X 100), straightness (STR = VSL divided by VAP X 100), and wobble (WOB = VAP divided by VCL X 100); and also for the amplitude of lateral head displacement (ALH) and the beat/cross frequency (BCF). Algorithms produced from these motion characteristics allowed distinctions to be made between cell motility patterns. Spermatozoa with straight-line velocity (VSL) greater than or equal to 40 microns/s, linearity (LIN) greater than or equal to 60% and amplitude of lateral head displacement (ALH) less than 5 microns were FP or non-hyperactivated. Tracks with curvilinear velocity (VCL) greater than or equal to 100 microns/s, linearity (LIN) less than 60% and amplitude of lateral head displacement (ALH) greater than or equal to 5 microns showed concomitants of hyperactivation. Classical hyperactivated tracks also showed straightness (STR) less than 60% and straight-line velocity (VSL) less than 30 microns/s.     

The role of glucose in supporting motility and capacitation in human spermatozoa.

Glucose has been reported to be beneficial to human sperm for optimal capacitation and fertilization, although it is unclear whether glucose is required for providing extra metabolic energy through glycolysis, or for generating some other metabolic product. In this study, the effects of sugars on human sperm capacitation, motility, and energy production were investigated. The glucose concentration that supported the greatest number of acrosome reactions was 5.56 mmol L(-1). Compared with incubations with no added sugar, this concentration of glucose, fructose, mannose, or galactose appeared to slightly increase the number of acrosome reactions occurring after 18 hours of capacitation, or following induction by 2 micromol A23187 + 3.6 mmol pentoxifylline L 1, but only glucose had a statistically significant effect. Glucose supported increased penetration of zona-free hamster oocytes, but its advantage was not statistically significant. The addition of 5.56 mmol glucose or fructose L(-1) to sugar-free medium immediately increased the adenosine triphosphate (ATP) concentration and motility of sperm. These parameters were then stable for 3 hours, but declined markedly after 18 hours. In the absence of a glycolysable sugar, motility began to decline in the first hour and only 2% or 3% of sperm remained motile after 18 hours. Glucose or fructose was required to support hyperactivated motility. 2-Deoxyglucose was detrimental to the ATP concentration and motility of sperm, and supported fewer spontaneous or progesterone-stimulated acrosome reactions than were observed in the absence of a sugar. We conclude that glycolytic ATP production is required for vigorous motility and hyperactivation in human sperm. Other products of glucose metabolism are not essential to support capacitation, but they may have a small, enhancing effect.     

Hyperactivated motility in rhesus macaque (Macaca mulatta) spermatozoa

Macaque spermatozoa can be capacitated according to a defined protocol and exhibit hyperactivated motility similar to that described in other species. The aim of this study was to create a method for defining hyperactivation that could be routinely used in the laboratory alongside our existing sperm motility analysis protocol. Percoll-separated macaque spermatozoa were incubated for 2 hours (37 degrees C; 5% CO(2) in air) at a concentration of 20 x 10(6)/mL in bicarbonate (36 mmol)-buffered Biggers, Whitten and Whittingham medium (BWW) containing 30 mg/mL bovine serum albumin (BSA), followed by an additional 30 minutes with (capacitated) or without (incubated) caffeine (1 mmol) and dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP; 1.2 mmol). One hundred and fifty progressive and hyperactivated tracks were selected from each of three monkeys. Thresholds for hyperactivation were based on the 10th (amplitude of lateral head displacement, ALH) and 90th (linearity, LIN) percentiles of the hyperactivated kinematic data set and were LIN less than or equal to 69% and ALH greater than or equal to 7.5 microM; a threshold of greater than or equal to 130 microM/s was also included for curvilinear velocity (VCL). These thresholds were 91% effective at identifying hyperactivated tracks. Capacitation of macaque spermatozoa, by the addition of caffeine and dbcAMP, resulted in a significant increase in ALH, VCL, and beat cross frequency and a significant decrease in total and progressive motility, straight line velocity, straightness, and LIN when compared to incubated spermatozoa, suggesting the expression of hyperactivated motility. Utilizing the above thresholds, hyperactivation was expressed by 5% +/- 0.8% of the incubated sperm population vs 53 +/- 3.7% of the capacitated sperm population (P < .0001). Hyperactivation was not observed when dbcAMP and caffeine were added separately and was significantly (P < .005) reduced by the addition of H-89. The results of this paper demonstrate that hyperactivation can be reliably estimated for rhesus macaque spermatozoa.