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1.
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.  相似文献   

2.
Mammalian spermatozoa are sensitive to oxygen-induced damages mediated by lipid peroxidation of the cell membrane. The aim of this study was to evaluate whether reactive oxygen species (ROS) could also induce axonemal damage. When Percoll-separated spermatozoa were treated with hydrogen peroxide, or the combination xanthine and xanthine oxidase (X + XO), there was a progressive decrease, leading to a complete arrest, in sperm flagellar beat frequency. Once demembranated in a medium containing magnesium adenosine triphosphate (Mg.ATP), ROS-immobilized spermatozoa still reactivated motility; however, the percentage and duration of motility obtained in these tests gradually decreased to zero in the next hour. In 50% of the cases, motility of intact spermatozoa spontaneously reinitiated after 6 to 24 hours of immobilization due to ROS treatment, although with percentages and beat frequencies lower than those of untreated spermatozoa. Studies using ROS scavengers (such as catalase, superoxide dismutase, and dimethylsulfoxide) indicated that hydrogen peroxide was the most toxic of the ROS involved, but that .O2- and .OH probably also played a role in immobilization of spermatozoa by ROS. The data suggest that ROS induce a chain of events leading to sperm immobilization, that axonemes are affected, and that limited endogenous repair mechanisms exist to reverse these damages.  相似文献   

3.
Demembranated cauda epididymidal spermatozoa of hamster, following reactivation with 1 mm ATP, exhibited either a loop or planar type of motility. The spermatozoa with planar motility exhibited increased progressive velocity (VSL), straightness (STR), linearity (LIN) and beat cross frequency (BCF) compared to the spermatozoa with loop type motility. cAMP was observed to have differential effects on the motility parameters of the demembranated spermatozoa depending on the type of motility. For instance, in the loop type, average path velocity (VAP), curvilinear velocity (VCL) and VSL were increased in the presence of cAMP unlike in the planar type. Furthermore, in an attempt to understand the role of protein kinases and protein phosphatases in regulation of sperm motility, the effects of various inhibitors of these enzymes on the motility and phosphorylation of proteins of reactivated demembranated spermatozoa were studied. Inhibitors of PTKase (protein tyrosine kinase) and protein phosphatases inhibited the motility of reactivated demembranated hamster spermatozoa. The activity of the respective enzymes associated with demembranated spermatozoa was concurrently inhibited, thus providing evidence that the effect of the inhibitors on motility was mediated through their inhibitory effects on the activities of the enzymes. The results also demonstrated that two phosphotyrosinylated proteins of molecular weight 65 and 80 kDa showed reduced phosphorylation in the presence of PTKase inhibitors, thus indicating their possible role in reactivation of motility of demembranated hamster spermatozoa.  相似文献   

4.
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.  相似文献   

5.
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   

6.
目的:评价精子运动参数在研究豚鼠精子超激活运动中的作用,进一步确定豚鼠精子超激活运动的指标。方法:采用计算机辅助精液分析(CASA)系统检测豚鼠精子不同孵育时间(0、1、3、5、7h)的运动参数。结果:豚鼠精子孵育过程中精子曲线运动速度(VCL)、平均路线速度(VAP)、头部侧摆幅度(ALH)随孵育时间的增加而提高,并在5h达到最大值,而直线运动速度(VSL)、直线性(LIN)、向前性(STR)、鞭打频率(BCF)随孵育时间的增加而降低,并在5h达到最低值。结论:豚鼠精子获能培养过程中在发生超激活运动前精子的运动方式可发生很大的变化。  相似文献   

7.
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.  相似文献   

8.
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)  相似文献   

9.
The presence of motility inhibitors in seminal plasma and within spermatozoa from control and infertile men with poor sperm motility was investigated using demembranated reactivated human spermatozoa. No difference was found in the inhibitory capacities in seminal plasma of patients with poor sperm motility (less than 50%) when compared with that of fertile controls with motility above 50%. No correlation was observed between inhibitory capacity and sperm motility. However, when extracts of spermatozoa from these patients were tested for the presence of inhibitor, it was observed that three of nine patients had an inhibitor in their sperm extract. By contrast, all sperm extracts from fertile control subjects were devoid of inhibitor. It was concluded that the presence of a motility inhibitor in seminal plasma does not explain the poor sperm motility observed in patients. The presence of a motility inhibitor within spermatozoa, however, may represent an important factor in the etiology of the poor sperm motility observed in some patients.  相似文献   

10.
The effectiveness of several glass coating agents in preventing sperm adherence to glass surfaces for both intact and demembranated human spermatozoa was investigated. These agents included bovine serum albumin (BSA), Sigmacote, poly glu-lys, Collodion and Formvar. The presence of at least 5% of seminal plasma in sperm suspensions prevented sperm adhesion to glass surfaces. On the other hand, 56% of washed spermatozoa resuspended in an isoosmotic buffer containing 1 mg/ml of BSA attached to glass. Collodion, Formvar and Sigmacote reduced sperm attachment to glass to 2, 5 and 7%, respectively. BSA was partially effective, with 20% sperm adherence to glass, and poly glu-lys was totally ineffective. Whereas Sigmacote and BSA coatings lacked transparency, Collodion always achieved the best light transmission. Demembranated reactivated spermatozoa were all attached to glass within 90 seconds of contact. This adhesion was prevented by Collodion and Formvar. Other agents were less effective or interfered with motility. In contrast to intact spermatozoa, demembranated spermatozoa have a very low progressiveness ratio (vector speed/track speed), a wide beating amplitude and because of their whiplash flagellar movement, their motility resembles that of hamster capacitated spermatozoa.  相似文献   

11.
Human spermatozoa treated with 0.05% Triton X-100 to remove the cell membranes became immotile but flagellar movement was reinitiated by addition of 0.06 to 1 mM adenosine triphosphate (ATP). The percentage of spermatozoa showing flagellar movement 2 minutes after addition of 1 mM ATP from men with idiopathic asthenospermia (mean +/- SD, 34 +/- 15%), oligozoospermia (17 +/- 21%), sperm autoimmunity (17 +/- 12%), vasoepididymostomy (20 +/- 22%), or idiopathic zero motility (0%) was significantly lower than with spermatozoa from normal men (54 +/- 12%). The percentage of reactivated spermatozoa was correlated with the proportion of live cells (Eosin Y stain, r = 0.602, P less than 0.001), percentage of motile cells (r = 0.761, P less than 0.001), and motility index (r = 0.759, P less than 0.001) in the same semen samples. When expressed relative to initial sperm motility, the proportion reactivated was similar for idiopathic asthenospermia (85%) and normospermia (82%). Thus, failure to produce ATP does not appear to be a frequent cause of low sperm motility in man.  相似文献   

12.
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.  相似文献   

13.
For in vitro capacitation to occur in cynomolgus monkey (Macaca fascicularis) spermatozoa, there is an absolute requirement for exogenous stimulation with the sperm activators, caffeine (1 mM) and db-cyclic adenosine monophosphate (dbcAMP) (1 mM), which are known to induce capacitation-related hyperactivated motility. Tyrosine phosphorylation of sperm tail proteins is an integral component of this caffeine- and dbcAMP-stimulated hyperactivated motility. In both capacitated and noncapacitated human spermatozoa, progesterone (P4) has been reported to elicit an immediate, potent increase in intracellular calcium ion concentrations [Ca2+]i. The objective of this study was to examine the effects of progesterone on requisite events in macaque fertilization, including [Ca2+]i, hyperactivated motility, and the concomitant tyrosine phosphorylation of sperm tail (STTP) proteins after treatment with caffeine and dbcAMP. The effect of 1 microM of progesterone on [Ca2+]i was determined by spectrofluorometry with the fluorescent indicator, fura-2/AM, on hyperactivated motility using computer analysis (HTM-IVOS) with the sorting criteria lateral head amplitude (> or = 8.0 microm), curvilinear velocity (> or = 150 microm/s), linearity (< or = 60%), and on STTP by immunocytochemistry. The results showed that progesterone elicited a significant increase in [Ca2+]i in caffeine- and dbcAMP-activated macaque sperm with maximal stimulation at 30 minutes after activation. The response in nonactivated sperm was dramatically reduced compared with the response in activated sperm. Basal [Ca2+]i increased as a function of time in both activated and nonactivated control sperm although basal levels were significantly increased in activated sperm. Progesterone stimulation resulted in a small but significant increase in both hyperactivation and STTP when sperm were first pretreated with caffeine and dbcAMP. Our results provide evidence that macaque sperm activation with caffeine and dbcAMP is required for a progesterone-elicited response, which results in calcium influx, hyperactivated motility, and sperm tail tyrosine phosphorylation.  相似文献   

14.
Summary. Semen from men of proven fertility was compared with that of men with unexplained infertility to determine differences in spermatozoal functions such as hyperactivation and acrosome reaction and spermatozoal motility characteristics. The hyperactivated spermatozoa in both groups could be visualised on the monitor of the Computer Assisted Semen Analyser and they exhibited 'circling', 'thrashing', 'starspin' and 'helical' motility patterns and the mean hyperactivation rates were not significantly different. However, 20% of the men with unexplained infertility did not exhibit hyperactivation compared to only 4% in the fertile group. Furthermore, the semen from infertile men when evaluated for hyperactivation could be categorised into two groups with those having lower hyperactivation (<10% or <6% after 4 and 6 h of incubation respectively), forming the first group, and those having a higher hyperactivation rate constituting the second group. In the fertile men such distinct groups were not visible and the percentage hyperactivation ranged from 1 to 16%. No significant differences were observed in the rate of acrosome reaction of fertile and unexplained infertile men.
The non-hyperactivated spermatozoa from unexplained infertile men showed a significant increase in path velocity (VAP), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) and a decrease in linearity (LIN) and straightness (STR) compared to spermatozoa from fertile men. Furthermore, the hyperactivated spermatozoa from infertile men also showed an increase in progressive velocity (VSL) (only after 2 h of incubation) and LIN and decrease in ALH and beat cross frequency (BCF) compared to spermatozoa from fertile men. The results are discussed in the light of the importance of the above spermatozoal functions and spermatozoal parameters in fertilization.  相似文献   

15.
A number of studies have demonstrated that high calcium ion activities inhibit sperm motility, but little is known about the effect of different calcium activities close to the physiological range. Therefore, we investigated whether raising calcium activities within the submicromolar range would inhibit the motility of demembranated human spermatozoa. Spermatozoa were demembranated with Triton X-100 and motility was measured objectively by computer assisted semen analysis. Motility, reactivated by 1 mol adenosine 5'-triphosphate (AlphaTauP)/L, was short lived, with maximum activity only sustained for about 1 min. Reactivated motility was not affected by 50 micromol cAMP/L. The amplitude of lateral head displacement was significantly greater at room temperature than at 37 degrees C, but there were no significant differences between the percentage of sperm motile or their velocity at the two temperatures. The calcium buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) at 1 mmol/L was included in the demembranation-reactivation medium, and free calcium ion activities were calibrated using the fluorescent calcium probe Fura-2. Calcium ion activities of > or =500 nmol/L significantly inhibited the percentage of demembranated-reactivated spermatozoa that were motile, and the velocity and lateral head displacement of these cells. The range of intracellular calcium activities in spermatozoa from 24 cryopreserved ejaculates was 110-534 nmol/L; roughly twice the value in fresh spermatozoa. Therefore, calcium ion activities in the range observed in cryopreserved spermatozoa can inhibit the activity of demembranated human spermatozoa.  相似文献   

16.
A preliminary study reported finding higher sperm velocity in seminal plasma in males of partners that conceived female offsprings. The null hypothesis was that sperm velocity was not related to the offspring gender. The objectives were: (a) to expand the previous study, and (b) to correlate offspring gender results with motility parameters determined through the computer-aided sperm analyzer (CASA) system. In combined fresh and frozen cycles (N = 187), sperm from cases with all female offsprings displayed higher curvilinear (48 +/- 1.0 mu/sec versus male 46 +/- 1.0, P < 0.05) and average path velocities (36 +/- 0.7 mu/sec versus male 34 +/- 0.7, P < 0.01). A criteria of less than 30 mu/sec or over 41 mu/sec average path velocity predicted 73 or 72% of the male or female offspring cases, respectively. A curvilinear velocity of less than 49 mu/sec or over 55 mu/sec predicted 58 or 59 % of the male or female offspring cases, respectively. Semen viscosity reflected in sperm velocity was linked to predominantly male or female sperm populations. Paracrine signals from the gender-skewed sperm precursor populations controlling viscosity merit further exploration.  相似文献   

17.
The objectives of this study were to describe hyperactivated motility in boar sperm and to determine the incidence of hyperactivation among boar sperm flushed from the oviduct. Oviducts were surgically removed from 13 gilts 32 hours after mating them to fertile boars. The majority of the sperm flushed from the oviducts was immotile, weakly motile, or stuck to mucus or cellular debris. The mucus could not be penetrated by the sperm. The remaining 3% to 19% of the flushed sperm was free-swimming. Only five hyperactivated sperm were recovered, all from the ampulla of the oviduct. The remainder of the free-swimming sperm travelled in linear trajectories and possessed significantly higher flagellar curvature ratios (the flagella were less bent) than boar sperm measured in diluted semen. Hyperactivated motility was induced in washed ejaculated boar sperm, using a 1-minute pulse of 4 mumol/L calcium ionophore A23187. The ionophore-treated sperm had significantly lower straight-line velocities, linearities, and flagellar curvature ratios than controls, as would be expected for hyperactivated sperm. They were vigorous and swam in circles. It was concluded that, although few hyperactivated boar sperm could be recovered from the oviduct, boar sperm are capable of undergoing hyperactivation.  相似文献   

18.
Kinematics of human spermatozoa incubated under capacitating conditions   总被引:1,自引:0,他引:1  
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.  相似文献   

19.
The aims of the present study were, first to develop a new method for evaluating sperm hyperactivation (HA) based on sperm head axis angle deviation, and second to apply this method in scoring selected sperm populations during capacitation. This was made possible by improving our original superimposed image analysis system (SIAS). The new option on the system enables us to determine the real angular deviation of the entire head in successive superimposed frames. The entire procedure for each spermatozoon requires 10-20 sec, according to the frame/rate utilized. A clear cut-off between the values of hyperactivated, transitional and non-hyperactivated spermatozoa was found at 11 and 21 frames/sec. However, at 6 frames/sec, a partial overlap between the three classes was found. We defined sperm activity as follows. At 11 frames/sec, hyperactivated: angle sum > or = 405 degrees; transitional: 200 degrees < or = angle sum < 405 degrees. At 21 frames/sec, hyperactivated: angle sum > or = 760 degrees; transitional: 350 degrees < or = angle sum < 760 degrees. The results, in agreement with previous research, show the peaks of activity at 3 h, dropping to a minimum at 6 h. This phenomenon was significantly more evident in fertile compared with subfertile semen samples.  相似文献   

20.
The aim of this study was to determine the differences in motility of frozen and thawed bull spermatozoa under conditions of weightlessness compared with ground conditions. The tests were performed within a series of scientific and technologic experiments under microgravity using sounding rockets in the Technologische Experimente unter Schwerelosigkeit (TEXUS) program launched in Kiruna, North Sweden. Using a computerized sperm motility analyzer, significant differences were found in sperm motility under microgravity compared with sperm under gravitational conditions on earth. Computer analysis showed alterations in straight line and curvilinear velocity, as well as in linearity values. The amount of progressively motile spermatozoa, including all spermatozoa with a velocity > 20 microns/second, increased significantly from 24% +/- 9.5% in the reference test to 49% +/- 7.6% in the microgravity test. In conclusion, there is strong evidence that gravity influences sperm motility.  相似文献   

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