不同获能时间豚鼠精子运动参数分析

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。     

Effect of Urea and Detergents on the Ability of Human Spermatozoa to Penetrate Zona-Free Hamster Oocytes

Washing of human spermatozoa with either BWW alone or with the same buffer containing 0.1 M urea, 0.005% SDS or 0.001% NP40 affected the penetration ability of the gametes into zona-free hamster oocytes to various degrees. In contrast, human spermatozoa washed with BWW buffer containing 0.3 M urea had an increased ability to fuse with the heterologous oocytes when compared to controls capacitated with BWW/BSA. Moreover, the presence or absence of BSA in the insemination medium did not further modify this enhanced penetration pattern. The BWW, BWW/0.1 M urea and BWW/SDS treatments apparently mimicked some of the in vitro capacitation properties of albumin-containing media; the BWW/0.3 M urea treatment overpowered the capacitation and acrosomal reaction extent obtained with BWW+BSA. In all samples the motility of the spermatozoa washed with BWW buffer alone or containing various additives (but no albumin) was significantly decreased if compared to the motility of semen samples washed with albumin containing media. However, each sperm sample behaved differently when exposed to a given buffer.     

Phospholipids in Guinea Pig Spermatozoa Before and After Capacitation In Vitro

Phospholipids of guinea pigs spermatozoa were examined using high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Two types of spermatozoa were compared: fresh epididymal (uncapacitated) spermatozoa and epididymal spermatozoa preincubated in Ca2+-free medium for 17-18 hr (capacitated spermatozoa). Determination of lipid phosphorus revealed that the total phospholipid content of spermatozoa did not change significantly during capacitation of spermatozoa. HPLC analyses of choline and ethanolamine phosphatides, lysophosphatides, and sphingomyelin revealed that the relative amounts of these phospholipids remained unchanged during capacitation. In contrast to spermatozoa, phospholipids in the medium surrounding spermatozoa reduced by 20% during capacitation of spermatozoa, suggesting that spermatozoa may utilize extracellular phospholipids as possible energy sources during their capacitation.     

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.     

Cytochemical Study on Human Spermatozoa Metabolism During in Vitro Capacitation

Summary: In eutherian mammalian spermatozoa the capacitation is coupled to a specific type of metabolism, that is glycolysis or oxidative respiration. A cytochemical study was carried out on cytochrome oxidase and lactate dehydrogenase in human spermatozoa collected at different times during in vitro capacitation.
Human spermatozoa were incubated in Diggers, Whitten and Wittingham's medium supplemented with 15% heat-inactivated human serum. Both histoenzymological reactions based on oxidative polymerization of diaminobenzidine (cytochrome oxidase) or on tetrazolium salts reduction (lactate dehydrogenase) can be quantitated and have been evaluated by microdensitometric method (Vickers M85).
The results suggest that human spermatozoa depend almost quite on the anaerobic glycolysis during in vitro capacitation.
Zusammenfassung: Cytochemische Studie an menschlichen Spermatozoen und deren Stoffwechsei während der in-vitro-Capacitation
Bei Säuger-Spermatozoen ist die Capacitation an einen spezifischen Stoffwechsei gekoppelt, die Glykolyse oder die oxydative Respiration. Hierzu wurde eine cytochemische Untersuchung an der Cytochromoxydase und an der Lactadethydrogenase menschlicher Spermatozoen durchgeführt, die zu verschiedenen Zeiten während der in-vitro-Capacitation gewonnen wurden. Die Spermatozoen wurden in Biggers, Whitten und Wittingham Medium unter Zusatz von 15% hitzeaktiviertem menschlichem Serum inku-biert. Beide histochemischen Reaktionen basierten auf der oxydativen Polymerisation von Diaminobenzidin (Cytochromoxydase) oder auf Tetrazoliumsalz-Reduktion (Lactatdehy-drogenase) und konnten quantifiziert werden; sie wurden ausgewertet mittels der Mikrodensitometrie-Methode (Vickers M85). Die Ergebnisse legen nahe, daß menschliche Spermatozoen meistens vollständig von der anaeroben Glykolyse während der in-vitro-Capacitation abhängig sind.     

Analysis of the Changes of Movement Function and Viability in Human Spermatozoa Induced by Reactive Oxygen Species

Objective: To study the influence of reactive oxygen species (ROS) on the mobility and viability of human spermatozoa. Methods: Spermatozoa with normal function were selected from human semen samples by the Percoll gradient centrifugation technique. ROS were generated by the hypoxanthine-xanthine oxidase system and were incubated with the spermatozoa under aerobic environment. Movement parameters of spermatozoa were analyzed by the computer-assisted semen analysis (CASA) system. Results: Thirty min after incubation with ROS, the motility, curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) of the spermatozoa were significantly decreased (P<0.01), while the change in the amplitude of lateral head displacement (ALH) was insignificant (P>0.05). After 60 min incubation, the motility was almost lost with all the movement parameters close to zero. Conclusion: When normal spermatozoa were incubated with ROS, the mobility was decreased. It is believed that ROS may be one of t     

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.     

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.     

Ca2+ is Essential for the Motility of Plasma Membrane-Intact, but not of demembranated. Hamster Spermatozoa

Extracellular Ca2+ is essential for the flagellar motility of membrane-intact hamster spermatozoa. When suspended in a medium completely free of Ca2+, most spermatozoa quickly lost their motility, and remained motionless until they were transferred back to Ca2+-containing medium. The motility could not be restored after the spermatozoa had been in Ca2+-free medium for more than 2 hr. Unlike membrane-intact spermatozoa, demembranated spermatozoa (spermatozoa without plasma membranes) exhibited active movement in Ca2+-free medium, and their motility was inhibited by Ca2+. In view of these facts, we suggest that the "hyperactivated motility" which membrane-intact spermatozoa display upon capacitation may be due to the activation of a Ca2+-dependent adenylate cyclase (and the resultant increase in intracellular cAMP), rather than being a direct effect of a rise in the intracellular Ca2+ concentration.     

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   

白念珠菌体外感染对人精子运动和超微结构的影响

目的: 体外研究白念珠菌(Ca)对人精子的运动功能有无直接影响并观察精子超微结构变化,对其影响机制作初步探讨。　方法: 将从念珠菌性阴道炎患者的分泌物中分离纯化的Ca制成活菌悬液,对 10例健康青年男性手淫获得并经上游优化处理的精子进行体外感染。按细菌与精子比例不同分成A组 ( 1∶1 )、B组 ( 1∶10 )、C组(1∶100)、D组(1∶1 000)、E组(1∶10 000)、F组(空白对照)分别孵育,于 0、1、2、4h取样,计算机辅助精子分析系统(CASA)检测人精子运动参数(前向性运动百分率、直线速度、曲线速度、平均路径速度、头部侧摆幅度 )、精子存活率、精子形态及凝集情况。透射电镜观察孵育 4h后的各组精子的超微结构变化。　结果: Ca与精子体外孵育后,前向运动百分率所受影响最大,且与菌体密度及时间密切相关;其他参数与对照组相比也相继出现显著性差异。观察到精子黏附于菌体和凝集现象。精子超微结构产生变化:精子核空泡增多,顶体破裂,质膜破损,线粒体排列紊乱。　结论: Ca体外可显著降低精子存活率和抑制精子运动功能,其机制可能与Ca对人精子的黏附作用和超微结构的损伤有关。     

Effect of leptin on motility, capacitation and acrosome reaction of human spermatozoa

Leptin is a polypeptide hormone with important roles in reproduction. It has been detected in human seminal plasma as well as on human ejaculated spermatozoa. This study aimed at studying the possible role of leptin in regulating human sperm functions. Immunofluorescent staining was used to study the expression of leptin and its receptor. The correlation between the concentration of leptin and soluble leptin receptor (ObRs) in seminal plasma as measured by enzyme-linked immunosorbant assay and sperm motility parameters measured by computer-assisted sperm analyais (CASA) was determined. The effects of recombinant leptin on human sperm motility, capacitation and acrosome reaction as measured by chlortetracycline staing were also studied. Leptin immunoreactivity was demonstrated at the equatorial and neck regions of human spermatozoa, whereas that of ObRs was shown up on the tail. After Percoll separation, spermatozoa with high density had more intense leptin immunoreactivity compared with those with low density. No significant correlation was found between seminal plasma concentration of leptin/ObRs and sperm motility parameters. After incubation with recombinant human leptin for either 3 h or overnight, there was no change in all the CASA motility parameters determined and percentages of capacitated and acrosome-reacted spermatozoa. We concluded that leptin does not have a significant effect on motility and capacitation/acrosome reaction in human ejaculated mature spermatozoa. Its role in male reproduction is yet to be determined.     

Effect of spermine on sperm capacitation of guinea pig in vitro.

Spermine (Sp) 10(-5) mM had vigorous activity of guinea pig spermatozoa, while it completely abolished sperm forward motility (SFM) at a concentration of 10(-3) mM. There appeared to be a dose relationship to inhibition to motility. 2-Difluoromethylornithine 10 mM antagonized the Sp-induced inhibition of SFM after 3 h of incubation. Capacitation of a guinea pig sperm was inhibited by Sp in a concentration-dependent manner. The majority of acrosome-reacted sperm did not display hyperactivated motility. Precapacitated sperm were able to undergo the acrosome reaction (AR) in the presence of Sp. Moreover, Sp-mediated inhibition of capacitation was a reversible process. Once sperm capacitation was completed, Sp no longer inhibited AR. Before capacitation, the content of Sp in spermatozoa was 4.5 +/- 0.5 micrograms/5 x 10(7) cells, whereas in case of capacitated spermatozoa it was significantly decreased (2.1 +/- 0.4 micrograms/5 x 10(7) cells). The penetration of spermatozoa into the zona-free hamster eggs in the presence of Sp was markedly decreased, but it did not affect the fertilizability of ova as compared to the control. These results suggest that Sp may be an inhibitory agent of sperm capacitation in guinea pig in vitro, and it may also be involved in the modulation of capacitation.     

Effect of Cooling, Freezing and Thawing Rates and Storage Conditions on Preservation of Human Spermatozoa

Human spermatozoa was relatively resistant to cooling shock. However, when diluted semen was cooled faster than 10 degrees C per minute from room temperature (RT) to 5 degrees C and rewarmed to RT, percentage motility and percentage alive of spermatozoa decreased when compared to the slower cooling rates (less than 5 degrees C/min). The optimum cooling rate from RT to 5 degrees C resulting in maximum survival of human spermatozoa was found to be 0.5 to 1 degree per minute when cooled from RT to 5 degrees C and subsequently frozen-thawed in liquid nitrogen (LN2). The optimal freezing rate of 10 degrees C per minute, from 5 degrees to -80 degrees C, resulted in higher survival of human spermatozoa than slower (1.1 degrees C/min) or faster (87.1 degrees C/min) freezing rates. Slow thawing in 20 or 35 degrees C air, on a dry bench, resulted in better survival than the other slower or faster thawing methods used. The temperature at which human semen samples were transferred to LN2 significantly influenced spermatozoa survival. Survival was higher when transferred at -30 degrees C or lower when compared with samples transferred at -15 degrees C or higher. However, maximal spermatozoa survival was obtained when the samples were transferred at -80 degrees C or lower. Transfer of human semen from LN2 to -25 to -30 degrees C and storage for 24 hours significantly reduced spermatozoa viability when compared with storage at 196 degrees C or -80 to -85 degrees C. No significant differences were found between storage temperatures of -80 to -85 degrees C and -196 degrees C in the maintenance of spermatozoa viability for up to 90 days.     

Protein tyrosine phosphorylation of the human sperm head during capacitation： immunolocalization and relationship with acquisition of sperm-fertilizing ability

The occurrence of tyrosine phosphorylation （TP） in the sperm head during capacitation has been poorly investigated, and no data exist on the relationship of its dynamics with the acquisition of sperm fertilizing ability. This study localized TP of head proteins in human spermatozoa during capacitation and explored its relationship with acquisition of the ability to display progesterone （P）-stimulated acrosome reactions （ARs） and to penetrate zona-free hamster oocytes. By immunofluorescence, TP immunoreactivity was revealed in the acrosomal region of formaldehyde-fixed/unpermeabilized samples, whereas it was abolished in fixed/permeabilized samples, in which TP immunoreactivity was high in the principal piece. No TP immunoreaetivity was detectable in unfixed spermatozoa. Head TP immunoreactivity was localized externally to the acrosome, close to the cytoplasmic membrane, as assessed by transmission electron microscopy. The increase in head TP was an early event during capacitation, occurring within 1 h in capacitating conditions. At this time, the P-stimulated ARs were also increased, whereas egg penetration was as poor as in uncapacitated spermatozoa. At 5 h of capacitation, the extent of neither head TP nor the P-induced ARs were greater than that at 1 h, whereas egg penetration had significantly increased. Seminal plasma inhibited head TP, P-induced ARs and egg penetration. None of these inhibitory effects, unlike those on tail TP, were prevented by the cAMP analogue dbcAMP （N,2-O-dibutyryladenosine 3＇,5＇-cyclic monophosphate）. In conclusion, head TP is a subsurface event occurring early during capacitation and is closely related to acquisition of the ability to display P-stimulated ARs, whereas the ability to fuse with oolemma and to decondense is a later capacitation-related event.     

Effect of recombinant β-defensin 1 protein on human sperm motility and viability

The reduction of sperm motility and subsequently reduced ability to undergo capacitation and acrosome reaction are considered as common causes of male infertility. The β-defensin family is a group of well-known secretory proteins with antimicrobial activity that contribute to the process of “sperm maturation” during the passage of spermatozoa in the epididymis when spermatozoa attain its motility. One member of this family is “β-defensin 1” which is present in seminal plasma and spermatozoa. The aim of this study was the incubation of human processed spermatozoa with recombinant β-defensin 1 (500 ng/ml) for 1, 2 and 3 hr at 37°C under 5% CO₂ atmosphere and assessment of sperm viability and motility in 59 semen samples. The analysis of semen samples such as sperm concentration, motility, viability, morphology and semen volume was performed according to the World Health Organization (2010; World health organization laboratory manual for the examination and processing of human semen (p. 287). Geneva, Switzerland: World Health Organization) criteria. The result of the current study shows that the incubation of spermatozoa with recombinant β-defensin significantly maintained percentage of sperm viability and motility compared to processed spermatozoa incubate in the absence of β-defensin in the studied time intervals (p < .05). Therefore, we concluded that recombinant β-defensin 1 protein as an agent with antimicrobial activity can maintain sperm viability and motility in in vitro condition.     

优选技术对人精子染色体及超微结构的影响

目的 :了解优选技术对人精子染色体及超微结构的影响。　方法 :分别应用上游法、Percoll密度梯度离心法及双曲管优选法处理精液 ,观察优选前后精子染色体畸变率、精子性染色体比例及超微结构相对正常的精子百分率。　结果 :精子染色体畸变率、精子性染色体比例及超微结构相对正常的精子百分率在 3种优选方法优选前后均无显著改变 (P >0 .0 5 )。　结论 :3种优选方法不会增加精子超微结构的损害 ,对精子染色体没有产生影响     

Effect of procaine, pentoxifylline and trolox on capacitation and hyperactivation of stallion spermatozoa

Reasons for low in vitro fertilisation rates in the horse include the difficulties in inducing capacitation and/or hyperactivation of stallion spermatozoa. The aim of this study was to analyse the effect of noncapacitating and capacitating modified Whitten's (MW) and modified Tyrode's medium (MT) and treatment with procaine (5 mmol), pentoxifylline (3.5 mmol) and trolox (120 mmol) on motility (CASA), capacitation, acrosomal status, viability and mitochondrial membrane potential of stallion spermatozoa (n = 4). While there was no influence of MW and MT on sperm motility, a significant increase in the percentage of viable-capacitated spermatozoa was observed after incubation in capacitating MW (P < 0.05). Pentoxifylline showed no significant effect on the motility pattern but increased the proportion of live-capacitated spermatozoa (P < 0.05). Trolox had no detectable effect on either capacitation or hyperactivation. Procaine was the only agent that induced hyperactivation in terms of a reduced proportion of progressively motile spermatozoa, straight line velocity, straightness, linearity and beat-cross frequency and an increase in the amplitude of lateral head displacement (P < 0.05). The combination of capacitating Whitten's medium and procaine showed the best results for the induction of capacitation and hyperactivation in stallion spermatozoa; this was possible even after short-term incubation.     

The Investigation of the Motility of Spermatozoa

Zusammenfassung:  Bei 324 Ejakulatproben wurden gleichzeitig die subjektive Bestimmungsmethode der Spermatozoenmotilität, der Motilitätsleistungsindex (Ishii) und der Eosin-Vitalitätstest durchgeführt. Statistisch konnte eine sehr gute Korrelation nachge-wiesen werden (α = 0,1%). Die Bestimmungsmethode des Motilitätsleistungsindex wird als Standard für die Ejakulatuntersuchung empfohlen.
Summary:  The motility of spermatozoa on 324 ejaculates was investigated with the help of two different methods, the sperm motile efficiency (Ishii) and a subjective calculation of the velocity of spermatozoa. Simultaneously the eosin-vitality-test was done. There was a very good statistical correlation (α = 0,1%) and no difference could be observed among the three methods. Ishii's sperm efficiency will be recommended as a standard.