OEP及卵黄浓度对蓝狐冻融精子质量的影响

人工采取 6只优质芬兰雄性蓝狐精液 ,利用不同OEP及卵黄含量的Tris 果糖 -柠檬酸钠稀释液稀释 ,制成细管冻精 ,透射电镜下观察精子冷冻前后质膜和顶体超微结构 ,荧光免疫方法检测不同培养时间冻融精子的质量。结果表明 ,蓝狐精子顶体外膜双层膜的厚度为 0 0 2 0 μm ,冷冻 -解冻过程中易发生质膜膨胀、顶体外膜融合现象。顶体产生的囊泡分两种类型 ,一种是体积较大的中空囊泡 ,平均直径为 1 2 5 μm。另一种是体积较小的实体囊泡 ,内充满顶体内容物 ,平均直径为 0 83μm ,两种囊泡的数量不定。OEP能有效抑制顶体囊泡形成 ,影响顶体囊泡类型、体积大小及囊泡数量 ,添加适宜剂量OEP能使顶体囊泡的体积明显缩小 ,囊泡的总数及中空囊泡的数量显著降低。蓝狐冻融精子质量与OEP及卵黄剂量有关 ,在卵黄存在的前提下 ,OEP有利于维持冻融过程中质膜 (5 6 3% )、顶体的完整性 (5 7 8% ) ,显著提高冻融精子活力 (5 4 7% )。在蓝狐精液稀释液中 ,OEP、卵黄的适宜含量分别为 1 %、 2 0 %     

TELEOGRYLUS EMMA精子体外顶体反应的超微结构比较研究

采用相同种类卵水诱导的方法对直翅目,蟋蟀科,黄脸油葫芦的受精囊精子的顶体反应过程进行系统观察.发现黄脸油葫芦精子顶体反应可划分为3个阶段,第1阶段,精子质膜膨胀、断裂或丢失;第2阶段,顶体复合体的顶体外层与顶体本体外膜发生融合,囊泡化;第3阶段,顶体复合体大部分脱落,只留有短锥状的顶体位于核前端.据观察,蟋蟀精子质膜不参与囊泡形成,此结果与家蝇及哺乳类的猪、牛、绵羊、猕猴精子的顶体反应结果很相似.经过比较发现卵水对受精囊内精子的诱导率明显高于精巢内,据分析,可能与精子的生理成熟有关,即便受精囊内精子比精巢内精子更趋于成熟.与其他学者的实验结果相比,蟋蟀精子顶体反应率与家蝇的相似,但明显低于其他动物.这可能与动物的授精方式有关.     

圆斑星鲽精子的超微结构及核前区特殊结构

分别以扫描电镜和透射电镜方法研究了圆斑星鲽 (Veraspervariegates)精子的超微结构。头部呈圆形、中段不发达 ,具有多个未分化的圆形小线粒体、鞭毛结构极简单 ,具有 9 2型的轴丝等特征 ,均表示圆斑星鲽的精子属于简单的原始类型 ,与多数其它高等鱼类精子结构相似。但是 ,圆斑星鲽精子核前部的凹陷 ,即不含染色质的电子透明区 ,以及核前区的囊泡结构却未见在其它新鳍鱼类精子中报道。圆斑星鲽精子核前凹陷开口于核前端稍偏处 ,凹陷呈不规则状 ,可深入到核的中央。凹陷处与染色质区无界膜分隔。凹陷区的前部及其开口处常有多个小的具单层界膜的囊泡 ,有时可见在凹陷区的开口处汇集成一个大泡。在某些处理过程中 ,例如在冷冻 -解冻过程中 ,囊泡可能丢失 ,而在精子顶端留下一个凹坑。尽管顶体在现存最早的新鳍鱼类 ,如雀鳝鱼及弓鳍鱼中已经完全消失 ,但考虑甚至在某些纯真骨鱼 ,如鲑形目中的螈鱼精子上 ,顶体仍然存在 ,而在其它一些纯真骨鱼精子或精子细胞中也存在顶体或顶体遗迹等事实。我们推测在圆斑星鲽精子中的上述特殊结构也是顶体的一种遗迹 ,但也不排除其是向外释放核内物质的一种途径的可能性     

中国雨蛙精子结构及其在系统发育上的意义

研究了中国雨蛙（Ｈｙｌａｃｈｉｎｅｎｓｉｓ）精子的超微结构,并初步探讨其在系统发育上的意义,中国雨蛙精子由头部和尾部两部分组成,头部一有棒状的细胞核,核内染色质高度浓缩,细胞核前方有顶体。顶体圆锥状,顶体下腔之中一圆锥状的顶体下锥和细小的囊泡,精子尾部细长,主要由轴丝,致密纤维和线粒体组成,尾部没有波动膜。从蟾蜍科,雨蛙科和蛙科的精子结构看,无尾两栖类在进化过程中,精子结构趋向简单,雨蛙科精子的结     

小鼠顶体形成相关蛋白的研究进展

顶体形成过程分为高尔基期、帽期、顶体伸长期和成熟期四个主要阶段，其正确形成对精子结构和功能具有重要影响。顶体形成需要各种细胞器(内质网和高尔基体)和细胞质结构(顶体板和精子领)之间的积极和特异性协调。本文主要对顶体形成过程中参与反式高尔基体网络囊泡分类运输、顶体附着到细胞核等过程的蛋白质的功能及调控机制进行了总结与讨论。这些蛋白质大多相互作用，表现出复杂的分子调控机制，促进了这一事件的发生。对精子顶体形成相关蛋白的功能及机制的研究将为临床男性不育症的诊断和治疗提供新的思路和对策。     

豚鼠精子在发生及顶体反应过程中细胞内Ca~(2+)的定位研究

实验利用焦锑酸钾法对豚鼠精子在发生及顶体反应过程中的Ca~(2+)定位作了较详细的研究。在精母细胞及精子细胞上都有Ca~(2+)分布,但睾丸中的精子上则无Ca~(2+)。成熟精子中Ca~(2+)主要定位于顶体帽的整个腹面及背面的两个特定区域。发生顶体反应的精子上Ca~(2+)则位于顶体外膜上或囊泡内,已发生顶体反应的精子中Ca~(2+)则位于顶体内膜上。     

无蹼壁虎精子头部形成的研究

本文用透射电镜观察了无蹼壁虎精子头形成的过程。早期精细胞具有显著的高尔基复合体、线粒体集合及细胞质桥、接着高尔基体成熟面分泌出前顶体囊泡,并逐渐向核移动。以后精子形成可分四个时间：时间Ⅰ,当前顶体囊泡移至核膜时,核膜凹陷形成封闭的顶体囊泡,囊泡底部靠近核膜有一电子致密的顶体颗粒;时间Ⅱ,细胞核延长,顶体囊泡变扁平;时期Ⅲ,细胞核进一步延长,核内染色质纤维变粗并沿核纵轴方向排列有序;时间Ⅳ,精子发育     

猪精子体外获能与顶体反应的超微结构研究

用４种方法,检测了猪精子体外获得的效果。结果证明：高离子浓度的前培养液和猪镦泡液,具有促进获能过程的作用,实验还获得了获能后顶体反尖的一些重要的形态学变化资料,包括质膜的膨胀、断裂、顶体膨胀、顶体外膜内陷或原位局部囊泡化,质膜再全部丢失。顶体内膜直到与卵母细胞质膜融合,才发生可见的变化。受精过程无论体内或体外,都容易发生多精入卵,体外受精则更甚。在精子穿过卵丘细胞之间时,一方面开始进行顶体反应,另     

东方扁虾精子的超微结构

利用电镜研究了东方扁虾（Ｔｈｅｎｕｓ ｏｒｉｅｎｔａｌｉｓ）精子的形态和结构。精子由核、膜复合物区和顶体区３部分组成。核内含非浓缩的染色质、微管及细纤维丝,外被核膜;５～６条辐射臂自核部位伸出,臂内充满微管。膜复合物区位于核与顶体之间,由许多膜片层结构及其衍生的囊泡共同组成。顶体区由顶体囊和围顶体物质组成,顶体结构复杂,由顶体帽、内顶体物质和外顶体物质等构成;围顶体物质呈细颗粒状,主要分布于顶体囊     

糖在猕猴精子低温冷冻保存过程中的作用

TTE或TEST防冻液在冻存猕猴精子时产生不同结果,其主要不同在于防冻液中糖成分的不同。本实验利用透射电镜技术检测这2种防冻液冻存的猕猴精子冷冻前后超微结构的变化,以说明糖在低温冻存过程中的作用。结果表明,冷冻复苏过程对精子结构产生了影响。TTE法低浊保存的猕猴精子的头部的质膜出现少许皱褶或泡化现象,但精子的顶体、核或是精子尾部的结构与鲜精的结构基本相似。猕猴精子经TEST法低温保存后,大部分精子结构则发生了明显的变化。精子膜、顶体和精子核明显泡化、损伤或破裂,精子尾部不能分辨出正常的超微结构。这提示,可能由于TTE防冻液中复杂的糖成分在降温／复苏过程对精子起到了较好的协同冷冻保护作用;而TEST防冻液中单一的糖成分不能完全保护精子避免低温损伤,低温保存过程破坏了精子的结构,并影响了复苏后精子体外存活能力与受精能力。     

Plasma membrane structure of bat spermatozoa: observations on epididymal and uterine spermatozoa in Myotis lucifugus

Plasma membrane structure of bat spermatozoa was examined utilizing electron microscopy of thin sections and freeze-fracture replicas. Notable membrane features observed in replicas from cauda epididymal spermatozoa included specialized particle aggregates at the junction between the acrosomal and postacrosomal region of the head (a membrane structure not previously described in mammalian spermatozoa) and another row of rod-like particles just anterior to the posterior ring. Both of these specializations in fractured plasma membranes correspond with regions where the membrane is closely apposed to underlying structures when viewed in thin sections. The postacrosomal sheath appears to be composed of an array of longitudinally oriented filamentous components. Characteristic ordering of intramembranous particles was also noted in replicas from the midpiece region and the annulus. Major changes in plasma membrane structure were not seen in spermatozoa stored in the female reproductive tract; however, the appearance of linear particle aggregations in the principal piece membrane was noted. No evidence was obtained to suggest that an acrosome reaction had occurred in spermatozoa stored in females.     

Sperm membrane fatty acid composition in the Eastern grey kangaroo (Macropus giganteus), koala (Phascolarctos cinereus), and common wombat (Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t = 3.81; df = 4; p < or = 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels.     

Epididymal maturation and ejaculation are key events for further in vitro capacitation of boar spermatozoa

Mammalian spermatozoa acquire functionality during epididymal maturation, and the ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to assess the effects of epididymal maturation, ejaculation and in vitro capacitation on sperm viability, acrosome integrity, mitochondrial activity, membrane fluidity, and calcium influx, both as indicators of capacitation status and sperm motility. Results indicated that boar spermatozoa acquired the ability to move in the epididymal corpus; however, their motility was not linear until the ejaculation. Epididymal spermatozoa showed low membrane fluidity and intracellular calcium content; ejaculation led to an increased calcium content, while membrane fluidity showed no changes. Acrosome integrity remained constant throughout the epididymal duct and after ejaculation and in vitro capacitation. The frequency of viable spermatozoa with intact mitochondrial sheath was higher in caput and ejaculated samples than in corpus and cauda samples, whereas the frequency of spermatozoa with high membrane potential was significantly lower in cauda samples. In vitro capacitation resulted in a decreased frequency of viable spermatozoa with intact mitochondrial sheath and an increased frequency of spermatozoa with high membrane potential in ejaculated samples. These results indicated that both epididymal maturation and ejaculation are key events for further capacitation, because only ejaculated spermatozoa are capable of undergoing the set of changes leading to capacitation.     

Motility characteristics and membrane integrity of cryopreserved human spermatozoa.

The motility characteristics of washed spermatozoa from 50 normal ejaculates were measured by time-lapse photography, before and after cryopreservation. Plasma membrane integrity was assessed by the hypo-osmotic swelling test and with the supravital fluorescent dye bisbenzimide (H33258). There was a marked decline in the percentage of progressively motile spermatozoa after cryopreservation, the extent varying widely among donors. Results were, however, consistent between different ejaculates from the same individual. The ability of spermatozoa to survive cryopreservation could not be predicted from the properties of the semen beforehand. The mean velocity of the spermatozoa was significantly reduced after freezing, but the lateral head displacement was unaltered. There was a significant reduction in the proportion of spermatozoa with intact plasma membranes after cryopreservation and the results of the hypo-osmotic swelling test and H33258 tests correlated closely. There was no correlation between the declines in the percentage of motile spermatozoa, or intact spermatozoa and the sperm velocity. We conclude that membrane rupture is not the sole cause of loss of motile spermatozoa during freezing and that the decrease in the proportion of motile spermatozoa is caused, at least in part, by a separate process from that responsible for the decrease in the average swimming speed of spermatozoa.     

小鼠附睾头精子获得与卵子质膜融合能力的物质基础研究

随着精子在附睾中的转运,它们与卵子质膜的融合能力逐渐增加。怩证明２附睾体和附睾尾的精子均具有相当高的膜融合能力,而附睾头中的精了奶少能与卵子质膜融合,这是还说明附睾头中的精子不具备与云透明带卵子融合的物质条件呢？利用附睾结扎留并延长体外获能时间,可使附睾头远端精子的融合能力明显地提高;在精子培养液中加入ＡＴＰ,并延长精卵共培养时间,也可使一少部分附睾头近端的精子获得与卵子质膜融合的能力。这表明附睾     

Acrosin and the acrosome in human spermatogenesis

Using the indirect immunofluorescent staining technique, the developmental patterns of (pro) acrosin and the outer acrosomal membrane were studied in human spermatogenesis. Specific antibodies against purified acrosin and outer acrosomal membranes from boar spermatozoa were raised in the rabbit and were found to crossreact with (pro)acrosin and outer acrosomal membrane from human spermatogenic cells. It was concluded that (pro)acrosin as well as the molecules building up the outer acrosomal membrane have been highly conserved during mammalian evolution. In the course of human spermatogenesis (pro)acrosin as well as the outer acrosomal membrane first appear in the haploid spermatids; the fluorescent areas of the individual cells steadily increase during spermiogenesis. Staining for acrosin and the outer acrosomal membrane, respectively, was found in identical compartments of the spermatogenic cells in juxtaposition to the nucleus. Round-headed spermatozoa from an infertile patient did not stain for (pro)acrosin or outer acrosomal membrane. The lack of the acrosin system was further substantiated by the gelatin substrate film technique demonstrating the absence of a gelatinolytic protease in round-headed spermatozoa. Hence, round-headed spermatozoa lack the acrosome with its constituent membrane proteins and the acrosin system housed by the acrosome of normal spermatozoa.     

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.     

Nonbeneficial effects of glycerol on the oocyte penetrating capacity of cryopreserved and incubated human spermatozoa

The effect of cryopreservation on human spermatozoa in the presence or absence of glycerol was assessed by using sperm motility, functional integrity of sperm membrane, and denuded hamster oocyte penetration tests. Glycerol treated cryopreserved spermatozoa yielded a significantly higher (P less than 0.01) percentage of motile sperm and percentage of sperm with functionally intact membrane immediately after thawing than the spermatozoa not treated with glycerol but cryopreserved. However, no significant difference was observed between these cryopreserved spermatozoa (either treated or untreated with glycerol) on the percentage of motile sperm and the rate of oocyte penetration when the sperm were washed and incubated for 2 hr in a medium containing no glycerol. Thus, it appears glycerol may not be beneficial, since cryopreservation of spermatozoa either treated or untreated with glycerol essentially yields similar oocyte-penetrating capacity of sperm.     

Cold-induced ultrastructural changes in bull and boar sperm plasma membranes

The effect of low temperatures on the ultrastructure of the plasma membrane of bull and boar spermatozoa was investigated. Cold-induced changes in the organization of sperm plasma membrane components were demonstrated by the use of fast-freezing combined with freeze-fracture electron microscopy. This preparation technique ensures fixation without artifacts. At 38 degrees C bull and boar spermatozoa exhibited a random distribution of intramembranous particles over the plasma membrane of both head and tail. Exposure to 0 degree C resulted in redistribution of the intramembranous particles: on the head and principal piece of bull spermatozoa and on the principal piece of boar spermatozoa, particle-free areas were observed, whereas on the boar sperm head, particle aggregates were present. The original particle distribution was restored upon rewarming of bull and boar spermatozoa to 38 degrees C, as well as after freezing and thawing of bull spermatozoa. Dilution of bull and boar semen into Tris-dilution buffer and Beltsville Thaw Solution-dilution buffer, respectively, could not prevent cold-induced redistribution of intramembranous particles. The observed particle reorganization upon cooling was interpreted as the result of lateral phase separation in the plasma membrane. Species-dependent differences in cold-induced ultrastructural changes were considered to be determined by lipid composition and asymmetry of the plasma membrane, and might be related to differences in cold resistance between species.     

Capacitation-like changes in equine spermatozoa following cryopreservation

The primary objective of this study was to assess plasma membrane characteristics and activation of signal transduction pathways in equine spermatozoa during both in vitro capacitation and cryopreservation. Significant plasma membrane restructuring, as assessed by measurement of plasma membrane lipid disorder and phospholipid scrambling, was not observed until after cryopreservation and subsequent thawing (P < 0.05). Although in vitro capacitated cells also displayed increased plasma membrane lipid disorder and phospholipid scrambling (P < 0.05), it appeared that regulation of these events in in vitro capacitated versus cryopreserved equine spermatozoa was not identical. Addition of 5 microM staurosporine to the capacitation media reduced plasma membrane phospholipid scrambling (P < 0.05), but supplementation to the freezing extender prior to cryopreservation did not. Furthermore, progesterone was able to induce a greater degree of acrosomal exocytosis in in vitro capacitated versus frozen/thawed spermatozoa. Expression of phospholipid scramblase, a protein thought to be important in plasma membrane phospholipid scrambling, did not differ between treatments. Comparison of protein tyrosine phosphorylation patterns between in vitro capacitated and cryopreserved cells demonstrated a divergence in signal transduction. Cellular signaling in in vitro capacitated equine spermatozoa appeared to be in part dependent on activation of the cAMP/PKA pathway, whereas signaling in cryopreserved cells seemed to proceed predominantly through alternative pathways. Taken together, these data support the idea that capacitation and "cryocapacitation" are not equivalent processes.