小鼠和豚鼠精子在附睾成熟过程中Ca~(2 )分布变化规律的研究

小鼠附睾头精子,其头部Ca~(2 )在顶体前区顶体外膜内侧结合最多,Ca~(2 )沉淀反应颗粒于该处呈连续层状。附睾头豚鼠精子其头部结合Ca~(2 )含量很少,且主要结合于顶体前区腹面顶体外膜内侧。小鼠附睾体和附睾尾精子Ca~(2 )的分布特征基本上和附睾头精子相同。但豚鼠附睾尾精子顶体外膜内侧无Ca~(2 )结合。和附睾头、附睾尾的附睾液相比,附睾体附睾液基质内具有大量Ca~(2 )存在。附睾体柱状上皮细胞的微绒毛切面上也具有Ca~(2 )沉淀反应颗粒,微绒毛可能与附睾液Ca~(2 )含量的调节有关。精子尾部Ca~(2 )主要分布于线粒体内,在质膜内、外两侧和线粒体外膜外侧也结合有少量的Ca~(2 )。和小鼠精子相比,豚鼠精子尾部线粒体内具有大量的Ca~(2 )。     

三疣梭子蟹精子顶体反应前后胞内Ca~(2+)的变化

应用激光扫描共聚焦显微镜(LSCM)和Fluo-3/AM荧染技术对三疣梭子蟹精子顶体反应前后的胞内Ca2 变化进行了观察和检测.结果显示,在精子顶体反应过程中,胞内Ca2 主要分布在细胞核、穿孔器和胞质膜残存处,胞内Ca2 浓度([Ca2 ]I)总体上呈现先上升后下降的趋势.顶体反应前精子的平均荧光强度为35.95±5.71;穿孔器前伸、顶体囊膜翻转阶段精子的平均荧光强度为66.80±7.35;顶体囊膜脱落、顶体丝形成阶段精子的平均荧光强度为3.87±2.82;上述各阶段间精子荧光强度有极显著差异(P<0.01).顶体反应穿孔器前伸、顶体囊膜翻转阶段的精子相比顶体反应前精子,[Ca2 ]I显著提高;而在顶体囊膜脱落、顶体丝形成阶段,[Ca2 ]I则急剧下降,只在顶体丝基部胞质膜残存处有微量Ca2 存在.初步探讨了三疣梭子蟹精子顶体反应前后胞内Ca2 变化的功能.     

大熊猫精子获能和顶体反应过程中钙分布变化规律的研究

应用焦锑酸钾原位定位法对大熊猫精子获能和顶体反应过程中进行钙定位研究,发现未获能精子的 Ｃａ２＋主要结合于顶体前区和赤道段质膜外侧和顶体内膜内侧（核膜侧）;随着获能的进行,Ｃａ２＋进入精子内部并主要结合于顶体区质膜内侧和顶体外膜外侧;顶体反应的精子,Ｃａ２＋结合于顶体内膜外侧、顶体后区质膜外侧和分散存在于释放的顶体内容物中,有些顶体反应精子的顶体内膜外侧结合的Ｃａ２＋特别丰富。精子尾部的Ｃａ２＋主要分布于中段线粒体内,且其内所含Ｃａ２＋含量随着获能和顶体反应而增加。另外尾部致密纤维和轴丝处也有少量Ｃａ２＋分布。     

哺乳动物精子中的ZP3结合蛋白研究进展

哺乳动物卵透明带糖蛋白ZP3(zona pellucida3)是介导精卵初级结合、诱发精子发生顶体反应的关键分子。目前已在精子中发现多种ZP3结合蛋白。95kD酪氨酸激酶受体可能通过其酪氨酸激酶活性介导ZP3诱发的顶体反应。β—1,4—半乳糖基转移酶与ZP3的糖基结合后,通过激活下游信号分子诱发顶体反应。精子蛋白sp56可能介导了顶体反应期间顶体基质与ZP之间的相互作用。透明带粘附素(zonadhesin)也是在顶体反应发生之后才与ZP发生相互作用。这些精子蛋白介导的下游信号事件将是下一步研究的热点。     

三疣梭子蟹精子顶体反应过程中的形态和结构变化

用离子载体A2 3187和卵水人工诱导三疣梭子蟹精子的顶体反应 ,分别获得 75 33%和 84 83%的顶体反应率。应用光镜和电镜技术观察了顶体反应前后精子形态和结构的变化。未处理精子呈陀螺形 ,由顶体、核杯和 5 - 10条核辐射臂组成。顶体包括顶体囊和顶体管。顶体囊的伞形头帽拥有约 70条辐射肋。连续发生的精子顶体反应过程被人为地分为四个阶段 :(1)头帽鼓起 ;(2 )顶体囊外翻 ;(3)穿孔器前伸 ,顶体囊膜翻转 ;(4 )顶体囊膜脱落 ,顶体丝形成。直到第四阶段才观察到钉状精子的辐射臂开始收缩。探讨了辐射臂和穿孔器前冲在精子入卵中的功能     

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。     

虾夷扇贝精子的超微结构

用扫描和透射电镜研究了虾夷扇贝(Patinopecten yessoensis)精子的超微结构.虾夷扇贝精子为典型的原生型,全长50μm左右,头部长约3 μm.精子主要由头部、中段和尾部三部分组成.头部顶体突出,呈倒"V"形;顶体下方为精核,电子密度较高且占头部大部分,具有核前窝(anterior nuclear fossa)、核后窝(posterior nuclear fossa)和植入窝(implantation fossa);4～5个近圆形的线粒体围绕着中心粒复合体形成精子的中段.尾部细长,尾部鞭毛横切面为典型的"9 2"结构.     

小鼠精子睾丸后成熟过程中单克隆抗体MSH27抗原表位的显露

MSH2 7是一株抗小鼠精子的单克隆抗体 ,此抗体及其纯化的抗原分子在体外受精实验中均可以阻止精卵膜融合 ,因而推测其抗原在精卵膜融合过程中具有重要的作用 .这里探讨了抗原分子在精子睾丸后成熟过程中MSH2 7识别表位的显露条件 .MSH2 7抗原分子在睾丸中产生 ,定位于精子顶体后区的质膜上 ;但MSH2 7所识别的抗原表位只在顶体反应后的精子上表露 .在受精过程中 ,精子必须完成顶体反应并穿过卵子的透明带才能接近卵子质膜并与之融合 .同时还研究了顶体反应和穿透明带作用对MSH2 7表位显露的影响 .在附睾成熟过程中 ,随着精子在附睾中的转运 ,它们发生顶体反应的能力不断增加 ;精子抗原表位的显露呈现顶体反应依赖性 ,且两者呈现线性回归关系 .钙离子载体A2 3 187诱发顶体反应后 ,MSH2 7染色阳性的精子远远少于顶体反应的精子 ;但穿过透明带后绝大多数精子 ( 98% )呈现MSH2 7染色阳性 .总之 ,在睾丸后成熟 (附睾成熟、获能、顶体反应 )和精子穿透明带的过程中 ,对整个精子群体来讲 ,单抗MSH2 7所识别的抗原表位的显露和精子最终获得穿入卵子质膜的能力是同步发生的 .     

大鲵精子的超微结构研究

本文运用透射电镜和扫描电镜研究了大鲵(Andrias davidianus)精子的超微结构,大鲵精子由头部(head),中片(midpiece)和尾部(tail)三部分组成。头部有棒状细胞核,核内染色质高度浓缩,细胞核前方呈细丝状,但非顶体结构。头部后端凹陷,称为植入窝(implantation fossa),植入窝内有线粒体和中心粒等细胞器结构,此区域为精子的中片。精子尾部细长,主要由轴丝和附属纤维(accessory fiber)组成,轴丝的外面具有波动膜。     

挂榜山小鲵精子形态结构研究

应用扫描电镜和透射电镜对挂榜山小鲵(Hynobius guabangshanensis)精子的超微结构进行观察和研究.结果表明:1)挂榜山小鲵的精子形态具有小鲵科精子的共同特征:头部细直、有锥形顶体;颈部短而不明显,尾部细长、有波动膜;2)该种小鲵的精子超微结构具小鲵科物种精子的共同特征,即精子无顶体钩,顶体呈三叶草状,尾部无线粒体,轴纤维粗大呈圆柱状等;3)除精子全长(194.1±7.15μm)在小鲵科物种中属于中等外,挂榜山小鲵和本科其他物种的精子在形态量度学方面差异明显:其头部占全长的比例(22.98%)比其他已知小鲵科的物种都低,尾部占全长的比例(65.79%)比其他小鲵科物种都高.     

Targeted deletion of Tssk1 and 2 causes male infertility due to haploinsufficiency

Targeted deletion of Tssk1 and 2 resulted in male chimeras which produced sperm/spermatogenic cells bearing the mutant allele, however this allele was never transmitted to offspring, indicating infertility due to haploinsufficiency. Morphological defects in chimeras included failure to form elongated spermatids, apoptosis of spermatocytes and spermatids, and the appearance of numerous round cells in the epididymal lumen. Characterization of TSSK2 and its interactions with the substrate, TSKS, were further investigated in human and mouse. The presence of both kinase and substrate in the testis was confirmed, while persistence of both proteins in spermatozoa was revealed for the first time. In vivo binding interactions between TSSK2 and TSKS were established through co-immunoprecipitation of TSSK2/TSKS complexes from both human sperm and mouse testis extracts. A role for the human TSKS N-terminus in enzyme binding was defined by deletion mapping. TSKS immunoprecipitated from both mouse testis and human sperm extracts was actively phosphorylated. Ser281 was identified as a phosphorylation site in mouse TSKS. These results confirm both TSSK 2 and TSKS persist in sperm, define the critical role of TSKS' N-terminus in enzyme interaction, identify Ser 281 as a TSKS phosphorylation site and indicate an indispensable role for TSSK 1 and 2 in spermiogenesis.     

Dynamin 2 associates with complexins and is found in the acrosomal region of mammalian sperm

Previous data showed that complexin I, a SNARE regulatory protein, is localized in and/or around the acrosome and is necessary for the acrosome reaction in sperm. To understand how complexin I regulates the acrosome reaction, we used complexin-GST pulldown assays to identify interacting proteins. We showed that both complexins I and II bound mouse sperm dynamin 2. Dynamin 2 is a 100 kDa GTPase essential to many aspects of endocytosis but its potential role in exocytosis is unknown. Dynamin 2 is expressed in rat testis and widely expressed in other tissues; however, the function of dynamin 2 in germ cells is uncertain. Dynamin 2 protein was detected in mouse testis and was most abundant in or around the developing acrosome of spermatids. In addition, dynamin 2 was co-localized with complexin I in the acrosomal region of mammalian sperm. Its co-localization and interaction with complexin I suggest that dynamin 2 may play a role during acrosome formation and/or acrosomal exocytosis.     

Characterization of recombinant murine binder of sperm protein homolog 1 and its role in capacitation

Sperm capacitation is a maturation step that is deemed to be essential for sperm to fertilize an oocyte. A family of proteins, the binder of sperm (BSP), are known to bind choline phospholipids on sperm membranes and promote capacitation in bulls and boars. Recently, BSP-homologous genes have been identified in the epididymal tissues of human (BSPH1) and mouse (Bsph1, Bsph2). The aim of this study was to determine the binding characteristics of the murine binder of sperm protein homolog 1 (BSPH1) and evaluate its effects on sperm capacitation. Since it is not possible to purify the native BSP proteins from human and mouse in sufficient quantity, a murine recombinant BSPH1 (rec-BSPH1) was produced and used for the functional studies. Similarly to BSP proteins from other species, rec-BSPH1 bound to gelatin, heparin, phosphatidylcholine liposomes, and sperm. Both native BSPH1 and rec-BSPH1 were detected on the head and the midpiece region of sperm, although a stronger signal was detected on the midpiece region when sperm were incubated in a capacitating media containing bovine serum albumin. More importantly, murine rec-BSPH1 was able to capacitate sperm, but was unable to induce the acrosome reaction. These results show that murine epididymal BSPH1 shares many biochemical and functional characteristics with BSP proteins secreted by seminal vesicles of ungulates, and suggest that it might play a similar role in sperm functions.     

TSKS concentrates in spermatid centrioles during flagellogenesis

Centrosomal coiled-coil proteins paired with kinases play critical roles in centrosomal functions within somatic cells, however knowledge regarding gamete centriolar proteins is limited. In this study, the substrate of TSSK1 and 2, TSKS, was localized during spermiogenesis to the centrioles of post-meiotic spermatids, where it reached its greatest concentration during the period of flagellogenesis. This centriolar localization persisted in ejaculated human spermatozoa, while centriolar TSKS diminished in mouse sperm, where centrioles are known to undergo complete degeneration. In addition to the centriolar localization during flagellogenesis, mouse TSKS and the TSSK2 kinase localized in the tail and acrosomal regions of mouse epididymal sperm, while TSSK2 was found in the equatorial segment, neck and the midpiece of human spermatozoa. TSSK2/TSKS is the first kinase/substrate pair localized to the centrioles of spermatids and spermatozoa. Coupled with the infertility due to haploinsufficiency noted in chimeric mice with deletion of Tssk1 and 2 (companion paper) this centriolar kinase/substrate pair is predicted to play an indispensable role during spermiogenesis.     

The C-kit receptor and its possible signaling transduction pathway in mouse spermatozoa

The presence and role of the c-kit protein was investigated in the mature sperm of the mouse. The c-kit monoclonal antibody (mAb) ACK₂ reacted specifically with the acrosomal region and the principal piece of fixed noncapacitated sperm but did not react with the acrosome region in acrosome-reacted sperm. ACK₂ significantly inhibited the acrosome reaction; this inhibition was relieved by the calcium ionophore A23187. The kit ligand stem cell factor (SCF) significantly increased the percentage of sperm undergoing acrosome reaction. This increase was partially inhibited by the calcium channel inhibitor (verapamil), the PI3k inhibitor (wortmannin), and the PLC inhibitor (U-73122). ACK₂ predominantly recognized c-kit proteins of 33, 48, and 150 kDa by Western blotting of mouse sperm extracts. The 48- and 150-kDa protein bands were released into the media and tyrosine autophosphorylated at low basal levels during acrosome reaction. On stimulation with SCF, the level of c-kit phosphorylation increased significantly. These findings suggest that c-kit is present in mature sperm, and its binding to SCF may result in the activation of PLCγ1 and PI3K, leading to receptor autophosphorylation, and ultimately may play a role in capacitation and/or the acrosome reaction. Mol. Reprod. Dev. 49:317–326, 1998. © 1998 Wiley-Liss, Inc.     

Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa

Calmodulin has been postulated as a mediator in the calcium-dependent processes that culminate in the acrosome reaction. Changes in calmodulin compartmentalization as a consequence of the increased permeability to extracellular calcium during capacitation and acrosome reaction have been suggested. In the present study the temporal localization of calmodulin in guinea pig spermatozoa was studied during in vitro capacitation and acrosome reaction by indirect immunofluorescence. Capacitation was achieved by incubation in Tyrode medium supplemented with pyruvate, lactate, and glucose in the presence and in the absence of calcium. Acrosome reaction was elicited in three different conditions: 1) by transfer to minimal culture medium containing pyruvate and lactate (MCM-PL) after in vitro capacitation 2) by 0.003% Triton-X 100 treatment, and 3) by A 23187 addition to sperm samples incubated in MCM-PL. During capacitation, calmodulin was observed both in the acrosome and in the flagellum; this localization seemed to be independent of the presence of extracellular calcium and of exogenous substrates. Throughout the acrosome reaction, different stages of calmodulin compartmentalization were observed. It became clustered around the equatorial region just before or a little after the acrosome reaction had occurred. Later, it was observed around the postacrosomal region in the acrosome-reacted sperm. The changes in calmodulin distribution were found to be dependent on the stage in the acrosome reaction.     

Identification of mouse trp homologs and lipid rafts from spermatogenic cells and sperm.

Intracellular Ca(2+) has an important regulatory role in the control of sperm motility, capacitation, and the acrosome reaction (AR). However, little is known about the molecular identity of the membrane systems that regulate Ca(2+) in sperm. In this report, we provide evidence for the expression of seven Drosophila transient receptor potential homolog genes (trp1-7) and three of their protein products (Trp1, Trp3 and Trp6) in mouse sperm. Allegedly some trps encode capacitative Ca(2+) channels. Immunoconfocal images showed that while Trp6 was present in the postacrosomal region and could be involved in sperm AR, expression of Trp1 and Trp3 was confined to the flagellum, suggesting that they may serve sperm to regulate important Ca(2+)-dependent events in addition to the AR. Likewise, one of these proteins (Trp1) co-immunolocalized with caveolin-1, a major component of caveolae, a subset of lipid rafts potentially important for signaling events and Ca(2+) flux. Furthermore, by using fluorescein-coupled cholera toxin B subunit, which specifically binds to the raft component ganglioside GM1, we identified caveolin- and Trp-independent lipid rafts residing in the plasma membrane of mature sperm. Notably, the distribution of GM1 changes drastically upon completion of the AR.     

C-kit receptor and its possible function in human spermatozoa

The presence and role of the c-kit proto-oncogene protein was investigated in the mature sperm of the human. A polyclonal antibody against the c-kit peptide was used to perform immunohistochemical (IHC) staining, electron microscopy (EM) studies, and Western blot analysis. The acrosomal region of fresh sperm specifically stained with the antibody. No acrosomal staining or staining limited to the equatorial region was noted in the acrosome-reacted (AR) sperm. EM studies demonstrated immunogold label on the plasma membrane (PM) of the acrosome, and confirmed the lack of binding following the acrosome reaction. A 150 kDa band was detected by Western blot analysis. This protein was released from the sperm surface during sperm capacitation and the acrosome reaction. Antibody against the c-kit receptor significantly inhibited the acrosome reaction and increased sperm agglutination, but did not significantly inhibit sperm motility. These results suggest that the c-kit receptor protein is present in mature human sperm and is released during capacitation and/or the acrosome reaction. The assessment of the c-kit receptor may also be a useful assay for sperm function in male infertility.     

Binding of a murine proteinase inhibitor to the acrosome region of the human sperm head

Proteinase inhibitors are present in the various glands, tissues, and secretions of the male reproductive tract. Some of these inhibitors bind to the acrosomal region of the sperm, and their release during in vitro or in utero incubation suggests that they may play a role in capacitation. In the mouse, the binding site for a trypsin-acrosin inhibitor, the acceptor, has been implicated in capacitation, zona binding, and the acrosome reaction. This presentation demonstrates that a component, molecular weight ?T20,000, on the human sperm head may recognize the murine inhibitor. Furthermore, the acrosome reaction can be induced in capacitated human sperm by immunoaggregation of bound murine inhibitor. The data indicate that the proteinase inhibitor binding site on the human sperm head may, as with a similar site on murine sperm, play a role in the early events of fertilization. © 1993 Wiley-Liss, Inc.     

Immunoelectron microscopic localization of calmodulin and phospholipase A2 in spermatozoa. I

Using the Lowicryl K4M embedding technique, together with indirect immunoferritin or immunogold labeling on ultra-thin sections, tubulin, calmodulin and phospholipase A2 were distinctly localized in ejaculated bull spermatozoa. Calmodulin was concentrated on the plasma membrane, nucleus, post-acrosomal substance, and, in lesser amounts, between coarse fibers and axonemal microtubules of the flagellum. Phospholipase A2 was distributed evenly along the plasma membrane, nucleus, acrosome, post-acrosomal substance, and in the flagellum, on mitochondria, fibrous sheath, coarse fibers, between coarse fibers and axonemal microtubules. Antibodies to tubulin labeled only axonemal microtubules, including the central pair of microtubules. Patterns of tubulin labeling were identical when ferritin granule- or gold particle-conjugated antibodies were tested. In agreement with our previous biochemical studies demonstrating calmodulin binding to phospholipase A2, concomitant with enhancement of phospholipase A2 activity (Arch Biochem Biophys 241:413, 1985), the overlapping distribution of calmodulin and phospholipase A2 in several parts of the sperm suggests that these proteins may play a concerted role in male gamete function in preparation for or during fertilization. The distinct distribution of tubulin along flagellum microtubules indicates their special function in sperm mobility.