精子表面蛋白P34H与男性精子质量关系的研究进展

精子表面蛋白P34H作为附睾上皮分泌的一种物质,并作为精子在附睾内成熟的标志,与男性不育的发生密切相关,而男性不育多由于精子数量减少或精子质量降低。研究显示,精子表面蛋白P34H能介导精卵透明带的结合,并且与精子顶体反应率、精子顶体酶活性、精浆中性a-葡萄糖苷酶、精子透明质酸酶活性呈正相关,其表达水平低下可影响精子质量,阻碍精子成熟,从而引起男性不育症的发生。本文对精子P34H蛋白与男性精子质量的相关性做一综述。     

E-cadherin在精子发生及精卵融合过程中的作用

精子发生是在睾丸内由生精细胞经过一系列复杂和系统的变化,最终成为精子的过程。精子在曲细精管内生成,随睾丸分泌物进入附睾内逐渐成熟;排出的精子进入女性生殖道获能并粘附在输卵管壁上,等待卵细胞经过并与其相遇,精子通过其顶体酶作用穿过透明带,与卵细胞结合并融合完成受精过程。精子发生、成熟、获能与受精,任一环节出现障碍,均会影响男性生育能力。E-cadherin是钙离子依赖性的介导细胞间粘附和信号转导的跨膜糖蛋白,位于精子头部,覆盖于整个顶体赤道板和顶体后区,参与精子发生与精卵融合过程,保证男性的正常生育能力。     

血管内皮生长因子及其受体在大鼠附睾内精子上的表达

背景：血管内皮生长因子及其受体在男性生殖领域中占有重要地位,但其在生殖系统中表达的意义和调节机制仍不十分清楚。 目的：观察血管内皮生长因子及其受体类fms酪氨酸激酶在青春期大鼠附睾内精子上的表达定位情况。 方法：取10只青春期雄性SD大鼠双侧附睾,分离得到浓度为(30~40)×109 L-1的精子,涂片,免疫荧光法检测精子上血管内皮生长因子及其受体类fms酪氨酸激酶的表达定位情况。 结果与结论：免疫荧光结果显示,血管内皮生长因子及其受体类fms酪氨酸激酶阳性蛋白均定位于大鼠附睾精子头部的顶体、尾部的颈段、中段和主段,尾部末段和精子核未见阳性染色。提示,血管内皮生长因子及其受体类fms酪氨酸激酶可能参与了精子的成熟过程,与精子的运动能力、获能和顶体反应有关。      

附睾的胚胎发生、结构及其功能

本文就哺乳动物（包括人）附睾的解剖分区、胚胎发生、结构和功能进行了综述。附睾由输出小管和附睾管构成。前者包含高柱状纤毛细胞和低柱状非纤毛细胞；后者包含主细胞、基细胞、顶细胞、窄细胞、亮细胞和晕细胞等。最近，我们在３３周人胚胎附睾的头段观察到淋巴细胞，体段观察到可能有内分泌功能的细胞，同时对各期胎儿附睾做了ＡＫＰ、ＡＣＰ、ＳＤＨ和ＰＡＳ反应的组化研究。附睾具有吸收、分泌和参与精子成熟的功能。近年来，相继发现了数十种与精子成熟密切相关的蛋白成分，它们为揭开精子成熟的奥秘奠定了基础。     

水通道蛋白-4在大鼠睾丸和附睾中的表达

目的 观察水通道蛋白-4（AQP4）在雄性大鼠睾丸和附睾的表达及分布特点,为探讨水通道蛋白在睾丸和附睾中的作用提供形态学基础。方法 采用免疫组织化学的方法,检测AQP4在睾丸和附睾的表达分布。结果 AQP4在睾丸中表达于各级生精细胞、支持细胞以及间质细胞中;在附睾管内表达于柱状上皮细胞,且腔面表达更明显。结论 AQP4可能参与了精子的生成及成熟过程,在激素的分泌调节中发挥作用。     

衰老对小鼠精子成熟和受精能力的影响

目的：探讨衰老对附睾精子成熟、体外受精和胚胎发育的影响。方法： 取老龄小鼠（18月龄，n=15）和青年小鼠（6月龄，n=15）附睾头精子和附睾尾精子，分别检测精子密度、存活率、活动率、正常形态率和胞浆小滴率，并通过体外受精比较附睾尾精子受精率和各阶段胚胎发育率。结果：老龄小鼠附睾头精子和附睾尾精子活动率、精子密度显著低于青年组（P<0.01），胞浆小滴率和畸形精子率显著高于青年组（P<0.05），附睾尾精子的受精率下降（P<0.01），胚胎各阶段发育率降低（P<0.01）。结论：衰老影响小鼠精子功能及附睾精子成熟过程。小鼠可作为雄性生殖衰老研究的动物模型。     

兔附睾蛋白的分离提纯及其免疫酶细胞化学研究

目前对精子在附睾内成熟的机制了解得很少。附睾的分泌和重吸收,为精子成熟提供了一个理想的环境。研究附睾的分泌和生理,对揭示精子成熟的机制和调节雄性生育有重要意义。我们用10％的十二烷基磺酸钠聚丙烯酰胺垂直平板电泳,在0.025mol Tris-甘氨酸缓冲液(pH8.3),电流45mA,温度10℃条件下,对兔附睾尾液、头液和精子膜蛋白进行了分析。结果表明,附睾尾液约有20条多肽区带,分布在14kd和94kd分子量之间。其中有三个主要蛋白的分子量依次为67,42和20kd。用激光密度扫描仪测得它们各占附睾尾液总蛋白的32.00％,19.35％和25.80％。附睾头液和精子膜蛋白各有25条以上的区带。其中     

锌缺乏导致小鼠睾丸游离锌离子和附睾精子数量减少

目的研究锌缺乏对小鼠睾丸游离锌离子和附睾精子数量的影响。方法锌缺乏饲养小鼠5周后,应用ZnSe金属自显影技术对小鼠睾丸和附睾进行染色,观察睾丸和附睾锌离子的分布,同时计数附睾精子数量,并与对照组小鼠的精子数量做对比。结果缺锌喂养后的小鼠睾丸游离锌离子明显减少,并且睾丸生精小管管腔狭小,生殖细胞层厚度增加,此外,附睾精子数量也明显低于对照组。结论睾丸和附睾内游离锌离子减少是锌缺乏小鼠精子数量下降的原因,本结果有助于改善男性生殖健康的状况。     

附睾功能基因研究进展

附睾是精子成熟和储存的重要场所,近年来,由于大量免费数据库的开放及新技术的开展,功能基因研究已经取得一定的成绩。通过DNA微阵距、基因芯片、表达谱差异分析、生物信息学分析和基因克隆等途径获得候选基因,通过生物信息学处理,最后进行基因功能确证,目前为止,已经发现的附睾基因不仅可以了解精子成熟的分子机制,而且为男性避孕及不孕症的治疗提供新的靶点。     

附睾管上皮的组织结构和功能

<正> 一、前言哺乳类动物精子在睾丸内生成后要经过附睾,才能达到形态学、生理学、生物化学上的成熟,获得运动和授精能力。而精子的成熟主要取决于附睾管上皮提供的合适微环境。在此,对附睾管上皮的组织结构和功能的研究作一综述,以期对男性计划生育科研提供一些资料。     

Identification of epididymis-specific antigens using neonatal tolerization

PROBLEM: Conventional immunization using whole sperm containing multiple antigens as the immunogen followed by hybridoma technology usually gives antibodies to antigens invariably of testicular origin, probably because of the strong immunogenic nature of these antigens. Therefore, an alternate approach of neonatal tolerization or subtractive immunization has been utilized to raise antibodies specific to epididymis by suppressing immune response to testicular antigens. METHOD OF STUDY: Neonatal mice were tolerized with testicular sperm proteins on days 0 and 5. These animals were then immunized with epididymal sperm proteins on day 21, followed by two boosters at biweekly intervals. Sera from these mice were used to localize epididymis-specific antigens. RESULTS: Sera from mice that were tolerized to testicular sperm proteins and later immunized with epididymal sperm proteins reacted only with epididymal proteins. CONCLUSION: The results of this study demonstrate that neonatal tolerization with testicular sperm proteins, followed by immunization with epididymal sperm proteins, enhances the production of antibodies to proteins exclusively of epididymal origin.     

Expression of sperm antigens during spermatogenesis and maturation detected with monoclonal antibodies

The expression of hamster sperm antigens was investigated during spermatogenesis and sperm maturation with the use of monoclonal antibodies generated in culture from mice immunized with hamster cauda epididymal spermatozoa or sperm heads. Antigens were localized by immunofluorescence and immunoperoxidase techniques which were first visualized on isolated spermatids over the developing acrosome. In one case, antibody inhibited fertilization in vitro although localization on testicular or epididymal spermatozoa was minimal compared with the early spermatid. Antibodies also recognized surface antigens first expressed in the epididymis whose localization on the spermatozoon altered during epididymal transit or incubation in capacitating medium. The results were discussed in relation to the expression and function of surface determinants on the haploid germ cell.     

Immunofluorescence antigen localization on boar sperm plasma membranes: monoclonal antibodies reveal apparent new domains and apparent redistribution of surface antigens during sperm maturation and at ejaculation

Purified boar sperm plasma membranes (PM) and PM proteins were used as antigens to produce 58 monoclonal antibodies against surface antigens. Fluorescence labelling (biotin-avidin-FITC) was used to determine the distribution of antigens in caput and cauda epididymal and in ejaculated spermatozoa with hybridoma supernatants and/or 1:100 diluted ascites fluid after subcloning. Sixteen areas (subdomains) of apparent restricted antigen mobility were identified and significant differences in the localization of most antigens in caput, cauda, and ejaculated PM were recognized. While localization patterns were highly reproducible with a given protocol for sample preparation and immunolabelling, localization patterns were markedly affected by changes in protocols. Fluorescence patterns were affected by the manner in which sperm were labelled (live sperm or sperm labelled at various steps), by washing, and by temperature or by addition of seminal plasma. These results indicate that the dynamic properties of the sperm PM or the surrounding fluids can easily mask or unmask or reconfigure binding sites for highly site-specific monoclonal antibodies and that antigen distribution is probably under-estimated when these labelling techniques are used. Such changes in the accessibility of antigenic sites to monoclonal antibodies limited determining the extent of distribution of a given antigen on epididymal sperm. However, the reproducibility of patterns when a given protocol is used and the large number of antibodies (39/42) displaying marked differences in localization on caput, cauda, and ejaculated PM suggest that changes in the organization of the PM constituents, whether by addition or subtraction of antigen or through configurational changes in proteins, are a major consequence of sperm maturation in the epididymis.     

Is lithium essential for epididymal sperm maturation?

A wider biological role of ultratrace element lithium in the mammalian reproduction has been reported, however, presence of lithium in the epididymal luminal fluid (ELF) and its influence on sperm during maturation events in the epididymal regions are still unknown. A pilot study was carried out in Jamunapari buck which revealed that levels of lithium in the ELF diminished gradually and significantly (P < 0.01) from caput to cauda epididymis, concomitantly, a distinct increase (P < 0.01) in the spermatozoan motility, viability and hypo-osmotic reactive sperm were observed, except spermatozoan motility that was found absent in the caput epididymis. Therefore, we hypothesize that levels of lithium in the epididymal regions is one of the motility initiation and/or regulatory factor for epididymal sperm maturation essential for acquiring fertilizing competence of sperm cells, hence, lithium could also be considered as one of the biomarker of sperm maturation in any species.     

Influence of macrophage migration inhibitory factor (MIF) on the zinc content and redox state of protein-bound sulphydryl groups in rat sperm: indications for a new role of MIF in sperm maturation

The function of macrophage migration inhibitory factor (MIF) in sperm maturation was studied by investigating its role in the biochemical maturation of the outer dense fibres. Rat sperm obtained from the caput and cauda epididymis were stimulated overnight with either recombinant MIF or MIF-containing vesicles originating from epididymal fluid at various concentrations. The zinc content of both the sperm and the medium was determined by means of atomic absorption spectrometry. Incubation in both recombinant MIF and vesicular MIF resulted in a statistically significant decrease of the zinc content in stimulated caput sperm of approximately 50%. In parallel, the conditioned media showed a clear increase in the concentration of this trace metal. The effect of MIF was less marked in cauda sperm. In addition, we demonstrated a statistically significant increase of detectable free thiol groups in the sperm mid- and principle piece in isolated rat sperm after stimulation with MIF at concentrations of 25 and 50 ng/ml. Our data suggest that MIF plays an important role in the maturation process of rat sperm during epididymal transit by inducing the elimination of zinc and affecting the amount of free sulphydryl groups in the sperm flagella.     

Transport and rearrangement of the intra-acrosomal protein acrin1 (MN7) during spermiogenesis in the guinea pig testis

We have recently shown that a 90-kDa glycoprotein, acrin1 (MN7), is exclusively localized in the dorsal region of the acrosomal apical segment of mature guinea pig sperm, and that its location changes during epididymal maturation. The present study examined the process of transport and organization of this protein in the acrosome during spermatogenesis in the guinea pig testis. Immunoperoxidase electron microscopy showed stage-specific localization of acrin1 within the developing acrosome, as follows: acrin1 first appeared in the proacrosomic vesicles of the early Golgi phase spermatids, and it was then localized in the electron-lucent matrix region of the acrosomic vesicles of the late Golgi phase spermatids. During the cap phase, acrin1 was abundant in the electron-lucent matrix of the acrosomal apical segment and in the head-cap region (principal segment). acrin1 became more restricted to the peripheral region of the electron-lucent matrix of acrosome phase spermatids and it was localized in the electron-lucent dorsal matrix region of maturation phase spermatids. In the final step of spermiogenesis, acrin1 disappeared from the equatorial and principal segments, and it was finally confined to the dorsal matrix region of the acrosomal apical segment. In addition, Western blot analysis showed that acrin1 of testes and epididymal sperm was of the identical size, indicating that acrin1 is not proteolytically modified during epididymal sperm maturation. These results indicate that acrosome morphogenesis is closely associated with the rearrangement of acrosomal proteins.     

Cytochemical and electrophoretic study of the stallion epididymal glycoproteins

It has been suggested that proteins produced in specific regions of the epididymis, mostly androgen dependent glycoproteins, are involved in the sperm maturation process. In the present work, the glycoconjugated distribution pattern and the electrophoretic characteristics of the stallion epididymal proteins were examined using lectin probes. The identification in the luminal fluid of some new proteins, probably synthesized and secreted by the epididymis, is an important initial step to investigate their interaction with the stallion sperm membrane. The binding of FITC-lectins (ConA, WGA, LPA, UEA, RCA, HPA) confirmed the presence of macromolecules containing carbohydrate residues in the epithelial cells with a distribution and relative density that was dependent on the epididymal region analyzed. The epithelium displayed affinity for more than one lectin, indicating diversity in the exposed sugar residues. The electrophoretic pattern of proteins obtained from ductus efferentes, corpus and cauda epididymis differed not only from those of the homologous blood serum, but also among the different epididymal regions. The most prominent bands correspond to 66, 55, 45 and 14 kDa proteins, present in different relative concentrations, in the three analyzed regions. A major band of 36 kDa was observed in the cauda epididymis region. The relative concentrations of protein bands of Mw 45, 36, 32, 20 and 18 kDa were significantly increased towards the distal regions of the ductus. The proteins of Mw 66, 55 and 14 kDa showed a relative higher concentration in the efferent ducts, decreasing to 25-30% in the cauda epididymal regions. The Mw 70, 66, 55, 45, 36, 32, 29, 23, 21, 18 and 14 kDa protein bands gave positive PAS reaction indicating that it corresponds to glycoproteins. Mannose residues were detected in the 70, 66, 55, 45, 36 and 32 kDa proteins. WGA-FITC binds to protein bands of Mw 70, 55, 45, 36, 32, 29, 25 and 24 kDa, suggesting the presence of N-linked glycoproteins. However, based on the resistance to the neuraminidase treatment, we suggest that the stallion epididymis contains both O- and N-glycoconjugates, probably in the N-acetyl O-diacetyl form. Although sperm maturation is an androgen-dependent process, no striking differences were detected in the SDS-PAGE obtained from animals in breeding and non-breeding seasons.     

Transport and rearrangement of the intra‐acrosomal protein acrin1 (MN7) during spermiogenesis in the guinea pig testis

We have recently shown that a 90‐kDa glycoprotein, acrin1 (MN7), is exclusively localized in the dorsal region of the acrosomal apical segment of mature guinea pig sperm, and that its location changes during epididymal maturation. The present study examined the process of transport and organization of this protein in the acrosome during spermatogenesis in the guinea pig testis. Immunoperoxidase electron microscopy showed stage‐specific localization of acrin1 within the developing acrosome, as follows: acrin1 first appeared in the proacrosomic vesicles of the early Golgi phase spermatids, and it was then localized in the electron‐lucent matrix region of the acrosomic vesicles of the late Golgi phase spermatids. During the cap phase, acrin1 was abundant in the electron‐lucent matrix of the acrosomal apical segment and in the head‐cap region (principal segment). acrin1 became more restricted to the peripheral region of the electron‐lucent matrix of acrosome phase spermatids and it was localized in the electron‐lucent dorsal matrix region of maturation phase spermatids. In the final step of spermiogenesis, acrin1 disappeared from the equatorial and principal segments, and it was finally confined to the dorsal matrix region of the acrosomal apical segment. In addition, Western blot analysis showed that acrin1 of testes and epididymal sperm was of the identical size, indicating that acrin1 is not proteolytically modified during epididymal sperm maturation. These results indicate that acrosome morphogenesis is closely associated with the rearrangement of acrosomal proteins. Anat Rec 259:131–140, 2000. © 2000 Wiley‐Liss, Inc.     

Changes in the plasma membrane proteins of stallion spermatozoa during maturation in the epididymis

The present paper reports modifications in the electrophoretic and cytochemical characteristics of mature and immature stallion spermatozoa. Some sperm surface glycoproteins (36, 32, 29, 21, 20, 18 kDa) detected in cauda epididymidis spermatozoa, were either absent or present in a very low relative concentration in immature sperm cells. A major 14 kDa protein band, observed in sperm extracts obtained from ductus efferentes, progressively decreased along the epididymal ductus. The nature and distribution of carbohydrate residues on the sperm membrane, during epididymal maturation, was also studied by use of lectin probes. Some protein bands bound concanavalin A while others, as the 36, 32 and 20 kDa proteins, exhibited higher affinity for WGA lectin. The distribution and relative density of mannose-, galactose-, N-acetylglucosamine-, N-acetylgalactosamine-, fucose- and sialic acid-containing macromolecules showed a characteristic pattern depending on the sperm membrane domain and on its origin. Some sperm surface domains displayed affinity for more than one lectin, indicating a diversity in their exposed carbohydrate residues, whereas others bound only one or no lectin. The passage of spermatozoa through the epididymidis was accompanied by changes in the accessibility or abundance of lectin ligands. Some lectins (UEA, WGA, LPA) gave stronger reaction in mature spermatozoa, while others (RCA, WFH, PNA) stained better immature spermatozoa. This remodeling of sperm surface molecules is probably a consequence of interactions between spermatozoa and the epididymal secretions, and may reflect addition or adsorption of new molecules, space configurations changes or biochemical modifications of pre-existing compounds. Our results suggest that the distribution and density of terminal oligosaccharidic residues on the sperm plasma membrane have species-specific characteristics. These post testicular developmental changes may be of significance in the overall understanding of the stallion fertility.