AFM对黄精赞育胶囊优选方处理前后大鼠弱精子超微结构的观察研究

目的:应用原子力显微镜技术实现对大鼠精子超微结构的实时成像,对比观察中药作用前后弱精子超微结构的变化.方法:制作少弱精症大鼠模型;运用非接触模式原子力显微镜技术对比观察正常精子和病理性精子在中药作用前后超微结构的动态变化.结果:获得精子头体、颈部和鞭毛等部位的实时超微结构图像.结论:AFM可直接探测精子头体的各种畸形,实现对精子全貌的观测.通过修复弱精子超微结构的病理形态学缺陷可能是黄精赞育胶囊优选方改善弱精子质量的机制之一.     

AFM对黄精赞育胶囊优选方处理前后大鼠弱精子超微结构的观察研究（英文）

目的：应用原子力显微镜技术实现对大鼠精子超微结构的实时成像,对比观察中药作用前后弱精子超微结构的变化。方法：制作少弱精症大鼠模型;运用非接触模式原子力显微镜技术对比观察正常精子和病理性精子在中药作用前后超微结构的动态变化。结果：获得精子头体、颈部和鞭毛等部位的实时超微结构图像。结论：AFM可直接探测精子头体的各种畸形,实现对精子全貌的观测。通过修复弱精子超微结构的病理形态学缺陷可能是黄精赞育胶囊优选方改善弱精子质量的机制之一。     

用原子力显微镜观察人类精子的表面结构

目的:探讨用原子力显微镜观察精子表面结构的方法。方法:经常规洗涤正常人精液后进一步除去精子表面和生理溶液中的蛋白质,直接用原子力显微镜观察人类精子的表面形态。结果:获得了人类精子表面的细微结构和精子头的三维数据。精子全长约47μm,精子头约4.6μm,顶体位于精子头前端1/2~2/3,顶体前端扁平。精子赤道区有两圈环形凸起。结论:不需特殊处理,用原子力显微镜能直接观察精子表面的超微结构,并获得量化的三维数据。     

原子力显微镜及其在肿瘤研究中的应用

原子力显微术是一种利用原子、分子间的相互作用力来观察物体表面超微结构的新型实验技术.介绍了原子力显微镜作为一种显微探测和操纵工具的主要特点及其在肿瘤研究中的优势,评述了国内外有关原子力显微镜在肿瘤的诊断、治疗、抗肿瘤药物开发等研究中的应用情况,展望了原子力显微镜应用于肿瘤单细胞研究的前景.     

原子力显微镜对人羊膜上皮细胞的观察

目的:在单细胞水平上分析人羊膜上皮细胞的超微结构及其机械性能(粘弹力、杨氏模量、硬度等),为进一步认识细胞结构与功能的关系奠定基础.方法:应用原子力显微镜(AFM)高分辨率、高灵敏度的特点,对人的羊膜上皮细胞进行观察.结果:人羊膜上皮细胞呈椭圆形,由原子力显微镜力位移曲线测量系统,可得粘弹力:1034.375±294.21 pN.硬度:1.1815±0.326mN/m,杨氏模量:16.44±4.67Kpa.结论:AFM能对人羊膜上皮细胞表面超微结构清晰地成像及提供更多更确切的表面信息及机械性能,从而增加对羊膜上皮细胞的认识.     

原子力显微镜在双微体形态学研究中的应用

原子力显微术(atomic force microscopy,AFM)是一种新型的纳米显微技术,由于其拥有标本制备简单、分辨率高等优点,因此常用于细胞超微结构的观察。双微体(double minute chromosomes,DMs)是基因扩增的主要表现形式,经常出现在肿瘤细胞及耐药细胞中,可使肿瘤细胞获得生存优势或产生耐药性,因此对双微体进行研究可使人类了解肿瘤的生长特性及其抗药性的产生机理。为寻找一种研究双微体的有效方法,本实验利用原子力显微镜对小鼠耐氨甲喋呤细胞3T3R500中的双微体进行观察,在获得双微体高分辨AFM形态图的同时,还对双微体的大小进行了测量,发现细胞中双微体大小存在差异。此外,就原子力显微镜在双微体研究中的一些技术细节进行了探讨。实验结果表明原子力显微术是研究双微体的一种有效手段。     

蟋蟀精子顶体复合体形成的超微结构

顶体复合体是昆虫精子进行穿卵受精的重要细胞器,它位于精子的最前端.1997年我们对蟋蟀科一种类昆虫北京油葫芦(Gryllus mitratusburmeister)精子顶体复合体形成过程进行了超微结构的观察,发现与前人描述的蟋蟀科精子顶体复合体超微结构特征在某些方面有所不同,从而为准确认识和掌握该结构的特点提供一定的理论依据.     

青春期大鼠实验性精索静脉曲张对附睾超微结构的影响

目的：精索静脉曲张（VC）与男生不育密切相关,但其导致不育的确切机制尚不清楚。大量临床观察和实验研究证明VC可引起睾丸损害,但对附睾的影响研究较少,特别关于青春期动物VC对附睾影响的研究尚未见任何报道,为此,本研究在建立青春期大鼠实验性VC模型的基础上,试图通过实验性VC对附睾超微结构的影响。来阐明其在不育发生机理中的地位。方法：部分结扎青春期大鼠左肾静脉建立VC模型,分别于手术后4周和8周取左右侧附睾始段头,体,和尾部,在光镜研究的基础上,制作透射电镜标本并进行观察,结果：VC大鼠左右侧附睾各段上皮的超微结构都发生明显改变;如上皮基膜增厚;上皮微绒毛稀少且局部受损;主细胞内多形态溶酶体增加,内质网扩张,高尔基复合体空泡化,线粒体嵴模糊,胞质内出现大空泡;晕细胞数增加且胞质内含大量高电子密度的溶酶体;亮细胞内脂肪滴和溶酶体明显增多,细胞膨胀,常可见游离面突入官腔,此外,附睾官腔内精子残余体增多,精子头出现核大泡,精子尾线粒体,纤维柱排列紊乱等。结论：青春期可引起大鼠附睾超微结构受损,这可能也是VC导致不育的重要原因之一。     

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.     

电镜技术在植物病害研究中的应用

电子显微镜(electron microscopy,EM)是20世纪最重要的发明之一,其特有的高分辨率,在植物病害检测、超微结构及形态观察中发挥了重要作用。对目前常用的透射电子显微镜,扫描电子显微镜、环境扫描电子显微镜、扫描隧道显微镜及原子力显微镜等新型电镜的原理及其在植物病害研究中的应用作一介绍,旨在为更好的运用电镜技术提供参考。     

Atomic force microscopy: a powerful tool for high-resolution imaging of spermatozoa

Atomic force microscopy (AFM) has emerged as the only technique capable of real-time imaging of the surface of a living cell at nano-resolution. Since AFM provides the advantage of directly observing living biological cells in their native environment, this technique has found many applications in pharmacology, biotechnology, microbiology, structural and molecular biology, genetics and other biology-related fields. AFM has also proved to be a valuable tool for reproductive biologists. An exhaustive review on the various applications of AFM to sperm cells is presented. AFM has been extensively applied for determining the structural and topological features of spermatozoa. Unstained, unfixed spermatozoa in their natural physiological surroundings can be imaged by this technique which provides valuable information about the morphological and pathological defects in sperm cells as three-dimensional images with precise topographical details. Sperm head defects and the acrosome at the tip of the head responsible for fertilization, can be examined and correlated with the lack of functional integrity of the cell. Considerable amount of work is reported on the structural details of the highly condensed chromatin in sperm head using AFM. Detailed information on 3D topographical images of spermatozoa acquired by AFM is expected to provide a better understanding of various reproductive pathways which, in turn, can facilitate improved infertility management and/or contraceptive development.     

Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia

Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.     

Inhibition of tyrosine phosphorylation of sperm flagellar proteins,outer dense fiber protein‐2 and tektin‐2, is associated with impaired motility during capacitation of hamster spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor, tyrphostin‐A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin‐A47‐treated spermatozoa exhibited circular motility, which was associated with a distinct hypo‐tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000–60,000. In this study, we provide evidence on the localization of capacitation‐associated tyrosine‐phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo‐tyrosine phosphorylated major proteins of tyrphostin‐A47‐treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein‐2 and the 51 kDa protein as tektin‐2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo‐tyrosine‐phosphorylation status of outer dense fiber protein‐2 and tektin‐2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR‐tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa. Mol. Reprod. Dev. 77: 182–193, 2010. © 2009 Wiley‐Liss, Inc.     

Mammalian Fused is essential for sperm head shaping and periaxonemal structure formation during spermatogenesis

During mammalian spermatogenesis, the diploid spermatogonia mature into haploid spermatozoa through a highly controlled process of mitosis, meiosis and post-meiotic morphological remodeling (spermiogenesis). Despite important progress made in this area, the molecular mechanisms underpinning this transformation are poorly understood. Our analysis of the expression and function of the putative serine–threonine kinase Fused (Fu) provides critical insight into key steps in spermatogenesis. In this report, we demonstrate that conditional inactivation of Fu in male germ cells results in infertility due to diminished sperm count, abnormal head shaping, decapitation and motility defects of the sperm. Interestingly, mutant flagellar axonemes are intact but exhibit altered periaxonemal structures that affect motility. These data suggest that Fu plays a central role in shaping the sperm head and controlling the organization of the periaxonemal structures in the flagellum. We show that Fu localizes to multiple tubulin-containing or microtubule-organizing structures, including the manchette and the acrosome–acroplaxome complex that are involved in spermatid head shaping. In addition, Fu interacts with the outer dense fiber protein Odf1, a major component of the periaxonemal structures in the sperm flagellum, and Kif27, which is detected in the manchette. We propose that disrupted Fu function in these structures underlies the head and flagellar defects in Fu-deficient sperm. Since a majority of human male infertility syndromes stem from reduced sperm motility and structural defects, uncovering Fu?s role in spermiogenesis provides new insight into the causes of sterility and the biology of reproduction.     

Antifertility effects in rats actively immunized with 80 kDa human semen glycoprotein.

A 80 kDa human sperm antigen has been identified using the serum of an infertile woman having circulating antisperm antibodies. The antigen was then purified to homogeneity by gel permeation chromatography using HPLC (protein PAK-125 column) system and on FPLC (superose-12 column) system. The antigen was found to be a glycoprotein. The antigen was mainly localized in the postacrosomal region of the human sperm, while it was localized in the head region of the rat sperm as demonstrated by immunofluorescent staining. The presence of this antigen was also demonstrated in the human prostate and endometrium and in the rat testis; epididymis and the prostate by immunocytochemical staining. The purified protein upon active immunization in female rats caused infertility in 100 percent animals. While in male rats it caused infertility in 90 percent animals. On morphometric analysis of testicular tissue it was observed that there was no significant change in spermatogonia and spermatocytes, but significant decrease in spermatids and sperm number as well as daily sperm production in the immunized male rats. The epididymal spermatozoa were markedly reduced in number and were largely found to be agglutinated. The results suggest that 80 kDa human sperm antigen appears to be a suitable candidate for immunocontraception both in male and female.     

Morphology and fine structure of Barbus barbus (Teleostei: Cyprinidae) spermatozoa

Morphology and fine structure of Barbus barbus L 1758 spermatozoa were studied using scanning (SEM) and transmission (TEM) electron microscopy. The results confirm that spermatozoa exhibit morphological features typical to all teleost fishes. They are differentiated into a head, a midpiece and a flagellum with the typical '9 + 2' pairs of microtubules. Both dynein arms are present in the flagellum. The spermatozoa have spherical nuclei, 4–6 mitochondria located in the postnuclear cytoplasmic region and centriolar complex (proximal and distal centrioles). Total length, head width, length of midpiece and length of flagellum were measured to be 56.35 ± 7.42, 1.80 ± 0.06, 0.48 ± 0.14 and 54.30 ± 6.97 μm, respectively. Highly significant linear correlation was observed between posterior and anterior width of midpiece (P < 0.01). Principal component analysis (PCA) was used to explore which parameters can explain the individual variation of sperm morphology. About 44% of the total accumulated variance was absorbed by the analysis of the two first components, distinguishing different groups of parameters related to head and midpiece. The lengths of flagellum and head are more isolated; indicating that the individual variation of sperm morphology depends on these two parameters. Comparing the results of this study with information on cyprinids spermatozoa reveals that the number of mitochondria and the length of the flagellum are good characters to characterize spermatozoa of the Cyprinidae in a phylogenetic arrangement.     

Immunolocalization of multiple Galpha subunits in mammalian spermatozoa and additional evidence for Galphas

Like somatic cells, mammalian spermatozoa appear to contain several different heterotrimeric G protein alpha-subunits that could mediate specialized cell responses. However, the precise Galpha subunits present, their subcellular location and their possible roles are still incompletely defined. In this study, using commercially available specific antibodies, we have shown by immunoblotting that Galpha(s) is present in human and mouse sperm lysates. Immunolocalization using intact spermatozoa from both species revealed this protein to be in the acrosomal cap region and the flagellum, particularly the principal piece. Treatment of permeabilized mouse spermatozoa with cholera toxin led to enhanced ADP-ribosylation of a protein the same size as Galpha(s), as well as an increase in cAMP, providing further proof for Galpha(s). Evidence for the presence and distinct localizations of Galpha(i2), Galpha(i3), Galpha(o), Galpha(q/11), and Galpha(olf) was also obtained. Of particular interest was Galpha(i2) which, like Galpha(s), was present in the acrosomal cap region and flagellum, the same regions where stimulatory and inhibitory adenosine receptors are localized. These observations are consistent with our hypothesis that G proteins mediate adenosine receptor modulation of adenylyl cyclase, with consequent alterations in cAMP production, apparently crucial for the spermatozoon's acquisition and maintenance of fertilizing ability.     

Morphology and ultrastructure of Siberian sturgeon (Acipenser baerii) spermatozoa using scanning and transmission electron microscopy

BACKGROUND INFORMATION: Available data concerning the sperm morphology of teleost fishes demonstrate wide variation. In the present study, the spermatozoa of Siberian sturgeon (Acipenser baerii Brandt, 1869), a chondrostean fish, was investigated. In contrast with teleost fish, chondrostean spermatozoa have a head with a distinct acrosome, whereas other structures, such as a midpiece and a single flagellum, are present in spermatozoa of most species. RESULTS: The average length of the head including the acrosome and the midpiece was 7.01+/-0.83 microm. Ten posterolateral projections derived from the acrosome were present on a subacrosomal region, with mean lengths of 0.94+/-0.15 microm and widths of 0.93+/-0.11 microm. The nucleus consisted of electrodense homogeneous nuclear chromatin. Three intertwining endonuclear canals, bound by membranes, traversed the nucleus longitudinally from the acrosomal end to the basal nuclear fossa region. There were between three and six mitochondria, two types of centrioles (proximal and distal) in the midpiece and two vacuoles composed of lipid droplets. The flagellum (44.75+/-4.93 microm in length), originating from the centriolar apparatus, had a typical 9+2 eukaryotic flagellar organization. In addition, there was an extracellular cytoplasm canal between the cytoplasmic sheath and the flagellum. CONCLUSIONS: A principal components analysis explained the individual morphological variation fairly well. Of the total accumulated variance, 41.45% was accounted for by parameters related to the head and midpiece of the sperm and the length of the flagellum. Comparing the present study with previous studies of morphology of sturgeon spermatozoa, there were large inter- or intra-specific differences that could be valuable taxonomically.     

From the cellular to the molecular dimension: The actual challenge for human fertilization research

The form that rat spermatozoa assume when swelling in hyposmotic media depends on the position of the cytoplasmic droplet, previous exposure to hypertonic media, and the stiffness of the flagellum. Bending at the end of the midpiece occurs when the swelling droplet is situated at this site; this occurs in midcaput cells, but sperm from more proximal sites do not bend in this fashion. Stiffening of caput sperm stored in vitro reduces the incidence of such midpiece bending but looping at the tip of the tail still occurs, and previous exposure of caput spermatozoa to hypertonic media also prevents hairpin bend formation. Mature sperm from the cauda are too stiff to form hairpin loops when placed in hypotonic media unless first treated with a penetrating disulphydryl-reducing agent, after which swollen spherical vesicles can result from very flexible flagella confined within an intact membrane. Long-chain acylcarnitines are more potent lytic agents than acylcholines, but, for both, chain lengths of 16 carbon atoms is optimal for preventing the swelling of rat caput sperm.