日本血吸虫精子的超微结构

目的：了解日本血吸虫正常精子的超微结构。方法：半超薄切片定位雄虫睾丸和含有成熟精子的雌虫输卵管，常规透射电镜制样并观察。结果：日本血吸虫精子由头、尾两部分组成，头部呈长卵圆形，平均长6．2μm，宽1．4μm，无顶体构造，前端钝圆，后端尖细，质膜下有1圈纵行的微管，核1个，前端有少量线粒体，尾部鞭毛1根，在中、后段主体部分鞭毛轴丝外周为9组二联管，中央为一团弥散的电子致密物质，呈9×2＋《1》型；但在过渡区鞭毛轴丝中央无电子致密物质，呈9×2＋0型。结论：日本血吸虫精子超微结构与其他血吸虫相似，具有同源性，但明显区别于复殖目大多数吸虫的构造。     

带绦虫精子发生过程中精子变形的超微结构观察

目的：研究带绦虫在精子发生过程中的精子变形。方法：透射电镜。结果：32个玫瑰花样的次级精母细胞经第2次减数分裂后,产生64个精细原形体（64－spermatid－plasmodium）。精细胞的变形过程复杂,首先见精细胞胞质和核伸长,胞质增多由胞质桥“cytophore”相连;然后是核的进一步伸长,核染色质聚合成线束状,最后脱离胞质桥,形成成熟精子。成熟的精子呈细线状,长约16．2－18．6μm,宽0．35－0．45μm,可分为有核的头部及无核的尾部两部分。头部长约5－6μm,占体长的1／3,有一个较长的核缠绕着轴丝,无线粒体。尾部长约11．2－16．6μm。在尾部的前段及头部的后方,纵断面上见一些线粒体包绕着轴丝,全长约1．6－1．7μm。精子的尾部只含一条结构为“9＋1”的轴丝。精子横断面,质膜下见46条微管（周围微管）。结论：带绦虫精子子的变形是一个非常复杂的过程。     

Bypassing natural sperm selection during fertilization: the azh mutant offspring experience and the alternative of spermiogenesis in vitro

Molecular aspects of spermiogenesis can be studied using mouse mutants and spermatids developed in vitro. The azh/azh mutant is an attractive model system because structural abnormalities in the sperm head and the ectopic position of the manchette are associated with tail bending and looping. Spermatids, developing an axoneme in vitro and capable of cell motility, offer the possibility of the dynamic analysis of tail development. Offspring generated by intracytoplasmic injection of azh/azh sperm heads into normal mouse oocytes complement the mouse mutant approach. A central question of sperm tail development is the role of the manchette, a transient microtubular structure assembled soon after the organization of the axoneme. The fractionation of intact manchettes by gradient centrifugation has enabled a biochemical analysis of constitutive tubulin isotypes and transiently associated proteins. For example, keratins Sak57, Odf1, and Odf2 are initially stored in the manchette before being sorted to the outer dense fibers and fibrous sheath of the developing spermatid tail. Additional proteins associated with the manchette include two proteases, the 26S proteasome and N-arginine convertase (both sorted to the developing spermatid tail), a spermatid perinuclear RNA binding protein, Spag4, an Odf1-binding protein, and type 4 cAMP-specific phosphodiesterase D. Keratin 9 and delta-tubulin are two proteins found in the perinuclear ring of the manchette, the insertion site of the microtubular mantle. Available data indicate that the manchette is a highly dynamic structure providing microtubular tracks to structural proteins participating in the sperm tail development.     

Protein kinase C and Mammalian spermatozoa acrosome reaction.

The presence of protein kinase C (PKC) in mammalian sperm was demonstrated by enzymatic activity assay and immunohistochemistry at the light and electron microscopy levels. The sperm head PKC is localized in the acrosome, equatorial segment, and postacrosomal region. In the flagellum, PKC is associated with the segmented column of the neck and is distributed along the mid, principal, and end pieces. Immunoreactive sites are observed in patches along the axoneme and outer dense fibers and are evenly distributed between these regions. Functional studies suggest the involvement of PKC in flagellar motility and acrosome reaction. The cross-talk between the signaling cascades that operate during sperm activation is discussed. (Trends Endocrinol Metab 1997;8:337-341). (c) 1997, Elsevier Science Inc.     

斯氏狸殖吸虫精子形成的透射电镜观察

用透射电镜观察了斯氏狸殖吸虫精细胞的变化和精子的主要形态结构特征。从而揭示了该虫精子形成的过程。证实了斯氏狸殖吸虫的精细胞和精子有顶体结构。成熟的精子可分为头部和尾部。头部由核质充满,核质可延伸至精子尾部中段的前端。头尾之间为连接段。尾部又由中段和末段组成。尾部的中段起始部的两侧各有一条轴丝。每条轴丝各由9对外周微管和2个中央微管组成,在每2条中央微管的外围,均由一个纤维鞘包绕,其间形成一个车轮样的9 2微管系统结构,其腹侧排列着线粒体。尾部的末段,主要为2条紧靠的轴丝。     

Calmodulin localization in mammalian spermatozoa.

The location of calmodulin in rabbit and guinea pig spermatozoa was determined by indirect immunofluorescence techniques. Spermatozoa that had not undergone the acrosome reaction exhibited four distinct regions of calmodulin-specific immunofluorescence: around the acrosome, in a band across the lower third of the head, and in two localized areas at the base and tip of the flagellum. In contrast, after the acrosome reaction, although other features of calmodulin distribution remained the same, the fluorescence associated with the anterior half of the head was notably absent. Instead, fluorescence was associated with the membranes that had separated from the sperm head. These findings suggest a potential role for calmodulin in the Ca2+-dependent control of sperm activation, in sperm-egg fusion, and in microtubule disassembly processes in the flagellum.     

Structure of Trypanosoma brucei flagellum accounts for its bihelical motion

Trypanosoma brucei is a parasitic protozoan that causes African sleeping sickness. It contains a flagellum required for locomotion and viability. In addition to a microtubular axoneme, the flagellum contains a crystalline paraflagellar rod (PFR) and connecting proteins. We show here, by cryoelectron tomography, the structure of the flagellum in three bending states. The PFR lattice in straight flagella repeats every 56 nm along the length of the axoneme, matching the spacing of the connecting proteins. During flagellar bending, the PFR crystallographic unit cell lengths remain constant while the interaxial angles vary, similar to a jackscrew. The axoneme drives the expansion and compression of the PFR lattice. We propose that the PFR modifies the in-plane axoneme motion to produce the characteristic trypanosome bihelical motility as captured by high-speed light microscope videography.     

An electron microscopic study on a strain of Trypanosoma vivax maintained in cattle (author's transl)]

The ultrastructure of the blood forms of Trypanosoma vivax from its natural host, cattle, is described. The pellicula, consisting of an unit membrane with a superimposed surface coat, the structure and attachment of the flagellum and the sub-pellicular microtubules showed the usual structural and organizational features. Cell organelles and cytoplasmic inclusions such as cell nucleus, mitochondria, kinetoplast, Golgi-complex, endoplasmic reticulum and membrane-isolated vacuoles, which occur in trypanosomidae, are presented and described. The ultrastructure of the trypomastigote forms of T. vivax has been compared to that of other trypanosome species and similarities and deviations are discussed. The mitochondrion shows a striking difference in dimension and number of microtubules from other trypanosome species. The kinetoplast, the K-DNA containing part of this mitochondrion, differs in its relative position to the basal body and in the mitochondrion from other flagellate species. It is marginally situated and the K-DNA is frequently lying parallel to the basis of the flagellum.     

Ultrastructure of the mature spermatozoon of <Emphasis Type="Italic">Eubothrium rugosum</Emphasis> (Batsch, 1786) with a re-assessment of the spermatozoon ultrastructure of <Emphasis Type="Italic">Eubothrium crassum</Emphasis> (Bloch, 1779) (Cestoda: Bothriocephalidea)

The ultrastructure of the mature spermatozoon of the bothriocephalidean tapeworm Eubothrium rugosum, a parasite of the burbot, Lota lota (L.), was studied by transmission electron microscopy for the first time. In addition, spermatozoon ultrastructure of Eubothrium crassum has been re-assessed. New is the finding, that the mature spermatozoa of both species of the genus Eubothrium exhibit essentially the same general morphology. They are filiform cells tapering at both extremities, and they possess the two axonemes with 9+“1” pattern of Trepaxonemata, attachment zones, a nucleus, cortical microtubules (CMs), electron-dense granules, and a single crested body. Structural polymorphism of the CBs has been found within the two Eubothrium species for the first time. The anterior ring of electron-dense tubular structures surrounding a single axoneme marks the border between the two defined regions, region I and region II of the spermatozoon. This unique feature has only been observed in the Bothriocephalidea. The anuclear axoneme region II of Eubothrium spermatozoa fluently verges into a nuclear region III. The posterior part of the spermatozoon contains one-axoneme, few CMs and a posterior extremity of the nucleus that subsequently disappears. The posterior extremity of the male gametes of the genus Eubothrium exhibits elements of a disorganized axoneme which characterize also spermatozoa of the family Triaenophoridae. Discussed are interspecific similarities and differences between the spermatozoa of the two Eubothrium species as well as between these and other Eucestoda.     

Molecular Structures of Acetylcholinesterase from Electric Organ Tissue of the Electric Eel

Three purified molecular forms of acetylcholinesterase (EC 3.1.1.7) with sedimentation coefficients of 18 S, 14 S, and 11 S were studied by analytical ultracentrifugation and electron microscopy. The three species have molecular weights of (1.1 +/- 0.1) x 10(6), (7.5 +/- 1.5) x 10(5), and (3.35 +/- 0.25) x 10(5), respectively. Electron micrographs reveal that the 18S and 14S forms are asymmetric, composed of a head, containing a large number of subunits, and an elongated tail. The 11S form of acetylcholinesterase is apparently a tetrameric structure devoid of the tail. Maleylation of 18S and 14S acetylcholinesterases abolishes their tendency to aggregate at low ionic strength.     

Ultrastructure of cultured cells from Schistosoma japonicum

Ultrastructures and their dynamic changes of the cultured cells from Schistosoma japonicum were observed in the present experiments. Several types-including polygonal, round granular, deltaic fan-shaped and flagellated cells-were found in the cultures. The polygonal cells took a major ratio in the cultures from adult S. japonicum, while the majority from schistosomula was round granular cells. The ultrastuctures on the cell surface were different between the cells from adults and schistosomula. Some papilla-like tubercula, microvilli and pinocytotic vesicles were observed on the surface of adult cells, but none were found on schistosomula cells. However, more or less mitochondria, endoplasmic reticula, ribosomes and glycogen were observed in the cytoplasm of the cultured cells from both adults and schistosomula. Golgi complexes were rarely found. The nucleus was round, with round nucleolus inside and clear pores on the unit membrane. There was much lumpish heterochromatin located near to the nuclear membrane. Cells from different worm tissues had their own organelles. The germ cells, vitelline cells, flame cells, multinucleate subtegumental cells and nerve cells could be observed in the cultures from adults. The vitelline cells were the greatest in number and nerve cells were the least in number among them. Similarly, there were germ cells, sustentacular cells, flame cells, nerve cells, mast cells, muscle cells, multinucleate subtegumental cells, interstitial cells and penetration gland cells in the cultures from the schistomomula. In addition, a few division cells were also found. It indicated that the schistosomula cells had greater potential ability to proliferate than the adult cells in in vitro culture. Along with the prolongation of the culture time, degeneration of schistosomal cell occurred more and more. Generally, the electron density of cultures gradually got lower, the cristae of mitochondria blurred and disappeared and the mitochondria themselves swelled and finally vacuoled completely. Vitelline cells were most sensitive to the changes of the in vitro condition in all cultures. Their degeneration showed the following characteristics: (1) vitelline globules fused each other, the space between vitelline globules and the membrane surrounding them broadened gradually and vitelline globules were released and uncovered; (2) rough-surfaced endoplasmic reticula enlarged, vacuolated and the ribosomes dropped; and (3) the number and volume of lipid increased. The ultrastructural changes of most of the cultures from schistosomula had the following trends: (1) heterochromatin increased and euchromatin decreased gradually; and (2) endoplasmic reticula changed into short tubes and vacuoles and disappeared finally. The degenerative process of the cultures from S. japonicum consisted of necrosis according to the ultrastructural changes of the mitochondria, vitelline globules, chromatin and endoplasmic reticula within the cells. The changes of the above structures could be used to estimate whether the culture conditions were appropriate.     

Lack of Spem1 causes aberrant cytoplasm removal, sperm deformation, and male infertility

We identified a previously uncharacterized gene, spermatid maturation 1 (Spem1), encoding a protein exclusively expressed in the cytoplasm of steps 14-16 elongated spermatids in the mouse testis. This protein contains no known functional domains and is highly conserved across mammalian species. Male mice deficient in Spem1 were completely infertile because of deformed sperm characterized by a bent head wrapped around by the neck and the middle piece of the tail. We show that lack of Spem1 causes failure of the cytoplasm to become loose and detach from the head and the neck region of the developing spermatozoa. Retained cytoplasmic components mechanically obstruct the straightening of the sperm head and the stretching of the growing tail, leading to the bending of the head in the neck, followed by the wrapping of the head by the neck or the middle piece of the sperm tail. Our study reveals that proper cytoplasm removal is a genetically regulated process requiring the participation of Spem1 and that lack of Spem1 causes sperm deformation and male infertility.     

Ultrastructurally normal and motile spermatozoa in a fertile man with Kartagener's syndrome.

The findings in a 40-year-old man with Kartagener's triad (sinusitis, bronchiectasis, and situs inversus) and corrected transposition of the great vessels are presented. Electron microscopy revealed normal ultrastructure of the axoneme in both respiratory cilia and sperm tails. Light microscopic evaluation of the spermatozoa showed 50 percent motility, suggesting normal fertility. This assumption is confirmed, as the patient has two children. We suggest that an abnormal, uncoordinated motility pattern of the ultrastructurally normal respiratory cilia results in improper mucociliary clearance. This coordination is not needed in swimming spermatozoa, which could explain the apparent paradox between bronchopulmonary symptoms and normal fertility in our patient.     

日本血吸虫尾蚴cDNA文库的构建及分析

目的　构建日本血吸虫尾蚴 c DNA文库。　方法　从日本血吸虫尾蚴中提取总 RNA,运用“SMART c D-NA文库构建试剂盒”构建文库。　结果　所建文库的初始滴度为 1.8× 10 7pfu/ ml,经 1次扩增后的滴度为 2 .5× 10 7pfu/ ml,插入子的平均长度为 1.0 75 kb,重组率为 94 .4 % ,并从该文库中调出了日本血吸虫保守基因 TPI和 JF2的全长 c DNA片段。　结论　构建了日本血吸虫尾蚴 c DNA文库     

Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins.

Nuclear structural changes during fertilization and embryogenesis in mice and in sea urchins have been followed by using antibodies against the nuclear lamins A/C and B and against antigens at the periphery of nuclei and chromosomes. Lamins are found on all pronuclei and nuclei during mouse fertilization, but with a diminished intensity on the second polar body nucleus. On sperm in both systems, lamins are reduced and detected only at the acrosomal and centriolar fossae. In sea urchin eggs, lamins are found on both pronuclei. Unlike in other dividing cells, the mitotic chromosomes of sea urchin eggs and embryos retain an association with lamins. The peripheral antibodies delineate each chromosome and nucleus except the mature mouse sperm nucleus. A dramatic change from the expected lamin distribution occurs during early development. In mouse morulae or blastocysts, lamins A/C are no longer recognized, although lamin B remains. In sea urchins both lamins A/C and lamin B, as detected with polyclonal antibodies, are lost after the blastula stage, although a different lamin A/C epitope emerges as recognized by a monoclonal antibody. These results demonstrate that pronucleus formation in both systems involves a new association or exposure of lamins, that the polar body nucleus is largely restricted from the cytoplasmic pool of lamins, and that mitotic chromosomes in the rapidly proliferating sea urchin egg retain associated lamins. They also suggest that changes in the expression or exposure of different lamins are a common feature of embryogenesis.     

防蚴润肤霜防御日本血吸虫尾蚴感染的研究

目的：实验评价新型少肤防护剂一防蚴润夫霜防御日本血吸虫尾蚴感染的效果，并了解其对皮肤的急性刺激反应。方法：采用模拟现场试验方法，分别在小白鼠腹部和尾部涂布防蚴润肤霜，并经泥水浸泡洗刷4-8h后接种日本血吸虫尾蚴，饲养40d解剖小鼠观察感染情况。以小鼠感染率、平均虫荷数和减虫率为评价指标，同时进行皮肤急性刺激试验。结果：防蚴润肤霜涂布小鼠腹部、尾部后减虫率达100％；家兔皮肤急性刺激试验结果为阴性。结论：防蚴润肤霜涂布皮肤后4-8h具有完全防御日本血吸虫尾蚴感染的作用，对皮肤无刺激作用。     

Trypanosoma cruzi nucleoside triphosphate diphosphohydrolase 1 (TcNTPDase-1) biochemical characterization,immunolocalization and possible role in host cell adhesion

Previous work has suggested that Trypanosoma cruzi diphosphohydrolase 1 (TcNTPDase-1) may be involved in the infection of mammalian cells and serve as a potential target for rational drug design. In this work, we produced recombinant TcNTPDase-1 and evaluated its nucleotidase activity, cellular localization and role in parasite adhesion to mammalian host cells. TcNTPDase-1 was able to utilize a broad range of triphosphate and diphosphate nucleosides. The enzyme's K_m for ATP (0.096 mM) suggested a capability to influence the host's ATP-dependent purinergic signaling. The use of specific polyclonal antibodies allowed us to confirm the presence of TcNTPDase-1 at the surface of parasites by confocal and electron microscopy. In addition, electron microscopy revealed that TcNTPDase-1 was also found in the flagellum, flagellum insertion region, kinetoplast, nucleus and intracellular vesicles. The presence of this enzyme in the flagellum insertion region and vesicles suggests that it may have a role in nutrient acquisition, and the widespread distribution of TcNTPDase-1 within the parasite suggests that it may be involved in other biological process. Adhesion assays using anti-TcNTPDase-1 polyclonal antibodies as a blocker or purified recombinant TcNTPDase-1 as a competitor revealed that the enzyme has a role in parasite–host cell adhesion. These data open new frontiers to future studies on this specific parasite–host interaction and other unknown functions of TcNTPDase-1 related to its ubiquitous localization.     

Mislocalization of DNAH5 and DNAH9 in respiratory cells from patients with primary ciliary dyskinesia

RATIONALE: Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by recurrent infections of the airways and situs inversus in half of the affected offspring. The most frequent genetic defects comprise recessive mutations of DNAH5 and DNAI1, which encode outer dynein arm (ODA) components. Diagnosis of PCD usually relies on electron microscopy, which is technically demanding and sometimes difficult to interpret. METHODS: Using specific antibodies, we determined the subcellular localization of the ODA heavy chains DNAH5 and DNAH9 in human respiratory epithelial and sperm cells of patients with PCD and control subjects by high-resolution immunofluorescence imaging. We also assessed cilia and sperm tail function by high-speed video microscopy. RESULTS: In normal ciliated airway epithelium, DNAH5 and DNAH9 show a specific regional distribution along the ciliary axoneme, indicating the existence of at least two distinct ODA types. DNAH5 was completely or only distally absent from the respiratory ciliary axoneme in patients with PCD with DNAH5- (n = 3) or DNAI1- (n = 1) mutations, respectively, and instead accumulated at the microtubule-organizing centers. In contrast to respiratory cilia, sperm tails from a patient with DNAH5 mutations had normal ODA heavy chain distribution, suggesting different modes of ODA generation in these cell types. Blinded investigation of a large cohort of patients with PCD and control subjects identified DNAH5 mislocalization in all patients diagnosed with ODA defects by electron microscopy (n = 16). Cilia with complete axonemal DNAH5 deficiency were immotile, whereas cilia with distal DNAH5 deficiency showed residual motility. CONCLUSIONS: Immunofluorescence staining can detect ODA defects, which will possibly aid PCD diagnosis.     

改良人原生殖细胞培养基培养日本血吸虫生殖类细胞的初步研究

目的探讨改良人原生殖细胞（human primordial germ cells,HPGC）培养基培养日本血吸虫（Schistosoma ja-ponicum,Sj）生殖类细胞的可行性。方法用HPGC培养基和改良HPGC培养基,分别对日本血吸虫36d成虫全细胞进行选择性培养,比较观察培养细胞的生长特性和一般形态,对2种培养基培养4-6周的细胞进行超微结构和AKP染色鉴定,对改良HPGC培养基培养细胞用BrdU掺入法检测细胞增殖与染色体核型分析。结果HPGC培养基培养2周后出现形态和大小较均匀的细胞,6周时多数细胞死亡或崩解;培养4周的细胞超微结构显示异常形态,APK染色呈阳性反应。改良HPGC培养基培养细胞呈半悬浮集落状态,4周内生长较快,形态差异悬殊,随着培养期延长,细胞呈相似的圆形,核和核仁清晰;培养6周后,细胞生长速度减慢,细胞核逐渐消失,至第10周左右死亡;培养8周内均可见细胞分裂相。BrdU掺入法检测培养4周细胞可见细胞核酸合成;培养5周的细胞超微结构显示正常形态,具生殖类细胞特征（胞质中可见大小不等的囊泡）,细胞染色体核型表现血吸虫单倍体和双倍体特征;培养6周的细胞AKP染色呈强阳性反应。结论用HPGC改良培养基能使日本血吸虫成虫的某些类别的细胞增殖,该类细胞具有生殖类细胞的部分特征。