A heat-shock protein 40, DNAJB13, is an axoneme-associated component in mouse spermatozoa

DNAJB13 is a type II HSP40/DnaJ protein. Using a specific antibody raised against the recombinant DNAJB13 protein, we characterized DNAJB13 in mouse testes and epididymal spermatozoa. The expression of DNAJB13 protein in testis was undetectable until postnatal Week 4 revealed by Western blot analysis, whereas Dnajb13 mRNA was detectable as early as postnatal Week 1 by RT-PCR. Immunohistochemistry analyses showed that DNAJB13 was localized in the cytoplasm of spermatids from step 2 to 3 onward with the strongest expression in step 9-10, and in the spermatid flagella. In mature spermatozoa, DNAJB13 was present along the entire length of the sperm flagellum, but not in the SDS-resistant tail structures lacking the flagellar axoneme, strongly suggesting that DNAJB13 is an axoneme-associated component. In addition, we showed that the expression of Dnajb13 mRNA and DNAJB13 protein was unaltered after heat shock treatment, indicating that DNAJB13 was constitutively expressed in mouse testis. Taken together, the present study suggested that DNAJB13 might be involved in assembly and stability of axoneme during sperm flagellum development.     

多纤毛细胞纤毛精确组装的调控机制

高等动物体内气管、脑室管膜及输卵管等上皮组织具有一类富含运动纤毛的多纤毛细胞,通过其细胞表面运动纤毛的周期性摆动可以清洁气管、驱动脑脊液流动和受精卵运动.运动纤毛发生或功能的异常则可导致气管炎、脑积水、不孕不育等多种遗传疾病.然而,在多纤毛细胞分化过程中关于如何精确组装运动性纤毛复杂结构的分子机制仍不清楚.该研究运用蛋...     

七星瓢虫精母细胞内轴丝的形态发生(英文)

应用细胞整装技术研究了七星瓢虫精子轴丝的早期形态发生和超微结构。在精子发生期间,起源于中心粒的两对基体—轴丝复合体出现在精母细胞内,在分裂间期它们彼此完全分离。当基体—轴丝复合体附着于精细胞核的核膜上,中心粒附体开始发生于生长轴丝的近心端,在染色质凝聚前中心粒附体最大。生长着的轴丝伴随着凝聚细胞核伸长。一个早期基体—轴丝复合体的轴丝是由具有内、外动力蛋白臂的9个双微管组成,缺少中央微管。     

Real-time observation of microtubules attached to microtubule organizing centers in vitro*

Summary— The dynamics and organization of microtubules associated with axonemes and kinetochores in vitro were visualized using video microscopy techniques. Microtubules attached either at the ends of axonemes or to mitotic chromosomes behave accordining to dynamic instability in our conditions. Microtubules attached to kinetochores showed lower rates of elongation and shortening than those nucleated by axonemes in the same conditions. In addition, elementary bundles of microtubules appeared spontaneously in association with kinetochores, with microtubules elongating along previously attached microtubules at even lower rates. Such side interactions, either spontaneous or stabilized by factors such as MAPs, might affect microtubule dynamics directly.     

Structural and Functional Characterization of Paramecium Dynein: Initial Studies

ABSTRACT Dynein arms and isolated dynein from Paramecium tetraurelia ciliary axonemes are comparable in structure, direction of force generation, and microtubule translocation ability to other dyneins. In situ arms have dimensions and substructure similar to those of Tetrahymena. Based on spoke arrangement in intact axonemes, arms translocate axonemal microtubules in sliding such that active dynein arms are (-) end directed motors and the doublet to which the body and cape of the arms binds (N) translocates the adjacent doublet (N+1) upward. After salt extraction, based on ATPase activity, paramecium dynein is found as a 22S and a 14S species. the 22S dynein is a three-headed molecule that has unfolded from the in situ dimensions; the 14S dynein is single headed. Both dyneins can be photocleaved by UV light (350 nm) in the presence of Mg^2-, ATP and vanadate; the photocleavage pattern of 22S dynein differs from that seen with Tetrahymena. Both isolated dyneins translocate taxol-stabilized, bovine brain microtubules in vitro. Under standard conditions, 22S dynein, like comparable dyneins from other organisms, translocates at velocities that are about three times faster than 14S dynein.     

Outer dense fibers stabilize the axoneme to maintain sperm motility

Outer dense fibers (ODFs), as unique accessory structures in mammalian sperm, are considered to play a role in the protection of the sperm tail against shear forces. However, the role and relevant mechanisms of ODFs in modulating sperm motility and its pathological involvement in asthenozoospermia were unknown. Here, we found that the percentage of ODF defects was higher in asthenozoospermic samples than that in control samples and was significantly correlated with the percentage of axoneme defects and non‐motile sperm. Furthermore, the expression levels of ODF major components (Odf1, 2, 3, 4) were frequently down‐regulated in asthenozoospermic samples. Intriguingly, the positive relationship between ODF size and sperm motility existed across species. The conditional disruption of Odf2 expression in mice led to reduced sperm motility and the characteristics of asthenozoospermia. Meanwhile, the expression of acetylated α‐tubulin was decreased in sperm from both Odf2 conditional knockout (cKO) mice and asthenozoospermic men. Immunofluorescence and biochemistry analyses showed that Odf2 could bind to acetylated α‐tubulin and protect the acetylation level of α‐tubulin in HEK293T cells in a cold environment. Finally, we found that lithium elevated the expression levels of Odf family proteins and acetylated α‐tubulin, elongated the midpiece length and increased the percentage of rapidly moving sperm in mice. Our results demonstrate that ODFs are beneficial for sperm motility via stabilization of the axoneme and that hypo‐expression of Odf family proteins is involved in the pathogenesis of asthenozoospermia. The lithium administration assay will provide valuable insights into the development of new treatments for asthenozoospermia.     

Sperm tail differentiation in the nudibranch mollusc Hypselodoris tricolor (gastropoda,opisthobranchia)

The sperm axoneme of Hypselodoris tricolor forms from a single centriole that is located initially beneath the plasma membrane and then migrates to the nuclear surface. A conspicuous centriolar adjunct-like formation is present in the neck of midspermatids, but it becomes very reduced at the end of spermiogenesis. In spermatocyte and spermatid mitochondria, intracristal bodies originate from the accumulation of a dense material in some cristae. From our observations and foregoing reports, it may be concluded that the process of sperm tail differentiation in opisthobranchs resembles that in pulmonates, whereas it differs in many respects from that occurring in prosobranchs. The appearance of intracristal bodies in modified mitochondria seems to be a special feature of spermatogenesis in the opisthobranchs that does not occur in the two other groups of gastropod molluscs.     

Tektin 4 is located on outer dense fibers, not associated with axonemal tubulins of flagella in rodent spermatozoa

Tektins, which are thought to be the constitutive proteins of microtubules in cilia, flagella, basal bodies, and centrioles, have been reported to be involved in the stability and structural complexity of axonemal microtubules. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin 2 and Tektin 4, have been verified to be present in sperm flagella. To elucidate the molecular localization of Tektin 4 in flagella of rodent spermatozoa, we performed immunocytochemistry, fractionation study followed by immunoblot analysis, and immunogold electron microscopy. Confocal laser scanning microscopy and immunogold electron microscopy indicated that Tektin 4 was associated with outer dense fibers (ODFs) in both the middle and principal piece of flagella in rat and mouse spermatozoa. Tektin 4 in rat spermatozoa is completely released by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Pre-embedding immunoelectron microscopy demonstrated that Tektin 4 located on the abaxial (convex) surface of ODFs in flagella, not associate with axonemal microtubules. Our data strongly suggested that Tektin 4 is not associated with axonemal tubulins but an ODFs-affiliated molecule in rodent spermatozoa.