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1.
ODF2 was first identified as the major component of the sperm tail outer dense fibers. Additionally, ODF2 is a critical component of the mature centriole of the animal centrosome where it locates to the distal appendages. Moreover, generation of primary cilia strictly depends on ODF2. The mature centriole is characterized further by recruitment of Cenexin. Albeit highly similar in sequence the relationship between ODF2 and Cenexin has not been investigated. We demonstrate here that ODF2 and Cenexin are alternative splice products by identifying a novel exon 3b encoding Cenexin specific amino acids. Even though ODF2 is the main isoform in testicular tissue RT-PCR analyses revealed that isoforms are not restricted to specific tissues. 相似文献
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ODF2 (outer dense fiber 2) was first described as the main protein component of the sperm tail cytoskeleton, the outer dense fibers, but was shown recently to be a component of the centrosomal scaffold in chicken. In mouse two related ODF2 cDNA clones were isolated which have been suggested to be most likely the result of alternative splicing. We show here the exon/intron organisation of mouse ODF2 and demonstrate that alternative splicing results in related cDNA sequences and most likely explains, at least partially, the highly complex protein pattern detected on Western blots. ODF2 was mapped to rat chromosome 3 and more specifically by FISH analysis at bands 3q11-->3q12. In addition, we demonstrate that ODF2 is indeed a component of the centrosome and the mitotic spindle poles in mammals. 相似文献
4.
Outer dense fiber 2 (Odf2) is highly expressed in the testis where it encodes a major component of the outer dense fibers of the sperm flagellum. Furthermore, ODF2 protein has recently been identified as a widespread centrosomal protein. While the expression of Odf2 highlighted a potential role for this gene in male germ cell development and centrosome function, the in vivo function of Odf2 was not known. We have generated Odf2 knockout mice using an Odf2 gene trapped embryonic stem cell (ESC) line. Insertion of a gene trap vector into exon 9 resulted in a gene that encodes a severely truncated protein lacking a large portion of its predicted coil forming domains as well as both leucine zipper motifs that are required for protein-protein interactions with ODF1, another major component of the outer dense fibers. Although wild-type and heterozygous mice were recovered, no mice homozygous for the Odf2 gene trap insertion were recovered in an extended breeding program. Furthermore, no homozygous embryos were found at the blastocyst stage of embryonic development, implying a critical pre-implantation role for Odf2. We show that Odf2 is expressed widely in adults and is also expressed in the blastocyst stage of preimplantation development. These findings are in contrast with early studies reporting Odf2 expression as testis specific and suggest that embryonic Odf2 expression plays a critical role during preimplantation development in mice. 相似文献
5.
Christoph Petersen Gerhard Aumüller Masoud Bahrami Sigrid Hoyer-Fender 《Molecular reproduction and development》2002,61(1):102-112
The outer dense fibers (ODF) are the main cytoskeletal structures of the sperm tail found in animals with internal fecundation. They consist of at least 14 polypeptides from which only a few are identified due to difficulties in isolation of the protein components. Here we report the isolation and molecular characterization of Odf3, encoding a novel protein of rat sperm ODF. Odf3 is transcribed in testes and more specifically in spermatids but it is also expressed in epididymides and brain suggesting a possible involvement in building of the cellular cytoskeleton. Odf3 encodes a putative protein of approximately 110 kDa. Secondary structure predictions indicated that ODF3 is a coiled-coil protein. The identification of coiled-coil proteins as constituents of outer dense fibers reveals a model for ODF formation. 相似文献
6.
Heide Tarnasky Min Cheng Young Ou Jacob C Thundathil Richard Oko Frans A van der Hoorn 《BMC developmental biology》2010,10(1):67
Background
Outer dense fiber protein 2, Odf2, is a major component of the outer dense fibers, ODF, in the flagellum of spermatozoa. ODF are associated with microtubule doublets that form the axoneme. We recently demonstrated that tyrosine phosphorylation of Odf2 is important for sperm motility. In the course of a study of Odf2 using Odf2 mouse knockout lines we observed that males of a high percentage chimaerism, made using XL169 embryonic stem cells, were infertile, whereas mice of low-medium percentage chimaerism were fertile. 相似文献7.
Human outer dense fiber gene, ODF2, localizes to chromosome 9q34 总被引:6,自引:0,他引:6
We have isolated the human homolog of the rat Odf2 gene. In rat, Odf2, the 84-kDa major outer dense fiber protein, interacts strongly and specifically with Odf1, the 27-kDa major outer dense fiber protein. The interaction is mediated by leucine zippers during ODF assembly along the sperm axoneme. We compared homology and genomic structure to rat and mouse Odf2 genes. Using fluorescence in situ hybridization, we mapped the human Odf2 gene (ODF2) to chromosome 9q34. 相似文献
8.
Odf2 (outer dense fiber 2) is the major protein of the cytoskeleton of the sperm tail. In somatic cells, it is a component
of the centrosome in which it is located in the appendages of the mother centriole. Additionally, as shown previously by forced
expression in cultured cells, Odf2 localizes to centrioles, basal bodies, and primary cilia, which are all structurally and
functionally interconnected. The importance of Odf2 has become obvious by the absence of primary cilia in Odf2-deficient cells
and by the embryonic lethality of the Odf2 gene trap insertional mouse. However, nothing is known about the endogenous localization of Odf2 in the tissues of adult
mice. We show here that Odf2 protein localizes to centrosomes, to photoreceptor primary cilia, and to basal bodies of ciliated
cells of the respiratory epithelium and of the kidney. Our results thus suggest that Odf2 contributes to assorted ciliopathies. 相似文献
9.
Yang K Meinhardt A Zhang B Grzmil P Adham IM Hoyer-Fender S 《Molecular and cellular biology》2012,32(1):216-225
Sperm motility and hence male fertility strictly depends on proper development of the sperm tail and its tight anchorage to the head. The main protein of sperm tail outer dense fibers, ODF1/HSPB10, belongs to the family of small heat shock proteins that function as molecular chaperones. However, the impact of ODF1 on sperm tail formation and motility and on male fecundity is unknown. We therefore generated mutant mice in which the Odf1 gene was disrupted. Heterozygous mutant male mice are fertile while sperm motility is reduced, but Odf1-deficient male mice are infertile due to the detachment of the sperm head. Although headless tails are somehow motile, transmission electron microscopy revealed disturbed organization of the mitochondrial sheath, as well as of the outer dense fibers. Our results thus suggest that ODF1, besides being involved in the correct arrangement of mitochondrial sheath and outer dense fibers, is essential for rigid junction of sperm head and tail. Loss of function of ODF1, therefore, might account for some of the cases of human infertility with decapitated sperm heads. In addition, since sperm motility is already affected in heterozygous mice, impairment of ODF1 might even account for some cases of reduced fertility in male patients. 相似文献
10.
Outer dense fibers (ODFs) and the fibrous sheath (FS) are unique structures of the mammalian sperm tail. Recently, progress has been made in the molecular cloning of ODF and FS proteins, and because of this, questions addressing the morphogenesis and underlying protein network that make up sperm tail structures and their function can now be addressed. Using the N-terminal leucine zipper motif of the major ODF protein ODF1, we had previously isolated interacting proteins Odf2, Spag4, and Spag5. We report here a yeast two-hybrid strategy to isolate a novel rat testicular protein, OIP1, that binds to the evolutionarily conserved Cys-Gly-Pro repeats in the C-terminus of ODF1. OIP1 is expressed in round spermatids as well as in spermatocytes and several somatic tissues, albeit at a lower level. No expression was detectable in epididymis, heart, and smooth muscle. OIP1 protein localizes to the sperm tail in a pattern expected for an ODF1-interacting protein. OIP1 belongs to the family of RING finger proteins of the H2 subclass. Deletion of the putative RING motif significantly decreased binding to ODF1. Genomic analysis of rat Oip1 and Oip1 homologs indicates that Oip1 is highly conserved. Oip1 is subject to differential splicing and alternative polyadenylation events. It is interesting that Oip1 mRNAs have been reported that lack the exon encoding the putative RING finger. 相似文献
11.
Wenlong Zhao Zhengzheng Li Ping Ping Guishuan Wang Xiaobing Yuan Fei Sun 《Journal of cellular and molecular medicine》2018,22(3):1755-1768
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. 相似文献
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13.
Outer dense fibers (ODF) and the fibrous sheath (FS) are major cytoskeletal structures in the mammalian sperm tail. The molecular mechanisms underlying their morphogenesis along the axoneme or their function are poorly understood. Recently, we reported the cloning and characterization of Odf2, a major ODF protein, and Spag4, an axoneme-binding protein, by virtue of their strong interaction with Odf1, the 27 kDa major ODF protein. We proposed a crucial role for leucine zippers in molecular interactions during sperm tail morphogenesis. Here we report the cloning and characterization of a novel gene, Spag5, which encodes a 200 kDa testicular protein that interacts strongly with Odf1. Spag5 is transcribed and translated in pachytene spermatocytes and spermatids. It bears 73% similarity with the mitotic spindle protein Deepest of unknown function. We identified two putative leucine zippers in the C-terminal part of the Spag5 protein, the downstream one of which is involved in interaction with Odf1. Interestingly, these motifs are present in Deepest. These results highlight the importance of the leucine zipper in sperm tail protein interactions. Mol. Reprod. Dev. 59: 410-416, 2001. 相似文献
14.
Outer Dense Fiber 2 Is a Widespread Centrosome Scaffold Component
Preferentially Associated with Mother Centrioles: Its Identification
from Isolated Centrosomes
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Yoshio Nakagawa Yukari Yamane Takeshi Okanoue Shoichiro Tsukita Sachiko Tsukita 《Molecular biology of the cell》2001,12(6):1687-1697
Because centrosomes were enriched in the bile canaliculi fraction from the chicken liver through their association with apical membranes, we developed a procedure for isolation of centrosomes from this fraction. With the use of the centrosomes, we generated centrosome-specific monoclonal antibodies. Three of the monoclonal antibodies recognized an antigen of ~90 kDa. Cloning of its cDNA identified this antigen as a chicken homologue of outer dense fiber 2 protein (Odf2), which was initially identified as a sperm outer dense fiber-specific component. Exogenously expressed and endogenous Odf2 were shown to be concentrated at the centrosomes in a microtubule-independent manner in various types of cells at both light and electron microscopic levels. Odf2 exhibited a cell cycle-dependent pattern of localization and was preferentially associated with the mother centrioles in G0/G1-phase. Toward G1/S-phase before centrosome duplication, it became detectable in both mother and daughter centrioles. In the isolated bile canaliculi and centrosomes, Odf2, in contrast to other centrosomal components, was highly resistant to KI extraction. These findings indicate that Odf2 is a widespread KI-insoluble scaffold component of the centrosome matrix, which may be involved in the maturation event of daughter centrioles. 相似文献
15.
The leucine zipper motif has been identified as an important and specific interaction motif used by various sperm tail proteins that localize to the outer dense fibers. We had found that rat Odf1, a major integral ODF protein, utilizes its leucine zipper to associate with Odf2, another major ODF protein, Spag4 which localizes to the interface between ODF and axonemal microtubule doublets, and Spag5. The rat Spag5 sequence indicated a close relationship with human Astrin, a microtubule-binding spindle protein suggesting that Spag5, like Spag4, may associate with the sperm tail axoneme. RT PCR assays indicated expression of Spag5 in various tissues and in somatic cells Spag5 localizes to endoplasmic reticulum and microtubules, as expected for an Astrin orthologue. MT binding was confirmed both in vivo and in in vitro MT-binding assays: somatic cells contain a 58 kDa MT-associated Spag5 protein. Western blotting assays of rat somatic cells and male germ cells at different stages of development using anti-Spag5 antibodies demonstrated that the protein expression pattern changes during spermatogenesis and that sperm tails contain a 58 kDa Spag5 protein. Use of affinity-purified anti-Spag5 antibodies in immuno electron microscopy shows that in rat elongated spermatids and epididymal sperm the Spag5 protein associates with ODF, but not with the axonemal MTs. This observation is in contrast to that for the other Odf1-binding, MT-binding protein Spag4, which is present between ODF and axoneme. Our data demonstrate that Spag5 has different localization in somatic versus male germ cells suggesting the possibility of different function. 相似文献
16.
Two appendages homologous between basal bodies and centrioles are formed using distinct Odf2 domains
Kazuhiro Tateishi Yuji Yamazaki Tomoki Nishida Shin Watanabe Koshi Kunimoto Hiroaki Ishikawa Sachiko Tsukita 《The Journal of cell biology》2013,203(3):417-425
Ciliogenesis is regulated by context-dependent cellular cues, including some transduced through appendage-like structures on ciliary basal bodies called transition fibers and basal feet. However, the molecular basis for this regulation is not fully understood. The Odf2 gene product, ODF2/cenexin, is essential for both ciliogenesis and the formation of the distal and subdistal appendages on centrioles, which become basal bodies. We examined the effects of Odf2 deletion constructs on ciliogenesis in Odf2-knockout F9 cells. Electron microscopy revealed that ciliogenesis and transition fiber formation required the ODF2/cenexin fragment containing amino acids (aa) 188–806, whereas basal foot formation required aa 1–59 and 188–806. These sequences also formed distal and subdistal appendages, respectively, indicating that the centriole appendages are molecularly analogous to those on basal bodies. We used the differential formation of appendages by Odf2 deletion constructs to study the incorporation and function of molecules associated with each appendage type. We found that transition fibers and distal appendages were required for ciliogenesis and subdistal appendages stabilized the centrosomal microtubules. 相似文献
17.
Outer dense fibre 2 (Odf2; also known as cenexin) was initially identified as a main component of the sperm tail cytoskeleton, but was later shown to be a general scaffold protein that is specifically localized at the distal/subdistal appendages of mother centrioles. Here we show that Odf2 expression is suppressed in mouse F9 cells when both alleles of Odf2 genes are deleted. Unexpectedly, the cell cycle of Odf2(-/-) cells does not seem to be affected. Immunofluorescence and ultrathin-section electron microscopy reveals that in Odf2(-/-) cells, distal/subdistal appendages disappear from mother centrioles, making it difficult to distinguish mother from daughter centrioles. In Odf2(-/-) cells, however, the formation of primary cilia is completely suppressed, although approximately 25% of wild-type F9 cells are ciliated under the steady-state cell cycle. The loss of primary cilia in Odf2(-/-) F9 cells can be rescued by exogenous Odf2 expression. These findings indicate that Odf2 is indispensable for the formation of distal/subdistal appendages and the generation of primary cilia, but not for other cell-cycle-related centriolar functions. 相似文献
18.
Requirement of hCenexin for proper mitotic functions of polo-like kinase 1 at the centrosomes
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Soung NK Kang YH Kim K Kamijo K Yoon H Seong YS Kuo YL Miki T Kim SR Kuriyama R Giam CZ Ahn CH Lee KS 《Molecular and cellular biology》2006,26(22):8316-8335
Outer dense fiber 2 (Odf2) was initially identified as a major component of sperm tail cytoskeleton and later was suggested to be a widespread component of centrosomal scaffold that preferentially associates with the appendages of the mother centrioles in somatic cells. Here we report the identification of two Odf2-related centrosomal components, hCenexin1 and hCenexin1 variant 1, that possess a unique C-terminal extension. Our results showed that hCenexin1 is the major isoform expressed in HeLa cells, whereas hOdf2 is not detectably expressed. Mammalian polo-like kinase 1 (Plk1) is critical for proper mitotic progression, and its association with the centrosome is important for microtubule nucleation and function. Interestingly, depletion of hCenexin1 by RNA interference (RNAi) delocalized Plk1 from the centrosomes and the C-terminal extension of hCenexin1 was crucial to recruit Plk1 to the centrosomes through a direct interaction with the polo-box domain of Plk1. Consistent with these findings, the hCenexin1 RNAi cells exhibited weakened gamma-tubulin localization and chromosome segregation defects. We propose that hCenexin1 is a critical centrosomal component whose C-terminal extension is required for proper recruitment of Plk1 and other components crucial for normal mitosis. Our results further suggest that the anti-Odf2 immunoreactive centrosomal antigen previously detected in non-germ line cells is likely hCenexin1. 相似文献
19.
Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme. 总被引:1,自引:0,他引:1
Outer dense fibers are structures unique to the sperm tail. No definite function for these fibers has been found, but they may play a role in motility and provide elastic recoil. Their composition had been described before, but only two of the fiber proteins, Odf1 and Odf2, are cloned. We cloned Odf2 by virtue of its functional and specific interaction with Odf1, which, we show, is mediated by a leucine zipper. Further work demonstrated that the 84-kDa Odf2 protein localizes to both the cortex and the medulla of the fibers, whereas the 27-kDa Odf1 protein is present only in the medulla. Here we report the cloning and characterization of a new Odf1-interacting protein, Spag4. Spag4 mRNA is spermatid specific, and the 49-kDa Spag4 protein complexes specifically with Odf1, but not Odf2, mediated by a leucine zipper. It also self-associates. In contrast to Odf1 and Odf2, Spag4 protein localizes to two microtubule-containing spermatid structures. Spag4 is detectable in the transient manchette and it is associated with the axoneme in elongating spermatids and epididymal sperm. Our data suggest a role for Spag4 in protein localization to two major sperm tail structures. 相似文献
20.
Recent work shows that two groups of keratins are expressed during mammalian spermatogenesis. One group, belonging to the classic epidermis-type keratins, is present in spermatogonia, spermatocytes, and spermatids. A member of this group, Sak57, a keratin 5 homologue, has been shown to co-align with microtubules and provide a scaffolding shell while also strengthening intercellular cytoplasmic bridges conjoining members of spermatogonial and spermatocyte cohorts. The other, keratin 9, is a component of the perinuclear ring of the manchette, a microtubular structure developed during the elongation and condensation of the spermatid nucleus. The second group, the outer dense fiber (Odf) proteins, is expressed preferentially during mammalian spermiogenesis. The family of Odf proteins-Odf1, Odf2, and Odf3-includes an expanding group of proteins co-assembled along the axoneme during the development of the sperm tail. Investigations on the assembly of epidermis-type and Odf sperm tail-targeted keratins are now focused on a group of chaperone-like Odf-binding molecules, designated Spags. Spags appear to drive Odfs to a precise destination. A daunting task is to determine how members of the family of keratins get the signal to produce linear scaffolds in specific spermatogenic cell populations and transport keratins to microtubule-containing structures such as the manchette and axoneme. 相似文献