Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme.

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.     

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.     

Structural and biochemical features of fractionated spermatid manchettes and sperm axonemes of the azh/azh mutant mouse

The tubulin-containing axoneme and manchette develop consecutively during mammalian spermiogenesis. The nature of their molecular components and developmental sequence are not completely known. The azh/azh (for abnormal sperm headshape) mouse mutant is an ideal model for analyzing tubulin isotypes and microtubule-associated proteins of the manchette and axoneme in light of a potential role of the manchette in the shaping of the sperm head and formation of the tail. We have searched for possible differences in tubulin isotype variants in fractionated manchettes and axonemes of wildtype and azh/azh mutant mice using isotype-specific tubulin antibodies as immunoprobes. Manchettes from wild-type and azh/azh mutant mouse spermatids were fractionated from spermatogenic stage-specific seminiferous tubules and axonemes were isolated from epididymal sperm. We have found that: (1) Fractionated manchettes of azh/azh mutants are longer than in wild-type mice; (2) Manchette and sperm tail axonemes display a remarkable variety of posttranslationally modified tubulins (acetylated, glutamylated, tyrosinated, alpha-3/7 tubulins). Acetylated tubulin was more abundant in manchette than in axonemes; (3) An acidic 62 kDa protein was identified as the main component of the perinuclear ring of the manchette in wild-type and azh/azh mice; (4) Bending and looping of the mid piece of the tail of azh/azh sperm, accompanied by a dislocation of the connecting piece from head attachment sites, were visualized by phase-contrast, immunofluorescence and transmission electron microscopy in about 35% of spermatids/sperm; and (5) A lasso-like tail configuration was predominant in epididymal sperm of azh/azh mutants. We speculate that spermatid and sperm tail abnormalities in the azh/azh mutant could reflect structural and/or assembly deficiencies of peri-axonemal proteins responsible for maintaining a stiffened tail during spermiogenesis and sperm maturation.     

Human outer dense fiber gene, ODF2, localizes to chromosome 9q34

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.     

Characteristics of the sperm tail of the velvet worm, Euperipatoides leuckarti (Onychophora)

The onychophoran sperm tail contains several kinds of microtubulcs; probably more than that of any other animal group. There are thus a peripheral manchette consisting of many tightly spaced microtubules, a ring of nine 'peripheral singlets' and a central axoneme of the classical 9 + 2 type (nine doublets and two central singlets). The protofilament organization of these various microtubules was examined and compared to the structure and mode of formation of the peripheral singlets with that of its analogues in other animal groups. The onychophoran peripheral singlets were found to differ in two respects from those in insects: they are formed from the manchette rather than from the axonemal doublets and their transient connection to the axoneme is to the A-subtubules of the doublet rather than to the B-subtubules. The manchette microtubules as well as the peripheral singlets consist of 13 protoh'laments. The manchette may serve a mechanical function (to strengthen the unusually thick sperm tail) hut the role of the peripheral singlets remains unknown.     

Sak57, an acidic keratin initially present in the spermatid manchette before becoming a component of paraaxonemal structures of the developing tail

We have previously reported that Sak57 (for Spermatogenic cell/Sperm-associated keratin of molecular mass 57 kDa) is an acidic keratin found in rat spermatocytes, spermatids, and sperm. Sak57 displays conserved amino acid sequences found in the 1A and 2A regions of the α-helical rod domain of keratins in human, rat, and mouse. We now report indirect immunofluorescence, confocal laser scanning microscopy and immunogold electron microscopy data showing that Sak57 is associated with the microtubular mantle of the manchette, a transient microtubular structure largely regarded as formed by tubulin and microtubule-associated proteins. The immunocytochemical localization of Sak57 was detected with a polyclonal antiserum to a multiple antigenic peptide (MAP) containing an amino acid sequence known to be present in the 2A region of the α-helical rod domain. During spermiogenic steps 8–12, Sak57 immunoreactive sites were restricted to microtubular mantle of the manchette which encircles the spermatid nucleus during shaping and chromatin condensation. At later stages (spermiogenic steps 12–14), Sak57 immunoreactive sites in the spermatid head region disappeared gradually as specific immunoreactivity appeared along the already assembled axoneme of the developing spermatid tail. Immunogold electron microscopy confirmed the presence of Sak57 immunoreactivity among microtubules of the manchette and on outer dense fibers and the longitudinal columns linking the ribs of the fibrous sheath. Mature spermatids (spermiogenic step 19) displayed tails with an immunofluorescent banding pattern contrasting with the lack of Sak57 immunoreactivity in the head region. Results from this study suggest that, during early spermiogenesis, a microtubular-Sak57 scaffolding is associated with the spermatid nucleus during shaping and chromatin condensation. During late spermiogenesis, the dispersion of the manchette coincides with the progressive visualization of Sak57 in the paraaxonemal outer dense fibers and longitudinal columns of the fibrous sheath in the developing spermatid tail. © 1996 Wiley-Liss, Inc.     

Testicular protein Spag5 has similarity to mitotic spindle protein Deepest and binds outer dense fiber protein Odf1

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.     

Characterisation and tissue-specific expression of the two keratin subfamilies of intermediate filament proteins in the cephalochordate Branchiostoma

The cloning of three intermediate filament proteins expressed at the gastrula stage (kl, Y1, X1) extends the size of the IF multigene family of Branchiostoma to at least 13 members. This is one of the largest protein families established for the lancelet. Sequence comparisons indicate five keratin orthologs, three of type I (E1, k1, Y1) and two of type II (E2, D1). This assignment is confirmed by the obligatory heteropolymeric polymerisation behaviour of the recombinant proteins. In line with the hetero-coiled-coil principle IF are formed by any stoichiometric mixture of type I and II keratin orthologs. In spite of the strong sequence drift chimeric IF are formed between K8, a human keratin II, and two of the lancelet type I keratins. We discuss whether the remaining 8 IF proteins reflect three additional and potentially cephalochordate-specific subfamilies. The tissue-specific expression patterns of the 5 keratins and some other IF proteins were analysed by immunofluorescence in the adult. Keratins are primarily present in ectodermally derived tissues. Developmental control of the expression of some IF proteins is observed, but three keratins (k1, Y1, D1) and an additional IF protein (X1) detected at the gastrula stage are expressed throughout the life cycle.     

Ultrastructural localization of hair keratin homologs in the claw of the lizard Anolis carolinensis

The claw of lizards is largely composed of beta‐keratins, also referred to as keratin‐associated beta‐proteins. Recently, we have reported that the genome of the lizard Anolis carolinensis contains alpha keratin genes homologous to hair keratins typical of hairs and claws of mammals. Molecular and immunohistochemical studies demonstrated that two hair keratin homologs named hard acid keratin 1 (HA1) and hard basic keratin 1 (HB1) are expressed in keratinocytes forming the claws of A. carolinensis. Here, we extended the immunocytochemical localization of the novel reptilian keratins to the ultrastructural level. After sectioning, claws were subjected to immunogold labeling using antibodies against HA1, HB1, and, for comparison, beta‐keratins. Electron microscopy showed that the randomly organized network of tonofilaments in basal and suprabasal keratinocytes becomes organized in long and parallel bundles of keratin in precorneous layers, resembling cortical cells of hairs. Entering the cornified part of the claw, the elongated corneous cells fuse and accumulate corneous material. HA1 and HB1 are absent in the basal layer and lower spinosus layers of the claw and are expressed in the upper and precorneous layers, including the elongating corneocytes. The labeling for alpha‐keratin was loosely associated with filament structures forming the fibrous framework of the claws. The ultrastructural distribution pattern of hard alpha‐keratins resembled that of beta‐keratins, which is compatible with the hypothesis of an interaction during claw morphogenesis. The data on the ultrastructural localization of hair keratin homologs facilitate a comparison of lizard claws and mammalian hard epidermal appendages containing hair keratins. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

Rat Spag5 associates in somatic cells with endoplasmic reticulum and microtubules but in spermatozoa with outer dense fibers

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.     

Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression

Human epidermal keratinocytes express under various growth conditions a total of at least nine keratins that can be divided into two subfamilies. Subfamily A comprises 40-, 46-, 48-, 50-/50'-, and 56.5-kilodalton (kd) keratins which are relatively acidic (pI less than 5.5) and, with the exception of 46-kd keratin, are recognized by AE1 monoclonal antibody. Subfamily B comprises 52-, 56-, 58-, and 65-67-kd keratins which are relatively basic (pI greater than 6) and are recognized by AE3 monoclonal antibody. Within each keratin subfamily, there is a constant member (50-/50'- and 58-kd keratins of the subfamilies A and B, respectively) that is always expressed. The other seven keratins of both subfamilies are variable members whose expression depends upon the cellular differentiated state, which is in turn modulated by the growth environment. The 56.5-kd keratin (subfamily A) and the 65-67-kd keratins (subfamily B) are coordinately expressed during keratinization. In contrast, the 40-, 46-, and 48-kd keratins (subfamily A) and the 52- and 56-kd keratins (subfamily B) are characteristic of cultured epidermal cells forming nonkeratinized colonies. These results demonstrate that human epidermal keratins can be classified according to their reactivity with monoclonal antikeratin antibodies, isoelectric point, and mode of expression. The classification of keratins into various subgroups may have important implications for the mechanisms of epidermal differentiation, the evolution of keratin heterogeneity, and the use of keratin markers for tumor diagnosis.     

Linkage of human keratin genes

Two families of keratins, type I and type II, can be distinguished within the intermediate filament family of proteins, and at least 20 genes in the human genome code for the 20 known keratin proteins. In epithelial intermediate filaments, keratins from both families appear to be coordinately expressed. We have screened a library of human genomic DNA and have identified several cases of linkage among homologous and heterologous pairs of keratin genes. Genes coding for type I keratins were found linked to those coding for type II keratins. Linkage was discovered also among homologous genes coding for type I keratins and among genes encoding type II keratins. In addition, we found genes coding for glycine-rich keratins linked to genes coding for those that do not contain glycine-rich regions. Our results raise the possibility that all keratin genes are linked in a single region of the human genome.     

Differential expression of keratin genes during mouse development

Suprabasal layers of the newborn mouse epidermis contain two mRNAs of 2.0 and 2.4 kb which are translated into keratins of 59 and 67 kDa, respectively. To study their expression during development, cDNA sequences corresponding to the 2.0- and the 2.4-kb mRNAs were cloned, characterized by hybridization selection assay, and used as probes to detect keratin sequences in polyadenylated RNA from Day 11, 13, 15, and 17 embryos. In RNA from Day 11 of gestation, two RNAs of 2.8 and 1.8 kb were identified. They were found to have homologies with both epidermal RNAs, suggesting that they are coding for proteins of the keratin family. These two sequences were not detected in sample of later stages. RNAs comigrating with the two epidermal keratin RNAs were identified only in Day 15 and 17 embryos indicating that their expression was induced between Day 13 and 15. Finally, the localization of the 59-kDa keratin mRNA was examined by in situ hybridization. The spinous and granulous cell layers were found to be heavily covered with grains while other regions of the tissue sections were unlabeled. All these results support the hypothesis of a sequential expression of keratins during differentiation of epidermal cells and suggest that proteins related to the keratins expressed specifically in keratinizing cells are expressed earlier during development.     

Concerted gene duplications in the two keratin gene families

Summary Evolutionary trees were derived from the keratin protein sequences using the Phylogeny Analysis Using Parsimony (PAUP) set of programs. Three major unexpected conclusions were derived from the analysis: The smallest keratin protein subunit, K#19 (Moll et al. 1982), is not the most primitive one, but has evolved to fulfill a highly specialized function, presumably to redress the unbalanced synthesis of keratin subunits. Second, the ancestors of keratins expressed in the early embryonic stages, K#8 and K#18, were the first to diverge from the ancestors of all the other keratins. The branches leading to these two keratins are relatively short, indicating a comparatively strong selection against changes in the sequences of these two proteins. Third, the two keratin families show extraodinary parallelism in their patterns of gene duplications. In both families the genes expressed in embryos diverged first, later bursts of gene duplications created the subfamilies expressed in various differentiated cells, and relatively recent gene duplications gave rise to the hair keratin genes and separated the basal cell-specific keratin from those expressed under hyperproliferative conditions. The parallelism of gene duplications in the two keratin gene families implies a mechanism in which duplications in one family influence duplication events in the other family.     

The catalog of human hair keratins. II. Expression of the six type II members in the hair follicle and the combined catalog of human type I and II keratins.

The human type II hair keratin subfamily consists of six individual members and can be divided into two groups. The group A members hHb1, hHb3, and hHb6 are structurally related, whereas group C members hHb2, hHb4, and hHb5 are rather distinct. Specific antisera against the individual hair keratins were used to establish the two-dimensional catalog of human type II hair keratins. In this catalog, hHb5 showed up as a series of isoelectric variants, well separated from a lower, more acidic, and complex protein streak containing isoelectric variants of hair keratins hHb1, hHb2, hHb3, and hHb6. Both in situ hybridization and immunohistochemistry on anagen hair follicles showed that hHb5 and hHb2 defined early stages of hair differentiation in the matrix (hHb5) and cuticle (hHb5 and hHb2), respectively. Although cuticular differentiation proceeded without the expression of further type II hair keratins, cortex cells simultaneously expressed hHb1, hHb3, and hHb6 at an advanced stage of differentiation. In contrast, hHb4, which is undetectable in hair follicle extracts and sections, could be identified as the largest and most alkaline member of this subfamily in cytoskeletal extracts of dorsal tongue. This hair keratin was localized in the posterior compartment of the tongue filiform papillae. Comparative analysis of type II with the previously published type I hair keratin expression profiles suggested specific, but more likely, random keratin-pairing principles during trichocyte differentiation. Finally, by combining the previously published type I hair keratin catalog with the type II hair keratin catalog and integrating both into the existing catalog of human epithelial keratins, we present a two-dimensional compilation of the presently known human keratins.     

Cornification in reptilian epidermis occurs through the deposition of keratin‐associated beta‐proteins (beta‐keratins) onto a scaffold of intermediate filament keratins

The isolation of genes for alpha‐keratins and keratin‐associated beta‐proteins (formerly beta‐keratins) has allowed the production of epitope‐specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha‐keratin range (40–70 kDa) or beta‐protein range (10–30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha‐proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin‐associated beta‐proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin‐associated beta‐proteins is typical for keratin‐associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine‐rich or sulfur‐rich proteins). In turtles and crocodilians epidermis, keratin‐associated beta‐proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine‐rich versus cysteine‐glycine‐rich keratin‐associated beta‐proteins in cells sequentially produced from the basal layer and not from the alternation of beta‐ with alpha‐keratins. The process gives rise to Oberhäutchen, beta‐, mesos‐, and alpha‐layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin‐associated proteins (KAPs) of the epidermis, the keratin‐associated beta‐proteins of sauropsids are capable to form filaments of 3–4 nm which give rise to an X‐ray beta‐pattern as a consequence of the presence of a beta‐pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Molecular cloning of Odf3 encoding a novel coiled-coil protein of sperm tail outer dense fibers.

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.