Human Molecular Genetics, 2002, Vol. 11, No. 14 1647-1658
© 2002 Oxford University Press
The Hook1 gene is non-functional in the abnormal spermatozoon head shape (azh) mutant mouse
1Institute of Human Genetics, 2Department of Zoology and Developmental Biology, University of Göttingen, 37073 Göttingen, Germany and 3Department of Anatomy and Cell Biology, University of Giessen, 35392 Giessen, Germany
Received March 11, 2002; Accepted May 3, 2002
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ABSTRACT |
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In mice carrying the autosomal recessive mutation ‘abnormal spermatozoon head shape’ (azh) all spermatozoa display a highly abnormal head morphology that differs drastically from the compact and hook-shaped head of the normal murine sperm. Moreover, the azh mutation causes tail abnormalities often resulting in coiled sperm tails or in the decapitation of the sperm head from the flagellum. We have isolated and characterized murine Hook1 cDNA and analyzed the corresponding genomic structure. Furthermore, the Hook1 gene was mapped to the same region on chromosome 4 to which the azh locus was previously linked. The Hook1 gene is predominantly expressed in haploid male germ cells, and immunohistochemical analysis revealed that Hook1 is responsible for the linkage of the microtubular manchette and the flagellum to cellular structures. Here, we report that the azh mutation is due to a deletion of exons 10 and 11 in the murine Hook1 gene leading to a non-functional protein. Our results indicate that loss of Hook1 function results in ectopic positioning of microtubular structures within the spermatid and causes the azh phenotype. Therefore, the human HOOK1 gene could serve as a candidate gene for male infertility due to teratozoospermia or decapitation defects.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spermatogenesis is a highly complex process during which diploid spermatogonia differentiate into motile spermatozoa that are able to fertilize the oocyte. During spermiogenesis, shaping and condensation of the nucleus as well as formation of the acrosome and the tail take place. Several mutations in the mouse are known that lead to abnormalities of the shape of the sperm head and the overlaying acrosome (1,2). However, most of these mutations have pleiotropic effects on spermiogenesis and on other tissues. In contrast, the azh mutation, which was first described in 1984 by Hugenholtz and co-workers (3), seems to be specific for the sperm head shape. The azh mutation was demonstrated to display an autosomal recessive pattern of inheritance, and the azh locus was mapped to mouse chromosome 4 (4). In the homozygous state, 100% of spermatozoa display abnormal head morphology, and these sperm show a strongly decreased ability in fertilization. It was suggested that this defect is manifested at the level of penetration into the oocyte (5). Furthermore, the offspring from normal mouse oocytes injected with azh sperm heads showed more severe tail defects than the original mutant (6). The nuclear abnormalities appeared to be related to disturbances in the spermatid microtubular manchette (7). Electron-microscopic analyses revealed that the first visible defect was an abnormal positioning of manchette microtubules, often resulting in ectopic positions of the microtubules along the plasma membrane (8). This leads to a cylindrical or conical posterior portion of the sperm head that tapers to a relatively small diameter where the flagellum is attached. The azh spermatozoa are sensitive to mechanical forces, which often causes tail detachment.
We have identified a new gene, mapped to the region of mouse chromosome 4 where the azh locus was suggested. The predicted amino acid sequence revealed high similarity to the hook protein of Drosophila melanogaster. The hook mutation had already been reported in 1927 by Mohr (9), who described a hook-like bristle phenotype. Additionally, mutations in the Drosophila hook gene lead to pleiotropic phenotypes, including eye degeneration (10). It was suggested that the hook gene product plays a role in the endocytosis of transmembrane ligands or their transport to multivesicular bodies. Further studies revealed that hook belongs to a new protein family, which is conserved between flies and humans (11). The different members of the human gene family were denoted as HOOK1, HOOK2 and HOOK3, respectively. Different domains were identified in the three human HOOK proteins, and it was demonstrated that the highly conserved NH2-domain mediates attachment to microtubules, whereas the central coiled-coil motif mediates homodimerization and the more divergent C-terminal domains are involved in binding to specific organelles (organelle-binding domains). Walenta and co-workers (12) demonstrated that endogenous HOOK3 binds to Golgi membranes, whereas both HOOK1 and HOOK2 are localized to discrete but unidentified cellular structures.
In the present study, we report the isolation and characterization of the murine Hook1 gene and its predominant expression in the testis. Furthermore, we demonstrate that two exons of the Hook1 gene are deleted in the azh mutant mouse, leading to a putative truncated protein that lacks both the conserved homodimerization domain and the putative organelle-binding domain. Our results further suggest that Hook1 function is necessary for the correct positioning of microtubular structures within the haploid germ cell. Moreover, disruption of Hook1 function in the azh mutant mouse causes abnormal sperm head shape and fragile attachment of the flagellum to the sperm head.
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RESULTS |
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Isolation and characterization of murine Hook1 cDNA
In order to identify new genes that could be involved in intracellular transport processes in the testis, a murine testicular cDNA library was screened under low-stringency conditions using a cDNA probe of the murine dynein light-chain gene Dlc1. Several clones were isolated and sequenced, and one clone revealed similarity to the Drosophila hook and human HOOK1 genes. The isolated murine cDNA clone comprises 1806 bp and contains a poly(A) tail at the 3' end together with an atypical polyadenylation signal AAGAAA. Comparison with human and Drosophila cDNA indicated that the murine cDNA was incomplete. Therefore, a testicular cDNA library from the RZPD (Resource Center and Primary Database, Berlin) was screened using the partial cDNA clone as a probe, and 12 new clones were identified. Five of these clones contained an insert spanning over 1.8 kb and were further analyzed by sequencing. The sequence of the largest clone comprises 2478 bp of the Hook1 cDNA sequence, including a 5'-untranslated region (UTR) of 197 bp and a 3'-UTR of 97 bp. A putative start codon ATG was identified at position 198 bp of the cDNA surrounded by the Kozak consensus sequence for initiation of translation (13). At the nucleotide level, the murine Hook1 cDNA sequence (GenBank accession no. AF487912) revealed 92% identity in the coding region to the previously reported human HOOK1 cDNA sequence (GenBank accession no. AF044923) (11).
Expression analyses of the murine Hook1 gene
Northern blot analyses were performed to elucidate in which tissues Hook1 is expressed. By using the complete Hook1 cDNA as a probe, a hybridization signal of 2.6 kb was exclusively obtained in testicular RNA, while no signals were detected in brain, heart, liver, spleen, lung, placenta, ovary muscle and eye (Fig. 1A). The integrity of the RNA was confirmed by hybridization of the blot with a cDNA probe of the ubiquitously expressed human elongation factor (14). Additionally, these tissues were also studied for Hook1 expression by RT–PCR analyses. A 715 bp Hook1 fragment corresponding to the 5' end of the murine cDNA could be amplified from all samples examined, with the exception of spleen RNA, and simultaneously the integrity of the RNA used for RT–PCR was proven by amplification of a fragment of the GAPDH cDNA (Fig. 1B). The specificity of the PCR product was verified by hybridization using the murine Hook1 cDNA as a probe (Fig. 1B, lower panel).
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To examine Hook1 expression in the murine testis in more detail, RNA from developing postnatal testes were extracted and used for northern blot analysis. Hybridization signals could be first detected in the RNA from testes of 25-day-old mice, which indicates that Hook1 expression starts in haploid germ cells (Fig. 1C). In addition, RT–PCR experiments were performed on these different testicular stages to detect Hook1 transcripts at a lower expression level. By using this more sensitive approach, Hook1 transcripts were observed in all testicular developmental stages analyzed (day 10 to day 25) (Fig. 1D), indicating that Hook1 is already transcribed at low levels in premeiotic germ cells and/or somatic cells of the murine testis. Interestingly, we detected an additional, but smaller, Hook1 transcript in RNA derived from 20- and 25-day-old mice, supporting the idea that in postmeiotic germ cells, alternative Hook1 transcript forms exist (Fig. 1D; Hook1sv). Subsequent sequence analysis of the RT–PCR products revealed that the smaller transcript is generated as a result of skipping of two exons (exons 17 and 18), leading to a change of the putative reading frame and a premature stop codon after 17 amino acids. To determine Hook1 testicular expression at the cellular level, in situ hybridization experiments using digoxigenized antisense and sense Hook1 riboprobes were performed. As can be seen in Figure 2A–C, strong staining was obtained with the antisense Hook1 probe predominantly in haploid spermatids, whereas no staining could be detected in any somatic cell types or by using the Hook1 sense control probe (Fig. 2D).
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Taken together, our expression analyses demonstrate that the murine Hook1 gene is alternatively spliced and is mainly expressed in haploid germ cell stages of the testis. However, Hook1 RNA is also present in most adult tissues, premeiotic germ cell stages and/or somatic cells at a lower transcript level.
The murine Hook1 gene consists of 22 exons and is localized on chromosome 4
To determine the genomic organization of the murine Hook1 gene, a cosmid library from the RZPD was screened, and two clones were isolated. The identity of these clones was proven by hybridization using the Hook1 cDNA, and several fragments of the cosmid clones were cloned and sequenced to characterize the exon–intron structure of the Hook1 gene. Additional information was obtained from different databases (http://www.ncbi.nlm.nih.gov/genome/seq/MmBlast.html) and compared with the Hook1 cDNA. Using this approach, 22 exons of the murine Hook1 gene were confirmed in the genomic sequence, ranging from 35 to 260 bp in size (Fig. 3).
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To analyze the gene copy number in the murine genome, a Southern blot analysis was performed using the complete Hook1 cDNA as a probe. Genomic DNA and DNA of the isolated cosmid clones were hybridized and similar hybridization patterns were obtained, indicating that the Hook1 gene is a single copy gene in the murine genome (data not shown). Furthermore, the chromosomal localization of the mouse Hook1 gene was elucidated by fluorescence in situ hybridization using the labelled Hook1 cosmid DNA as a probe. The Hook1 gene was mapped to mouse chromosome 4 region C5–D2 (data not shown). This region on mouse chromosome 4 is syntenic to human chromosome 1p32.1, where several genomic clones (GenBank accession nos AC068202, AL138845 and AL352744) are localized, containing parts of the human HOOK1 gene.
Histopathology of the azh/azh mouse
As described above, the murine Hook1 gene was mapped to chromosome 4, region C5–D2. Interestingly, the locus for the azh mutation (abnormal spermatozoon head shape) was found to be localized on mouse chromosome 4, region C7 (4). Owing to the predominant expression of the Hook1 gene in haploid male germ cells together with the mapping data, we considered the Hook1 gene as a candidate gene for mutational analysis in the azh/azh mouse.
The azh/azh mouse is a mutant that displays an abnormal sperm head morphology (7,8), the most common abnormalities being club-shaped and crescent forms (Fig. 4A, B). In addition, several spermatozoa reveal looping in the midpiece of the sperm flagellum, and detached sperm heads and tails are often seen (Fig. 4A). Histological analysis of testes from a azh/azh mouse was performed, displaying in approximately 25% of the seminiferous tubules an arrest of spermatogenesis at variable levels (Fig. 4C), ranging from tubules with an arrest in elongating spermatids to tubules containing only Sertoli cells. The remaining seminiferous tubules do not show signs of germ cell arrest, but different sperm maturation defects were observed (Fig. 4D). These abnormalities include focal spermatozoa formation, decreased efficiency in transition from round to elongated spermatids, and a remarkable number of spermatozoa with abnormal head shapes (Fig. 4D).
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Expression analyses of the Hook1 gene in the azh/azh mouse
To elucidate whether a mutation in the murine Hook1 gene is responsible for the azh phenotype, we studied the expression of Hook1 in the azh/azh mutant mouse. Northern blot analysis on total testicular RNA from adult mutant and wild-type mice using Hook1 cDNA as a probe revealed an approximately 400 nucleotides smaller transcript in the azh/azh mouse (data not shown). This result indicates that in the azh allele, part of the Hook1 gene is deleted or the correct splicing of the Hook1 mRNA precursor is affected. To characterize the putative deletion at the RNA level, RT–PCR experiments were performed using three different primer combinations (Fig. 5A). On the one hand, no differences in the size of the amplification products of the wild-type and azh RNA were detected using primers for amplification of both the 5' or 3' fragments of the Hook1 cDNA. On the other hand, an approximately 400 bp smaller PCR product was obtained for the central fragment of the Hook1 cDNA in both the +/azh and azh/azh mouse. Subsequent cloning and sequencing of the PCR products revealed that a fragment of the Hook1 cDNA was missing in the RT–PCR product obtained from the azh testicular RNA corresponding exactly to exons 10 and 11 of the Hook1 gene.
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In azh/azh mice, two exons of the Hook1 gene are deleted
To determine the size of the mutation at the genomic DNA level, PCR experiments were performed to amplify the region around exons 10 and 11 of the Hook1 gene. Primers were used that were located in exons 9 and 12, respectively. The fragment obtained using wild-type DNA was approximately 3.4 kb in length, whereas the product resulting from azh/azh DNA was only 1.4 kb in size. Both PCR products were entirely sequenced and the size of the deletion (2021 bp) in the azh allele was determined. Furthermore, PCR analysis was performed using one primer located in a region of intron 9 that is not deleted in the azh allele and one primer in intron 9 absent in the azh allele (Fig. 5B). In addition, a third primer was included that is located in intron 11. While in DNA of wild-type and heterozygous mice, the expected 505 bp fragment was amplified, a smaller PCR product of 343 bp in size was obtained from DNA of the homozygous azh mouse (Fig. 5B). In summary, these results strongly suggest that this deletion in the Hook1 gene is the underlying cause of the azh mutation.
Owing to this mutation, the deduced amino acid sequence of the Hook1 gene product is changed after amino acid 263, and followed by a stop codon after 17 amino acids, resulting in a putative truncated protein of 280 amino acids in the azh/azh mouse. To prove the effect of the mutation at the protein level, a western blot experiment was performed using monospecific Hook1 antibodies. It is worth noting that these Hook1-specific antibodies recognize the C-terminal region (amino acid positions 480–662). While in testicular protein extracts of wild-type and +/azh mice, an approximately 84 kDa protein band was detected, no signal was obtained in the protein extract of the azh/azh mouse (Fig. 5C). The amount and quality of the loaded protein samples were proven by detection of 
-tubulin. These results clearly demonstrate that in the azh/azh mouse, no functional Hook1 protein is synthesized.
The Hook1 protein co-localizes to microtubular structures in male germ cells
In order to elucidate the effects of the loss of a functional Hook1 protein in male germ cells of azh mutant mice, we next investigated the subcellular distribution of Hook1 in differentiating spermatids. Therefore, indirect immunofluorescence analyses were performed using both Hook1- and -tubulin-specific antibodies. First, tubulin antibodies together with FITC-labelled secondary antibodies (green fluorescence) were used to visualize the tubulin network of the germ cells at different stages of spermatogenesis (Fig. 6A, D, G, J). During spermiogenesis from step 8 onwards, a germ-cell-specific microtubular structure termed the manchette becomes visible, displaying a V-shaped structure (Fig. 6A, D). Second, Hook1-specific antibodies detected by Cy3-coupled secondary antibodies (red fluorescence, Fig. 6B, E, H, K) were used to localize Hook1 at the subcellular level. Interestingly, Hook1 protein localizes to the manchette in spermatids from steps 8–10 (Fig. 6E), whereas in premeiotic germ cells, no specific Hook1 staining was observed. In the overlay image (Fig. 6C), the yellowish color represents -tubulin and Hook1 co-localization together with DAPI-stained nuclei. At a higher magnification level, it is revealed that additional Hook1 staining is also present between the microtubule manchette and the nucleus (Fig. 6C). During manchette elongation, the Hook1 staining pattern changes to a preferential labelling of the nuclear ring of the manchette (Fig. 6E, F, H and I), whereas the strong labelling of the manchette decreases. Additionally, a punctuate Hook1 staining is found at the proximal edges of the microtubular manchette (Fig. 6F), and an accumulation of Hook1 protein becomes apparent at the distal region of the microtubules (Fig. 6F). In more mature spermatids, the manchette migrates posteriorly and the broad Hook1 staining of the manchette is replaced by a prominent punctuated staining (Fig. 6H, I, K and L). On the one hand, at the proximal end of the manchette, the nuclear ring is stained (Fig. 6H and I) and one selective Hook1 signal is localized at the putative attachment site of the flagellum to the nucleus of the spermatid (Fig. 6H, I, K and L), and, on the other hand, distal ends of microtubule fibers are decorated by the Hook1-specific antibody (Fig. 6K, L). Furthermore, even at later stages of spermatid differentiation, the punctuate Hook1 staining pattern is found at both the attachment site and the proximal end of the elongated manchette (Fig. 6K, L). In contrast, in mature spermatozoa, Hook1 protein could not be detected either by indirect immunofluorescence or by western blot analysis (Fig. 5C), indicating that the definite role of Hook1 is restricted to spermatid differentiation.
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DISCUSSION |
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Here we show that in the azh (abnormal spermatozoon head shape) mutant mouse, a deletion of two exons in the Hook1 gene occurred, resulting in a smaller Hook1azh transcript and a putative truncated polypeptide. Because our antibody recognizes the C-terminal part of the protein, the truncated mutant form of the Hook1 protein was not detected in testicular protein extracts of the azh mouse, clearly demonstrating that no functional Hook1 protein is produced in homozygous azh mice.
Hook1 belongs to a small gene family with at least three members in mammals and one gene in Drosophila. The Drosophila hook gene was first to be identified and was so-named because the bristles of the fly displayed a hook-like structure (9). Mutations in the hook gene revealed that the Hook protein is of functional importance in the uptake of transmembrane ligands into multivesicular bodies (10). On the one hand, loss of hook function leads to pleiotrophic effects in Drosophila (FlyBase ID FBgn0001202), but fertility is not disturbed. On the other hand, in the homozygous azh mouse, loss of Hook1 function causes severe reduced fertility, whereas other organs are not affected. One possible explanation for these differences could be that – at least in human and mouse – three different HOOK genes exist (12 and our database research). The human HOOK3 gene product was shown to be involved in defining the architecture and localization of the Golgi complex; however, human HOOK1 and HOOK2 proteins seem to have a different function, because HOOK1 and HOOK2 did not co-localize with the Golgi complex (12). Our results from indirect immunofluorescence support this observation, because during early steps of spermatid differentiation, Hook1 and the Golgi complex are found at different positions within the germ cell.
The main morphological disturbance in the azh mutant mouse is the abnormal head shape of the spermatozoa. Additionally, abnormal bending and looping of the sperm flagella as well as a highly fragile connection between the tail and the head of the sperm is often observed. Furthermore, Hook1 protein was not detected in wild-type spermatozoa of the epididymis by western blot analysis or by indirect immunofluorescence using specific antibodies. These results suggest that a complete or partial loss of Hook1 function indirectly causes the phenotype in azh mutant mice. This hypothesis is strengthened by the fact that in both wild-type and azh homozygous males, the development of germ cells proceeds similarly until step 8 (7). At this stage, spermatid nuclei display a round shape and microtubules of the manchette are close to the nuclear membrane. However, in azh spermatids, the microtubules of the manchette are often in ectopic positions and at an excessive distance from the nucleus (7,8). In the present study, the indirect immunofluorescence analyses demonstrate co-localization of the manchette and the Hook1 protein in wild-type spermatids. Therefore, it is likely that Hook1 interacts directly with the manchette via its microtubule-binding domain localized in the N-terminal region of the protein. Microtubular binding was demonstrated for all three human HOOK proteins (12). Comparison of the deduced amino acid sequences of the murine and human Hook genes revealed high similarity over the whole sequence, and, especially in the N-terminal regions of the different Hook proteins, conserved amino acid stretches are found (88% identical amino acids between human and murine Hook1). In a recent study, this domain was determined to be sufficient to bind directly to microtubules (12), and it can be assumed that the murine Hook1 protein also binds to microtubules (see also Fig. 7). Moreover, HOOK proteins with a C-terminal truncation were also found to be associated with microtubules. This indicates that in the azh mutant mouse, a truncated Hook1 protein could bind to microtubules; however, it cannot establish the connection to other cell organelles (e.g. the nuclear envelope). The C-terminal domain could be responsible for mediating the specific binding to organelles. It was demonstrated that human HOOK3 interacts with the membrane of the Golgi apparatus in vitro (12), and it can be assumed that the murine Hook1 protein establishes the contact between the manchette and the nuclear envelope, because the direct links between the nuclear membrane and the microtubules of the manchette were described. In previous studies, rod-like linkers were observed using electron microscopy, and represent components of the amorphous material between the manchette and the nuclear envelope (15,16). The presence of fuzzy material connecting the nuclear membrane with the microtubules of the manchette indicates that the manchette and the nuclear envelope have a structural relationship through which they may exert forces on each other (16). Therefore, loss of the C-terminal domain in the Hook1azh protein (Fig. 7) would lead to disruption of the connection between the manchette and the nuclear envelope and subsequently to an abnormal position of the microtubules of the manchette, resulting in an abnormal sperm head shape.
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Furthermore, the central domain of the Hook proteins contains a leucine zipper that is embedded in a coiled-coil domain (Fig. 7). This domain is responsible for the homo-dimerization of hook proteins in Drosophila and human (10,12). Interestingly, we detected an alternative splice variant of the Hook1 gene. Owing to the skipping of exons 17 and 18, the putative Hook1sv protein lacks the C-terminal domain, but still contains the microtubule-binding region as well as the dimerization domain (Fig. 7). It is an attractive suggestion that dimeric Hook1sv proteins link the microtubules with each other, because it was observed in the azh mutant spermatid that the microtubules of the manchette tend to split (7), and both the dimerization domain and the organelle-binding domain are absent from the mutant Hookazh protein.
Moreover, Hook1 function is necessary for further spermatid development. During late stages of spermatid maturation, the cytoplasm and the manchette elongate and surround the middle part of the tail. In a high percentage of azh spermatozoa, structural abnormalities in the midpiece were observed. However, the axoneme as well as the outer dense fibers were intact (17,6). Electron-microscopic analysis demonstrated structural and assembly deficiencies of peri-axonemal proteins and abnormal accumulation of electron-dense material (17). The elongated manchette is thought to be used as a temporary storage and for transport of structural and signalling proteins (18–20). During these steps of spermatid differentiation, Hook1 is found to be localized at the distal tip of microtubules of the elongating manchette, indicating that Hook1 function is necessary for the correct positioning and elongation of the manchette within the spermatid. In this process, Hook1 must interact with (unknown) cellular structures other than the nuclear envelope. Disruption of this interaction in azh mice results in disposition of the microtubules and leads to a dislocation or abnormal accumulation of material essential for proper assembly of the midpiece tail structures.
Interestingly, Hook1-specific antibodies detect an approximately 84 kDa protein in human testicular extracts. This result supports the idea that the human HOOK1 protein displays a similar function in male germ cell differentiation. Several reports in the literature describe men with infertility due to abnormal spermatozoon head shape or to decapitation defects of their sperm. In patients with the decapitation defects, ultrastructural analyses of the spermatozoa revealed that the sperm tails were normal; however, several abnormalities were observed in the sperm head, including absence of the implantation fossa and basal plate (21–24). Moreover, in spermatozoa of these patients, degeneration of the tail midpiece with a large cytoplasmic droplet was observed (25). These defects are similar to that described for the azh spermatozoa (17). Although the functional relevance of Hook1 in the process of the attachment of the flagellum to the spermatid head is unknown, the results of our indirect immunofluorescence experiments indicate that Hook1 is involved in this process. This assumption is also supported by the observation that in azh mice, occasional sperm cells with two flagella and two sites of implantation in the nucleus were found (8). The authors speculated that the azh mutation affects the localization of multiple microtubule systems. Our results indicate that Hook1 is involved in the positioning of the microtubules of the manchette and the flagellum in relation to the membrane skeleton. Disruption of this interaction results in morphologically abnormal spermatozoa and male infertility. Our results also suggest that mutations in the human HOOK1 gene could cause male infertility in humans. In addition, there have been several reports of infertile men with chromosomal breakpoints at 1p32–33, where the genomic clones containing the human HOOK1 gene are located (26–29).
In summary, we have isolated and characterized the murine Hook1 gene, and, furthermore, our results strongly suggest that a deletion in the Hook1 gene causes the azh phenotype. Moreover, our results indicate that the human HOOK1 gene could serve as a candidate gene for mutational analysis in infertile patients with teratozoospermia or decapitation defects.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Identification and cloning of murine Hook1 cDNA
A mouse testis cDNA library (Uni-ZAP XR, Stratagene; CD-1 10–12 weeks old) was screened under low-stringency conditions using a cDNA probe for the murine dynein light chain 1. Positive phagemid clones were purified and plasmid clones were obtained by in vivo excision after the manufacturer's protocol. One of these clones comprises an insert of 1806 bp length with a poly(A) tail. Comparison with sequences reported in the database revealed similarity to the human HOOK1 cDNA and indicated that the murine Hook1 cDNA was incomplete. To identify missing 5' sequences, a testicular cDNA library from the RZPD (Resource Center and Primary Database, Berlin) was screened with the Hook1 partial cDNA and several clones were isolated. Clones with inserts larger than 1.8 kb were sequenced. Finally, two cDNA clones were identified that comprise the 2478 bp sequence of the murine Hook1 cDNA.
Southern and northern blot analyses
Genomic DNA was extracted from azh and wild-type mice tissues or tails using standard protocols and digested with the corresponding restriction enzyme. After electrophoresis, the DNA was transferred onto Hybond C membranes (Amersham, Piscataway, NJ, USA) and hybridized with a 32P-labelled Hook1-cDNA probe.
Total RNA was extracted from different mouse tissues by the RNA-NOW reagent (ICN, Frankfurt, Germany) and separated in an agarose gel under denaturing conditions. The RNA was transferred onto Hybond C membranes and hybridized with a 32P-labelled Hook1-cDNA probe.
In situ hybridization
Detection of Hook1 transcripts on paraffin sections was performed as previously described (30). Briefly, 7 µm testicular paraffin sections were dewaxed and hybridized with digoxigenized riboprobes. For detection, anti-digoxigenin antibodies coupled with alkaline phosphatase were used.
RT–PCR analyses
The RT–PCR experiments were performed with Hook1-specific primers H-5 (5'-AAG CTT GAC CAG TTG GAT GGC TCT-3') and H-D (5'-GCC AGC TGC TTT CTG AGT AA-3'), or with H-9 (5'-TAC AGC AAG AAG GGA CGG AGA ATG AAC-3') and H-III (5'-CTT CCG TTG TGT TTG CTT TGC-3') or with H-5p (5'-GCC ACA GTA CGA GCT GCC GCT-3') and H-PE (5'-GCC ATC CAA CTG GTC AAG CT-3'). For RT–PCR analyses, the One Step RT–PCR Kit (QIAGEN, Hilden, Germany) was used.
As controls for the integrity of the RNA, fragments of the murine glyceraldehyde-3-phosphate dehydrogenase were amplified using primers GAPDH-f (5'-CATCACCATCTTCCAGGAGC-3') and GAPDH-r (5'-ATGACCTTGCCCACAGCCTT-3').
Generation of Hook1-specific antibodies
For the generation of the Hook1 fusion protein the Strep-tag kit (Institut für Bioanalytik, Göttingen) was used. A 546 bp fragment of the Hook1 cDNA (1638–2184 bp) was amplified using primers H-PF (5'-GTA TTC GGT CTC TGG CCC AAG AAG GGA CGG AGA ATG A-3') and H-PR (5'-GTA TAC GGT CTC TGC GCT TTC CTC ATA ATC CCT CAA TT-3'). Both primers contained an extra 5' sequence with a BsaI restriction site. The Strep-tag vector DNA and the PCR product were ligated after digestion with BsaI. The fusion protein was purified and used for immunization of two New Zealand rabbits. To obtain monospecific Hook1 antibodies, the resulting antisera were affinity-purified by western blotting using a Strep-tag II Hook1 fusion protein.
Western blot analyses
Tissues were homogenized in 10 volumes of SEM buffer (0.32 M sucrose, 1 mM EDTA and 0.1% v/v 2-mercaptoethanol) and adjusted to a final protein concentration of 10 µg/µl. Twenty micrograms of each homogenate were loaded onto a precast 4–12% Bis–Tris gel (Biozym, Oldendorf, Germany). After electrophoresis, the proteins were blotted onto PVDF membranes (Macherey Nagel, Düren, Germany) as described previously (31). The Hook1 protein was probed with the affinity-purified anti-Hook1 antibodies. The -tubulin was detected using a commercially available antibody (Sigma-Aldrich Chemie, Deisenhofen, Germany). For detection of bound antibodies, filters were incubated with a 1 : 10 000 dilution of alkaline phosphatase-conjugated goat anti-rabbit or goat anti-mouse IgG (Sigma-Aldrich Chemie), and proteins were visualized with 0.35 mg/ml nitroblue tetrazolium salt (NBT) and 0.18 mg/ml 5-bromo-4-chloroindolylphosphate (BCIP) substrate.
Immunohistochemical analyses
Testicular tissue was fixed in Bouin's fixative and subsequently dehydrated in alcohol. Fixed testes were embedded in paraffin. Sections of 5 µm were hydrated by descending alcohol concentrations after removing the paraffin using roticlear solution (Roth, Karlsruhe). The sections were incubated in PBS/0.02% Tween-20 (2x10 min) and blocked for 1 h in PBS containing 2% goat normal serum, 3% BSA and 1xroti-block solution (Roth). Thereafter, testicular sections were covered with 20 µl purified anti-Hook1 antibody solution and incubated overnight in a humidified chamber at 4°C. For detection of bound antibodies, slides were incubated with a 1 : 100 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma-Aldrich Chemie), and were visualized with 0.35 mg/ml NBT and 0.18 mg/ml BCIP substrate.
For squeeze and suspension preparations, testes were isolated and placed into prewarmed M2 medium (Sigma-Aldrich Chemie). The testis was dissected and single seminiferous tubules were placed on a slide, squeezed with little pressure and air-dried. The remaining material was sucked several times through a 1 ml pipette tip to obtain a cell suspension. Drops of 20 µl were given on coated slides and air-dried. The tissues were fixed for 10 min in ice-cooled acetone (-20°C) and dried again. They were then incubated in PBS/0.02% Tween-20 (2 x10 min). After a blocking step for 1 h in PBS containing 5% goat normal serum, 3% BSA and 1xroti-block solution, testicular cells were covered with 20 µl purified anti-Hook1 antibody blocking solution and incubated overnight in a humidified chamber at 4°C. Additionally, a monoclonal antibody (anti--tubulin) was added in a final dilution of 1 : 50. After four washes with PBS/0.2% Tween-20, the slides were incubated with secondary antibodies (goat anti-rabbit-Cy3, goat anti-mouse–FITC) in a final concentration of 1 : 50 in PBS for 1 h at room temperature. Subsequently, they were washed again in PBS/0.2% Tween-20 four times and covered with a drop of vector shield solution containing DAPI stain. Slides were examined using a BX60 microscope (Olympus, Hamburg) with fluorescence equipment and the Analysis software program (Soft Imagin System, Münster).
Animals
Adult mice homozygous or heterozygous for the azh mutation were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Heterozygous males were bred with heterozygous or homozygous azh females to establish a stock. All mice were maintained in a temperature- and light-controlled room. Wild-type mice (C57BL/6 or CD1 strain) were used for control experiments.
Genomic PCR analyses
For the determination of the size of the deletion in the azh allele, PCR was performed using primers H-5 (5'-AAG CTT GAC CAG TTG GAT GGC TCT-3') and H-8a (5'-GTG TAT CTG CCC TTT TGG ATT CAG-3') and Titanium-Taq polymerase (Clontech, Heidelberg).
The genotype of the offspring was determined by morphological analyses of the sperm head shape or by PCR analysis using primers Hk-
-for (5'-GCC AGA TGT TGG TCA GAG GCA GTA A-3'), Hk--rev (5'-GGC CAA ATC TAG GAG AGC GGA GCA T-3') and Hk--rev2 (5'-CAG GGA AAC CTG AAG AGC TCA GTA A-3'). For amplification, the DNA was denatured for 15 min at 96°C, then 35 cycles followed, each with 30 s at 94°C, 30 s 50°C and 45 s at 72°C. After a final step at 72°C for 10 min, the amplification products were analyzed on ethidium bromide-stained agarose gels.
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ACKNOWLEDGEMENTS |
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We thank Nadine Dörwald and Stefan Wolf for providing expert technical assistance. This work was supported by the DFG through Grant NE 756/1-1 and by SFB 271.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Institute of Human Genetics, University of Göttingen, Heinrich-Dueker-Weg 12, 37073 Göttingen, Germany. Tel: +49 551 397598; Fax: +49 551 399303; Email: jneesen{at}gwdg.de
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REFERENCES |
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