Human Molecular Genetics, 2001, Vol. 10, No. 11 1117-1128
© 2001 Oxford University Press
Disruption of an inner arm dynein heavy chain gene results in asthenozoospermia and reduced ciliary beat frequency
1Institute of Human Genetics, University of Goettingen, Heinrich-Dueker-Weg12, 37073 Goettingen, Germany, 2Division of Electron Microscopy, Center of Anatomy, University of Goettingen, 37073 Goettingen, Germany, 3Department of Andrology, Clinical Training Center of the European Academy of Andrology, School of Medicine, Philipps University, 35039 Marburg an der Lahn, Germany, 4Institute of Anatomy, University of Hamburg, 20246 Hamburg, Germany and 5Children’s Hospital of the Ruhr—University Bochum, 44791 Bochum, Germany
Received 27 February 2001; Revised and Accepted 17 March 2001.
DDBJ/EMBL/GenBank accession no. HF312721.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Impaired ciliary and flagellar functions resulting in male infertility and recurrent respiratory tract infections are found in patients suffering from primary ciliary dyskinesia (PCD). In most cases, axonemal defects are present, i.e. PCD patients often lack inner and/or outer dynein arms in their sperm tails and cilia, supporting the hypothesis that mutations in dynein genes may cause PCD. However, to date it is unclear whether mutations in dynein heavy chain genes are responsible for impaired flagellar and ciliary motility in mammals. To elucidate the role of the mouse dynein heavy chain 7 (MDHC7) gene, which encodes a component of the inner dynein arm, we have generated mice lacking this dynein heavy chain isoform. Both MDHC7+/– and MDHC7–/– mice are viable and show no malformations; however, homozygous males produce no offspring. In comparison to MDHC7+/– and wild-type mice the spermatozoa of MDHC7–/– mice revealed a dramatic reduced straight line velocity and progressive movement, resulting in the inability of MDHC7-deficient sperm to move from the uterus into the oviduct. Additionally, we measured the beat frequency of tracheal cilia and observed a decrease in the beat frequency of
50% in MDHC7–/– mice. The reduction in both ciliary and flagellar motility is not correlated with any gross defects in the axonemal structure. The phenotype of MDHC7–/– mice is similar to that observed in some patients suffering from PCD, and our data strongly suggest that in some patients this disease could be due to mutations in the homologous human gene DNAH1 (HDHC7). |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Axonemal dyneins are large motor protein complexes that generate the force for the movement of eukaryotic cilia and flagella (for reviews, see refs 1–3). The dynein complexes are connected to the A-tubule of the nine peripheral microtubule doublets of the axoneme at regular distances. According to their position relative to the central microtubules of the axoneme they are called inner or outer dynein arms. Each dynein arm consists of two or three heavy chains, two to four intermediate chains and several light chains (4–6). Dynein motor activity is due to the action of the heavy chains. The central part of the heavy chain contains at least four putative nucleotide bindings motifs called P-loops (P1–P4). The P1-loop contains the ATP hydrolytic site and was found to be highly conserved in all dynein heavy chains examined so far. The N-terminal part of the heavy chain forms a stem structure and is required for assembly of the dynein complexes and cargo attachment (7). It not only seems to play a role in the interaction with the complex of intermediate and light chains but also mediates the dimerization of the heavy chains. The microtubule-binding region is located in a small globular domain in the C-terminal segment of the heavy chain (8–10).
In the biflagellate green alga Chlamydomonas reinhardtii 16 different dynein heavy chains were identified of which 14 belong to the axonemal type (11). While the outer dynein arm consists of three heavy chains (
, ß and 
), the inner arms are organized in a more complex manner. Different axonemal heavy chains are combined to form several distinct inner dynein arms (12). By mutation analysis, alterations in flagellar motility and arm assembly could be correlated to mutations in dynein heavy chain genes and linked to different functions of outer and inner arm complexes (7,13–15). Mutations which affect the -heavy chain of the outer dynein arm reduce the swimming velocity but do not influence the assembly of outer arm. In contrast, mutations of ß- and -heavy chain genes decrease the beat frequency and additionally, the complete outer arm is lacking in these mutants. However, Chlamydomonas mutants lacking some inner dynein arms have nearly normal beat frequency, but the amplitude of the flagella beat is altered (16).
In mammals much less is known about the function of dynein heavy chains, although more than 15 different dynein heavy chain isoforms have been identified in rat, mouse and human (17–21). In a previous study it was shown for the left right dynein (lrd) gene that dynein heavy chain function is necessary for the establishment of the left-right body axis in mammals (22). Further studies have demonstrated that this mutation leads to immotile nodal cilia in the developing embryo. However, function of the lrd gene seems to be required only for proper embryonic mouse development, because iv mutants are fertile and reveal no functional disorder in lung cilia (23,24).
Impaired ciliary and flagellar functions resulting in male infertility and recurrent respiratory tract infections are found in patients suffering from primary ciliary dyskinesia (PCD, OMIM 242650) (25). In 50% of PCD patients, the autosomal recessive disorder is correlated with situs inversus, denoted as Kartagener Syndrome (OMIM 244400). In most cases axonemal defects were present, i.e. PCD patients often lack inner and/or outer dynein arms in their sperm tails and cilia, supporting the hypothesis that mutations in dynein genes may cause PCD. Further confirmation of this theory comes from a recent finding that loss-of-function mutations of the DNAI1 gene, which encodes a dynein intermediate chain, were identified in a PCD patient (26). However, until now it is unclear whether mutations in dynein heavy chain genes are responsible for impaired flagellar and ciliary motility in mammals.
To identify dynein heavy chain sequences, we have performed PCR experiments using reversed-transcribed mouse and human testicular RNA (19). One of the identified genes was named MDHC7 (mouse dynein heavy chain 7), which encodes a putative heavy chain of the inner arm type. The gene is a single copy gene in the murine genome and was assigned to chromosome 14. The human homologous gene HDHC7 (renamed as DNAH1) has also been identified and maps to 3p21.3 (19). To elucidate the function of this dynein heavy chain isoform, we have disrupted the MDHC7 gene by homologous recombination. Due to the replacement of the ATP-binding site (P1-loop) by the neomycin resistance gene, the MDHC7 gene was inactivated. No functional gene product could be detected in MDHC7–/– animals. Loss of dynein heavy chain function results in male infertility and a reduction of ciliary beat frequency (CBF), but not in structural defects of tracheal cilia or sperm flagella.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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MDHC7 is expressed in different adult murine tissues
To study the expression pattern of the MDHC7 gene both northern blot and RT–PCR analyses were performed using RNA from different adult tissues (testis, lung, heart, brain, kidney, spleen, liver and muscle). By northern blot analysis, using 20 µg of total RNA in each lane, 15 kb MDHC7 transcripts could be observed in RNA from testis and after long exposition in lung and brain, whereas no hybridization signals could be detected in spleen, muscle, liver and kidney RNA (data not shown). By RT–PCR a 260 bp MDHC7 amplification product could be observed in all tissues analyzed (Fig. 1A). The integrity of the RNA was demonstrated by amplification of a 100 bp fragment of the S16 ribosomal protein.
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The intratesticular sites of MDHC7 expression were determined by northern blot experiments using RNA from different postnatal testis stages and by in situ hybridization. As shown in Figure 1B, MDHC7 expression starts at day 15 of postnatal testis development, which corresponds to the presence of pachytene spermatocytes. To answer the question of whether MDHC7 is specifically expressed in male germ cells, we performed in situ hybridization using sense and antisense MDHC7-ribo-probes. With the digoxigenized antisense probe, MDHC7 transcripts could be visualized in the cytoplasm of spermatocytes (Fig. 1C and D, arrowheads) and weaker staining was observed in round spermatids, whereas no MDHC7 transcripts could be detected using sense MDHC7-ribo-probes (Fig. 1E).
Generation of MDHC7-deficient mice
MDHC7 cDNA was used to screen a Sv129 mouse genomic library as described previously (19). The exon positions were determined by restriction enzyme mapping, Southern blot analyses and sequencing. Four MDHC7 exons encoding the putative ATP-binding site (Fig. 2A) were selected for substitution with the neomycin resistance gene of the targeting vector.
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The targeting vector was linearized and used for transfection of R1 embryonic stem (ES) cells. ES cells containing the disrupted allele were selected using Southern blot analysis. Three independent ES cell lines carrying the mutated allele were identified. One clone produced germ line-transmitting chimeras after aggregation with morula derived from CD1 females. The chimeras were bred with CD1 and 129/Sv females, respectively, to establish the dynein-disrupted allele on CD1 x 129/Sv hybrid and on 129/Sv inbred genetic background. Genotypes of the offspring were determined by Southern blot and PCR analyses (Fig. 2B and C).
Both female and male mice heterozygous for the MDHC7 mutation were normal and fertile. Heterozygous animals were mated to generate homozygous offspring. Approximately 25% of the offspring were homozygous for the mutated allele, indicating the absence of an increased embryonic or postnatal lethality in MDHC7–/– mice.
Recent investigations have demonstrated that dynein heavy chain function is involved in the left-right specification (22,24). To validate whether MDHC7 is also involved in this developmental process, we have analyzed neonates for inverted right-sided stomachs and 10 homozygous adult animals were examined for laterality of heart, lung and spleen. We could not detect any defect of organ position in MDHC7–/– mice. These results strongly suggest that MDHC7 is not involved in determination of left-right body axis in mice.
MDHC7 gene product is absent in knock-out mice
In order to assess whether the MDHC7 gene product is absent in MDHC7–/– mice, northern blot experiments were performed using testis RNA from wild-type, heterozygous and homozygous animals. A 2.6 kb cDNA fragment spanning V the P1-loop region and 5' sequence of MDHC7 was used as a hybridization probe. In testicular RNA of wild-type and heterozygous mice, 15 kb MDHC7 transcripts could be detected (Fig. 2D), but no hybridization signal was observed in RNA of MDHC7–/– mice (Fig. 2D). We also did not discover any truncated MDHC7 transcripts. To prove the integrity of the RNA, the same blot was rehybridized using a MDHC1 cDNA probe. MDHC1 encodes a putative outer dynein arm heavy chain. MDHC1 transcripts were present in all three tested RNAs (Fig. 2D).
The inactivation of MDHC7 in homozygous MDHC7–/– mice was also verified at the protein level by western blot experiments. Whole testis and oviduct protein extracts were probed with anti-MDHC7 monospecific antibodies. In protein extracts of wild-type and heterozygous animals a protein band of 400 kDa was detected by the antibodies, but no MDHC7 protein was observed in protein extracts of dynein knock-out mice (Fig. 2E). The loading of equal amounts of protein was controlled by using anti -tubulin and anti neomycin phosphotransferase II antibodies (Fig. 2E). The presence of the MDHC7 protein in oviduct and testis tissues supports the idea that the MDHC7 dynein heavy chain is a component of cilia as well as of sperm flagella. The results also show that knock-out mice have no functional MDHC7 heavy chain.
MDHC7 homozygous mutant male mice are infertile
The genotype of the offspring was determined by Southern blot analyses and PCR studies (Fig. 2B and C). Breeding of heterozygous offspring was performed to obtain homozygous animals lacking the MDHC7 gene product. MDHC7–/– males and females were bred with wild-type animals to test their fertility. All MDHC7 females lacking were found to be fertile; however, from a total of 30 MDHC7–/– males no offspring were obtained.
MDHC7-deficient spermatozoa are not able to move from the uterus into the oviduct
To investigate the cause for infertility of male MDHC7–/– mice we determined the number of spermatozoa in the epididymis and in uteri or oviducts of mated wild-type females. At least three males were analyzed. As shown in Table 1, the sperm number in the epididymis is similar in wild-type and MDHC7–/– males, indicating that production of spermatozoa is not affected in knock-out mice. Uteri and oviducts of vaginal plug positive females were extracted and examined for the presence of spermatozoa (Table 1). In the uterus of females which were bred with MDHC7–/– males, a normal amount of spermatozoa was ascertained, but nearly all of these sperms were either immotile or showed a strongly decreased motility in comparison to wild-type spermatozoa. This result suggests that the motility or the motility endurance of the MDHC7–/– sperms is reduced. This suggestion was further supported by the observation that no spermatozoa of MDHC7–/– mice were found in the oviduct (Table 1). No variance in sperm number or movement was observed for MDHC7+/– males in comparison to wild-type animals. These findings sustain the assumption that loss of MDHC7 function results in an abnormal flagella motility which prevents the migration of MDHC7–/– spermatozoa from the uterus into the oviduct.
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We next assessed whether MDHC7–/– spermatozoa are able to bind and penetrate oocytes. In vitro fertilization experiments were performed with eggs containing or lacking cumulus cells. In both cases, MDHC7–/– sperms penetrated the oocytes and fertilized them, although a reduced number of 8-cell-stage embryos were obtained compared with the number with spermatozoa of heterozygous mice (Table 2).
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MDHC7-deficient spermatozoa show decreased motility
To characterize sperm motility in more detail, we used a computer-assisted sperm analyzer. For all investigated parameters we did not observe significant differences between spermatozoa of wild-type and heterozygous mice. Approximately 70% of spermatozoa of these mice were motile and 20–30% of the spermatozoa showed progressive motility (Table 3). In contrast only 38% of the spermatozoa of MDHC7–/– mice were motile and, more importantly, only 1% of these spermatozoa revealed a progressive movement (Table 3).
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Some parameters were evaluated in more detail (Fig. 3). Curvilinear velocity represents the total distance passed through by a sperm in a time unit (Fig. 3A). Spermatozoa of wild-type and heterozygous males have a velocity of
260 µm/s, whereas the speed of MDHC7–/– sperm was decreased to 100 µm/s. Furthermore, a drastic reduction in the average path velocity (Fig. 3B) and the straight line velocity (Fig. 3C) in MDHC7–/– spermatozoa was observed; these spermatozoa have only 20–25% of the velocities estimated for the spermatozoa of MDHC7+/+ and MDHC7+/– animals. The straight line velocity represents the straight line progressive movement of a sperm between the beginning and the end of the measurement divided by the time elapsed. On the other hand, no significant alterations were observed for the straight forward movement (Fig. 3D) or the linearity (Fig. 3F) of motile MDHC7–/– spermatozoa. These parameters were only slightly decreased in MDHC7-deficient spermatozoa.
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More important was the finding that the loss of MDHC7 function is correlated with a reduction in the amplitude of the sperm tail beat. In MDHC7+/+ and MDHC7+/– animals the lateral amplitude of spermatozoa flagella was 13 µm, whereas the amplitude in MDHC7-deficient spermatozoa decreased to
7 µm (Fig. 3F). In contrast to these observations, we measured a slight increase in the flagellar beat frequency in motile MDHC7–/– sperm (38 Hz) compared with spermatozoa of wild-type mice (28 Hz) (Fig. 3G).
Tracheal beating frequency is reduced in mice lacking MDHC7
The results of sperm motility analyses raised the question of whether cilia are also affected by the loss of MDHC7 function. By western blot analysis it was demonstrated that the dynein heavy chain protein band is missing in protein extracts of oviduct tissue from MDHC7–/– mice (Fig. 1E), but microscopical investigations using freshly isolated tissues revealed that cilia of the oviduct and trachea of such mice were motile. Therefore, we analyzed the beat frequency of tracheal cilia. Using a photo-electrical method we could demonstrate that tracheal cilia of CD1 wild-type mice had an average CBF of 13.7 ± 2.3 Hz, whereas heterozygous mice presented a slightly increased value of 16.0 ± 4.7 Hz. On the contrary, we observed a 50% CBF reduction in mice lacking functional MDHC7. In these animals the obtained CBF was 7.6 ± 2.4 Hz (Table 4).
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In the analyzed tracheal rings of MDHC7–/– mice some segments were present in which cilia movement took place in a coordinated manner and a directed particle transport was visible. This suggests that loss of MDHC7 does not result in dyskinetic beating of cilia. The main defect consists of a decrease in CBF.
MDHC7 is detectable along the whole flagella of human and mouse spermatozoa
To elucidate the distribution of MDHC7 along the flagella, human and mouse spermatozoa were fixed on glass slides and probed with monospecific anti-MDHC7 antibodies. The complete flagella were labeled by the anti-MDHC7 antibodies (Fig. 4A and C). These results indicate that the MDHC7 dynein heavy chain is equally distributed along the flagella of human and mouse spermatozoa. Furthermore, spermatozoa of MDHC7+/– and MDHC7–/– mice were analyzed using anti-MDHC7 antibodies. We observed no difference in the intensity of the staining of flagella from heterozygous animals (Fig. 4E) in comparison to wild-type spermatozoa (Fig. 4C). In western blot analysis no truncated MDHC7 polypeptide could be detected in testicular protein extracts of MDHC7–/– mice. On the other hand flagella of MDHC7–/– spermatozoa were barely labeled by using MDHC7 antibodies (Fig. 4G). The results obtained from immunocytochemistry indicate that low amounts of truncated MDHC7 polypeptides are generated in MDHC7–/– mice.
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No structural defects were observed in the axonemal structure of sperm flagella or cilia
In order to investigate whether impaired ciliary and flagellar motility in MDHC7–/– mice is correlated with structural defects of the axoneme, electron microscopy analyses of lung (Fig. 5A and B) and testis tissues as well as of tracheal cilia (Fig. 5C) and spermatozoa flagella (Fig. 5D) were performed. We could not detect any gross defects either in tissues or in the axonemal structure of sperm tails or cilia. In all analyzed axonemal cross sections inner and outer dynein arms were present. These findings strongly suggest that deletion of the central and C-terminal part of the MDHC7 heavy chain did not affect the assembly of other components of the inner dynein arm.
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Chlamydomonas mutants have demonstrated that the N-terminal part of the dynein heavy chain is important for dimerization of heavy chains and assembly of the whole dynein complex (7). The finding that no structural defects were found in the axoneme of MDHC7-deficient cilia and flagella suggest that in these mice a truncated MDHC7 gene product is generated. By using both northern and western blot analyses we were not able to detect such truncated gene products. Because we could not exclude the possibility that these methods were not sensitive enough, we performed RT–PCR experiments. We used different MDHC7-specific primer combinations to amplify fragments of either the central domain (P1-loop) which was deleted in knock-out mice or the 5'-region of the MDHC7 transcript. In testis RNA from heterozygous and wild-type mice all fragments (control, P1-loop and 5'-region) were detected (Fig. 6). As expected, the P1-loop fragment was not amplified in MDHC7–/– mice; however, a PCR product covering the 5'-region of the MDHC7 transcript was observed. This result demonstrates that a truncated MDHC7 transcript is produced in MDHC7–/– mice and strengthens the assumption that a truncated MDHC7 protein is inserted into the inner arm dynein complex of MDHC7–/– mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In the last few years more than 12 axonemal dynein heavy chain isoforms were identified in different species but, particularly in mammals, the specific functions of most of these isoforms are still unknown (17–20,22,24). Only for the lrd gene, which encodes a putative outer arm dynein heavy chain, was a role in the establishment of the left-right body axis demonstrated (22,24).
The present study is the first report of the disruption of an inner dynein arm heavy chain gene in vertebrates which results in male infertility and reduced CBF. MDHC7 was identified by an RT–PCR approach using testicular RNA. Sequence alignment confirmed that it is closely related to dynein heavy chain isoforms of the inner arm (19). Comparison of additional amino acid sequence information with the five complete known heavy chain sequences (outer arm , ß, and inner arm 1, 1ß) of Chlamydomonas corroborated this result and gave highest similarity to the inner arm heavy chains (27–29).
MDHC7 is mainly expressed in testis and somatic tissues containing ciliated epithelia, implying a role in ciliary and flagellar motility. Similar expression data were obtained for the MDHC7 homologous rat gene Dnahc1. Dnahc1 expression was reported in heart, spleen, lung and testis using a multiple-tissue northern blot (21). In contrast, for other dynein heavy chain genes a testis-specific expression was suggested (19,30). The different expression patterns of individual dynein heavy chain genes might indicate that in mammals dynein heavy chain genes have adapted specific functions for ciliary and/or flagellar beating during embryonic development as well as in the adult organism. MDHC7 belongs to the group of dynein heavy chains whose function is mainly necessary for both ciliary and flagellar motility during postnatal life, but it seems not to be involved in the establishment of the left-right body axis.
To elucidate the function of MDHC7 in mice, a targeted mutation of the gene was generated deleting the ATP-binding site (P1-loop) of the MDHC7 gene. Several studies have indicated that the P1-loop is the motif which is responsible for ATP hydrolysis and that this domain is essential for the function of the heavy chain (24,31). We selected four exons of MDHC7 encompassing the P1-loop for deletion by homologous recombination in ES cells. In heterozygous animals that were phenotypically normal we could not detect any reduction in MDHC7 gene expression. Furthermore, the CBF and sperm velocity are not decreased in heterozygous animals, which makes it unlikely that non-functional truncated MDHC7 protein is incorporated into cilia or flagella in these mice.
From analyses of Chlamydomonas mutants it is well known that inner and outer dynein arms are functionally different. Mutations which affect the outer dynein arms cause a reduction in beat frequency, but the waveform is not altered (14,16). In contrast, mutants with defective inner dynein arms have almost normal beat frequency, but swim with an altered amplitude.
The analysis of the MDHC7-deficient spermatozoa motility gave similar results to those reported for Chlamydomonas inner dynein arm mutants. Loss of MDHC7 functions results in an 50% decrease of the lateral amplitude of the flagella beat. This alteration of the flagellar waveform could be the reason for the strongly reduced swimming velocities of these spermatozoa. Interestingly, the beat frequency is not decreased in these spermatozoa. However, the measurement of the beat frequency of tracheal cilia in MDHC7–/– mice clearly shows that the loss of an inner dynein heavy chain function is correlated with a decrease in beat frequency in cilia.
In Chlamydomonas different mutants were described which cause structural defects in the axoneme (7,15,32,33). The analyses of these mutants have also demonstrated that the first 150 kDa of the N-terminal part of the heavy chain are essential for the assembly of the dynein arm. This was shown for both outer and inner arm heavy chains. Mutants with a truncated inner arm 1 heavy chain (lacking the P1-loop) are able to assemble a partially functional inner arm complex with the 1ß heavy chain and the other components (27). On the other hand, loss of the complete 1 heavy chain leads to a missing I1 inner arm and these mutants showed a strongly reduced swimming velocity. In MDHC7–/– mice the situation seems to be similar, because the loss of the P1-loop domain induces a decreased flagellar and ciliary motility, but is not correlated to structural defects in the axoneme. No truncated MDHC7 polypeptides or transcripts were detected by northern or western blot experiments. However, by using RT–PCR approaches truncated MDHC7 transcripts were observed. Furthermore, immunological staining of spermatozoa from MDHC7–/– mice using MDHC7-specific antibodies revealed the presence of truncated MDHC7 polypeptides. On the basis of these observations it can be suggested that the targeted deletion did not affect the N-terminal part of the MDHC7 polypeptide which might be sufficient for the assembly of the inner arm complex. However, we cannot rule out the possibility that a complete loss of the MDHC7 heavy chain does not influence the assembly of the other components of the inner dynein arm in mice.
Although we could not detect any structural defects in the axoneme of cilia or sperm flagella of MDHC7–/– mice, the loss of MDHC7 function was found to lead to male infertility. The infertility of MDHC7–/– mice is caused by an altered sperm motility, because MDHC7-deficient spermatozoa do not migrate into the oviduct. However, these spermatozoa can fertilize oocytes in vitro, which demonstrates that the binding to the oocyte and development of the zygote is not affected by this mutation. The loss of MDHC7 function leads to a higher amount of complete immotile spermatozoa and both the velocity and progressive motility of the remaining motile MDHC7-deficient spermatozoa are reduced, which results in asthenozoospermia. Reduced sperm motility has also been implicated in human male infertility and moreover, asthenozoospermia is considered to be a major cause of male subfertility (34). In several studies patients with unexplained asthenozoospermia were analyzed for ultrastructural defects of their sperm (35,36) and in 20–30% of these patients no abnormalities of their sperm were observed. The phenotype of MDHC7 knock-out mice strengthens the assumption that loss of the DNAH1 (HDHC7) dynein heavy chain function could cause asthenozoospermia in humans. Furthermore, there is a clear correlation between asthenozoospermia and respiratory disease in some infertile patients (37).
The correlation between asthenozoospermia and respiratory disease becomes more obvious in patients suffering from PCD. PCD or immotile cilia syndrome is an autosomal recessive disorder with an incidence of 1 in 15 000–20 000 and is genetically heterogeneous. Due to impaired ciliary motility, PCD patients suffer from recurrent respiratory and pulmonary tract infections leading to chronic bronchitis and/or bronchiectasis, chronic rhinosinusitis and otitis media. Most of the male PCD patients are infertile due to immotile spermatozoa and for females subfertility is suggested. In 50% of patients situs inversus is observed. However, in some PCD patients cilia and sperm flagella are not completely immotile (38), similar to the phenotype of MDHC7 knock-out mice. MDHC7 was assigned to chromosome 14, region B-C, and the homologous human gene HDHC7 (DNAH1) was localized to 3p21.3, which is syntenic to mouse chromosome 14 region B-C (19). Interestingly, in a previous study a potential PCD locus was assigned to 3p21 (39). In contrast to PCD patients, the reduced CBF in MDHC7–/– mice is not correlated with a higher incidence of infections of the respiratory tract. Homozygous MDHC7–/– mice do not show respiratory distress or reduced vitality. However, this finding seems to be a general feature of the murine species. Both for hpy/hpy (hydrocephalic-polydactyl) mice and for WIC-Hyd mutant rats no respiratory problems were reported, even though both mutants have immotile cilia (40,41). Even in mice lacking all cilia and flagella due to a disrupted hepatocyte nuclear factor/forkhead homolog 4 gene (hfh-4) no respiratory distress is observed (42).
It is reasonable to hypothesize that the position of mutations in dynein heavy chain genes either results in a structural defect of the axoneme or leads only to a reduction of ciliary and flagellar motility. Our results indicate that mutations which affect the central and C-terminal part of an inner arm dynein heavy chain do not result in axonemal abnormalities. This would imply that in patients with structural defects of cilia, mutations in dynein genes should be located in the N-terminal part of the heavy chain.
Taken together, we have generated mice lacking a functional inner arm dynein heavy chain, which could be used as an animal model for PCD or asthenozoospermia. Our results also strongly support the hypothesis that the homologous human gene HDHC7 (DNAH1) is a candidate gene for some cases of PCD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of the MDHC7 gene disruption vector
For isolation of an MDHC7 genomic clone, a J1 129/Sv mouse genomic library was screened using MDHC7 cDNA as a probe (19). Phage DNA was restricted with NotI and the genomic mouse fragment was cloned into the NotI restriction site of pZErO-TM-2 vector (Invitrogen). A 4.5 kb BamHI/BamHI fragment and a 3.5 kb SmaI/NotI fragment flanking the P1-loop sequence of MDHC7 were subcloned into BamHI and SmaI/NotI restricted pBluescript vector (Stratagene), respectively. The 4.5 kb BamHI fragment was used as 5' homologous arm and was inserted into the BamHI digested pPNT vector (Fig. 2) (43). The correct orientation was ascertained by restriction analysis using the SpeI restriction enzyme. The 3.5 kb SmaI/NotI fragment was isolated from pBluescript by NotI/XhoI digestion and cloned into NotI/XhoI restricted pPNT vector containing the 4.5 kb genomic fragment. The resulting 14.5 kb targeting vector was linearized with NotI and used for transfection of ES cells.
ES cell culture
The ES cell line R1 was cultured as described previously (44). Confluent plates were washed in PBS buffer and trypsinized. The cells were suspended in PBS buffer at 2 x 107 cells/ml. One millilitre aliquots were mixed with 50 µg of linearized targeting vector and electroporated at 250 V and 500 µF using a Bio-Rad Gene Pulser apparatus. Cells were plated onto non-selective medium in the presence of G418-resistant embryonic mouse fibroblasts. After an incubation step of 36 h the medium was changed containing 350 µg/ml G418 and 2 µM ganciclovir. Ten days later resistant clones were picked and transferred into 24-well trays.
Generation of chimeric mice
Chimeric mice from ES cells carrying the disrupted MDHC7 allele were generated by aggregating 10–15 compact ES cells and two 2.5-day-old embryos of the CD1 mouse strain as described by Wood et al. (45). Female and male chimeras were mated with CD1 mice. Offspring were analyzed by PCR and Southern blot analyses to identify ES cell-derived offspring. Heterozygous offspring were bred to obtain homozygous mice.
DNA and PCR analyses
Genomic DNA was extracted from ES cells (46) and mouse tails (47) and digested with XbaI. After electrophoresis, the DNA was transferred onto Hybond N membranes (Amersham) and hybridized with a 32P-labeled 1 kb BamHI/XbaI fragment (Fig. 2A). Absence of additional random integration of the targeting vector was checked by hybridization using a neomycin phosphotransferase II probe. Hybridization was carried out as described previously (19). Due to the introduction of the neomycin resistance gene cassette an additional XbaI restriction site was generated and the mutant XbaI fragment has a size of 6 kb while the corresponding wild-type allele has a size of 10 kb.
For PCR analysis 1 µg of genomic DNA and 10 pmol of each primer (M7-f, 5'-GCAGCGATGGTGCTTTCGGCACTCAATTGTC-3'; M7-r, 5'-GACCCTTGAAGAACTTTCCCATGGCCATGAAG-3'; and Neo2F, 5'-CGCAGCGCATCGCCTTCTATCGCCTTCTTG-3') were used. Cycling conditions were 1 min at 94°C, 1 min at 55°C and 2 min at 72°C. After 35 cycles the products were separated in a 1% agarose gel. The wild-type allele (primers M7-f–M7-r) has a size of 926 bp while the mutated allele (primers M7-f–Neo2F) has a size of 800 bp.
Generation of MDHC7-specific antibodies
A 1984 bp cDNA fragment (5' of the P1-loop coding region, GenBank accession no. AF312721) of MDHC7 was cloned into pQE30 expression vector (Qiagen). A His-tag fusion protein was purified according to the manufacturer’s instructions. Approximately 100 µg/ml of purified fusion protein was mixed with an equal volume of Freund’s adjuvant (complete or incomplete) and injected three times subcutaneously into female New Zealand rabbits.
Enrichment of monospecific antibodies were performed by eluting immunoglobulin bound to filter fixed His-tag fusion protein as described by Lemaire et al. (48).
Western blot analysis and immunocytochemistry
Tissues were homogenized in 10 vol of SEM-buffer [0.32 M sucrose, 1 mM EDTA and 0.1% (v/v) mercaptoethanol] and adjusted to a final protein concentration of 10 µg/µl. Twenty micrograms of each homogenate was loaded onto a pre-cast 3–8% NuPAGE Tris-Acetate gel or 4–12% NuPAGE Bis-Tris gel (Invitrogen). After electrophoresis, the proteins were blotted to PVDF membranes (Machery Nagel) as described by Lemaire et al. (48). Dynein was probed with MDHC7-specific antibodies. Neomycin phosphotransferase II and -tubulin were detected using commercially available antibodies (5'
3'; Sigma-Alderich Chemie). For detection of bound antibodies, the ‘WesternBreeze Chemiluminescent’ detection kit (Invitrogen) was used.
Freshly prepared spermatozoa were washed three times with PBS and a drop of the sperm solution was fixed on a glass slide by air-drying. Fixed spermatozoa were treated in PBS containing 1% Triton-X-100 for 5 min. After a blocking step in PBS with 5% sheep normal serum (Sigma-Alderich Chemie) for 1 h the spermatozoa were incubated with monospecific anti-MDHC7 antibodies overnight at 4°C. After four washes in PBS spermatozoa were incubated with sheep anti-rabbit IgG Cy3 conjugated for 2 h. Spermatozoa were washed again four times in PBS and embedded with 20 µl of Vectashield solution (Vector Laboratories) containing 1.5 µg/ml DAPI (4'-6'-diamidino-2-phenylindole).
Fertility test
To examine the fertility of dynein-deficient mice, 30 mature males were mated, each with two CD1 females for at least 12 weeks. Females were checked for the presence of vaginal plugs.
For the in vitro fertilization assays adult CD1 females were superovulated by intraperitoneal injections of 5 U eCG followed by 5 U hCG 46–48 h later. Oocytes were collected 10–12 h after hCG administration.
Epididymal spermatozoa were capacitated for 1.5 h and added to the oocytes in 400 µl drops of fertilization medium and incubated for 6 h at 37°C in 5% CO2. The eggs were washed and cultured in M16 medium covered with mineral oil. Progression of the fertilized oocyte development was analyzed microscopically over a period of 3 days.
Sperm and tracheal cilia motility analyses
Testes and epididymes of wild-type, heterozygous and mutant homozygous mice were dissected in M2 media (Sigma). Spermatozoa were allowed to swim out of the epididymis for 20 min at 37°C. A drop of the sperm solution was transferred to the incubation chamber which was set at a temperature of 37°C. Sperm movement was quantified using the computer-assisted semen analysis (CASA) system (CEROS version 10, Hamilton Thorne Research). At least 250 motile sperms each from MDHC7+/+, MDHC7+/– and MDHC7–/– mice were analyzed. Mean, standard deviation, median and range were calculated using the Microsoft Excel 97 computer program.
Measurements of CBF were performed using a photo-electrical method (49–51). In brief, the tracheas of wild-type, heterozygous and MDHC7-deficient mice were dissected in M2 media. Tracheal rings were cut using micro dissection scissors and 200 µl of the cell suspension was pipetted into micro-slides with one cavity which were positioned in a phase contrast microscope (Diavert, Leitz). A heating platform was used to maintain a temperature of 37°C throughout the investigation. Moving cilia were positioned in the light beam of the microscope, which was reduced to an area of 1 µm2. Single cells were not taken into account for analyses. Rhythmic movements of the cilia resulted in a variation of light intensity which was registered by a photometer (MPV compact, Leitz) positioned in the light beam. This undulating signal was transferred to a printer (Cardiostat T, Siemens). The beat frequency was calculated from the number of peaks per time unit. From three animals of each genotype different probes were examined in this manner for 
10 s and the median of these probes was taken as the CBF of this sample.
Electron microscopy analyses
Testis tissue and spermatozoa were fixed by immersion in 5% glutaraldehyde in 0.2 M phosphate buffer, postfixed with 2% osmium tetraoxide and embedded in epoxy (Epon) resin. Fixation, sampling and embedding for transmission electron microscopical analysis of cilia was performed as described previously (52). Briefly, lungs were fixed by intra-tracheal instillation of a mixture of 1.5% paraformaldehyde and 1.5% glutaraldehyde in 0.15 M HEPES buffer. Tissue blocks were osmicated, stained in 1.5% aqueous uranyl acetate overnight, dehydrated in acetone and finally embedded in Araldite. From each animal more than 100 cilia were examined and five consecutive cuts, each 70 nm thick, were analyzed.
In situ hybridization, northern blot analysis and RT–PCR experiments
Detection of MDHC7 transcripts on paraffin sections was performed with digoxigenized ribo-probes as previously described (53). Northern blot experiments were performed according to standard methods (54) using a 2.6 kb MDHC7 cDNA probe spanning the P1-loop region and 5' MDHC7 sequence. Sequence of the 2.6 kb cDNA fragment is stored in the GenBank data library under accession no. AF312721.
For RT–PCR experiments, cDNA synthesis was performed using the MDHC7-specific primer M7rSCA (5'-TACAGCGCAGGATGGTACAAGGGG-3') and 5 µg of total testicular RNA. Superscript reverse transcriptase (Life Technologies) was used to synthesize the first cDNA strand. One microliter of cDNA was subjected to a PCR reaction with the gene-specific primers M7KALA (5'-GACCTGGGGAAGGCCTTAGCCATAC-3'), M7rSCA or M7ERV (5'-GAGGACTTTCAGGAGCGTGTGGAGCA-3') and M7rDCR (5'-CGGTGGGAGTCCGGCAATCAAACTC-3'). The resulting 260 or 352 bp PCR fragments were separated on a 1.5% agarose gel. As controls for the integrity of the RNA, fragments of the S16 ribosomal protein RNA using primers S16F (5'-AGGAGCGATTTGCTGGTGTGGA-3') and S16R (5'-GCTACCAGGCCTTTGAGATGGA-3'), or of the murine glyceraldehyde-3-phosphate dehydrogenase RNA using primers GAPDH-f (5'-CATCACCATCTTCCAGGAGC-3') and GAPDH-r (5'-ATGACCTTGCCCACAGCCTT-3'), were amplified.
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ACKNOWLEDGEMENTS |
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We thank Anne Greiwe and Stefan Wolf for providing expert technical assistance and Irene Mendoza, Stefan Bohlander and Peter Burfeind for helpful discussion and critical reading of the manuscript. This work was supported by the BMBF through grant 01KY9504 and by the DFG through grant NE 756/1-1.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +49 551 397598; Fax: +49 551 399303; Email: jneesen@gwdg.de
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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