Disruption of the murine dynein light chain gene Tcte3-3 results in asthenozoospermia

in Reproduction

To elucidate the role of the mouse gene Tcte3 (Tctex2), which encodes a putative light chain of the outer dynein arm of cilia and sperm flagella, we have inactivated this gene in mice using targeted disruption. Breeding of heterozygous males and females resulted in normal litter size; however, we were not able to detect homozygous Tcte3-deficent mice using standard genotype techniques. In fact, our results indicate the presence of at least three highly similar copies of the Tcte3 gene (Tcte3-1, Tcte3-2, and Tcte3-3) in the murine genome. Therefore, quantitative real-time PCR was established to differentiate between mice having one or two targeted Tcte3-3 alleles. By this approach, Tcte3-3−/− animals were identified, which were viable and revealed no obvious malformation. Interestingly, some homozygous Tcte3-3-deficient male mice bred with wild-type female produced no offspring while other Tcte3-3-deficient males revealed decreased sperm motility but were fertile. In infertile Tcte3-3−/− males, spermatogenesis was affected and sperm motility was reduced, too, resulting in decreased ability of Tcte3-3-deficient spermatozoa to move from the uterus into the oviduct. Impaired flagellar motility is not correlated with any gross defects in the axonemal structure, since outer dynein arms are detectable in sperm of Tcte3-3−/− males. However, in infertile males, deficient Tcte3-3 function is correlated with increased apoptosis during male germ cell development, resulting in a reduction of sperm number. Moreover, multiple malformations in developing haploid germ cells are present. Our results support a role of Tcte3-3 in generation of sperm motility as well as in male germ cell differentiation.

Abstract

To elucidate the role of the mouse gene Tcte3 (Tctex2), which encodes a putative light chain of the outer dynein arm of cilia and sperm flagella, we have inactivated this gene in mice using targeted disruption. Breeding of heterozygous males and females resulted in normal litter size; however, we were not able to detect homozygous Tcte3-deficent mice using standard genotype techniques. In fact, our results indicate the presence of at least three highly similar copies of the Tcte3 gene (Tcte3-1, Tcte3-2, and Tcte3-3) in the murine genome. Therefore, quantitative real-time PCR was established to differentiate between mice having one or two targeted Tcte3-3 alleles. By this approach, Tcte3-3−/− animals were identified, which were viable and revealed no obvious malformation. Interestingly, some homozygous Tcte3-3-deficient male mice bred with wild-type female produced no offspring while other Tcte3-3-deficient males revealed decreased sperm motility but were fertile. In infertile Tcte3-3−/− males, spermatogenesis was affected and sperm motility was reduced, too, resulting in decreased ability of Tcte3-3-deficient spermatozoa to move from the uterus into the oviduct. Impaired flagellar motility is not correlated with any gross defects in the axonemal structure, since outer dynein arms are detectable in sperm of Tcte3-3−/− males. However, in infertile males, deficient Tcte3-3 function is correlated with increased apoptosis during male germ cell development, resulting in a reduction of sperm number. Moreover, multiple malformations in developing haploid germ cells are present. Our results support a role of Tcte3-3 in generation of sperm motility as well as in male germ cell differentiation.

Introduction

The microtubule-based motility of cilia and flagella plays a critical role in fertility and viability in mammals. The motive force to power sperm flagella is generated by dynein motors that are attached to the nine outer-doublet microtubules of the axoneme. Apart from inner and outer axonemal dynein arms, other classes of motor proteins are also expressed in the testis. Cytoplasmic dynein motors are involved in a wide variety of fundamental cellular processes including mitotic spindle formation and orientation, vesicle transport, formation, and localization of the Golgi complex, assembly and motility of cilia and flagella or spermatid translocation (Karki & Holzbaur 1999, Miller et al. 1999, King 2000a,b). In general, all dynein motors are multisubunit complexes and consist of heavy, intermediate, and light chains, which differ in their molecular weight and function (Gibbons 1995, King 2000a,b). Dynein heavy chains harbor the motor domain and generate the force for sliding of microtubules, while intermediate and light chains are involved in the assembly and regulation of the whole complex. Most of the dynein polypeptides are specific either for axonemal or cytoplasmic complexes. However, for a few light chains (TCTEX1, TCTE3, DCL1 or DNALI1), it was suggested that they could play a role in both dynein subclasses (Harrison et al. 1998, Bowman et al. 1999, DiBella et al. 2001, Rashid et al. 2006).

In mammals, an increasing number of genes encoding components of axonemal and cytoplasmic dynein complexes were identified (Tanaka et al. 1995, Neesen et al. 1997, Grissom et al. 2002) and the function of some of these genes was analyzed by the generation of dynein-deficient mice. These models demonstrate that the loss of the dynein function can result in embryonic lethality, disturbed body axis formation, hydrocephalus, or male infertility (Supp et al. 1997, Neesen et al. 2001, Ibanez-Tallon et al. 2002, Rana et al. 2004). In contrast, to date no mouse model for targeted inactivation of a dynein light chain is available.

Here we report the functional analysis of the dynein light chain Tcte3-3 by targeted disruption in mice. The Tcte3 (Tctex2) gene was initially identified in the testis of mice bearing the t-haplotype as a candidate for involvement in nonMendelian transmission (Lader et al. 1989, Huw et al. 1995). It was described as a putative sperm membrane protein (Huw et al. 1995) but subsequent identification of the Chlamydomonas homolog LC2 revealed that this protein is a component of outer dynein arm complexes (Patel-King et al. 1997). Complete deletion of the LC2/oda12 gene in Chlamydomonas causes loss of all outer dynein arms and a slow jerky swimming phenotype (Pazour et al. 1999), indicating that LC2 is required for the outer arm assembly in Chlamydomonas, while another mutant lacking the 3′-end of the LC2 gene is partially functional and allows for the assembly of outer dynein arms. In the outer dynein arm, LC2 is associated with an intermediate chain at the base of the dynein particle. More recently it was demonstrated that the Tcte3 expression is regulated in a tissue-specific manner. Interestingly, TCTE3 could also be detected in cytoplasmic dynein samples derived from rat kidney, and spleen, supporting that TCTE3 could act as a component of the cytoplasmic dynein complex in these tissues. It was proposed that the heterogeneity in the polypeptide composition that build the cytoplasmic dynein motor could reflect specific requirements for a precise temporal and spatial regulation of motor cargo-binding within different cell and tissue types (DiBella et al. 2001).

The human orthologous TCTE3 gene was mapped on chromosome 6q27 (Neesen et al. 2002). Both the murine and human genes comprise of four exons and their encoded proteins share 87% homology. Expression studies demonstrate that human and murine TCTE3 genes are mainly expressed during male germ cell development; however, weaker expression of Tcte3/TCTE3 is observed in several tissues including brain, lung, and trachea (DiBella et al. 2001, Neesen et al. 2002). In addition, these analyses reveal that two transcript variants are expressed that differ by the presence of exon 3. The expression in tissues containing cilia and the putative role of TCTE3 in the assembly and function of outer dynein arms raised the hypothesis, that TCTE3 could be a candidate gene mutated in patients suffering from primary cilia dyskinesia (PCD). This disease is characterized by recurrent infections of the respiratory tracts and male infertility due to decreased sperm motility. A mutational analysis of 25 PCD patients revealed no disease causing mutation in the coding sequence of the human TCTE3 gene (Neesen et al. 2002). However, because PCD is a genetically heterogeneous disease, TCTE3 cannot be excluded as candidate gene. To gain further insight into the function of Tcte3, we have generated mice carrying a targeted disruption of the Tcte3-3 gene by replacing the exon 2 with a neomycin resistance gene fragment. The homozygous targeted mutation caused reduced sperm motility and resulting in some males in infertility. In infertile males, reduction or deficiency of the Tcte3-3 gene product leads to a decreased sperm number and an increased rate of apoptosis in male germ cells. Furthermore, different abnormalities were observed in developing germ cells, supporting a basic role of Tcte3-3 in spermatogenesis.

Results

Expression of the murine Tcte3 gene

Several studies have demonstrated Tcte3 expression in male germ cells as well as in somatic tissues (Huw et al. 1995, DiBella et al. 2001). We determined the intratesticular sites of Tcte3 expression by northern blot experiments using RNA from different postnatal testis stages and by in situ hybridization. Tcte3 expression can be detected by northern blot analysis on day 11 of postnatal testis development (Fig. 1A), which corresponds to the appearance of leptotene spermatocytes. An increased expression is observed on day 15 when pachytene spermatocytes appear. Using RT-PCR Tcte3 transcripts were found in testicular RNA from 7-day-old mice (data not shown), indicating a weak expression of Tcte3 in spermatogonia or somatic testicular cells. To further characterize Tcte3 expression in male germ cells we performed in situ hybridization using sense and antisense Tcte3-ribo-probes. With the digoxigenized antisense probe, Tcte3-transcripts could be visualized in the cytoplasm of spermatocytes (Fig. 1B and C) and weaker staining was observed in round spermatids. In contrast to RT-PCR results no Tcte3-transcripts could be detected in spermatogonia or using sense Tcte3-ribo-probes (Fig. 1D).

Figure 1

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Figure 1

Analyses of testicular Tcte3 expression. (A) Northern blot analysis of testicular RNA from different postnatal developmental stages. Twenty micrograms of total testicular RNA of ten to fifteen day old mice were hybridized with a 32P labeled 0.7 kb Tcte3-3 cDNA fagment. The same blot was rehybridized with a human elongation factor II (EF-2) probe. Localization of Tcte3-transcripts by in situ hybridization. Digoxigenized labeled Tcte3-3 antisense (B and C) riboprobe was used for hybridization of paraffin embedded testicular cross section. Tcte3 transcripts were detected in the cytoplasm of primary spermatocytes (arrowheads) and in round spermatid stages (arrows). No staining of the cytoplasm was observed after hybridization using a sense Tcte3-3-riboprobe (D). Localization of the TCTE3 protein in male germ cells by indirect immunofluorescence (E–Q). TCTE3 specific antibodies were detected with Cy3-coupled antibodies (red color; E–G, K–N, and P), while FITC-coupled secondary antibodies were used to visualize microtubules (Tub), (green color; G, J, L, O, and P). Additionally, DAPI was used to stain the nucleus (blue color; E–Q). In spermatocytes the whole cytoplasm is stained (E and F). In spermatids at early elongation phase TCTE3 is concentrated in the cytoplasm while weaker staining is observed in the nucleus region (I–L). During further differentiation of spermatids intensity of TCTE3 staining decreases. Some protein can be detected around the nucleus and in the cytoplasm surrounding the microtubular manchette (G, arrows). Figure H shows round spermatids incubated with secondary antibodies only. (M–Q) Immunostaining of mouse spermatozoa with anti-TCTE3 and anti-tubulin antibodies. The TCTE3 polypeptide is detectable along the whole flagella (M, N and P). Negligible staining was observed in sperm incubated only with secondary antibodies (Q).

Citation: REPRODUCTION 139, 1; 10.1530/REP-09-0243

To determine the localization of TCTE3 in germ cells more precisely squeeze preparations were incubated with antibody combinations. Tcte3-specific antibodies were detected using a Cy3-conjugated immunoglobin (red fluorescence signal), whereas antibodies against tubulin were marked with FITC conjugated antibodies (green fluorescence signal). Additionally, the nuclei of testicular cells were stained with DAPI (blue signal). Consistent with RNA expression analyses TCTE3 signals were barely detected in spermatogonia. In spermatocytes, the TCTE3 protein staining spreads over the whole cytoplasm, without any specific accumulation (Fig. 1E). A similar staining pattern was observed in dividing spermatocytes, with the exception of a more intense fluorescence signal between nuclei (Fig. 1F). In round spermatids TCTE3 was predominantly found in the cytoplasm and overlapped with tubulin staining (Fig. 1I–P). In the region of the nucleus TCTE3 staining was weaker, but clearly visible. In elongated spermatids TCTE3 protein labeling was decreased, but was detectable in the cytoplasm (Fig. 1G). The microtubular manchette was clearly marked by antibodies against β-tubulin (Fig. 1G). To elucidate the distribution of TCTE3 along the flagella, mouse spermatozoa were fixed on glass slides and probed with specific anti-TCTE3 antibodies. Complete flagella were labeled (Fig. 1M–P) indicating that the TCTE3 dynein light chain is equally distributed along the flagella of mouse spermatozoa.

Generation of Tcte3-3-deficient mice

Tcte3-cDNA was used to screen a Sv129 mouse cosmid library and two positive clones were isolated. Positions of Tcte3-3 exons were determined by restriction enzyme mapping, Southern blot analyses, and sequencing. Four Tcte3-3 exons were identified (Fig. 2A) and exon 2 was selected for substitution with the neomycin resistance gene of the pPNT targeting vector (Tybulewicz et al. 1991). After homologous recombination, a total of 7.7 kb genomic region was deleted including the exon 2 of the Tcte3-3 gene (Fig. 2B).

Figure 2

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Figure 2

Targeted disruption of the mouse Tcte3-3 gene. (A) Schematic representation of the Tcte3 gene locus on murine chromosome 17. Positions of Tcte3 exons are indicated by grey boxes. (B) Schematic representation of the targeting strategy. Wild-type Tcte3-3 locus (top), targeting construct (middle) and targeted allele (bottom). Exon 2 (black box) was replaced by the neomycin resistance gene cassette. Arrows indicate direction of transcription for both the Tcte3-3 and the neomycin resistance gene. (C) Southern blot analysis for genotyping the ES cells. Genomic DNA was restricted using KpnI restriction enzyme. An external probe (gray box, 3′-probe) gave rise to a 9.2 kb (mutant), and a 5.4 kb (wild-type) hybridization signal. (D) Hybridization with the neomycin fragment probe proved homologous recombination event in ES cell clone 1 and 3. (E) Long-range PCR was done using DNA from ES cells 1 and 3. With primers Neo3F and Tc-probe-r a 5.1 kb fragment was amplified from the targeted allele. Amplification using primer pair Tc-geno-F and Tc-probe-r resulted in a 4.8 kb fragment derived from the wild-type allele. (F) PCR genotyping of mice. Wild-type allele yields a PCR product of 900 bp using the Tc-fin-F and Tc-fin-R primers. The targeted allele is expected to generate an ∼600 bp fragment using the primers Neo3F and Tc-fin-R. (G) Quantitative real-time PCR analysis of Tcte3 mice. The average value calculated for the quantity of neomycin for each sample was plotted versus mice identification number. Putative homozygous mice have a value of ∼1 indicating the presence of two neomycin copies, while heterozygous mice containing one neomycin copy have quantities of ∼0.5. E, EcoRI; K, KpnI; S, SalI; N, NotI; X, XbaI; Xh, XhoI; * marks restriction sites deriving from cloning vector. Arrows () indicate positions of primers used for long range PCR.

Citation: REPRODUCTION 139, 1; 10.1530/REP-09-0243

Embryonic stem (ES) cells containing the disrupted allele were selected using Southern blot analysis (Fig. 2C) and two clones (clone 1 and clone 3) were identified. Additional random integration of the targeting vector was excluded by Southern blot analysis using a neomycin gene fragment hybridization probe (Fig. 2D). Moreover, PCR amplification of a 5.1 kb fragment from ES cell DNA of clone 1 and 3 proved the correct integration of the construct (Fig. 2E). The two ES cell lines carrying the mutated allele were used to generate germ line transmitting chimeras after injection in blastocytes derived from C57BL/6 females. Chimeras were bred with C57BL/6 and 129/Sv females, respectively, to establish the Tcte3-3-disrupted allele on C57BL/6×129/Sv hybrid and on 129/Sv inbred genetic background. Genotypes of offspring were determined by Southern blot and PCR analyses (Fig. 2F). Both, female and male mice heterozygous for the Tcte3-3-mutated allele revealed no malformations and were fertile.

Heterozygous animals were bred to generate Tcte3-3-deficient mice. More than 100 offspring were genotyped at postnatal day 20 using Southern blot or PCR. Approximately 20% of analyzed offspring were genotyped as wild-type while the remaining 80% revealed a heterozygous pattern. This result indicates that either Tcte3-3-deficient animals are lethal or that the technique used to genotype mice is unable to discriminate between heterozygous or homozygous offspring.

Genomic organization of the Tcte3-locus

Huw et al. (1995) suggested the existence of more that one Tcte3 gene in the murine genome. We therefore performed Southern blot analyses using different enzymes and the Tcte3-3 cDNA as a probe (data not shown). Comparison with the restriction pattern of cosmid DNA used for the generation of the targeting vector revealed no differences, indicating that putative copies of the Tcte3-locus must be very similar. Using the Tcte3-3 cDNA, we performed a database search and identified two bacterial artificial chromosomes (BAC) clones (accession numbers AC182749 and AC173477) from gene bank, both derived from murine chromosome 17. Alignment with the Tcte3-3 cDNA revealed three repeat units of ∼31.5 kb each containing one copy of the Tcte3 gene. The three Tcte3 gene copies were designated as Tcte3-1, Tcte3-2, and Tcte3-3 (Fig. 2A). We next asked whether the three genes can be distinguished. Sequence comparison of the four exons of the three Tcte3 genes showed that in the nontranslated sequence of exon 1 of Tcte3-2 and Tcte3-3, a 7 bp sequence repeat (GGCGGAG) is missing. Moreover, in exon 3 of Tcte3-3 at position 535 of the corresponding cDNA cytosine is replaced by guanine which results in an amino acid exchange of arginine to glycine. To examine whether all three putative Tcte3 genes are transcribed, we searched the murine EST database for Tcte3 EST clones. We found EST clones matching the sequence of Tcte3-1, Tcte3-2, and Tcte3-3 indicating that all three genes are active. Sequence analyses of the cosmid DNA fragments used to generate the targeting vector revealed complete identity with the Tcte3-3 gene database information, however, single base substitutions were found for sequence information of Tcte3-1 and Tcte3-2 genomic fragments.

Because the three Tcte3 gene copies were highly similar, a quantitative PCR was established to genotype the offspring. By this technique, we were able to differentiate between animals with one or two targeted alleles (Fig. 2G). Breeding of heterozygous offspring was performed to obtain homozygous animals lacking the Tcte3-3 gene product. From forty-seven F2 generation animals 11 mice were genotyped by quantitative PCR as homozygous for the targeted allele while 10 mice were wild-type and 26 were heterozygous for the mutated allele.

We observed no differences in phenotypes of mice hetero- or homozygous for the Tcte3-3 mutated allele on different genetic backgrounds or derived from different chimeric progenitors, with the exception that number of offspring was lower on 129/Sv background. Therefore, experiments were done with mice on C57BL/6×129/Sv hybrid background.

Tcte3-3 gene product is detectable in knock-out mice

The expression of Tcte3-3 in homoygous Tcte3-3−/− mice was analyzed at the protein level by western blot experiments. Whole testis protein extracts were probed with anti-TCTE3-specific antibodies. In protein extracts of wild-type, heterozygous and homozygous animals a protein band of about 22 kDa was detected (data not shown). The loading of equal amounts of protein was controlled by using anti-tubulin antibodies.

Some Tcte3-3 homozygous mutant male mice are infertile

Tcte3-3−/− males and females of the F2 generation were bred with wild-type animals to test their fertility. All Tcte3-3 lacking females and 25 tested males were found to be fertile, however, from 10 Tcte3-3−/− males no offspring were obtained after three month of breeding with heterozygous or wild-type females.

To elucidate the cause of infertility we analyzed testicular morphology. Testes from infertile Tcte3-3−/− males (70.3±10.1 mg) were significantly smaller compared to wild-type littermates (90.2±9.3 mg; Fig. 3A). Subsequent histological analysis indicated no abnormalities in the basal part of the seminiferous tubules of Tcte3-3−/− animals (Fig. 3C and D), where spermatogonia and Sertoli cell nuclei are located. Type A spermatogonia with large spherical nuclei were located normally, at the base of epithelium. Spermatogonia containing round nuclei with a large number of heterochromatin clumps were also evident (Fig. 3C and D). In contrast, spermatocytes were significantly reduced in number (reduction was ∼30% compared to wild-type littermates), while postmeiotic stages; spermatids and spermatozoa were sometimes absent in mutant testis. Histological analysis of the epididymis revealed an overall reduced number of spermatozoa in comparison to wild-type epididymis (Fig. 3E, G and H). A considerable number of spermatozoa display head anomalies (Fig. 3E and G) and up to 40–50% of sperm showed malformation of the tail or the development of two tails (Fig. 3E and F).

Figure 3

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Figure 3

Histology of Tcte3-3−/− testes. (A) shows a testis of a wild-type and a Tcte3-3−/− male of 3 months of age. (B–H) Light microscopically analyses of testicular and epididymal semi-thin sections stained with toluidine blue. (B) In wild-type testis different stages of spermatogenesis are visible and tails of elongated spermatids reach into the lumen of tubules. (C and D) Tcte3-3−/− seminiferous tubules show hypospermatogenesis or only focal spermatogenesis with release of premature germ cells in the lumen (arrowheads). Elongated spermatids frequently display head anomalities (arrows). Percentage of spermatids with head deformations varies in different tubule cross sections and was estimated at ∼15–20% compared to unaffected sperm. (E–G) In the epididymis, biflagellate or bend tail spermatozoa (arrowheads) and spermatozoa with head abnormalities (arrows) are also frequently found. Moreover, fewer spermatozoa are observed in cross sections compared to wild-type epididymis (H).

Citation: REPRODUCTION 139, 1; 10.1530/REP-09-0243

Transmission electron microscopy (TEM) was used to further characterize defective cells in mutant testis (Fig. 4). Ultrastructural analysis indicated no abnormalities in the basal portion of seminiferous tubules (spermatogonia, primary spermatocytes) and in the interstitial Leydig cells. TEM provided evidence that substantial cell death in Tcte3-3−/− testis was occurring. Elongating spermatids exhibited various signs of degeneration, ranging from nuclear condensation to the formation of abnormal sperm heads resulting focally in an abrupt arrest of spermatogenesis in Tcte3-3−/− males (Fig. 4A and B). In the cytoplasm of Sertoli cells and elongated spermatids large vacuoles were observed (Fig. 4C). Spermatids, which had escaped from the degeneration, in considerable numbers harbored biflagellate pattern during the elongation step (Fig. 4E and F). Some spermatids exhibited a normal morphology with elongated nucleus. Putative spermatids in more advanced stages of degeneration could not unequivocally be identified. Small clumps of heterochromatin could be distinguished in the nuclei of cells in early stages of degeneration. In the later stages of degeneration, the areas of heterochromatin became larger. Condensed chromatin and irregular pattern of organelles were seen in latest stages of germ cell degeneration (Fig. 4). Axonemal tail sections of spermatozoa analyzed by TEM demonstrated the presence of normal 9+2 axonemes as well as inner and outer dynein arms (Fig. 4D).

Figure 4

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Figure 4

Transmission electron microscopy analyses of Tcte3-3−/− male germ cells. Testicular histology in Tcte3-3−/− males is normal until beginning of spermatid elongation. (A) shows a normal round spermatid (rSp), while elongating spermatid start to degenerate (deSp). In (B) spermatids with normal sperm head (eSp) are located beneath degenerating elongated spermatids (deSp). In spermatids and surrounding Sertoli cells, development of vacuoles (V) becomes obvious (C). The axonemal structure is not disturbed in Tcte3-3−/− sperm tails and outer dynein arms are detectable (arrows, D). In the epididymis often spermatozoa with two flagella or bend tails are observed (E, arrows indicate double tail formation; F, arrows indicate double tail formation or coiled flagella).

Citation: REPRODUCTION 139, 1; 10.1530/REP-09-0243

Germ cells of infertile Tcte3-3-deficient mice undergo apoptosis

Cellular degeneration found in Tcte3-3−/− testes of infertile males suggested that spermatogenic cell apoptosis increased in Tcte3-3−/− mice. Therefore, TUNEL assays were performed on Tcte3-3−/− testicular sections (aged 3 months) in order to assess the possible cell death rate. Those regions in the Tcte3-3−/− seminiferous tubules that contained predominantly cells in prophase I stages exhibited a high frequency (more than 50%) of cell death (Fig. 5A). At higher magnification, the majority of TUNEL-positive cells could be identified as spermatogonia and spermatocytes in most of the testicular tubules from Tcte3-3−/− mice (Fig. 5B). In contrast, cell death was rare in the wild-type testes (Fig. 5C).

Figure 5

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Figure 5

Apoptosis is increased in Tcte3-3−/− testes. (A and B) show testicular sections of a Tcte3-3−/− male after TUNEL reaction. TUNEL-positive staining is mostly evident in spermatogonia, however also primary spermatocytes and round spermatids are affected. Interstitial cells were not labeled. Only few cells are stained using the TUNEL assay in wild-type testis (C). (D) Quantification of the incidence of TUNEL-positive germ cells in wild-type (+/+) and Tcte3-3−/− males.

Citation: REPRODUCTION 139, 1; 10.1530/REP-09-0243

Quantification of apoptosis was carried out by counting apoptotic germ cells that were clearly stained by the TUNEL assay. A total of 25 randomly selected seminiferous tubule cross-sections were analyzed from each of two mice. The rate of germ cell apoptosis was expressed as the number of apoptotic cells per tubule. An average of 25–30 cells was found to be TUNEL positive in each of the Tcte3-3−/− seminiferous tubules (Fig. 5D). This result shows that germ cell development in the Tcte3-3−/− mice is impaired.

Tcte3-3-deficient spermatozoa reveal reduced motility

To investigate the causes for Tcte3-3−/− male infertility, we determined the number of spermatozoa in the epididymis of these males as well as in the uteri/oviducts of mated wild-type females. At least three males of each genotype were analyzed. As shown in Table 1, the sperm number in the epididymis is significantly reduced in infertile Tcte3-3−/− males in comparison to wild-type males, indicating that production of spermatozoa is affected in the knock-out mice. Moreover, uteri and oviducts of vaginal plug positive females were extracted and examined for the presence of spermatozoa (Table 1). In the uteri of females which were bred with Tcte3-3−/− males, a slightly reduced amount of spermatozoa was ascertained; however, nearly no spermatozoa of Tcte3-3−/− mice were found in the oviduct (Table 1). This result suggests that the motility of Tcte3-3−/− sperms is reduced. In contrast, fertile Tcte3-3−/− males did not reveal reduced number of sperm in the epididymis in comparison to wild-type males.

Table 1

Sperm analysis in Tcte3-3+/+ and infertile Tcte3-3−/− mice.

Genotype of male
Number of sperm+/+−/−
Epididymis×105190±0.4 (3)1.6±0.2 (4)
Uterus×1035.8±1.5 (3)1.5±1.2 (4)
Oviduct×10120±2.9 (3)0.1±0.7 (4)

Number of spermatozoa extracted from Tcte3-3+/+ and Tcte3-3−/− epididymis or from uterus and oviduct of mated females was determined. Data in sperm analysis represent the mean±s.d. of the number of individual measurements indicated in parenthesis.

To characterize sperm motility in more detail, we used a computer assisted sperm analyzer. Analyses were done after 1.5, 3.5 and 5.5 h after sperm extraction from the epididymis. We did not observe significant differences between spermatozoa of wild-type and heterozygous Tcte3-3+/− mice for any of the investigated parameters (data not shown). Approximately 60–76% of spermatozoa of these mice were motile and 40–54% of the spermatozoa showed progressive motility (Table 2). In contrast, only 20–31% of the spermatozoa of Tcte3-3−/− mice were motile and more importantly only between 11 and 18% of these spermatozoa revealed a progressive movement (Table 2).

Table 2

Analysis of the sperm motility of Tcte3-3+/+ and Tcte3-3−/− mice.

Genotype of maleIncubation time (h)Total number of measured spermatozoa (%)Number of motile spermatozoa (%)Number of spermatozoa with progressive movement (%)
+/+1.52895 (100)2196 (76)1575 (54)
3.53069 (100)2066 (67)1356 (44)
5.53681 (100)2245 (61)1520 (41)
−/−1.54259 (100)1326 (31)748 (18)
3.53974 (100)1054 (26)582 (14)
5.54204 (100)833 (20)453 (11)

Motility of spermatozoa from Tcte3-3+/+ and Tcte3-3−/− males was determined after 1.5, 3.5, and 5.5 h using a computer-assisted semen analysis system.

Certain functional parameters were evaluated in more detail (Fig. 6). Curvilinear velocity represents the total distance passed through by a sperm in a time unit (Fig. 6A and B). Most spermatozoa of wild-type males showed velocities of >200 μm/s, whereas the speed of Tcte3-3−/− sperm from infertile and fertile males was decreased to ∼100–150 μm/s. Furthermore, a reduction in the average path velocity (Fig. 6C and D) and in the straight line velocity (Fig. 6E and F) of Tcte3-3−/− spermatozoa was observed, which have only 50–60% of the velocities estimated for spermatozoa of wild-type 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. In contrast, no significant alterations were observed for beat frequency (Fig. 6I) or the linearity (Fig. 6J) of motile Tcte3-3−/− spermatozoa.

Figure 6

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Figure 6

Results of the computer assisted analysis of sperm motility. Different parameters, (A–F) sperm velocities (μm/s), (G and H) lateral amplitude of the sperm tail beat (μm), (I) beat frequency of the sperm flagella (Hz) and (J) forward movement (%) of wild-type spermatozoa (+/+) and Tcte3-3−/− sperm (−/−) were measured after 1.5 and 3.5 h incubation time. Tcte3-deficient spermatozoa showed significantly reduced swim velocities and a decreased lateral amplitude in comparison to spermatozoa of wild-type and heterozygous mice (P<0.001 by Mann–Whitney U test). No significant difference is observed in the linearity of sperm movement. The beat frequency of Tcte3-3−/− sperm is slightly increased. For all parameters the medians (25–75%) and percentiles (5–95%) are shown.

Citation: REPRODUCTION 139, 1; 10.1530/REP-09-0243

Interestingly, the loss of Tcte3-3 function is correlated with a slight reduction in the amplitude of the sperm tail beat (Fig. 6G and H). While in wild-type animals the lateral amplitude of most of spermatozoa flagella was more than 10 μm, whereas the amplitude in the majority of Tcte3-3-deficient spermatozoa was decreased to <8 μm.

Discussion

Over the past years, an increasing number of axonemal and cytoplasmic dynein genes were identified in different species, but particularly in mammals, specific functions of most of these polypeptides are still unknown. To our knowledge, the present study is the first report of the targeted disruption of a dynein arm light chain gene in mouse which can result in male infertility. Tcte3 genes are mainly expressed in testis and somatic tissues containing ciliated epithelia, implying a role in ciliary and flagellar motility (Rappold et al. 1987, DiBella et al. 2001, Neesen et al. 2002). Moreover, Tcte3 expression was also demonstrated in liver and spleen and it was suggested that Tcte3 expression in these tissues was due to its possible role as a component of the cytoplasmic dynein complex. In this regard Tcte3 could be involved in distinct cargo-binding activities (DiBella et al. 2001). The different expression patterns of individual dynein genes might indicate that mammalian dynein genes have adapted specific features for ciliary and/or flagellar beating and potentially other cellular processes in both embryonic development as well as in the adult organism. Our results support this hypothesis because loss of Tcte3-3 function in some males on the one hand resulted in decreased sperm motility supporting its function as an axonemal dynein, on the other hand increased apoptosis of male germ cells and multiple malformations within the differentiating spermatozoa are observed, indicating an important role of Tcte3-3 in spermatogenesis.

It is surprising that the inactivation of the Tcte3-3 gene copy has such phenotypic effects in some mice, considering that three putative Tcte3 gene repeat units were found on murine chromosome 17. Data derived from the EST libraries suggests that all three copies are transcribed, although the expression level of each individual gene is unknown. Alignment of sequence information available for Tcte3-1 and Tcte3-2 genomic fragments with our Tcte3 cosmid DNA used for the generation of the targeting vector revealed single nucleotide substitutions, whereas complete identity with the Tcte3-3 genomic fragments were found. Therefore, it is most probable that we have inactivated the Tcte3-3 gene by homologous recombination. Interestingly, the TCTE3-3 amino acid sequence differs in position 125 from the sequences of TCTE3-1 and TCTE3-2. An arginine is replaced by glycine. It is unclear whether this exchange is of any functional relevance for the TCTE3-3 function.

Our western blot analyses revealed no detectable differences in the amount of TCTE3 protein between testicular protein extracts from wild-type, heterozygous or homozygous Tcte3-3-deficient mice. A recent report indicating that Tcte3 expression is regulated by sense–antisense transcript pairs (Chan et al. 2006). The authors stated that different species of Tcte3 antisense transcripts exist and that some were not polyadenylated and capped. The Tcte3 antisense amplicons revealed 100% complementarity either to exons 3 and 4 or only to exon 4. Tcte3 antisense transcripts were only detected in testis, while Tcte3 sense transcripts were also observed in other adult tissues. This could explain that the phenotype in Tcte3-3−/− males seems to be restricted to male reproductive organs. The authors suggested that Tcte3 antisense transcripts serve as a noncoding regulatory RNA and are involved in the vigorous control of gene expression necessary for spermatogenesis (Chan et al. 2006). The homozygous inactivation of the Tcte3-3 gene copy by homologous recombination might disturb the balance between Tcte3 sense and antisense transcripts towards degradation of Tcte3 transcripts in male germ cells. In this content it should be mentioned that results of situ hybridization experiments using a sense Tcte3 probe argues against a strong expression of Tcte3 antisense transcripts within the murine testis. However, we cannot exclude that experimental conditions prevent efficient binding of the 76 bp of the sense probe covering exon 4 to Tcte3 exon 4-specific antisense transcripts. Moreover, the sensitivity of in situ hybridization could be insufficient to detect low amounts of antisense transcripts, because Tcte3 transcripts were also not visualized in spermatogonia although RT-PCR results indicated low expression of Tcte3 in these cells.

Orthologous genes to murine Tcte3 have been reported in several species including human (TCTE3), the green algae Chlamydomonas (LC2) and the rainbow trout (LC2; Patel-King et al. 1997, Inaba et al. 1999, Neesen et al. 2002). Loss of the complete LC2 gene in Chlamydomonas cause structural defects in the axoneme (Pazour et al. 1999). Owing to a defect in protein complex assembly all outer dynein arms are missing and mutants have a decreased flagellar beat frequency. Interestingly, another mutant with a deletion in the 3′-end of the LC2 gene is able to produce low amounts of complete LC2 protein which is sufficient for the assembly of a variable number of outer dynein arms. This mutant also revealed reduced swim velocities (Pazour et al. 1999). In mice the inactivation of one Tcte3 gene copy could have a similar effect. However, Tcte3 expression seems to be sufficient for the assembly of outer dynein arms, because defects in the axonemal structure were not detected by electron microscopy. Another putative explanation is that in mouse the TCTE3 polypeptide is not necessary for the assembly of outer dynein arms. Nevertheless, some Tcte3-3−/− males are infertile due to altered sperm motility. The majority of Tcte3-3-deficient spermatozoa are unable to migrate into the female oviduct. Thus, loss of Tcte3-3 function can lead to a higher percentage of completely immotile spermatozoa as well as reduction of both velocity and progressive motility of the remaining motile spermatozoa, which results in asthenozoospermia. In this context it is important to note that TCTE3 seems to play a central role in the activation of sperm motility. In rainbow trout, LC2 is phosphorylated in a cAMP-dependent manner when sperm motility is activated (Inaba et al. 1999). A putative sequence motif for phosphorylation by cAMP-dependent protein kinase is localized in the N-terminal region of salmon and trout LC2 polypeptides, but is also found in human and murine TCTE3 proteins (Inaba et al. 1999, Neesen et al. 2002). It is tempting to speculate that in Tcte3-3−/− spermatozoa-reduced TCTE3 polypeptide amounts result in an ineffective activation by the cAMP-dependent phosphorylation which causes less progressive movement of sperm. In this context, it should be mentioned that TCTEX4 which is encoded by an alternative splice variant of Tcte3 was found to interact with the protein kinase CSNK2B subunit. Casein kinase 2 (CSNK2B) is involved in phosphorylation of a variety of nuclear and cytosolic proteins and could play an important role in cell proliferation and transformation (Bai et al. 2003).

Reduced sperm motility has also been implicated in human male infertility and moreover, asthenozoospermia is considered to be a major cause of impaired male fertility. In several studies, patients with unexplained asthenozoospermia were analyzed for ultrastructural sperm defects (Wilton et al. 1992, Courtade et al. 1998) and in 20–30% of these patients no abnormalities of their sperms were observed. The phenotype of Tcte3-3 knockout mice strengthens the assumption that loss of the Tcte3 dynein light chain function could cause asthenozoospermia in humans.

Electron microscopical investigations demonstrate degeneration of germ cells mainly occurring in elongating spermatids. It is not known, how the loss of Tcte3-3 function results in degeneration of male germ cells and an increased apoptotic rate. However, several recent reports gave evidence for a correlation between Tcte3 expression and apoptosis. In Ovol1 deficient mice, germ cell degeneration and defective sperm production is observed. Ovol1 encodes a member of the ovo family of zinc-finger transcription factors. Microarray analysis indicated that in Ovol1−/− testis ∼200 genes are differentially expressed. One of the genes that are down regulated is Tcte3. Interestingly, down regulation of Tcte3 expression already occurs even if apoptosis is minimal (Li et al. 2005). Further support for the correlation between Tcte3 and apoptosis comes from studies using ethylene glycol monomethyl ether (EGME). EGME induces cell death via an apoptotic process (Ku et al. 1995). Spermatocytes in early and late pachytene stages seem to be more sensitive to the apoptotic stimulus of EGME (Chapin et al. 1984). Tcte3 was found to be downregulated early after EGME-induced testicular lesion (Wang & Chapin 2000). How Tcte3 is involved in the apoptotic process of male germ cells is not understood. However, Tcte3 could directly induce meiotic gene activation, because Tcte3 can bind to the IME2 promoter which is involved in the expression of meiotic genes in yeast (Malcov et al. 2004).

A recent study suggested an interaction between TCTE3 and CREB3L4 (ATCE1) by yeast two hybrid assay (Stelzer & Don 2002). Creb3l4 is a novel mouse cyclic adenosine monophosphate-responsive element-binding protein-like gene that is expressed in postmeiotic male germ cells. It has significant sequence homology to a subfamily of CREB genes with the LZIP peptide as a prototype. CREB plays a crucial role during spermatogenesis and activation of CREB leads to expression of many genes necessary for germ cell differentiation, whereas expression of an unphosphorylated dominant negative form of CREB in Sertoli cells induces apoptosis of spermatocytes (Scobey et al. 2001). CREB3L4 showed specific binding to the nuclear factor-κB-binding element and it was suggested that CREB3L4 could function as an activator of late haploid genes. The physiological correlation between Creb3l4 and Tcte3 is unknown; however, it is tempting to speculate that loss of Tcte3-3 affects Creb3l4 function. This could result in impaired activation of late haploid genes and could lead to spermatid degeneration. Interestingly, Creb3l4-deficient mice are fertile (Adham et al. 2005). However, males reveal reduced spermatogenesis, caused by an increased apoptosis of meiotic and postmeiotic germ cells similar to the disturbances in germ cell differentiation observed in Tcte3-3-deficient mice.

At present, we can only speculate why some Tcte3-3-deficient males are infertile while others are fully fertile. Because Tcte3-3−/− males older than one year are still fertile it is unlikely that infertility is an age-related problem. More likely, modifying genes exist that due to the mixed background could influence fertility in Tcte3-3-deficient males.

Taken together, we have generated mice lacking a functional Tcte3-3 gene supporting a vital role of Tcte3 for male germ cell survival and proper development.

Materials and Methods

Construction of the Tcte3-3 gene disruption vector

Tcte3-3 cDNA was isolated by RT-PCR experiment from murine testicular RNA using primers Tc3-5 5′-GCC CTG TGC TTC GCG GCA TCT GAG C-3′ and Tc3-3 5′-GCT GGT CCT GAG CTA TTC ACA ATA G-3′. The resulting PCR product was cloned into pGEM-T easy vector (Promega) and subsequently sequenced.

For isolation of a Tcte3-3 genomic clone a J1 129/Sv mouse genomic library was screened using Tcte3 cDNA as a probe. A 5 kb SpeI/KpnI fragment and a 3.8 kb XbaI fragment were isolated from the cosmid DNA and cloned into the pBlueScript vector (Stratagene, La Jolla, CA, USA) using the same restriction sites. The 5 kb fragment was restricted using XbaI and EcoRI restriction enzymes. The resulting 3.5 kb fragment was then ligated into XbaI/EcoRI restricted DNA of the pPNT vector (Fig. 2B). The 3.8 kb fragment was restricted by NotI and XhoI enzymes and was ligated into the NotI/XhoI sites of the pPNT vector (Fig. 2B). The resulting targeting vector was linearized with NotI and used for transfection of ES cells.

A 1.4 kb fragment was isolated from the 5 kb SpeI/KpnI fragment by cutting with EcoRI restriction enzyme and was used as a 3′-external probe for screening the DNA of recombinant R1 ES cell clones.

Generation of Tcte3-3 chimeric mice

The ES cell line R1 was cultured as previously described (Wurst & Joyner 1993). Confluent plates were washed in PBS buffer and trypsinized. The cells were suspended in PBS buffer at 2×107 cells/ml. One milliliter of aliquots were mixed with 50 μg linearized targeting vector and electroporated at 250 V and 500 μF. Cells were cultured in nonselective medium in the presence of G418-resistant embryonic mouse fibroblasts. After 36 h the medium was changed containing G418 at 350 μg/ml and ganciclovir at 2 μM. Ten days later resistant clones were picked and transferred into 24-well trays.

Two Tcte3-3 targeted 129/SvJ ES clones (designated as clone 1 and clone 3) were cultured and injected into 3.5 dpc blastocysts derived from C57BL/6J mice, which were then implanted into pseudo pregnant CD1 mice to generate chimeric mice. Nine chimeras were obtained by two independent injections of targeted ES clones. Chimeric offspring were bred with C57BL/6 and 129/SvJ female mice to generate F1 animals on respective genetic background (C57BL/6J×129/Sv and in 129/Sv). Heterozygous offspring were bred to obtain homozygous mice. All animal experimentations were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Goettingen, Germany.

DNA and PCR analyses

Genomic DNA was extracted from ES cells and mouse tails and digested with KpnI. After electrophoresis the DNA was transferred onto Hybond N membranes (Amersham) and hybridized with a 32P-labeled 1.4 kb – 3′-external probe (Fig. 2B). The targeted KpnI fragment has a size of 9.2 kb while the corresponding wild-type allele has a size of 5.4 kb. Absence of additional random integration of the targeting vector was checked by hybridization using a neomycin phosphotransferase II probe.

In order to confirm the successful targeting of the Tcte3-3 locus, a long-range PCR experiment was carried out using DNA from putative heterozygous ES cells and the TaKaRa LA Taq PCR kit (Cambrex, Verviers, Belgium). The first PCR covered 5.1 kb of the genomic sequence using primer set Neo3F and Tc-probe-r (5′-GTA CTG TGC TCC AGA CCA TGA TAA G-3′) to amplify the targeted allele fragment. The Tc-probe-r primer was located outside the 5 kb SpeI/KpnI fragment (Fig. 2B). In a second PCR the primer pair Tc-geno-F (5′-ATG TCT GTT CTA ATC ATG CCT TTT T-3′) and Tc-probe-r was used to amplify the wild-type Tcte3-3 locus fragment of 4.8 kb size (Fig. 2E).

For PCR analysis 1 μg genomic DNA and 10 pmol of each primer Tc-fin-F 5′-CAC AGA TCT TAA GAG AAA GAC TGA GAG AGT C-3′, Tc-fin-R 5′-GGT AGA GAG GTT CAG AGT ATG CTA CCT T-3′ and Neo3F 5′-CCT TCT ATC GGC TTC TTG ACG AG-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% (w/v) agarose gel. The wild-type allele (primer combination Tc-fin-FTc-fin-R) has a size of 900 bp while the mutated allele (primer combination Tc-fin-RNeo3F) has a size of ∼600 bp (Fig. 2F).

Genotyping by quantitative real-time PCR

Owing to the putative presence of highly similar copies of the Tcte3 gene in the mouse genome, genotyping of Tcte3-3 mice was based on quantitative PCR amplification of the neomycin gene (neo) using specific primers Neo-447f 5′-C TTG TCG ATC AGG ATG ATC TGG-3′ and Neo-597r 5′-G GCC ATT TTC CAC CAT GAT ATT-3′. Serial dilutions of sample and standard DNA's were made with buffer AE (Qiagen). DNA of pelota heterozygous mice (Adham et al. 2003) containing one neomycin copy at concentrations of 20, 10, 5, 2.5, and 1.25 ng/μl was used as standard while each sample DNA (Tcte3-3 mice) was used at a concentration of 10 ng/μl. Primers and probes were designed accordingly to generate amplicons <150 bp in length, enhancing the efficiency of PCR amplification.

Real-time quantitative PCR was performed using double stranded DNA binding dye Syber Green PCR Master Mix (Applied Biosystems, Darmstadt, Germany) in an ABI GeneAmp 7000 Sequence Detection System. Each reaction was run in triplicate and melting curves were constructed by using the Dissociation Curves Software (Applied Biosystems) to ensure that only a single product was amplified. Quantitative real-time PCR of DNA specimens and standards were conducted in a total volume of 10 μl with 5 μl of 1×TaqMan Master Mix, 2.5 μl of each forward and reverse primer in a final concentration of 1 μM and 2.5 μl DNA. Thermal cycler parameters were 2 min at 50 °C, 10 min at 95 °C and 40 cycles involving denaturation at 95 °C for 15 s and annealing/extension at 58 °C for 1 min. Standard curves of the threshold cycle number versus the log number of gene copies were generated and were used to extrapolate the number of neomycin copies. Quantitative real-time PCR results were reported as the number of copies for Pelota/neomycin and the mean were calculated.

In situ hybridization, northern blot analysis and RT-PCR experiments

Detection of Tcte3-1, Tcte3-2, and Tcte3-3 transcripts on paraffin sections was performed using digoxigenized ribo-probes as previously described (Neesen et al. 1994). For in situ hybridization a 339 bp Tcte3 cDNA probe was used covering part of exon 2, exon 3, and 76 bp of exon 4 of the Tcte3 gene.

For northern blot experiments total RNA was isolated from adult mouse tissues and from different testicular postnatal stages using the RNA-NOW reagent (ICN, Frankfurt, Germany) according to the manufacturer's instructions. Approximately 20 μg total RNA of each tissue was loaded on a 1.2% (w/v) agarose-formaldehyde gel, electrophoretically separated and transferred onto Hybond C membrane (Amersham). Filters were hybridized with a 32P-labeled 0.7 kb Tcte3-3 cDNA fragment. Integrity and equal amounts of loaded RNA were tested by rehybridizing the blots using a 32P-labeled 1.6 kb BamHI/BglII fragment of the human elongation factor 2 cDNA or a β-actin probe. Hybridization was carried out at 65 °C using Rapid-hyb hybridization solution (Amersham), containing 300 μl denatured salmon sperm DNA (10 μg/μl). Filters were washed 15 min in 2×SSC (0.3 M NaCl, 0.15 M Tris), 0.2% (w/v) SDS at 65 °C and for 10 min in 0.2×SSC, 0.2% (w/v) SDS at 65 °C.

For RT-PCR experiments the OneStep RT-PCR kit (Qiagen) was used. The cDNA synthesis was performed with Tcte3-specific primers Tc3-5 and Tc3-3 and 5 μg total testicular RNA. The resulting 690 bp PCR fragments were separated on a 1% (w/v) agarose gel. As controls for the integrity of the RNA, fragments of the murine glyceraldehyde-3-phosphate dehydrogenase RNA using primers Gapdh-f (5′-CAT CAC CAT CTT CCA GGA GC-3′) and Gapdh-r (5′-ATG ACC TTG CCC ACA GCC TT-3′) were amplified.

Generation of Tcte3-specific antibodies

The Tcte3-3 cDNA was isolated from pGEM-T plasmid DNA by EcoRI digestion and was ligated into EcoRI restricted pET 41b vector DNA (Novagen, Darmstadt, Germany). TCTE3-3-GST fusion protein was purified according to the manufacturer's instructions. Approximately 100 μg/ml 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 specific TCTE3 antibodies was performed by eluting immunoglobulin bound to filter fixed fusion protein as described (Rashid et al. 2006). Moreover, we used polyclonal goat anti-TCTE3 antibodies (Santa Cruz Biotechnology, Heidelberg, Germany).

Fertility test and sperm motility analyses

To examine the fertility of Tcte3-3-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.

Testes and epididymides of wild-type, heterozygous and mutant homozygous mice were dissected in M2 media (Sigma–Aldrich Chemie). Spermatozoa were allowed to swim out of the epididymis for 20 min at 37 °C. A drop of the sperm solution was transferred to an incubation chamber which was set at a temperature of 37 °C. Sperm movement was quantified using the computer-assisted semen analysis system (CEROS version 10, Hamilton Thorne Research, Beverly, MA, USA). At least 2500 motile sperms each of five Tcte3-3+/+, five Tcte3-3+/−, and seven Tcte3-3−/− mice were analyzed.

Statistical analysis was performed using the Statistica software program (StatSoft Inc., Tulsa, OK, USA). Data of fertility and sperm analysis were compared using the Mann–Whitney U test and Student's t-test.

Apoptotic assay

Slides containing testicular paraffin thin sections were processed for TUNEL assay to assess the number of cells undergoing apoptosis using an ApopTag in situ detection kit (Chemicon International, Inc., Temecula, CA, USA). Slides were pre-treated with H2O2 and incubated with the reaction mixture containing TdT and digoxigenin-conjugated dUTP for 1 h at 37 °C. Labeled DNA was visualized with a peroxidase-conjugated antidigoxigenin antibody and 3,3′-diaminobenzidene as chromagen. The percentage of cell death was determined by the number of cells exhibiting brown nuclear staining per section and results were compared to wild-type littermate.

Electron microscopy and immunohistochemical analyses

Testicular and epididymal tissues were cut into small pieces and fixed by immersion in 3.5% (v/v) glutaraldehyde and 1% (w/v) paraformaldehyde in 0.1 M cacodylate buffer (ph 7.4). Specimens were washed in 0.1 M cacodylate buffer containing 0.1 M sucrose and postfixed in 1% (w/v) osmium tetroxide. Semithin sections were stained with toluidine blue and photographed using a Zeiss light microscope. Ultrathin sections were contrasted with 1% (w/v) uranyl acetate in cacodylate buffer and examined by electron microscopy.

For squeeze preparations, testes were isolated and placed into RPMI medium (Sigma–Aldrich Chemie). The testis was dissected and a small piece of a seminiferous tubule was transferred onto a slide. The tissue was covered by a coverslip, squeezed with little pressure and frozen in liquid nitrogen. Thereafter, the coverslip was removed. The slide was incubated for 3 min in 90% (v/v) ethanol and then air-dried. 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. Cells were treated with PBS containing 1% (v/v) Triton X-100 for 5 min. After a blocking step for 1 h in PBS containing 5% (v/v) normal goat serum, 3% (w/v) BSA and 1× roti-block-solution (Roth, Karlsruhe, Germany), cells were covered with 20 μl purified anti-TCTE3 antibody solution (diluted 1:5 with PBS) and incubated overnight in a humidified chamber at 4 °C. Additionally, anti-tubulin MABs were added in a final dilution of 1:50. After four washes with PBT, slides were incubated with secondary antibodies (goat-anti-rabbit-Cy3 and goat-anti-mouse-FITC) in a final concentration of 1:50 in PBS for one hour at room temperature. Finally, slides were washed four times in PBT, covered with a drop of vector shield solution containing DAPI stain and examined using a B×60 microscope (Olympus, Hamburg, Germany) with fluorescence equipment and ‘Analysis’ software program (Soft Imaging System, Muenster, Germany).

Declaration of interest

The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

Funding

This work was supported by the DFG through grant NE 756/1-2.

Acknowledgements

We thank M Schindler, H Riedesel, and S Wolf for help in the generation and breeding of knockout mice. We also thank N Dörwald for providing expert technical assistance. Parts of this work are components of the dissertation: Functional Analysis of the Dynein Light Chain Genes, Dnali1 and Tcte3 of Sajid Rashid and are available online (http://webdoc.sub.gwdg.de/diss/2006/rashid/rashid.pdf).

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    Analyses of testicular Tcte3 expression. (A) Northern blot analysis of testicular RNA from different postnatal developmental stages. Twenty micrograms of total testicular RNA of ten to fifteen day old mice were hybridized with a 32P labeled 0.7 kb Tcte3-3 cDNA fagment. The same blot was rehybridized with a human elongation factor II (EF-2) probe. Localization of Tcte3-transcripts by in situ hybridization. Digoxigenized labeled Tcte3-3 antisense (B and C) riboprobe was used for hybridization of paraffin embedded testicular cross section. Tcte3 transcripts were detected in the cytoplasm of primary spermatocytes (arrowheads) and in round spermatid stages (arrows). No staining of the cytoplasm was observed after hybridization using a sense Tcte3-3-riboprobe (D). Localization of the TCTE3 protein in male germ cells by indirect immunofluorescence (E–Q). TCTE3 specific antibodies were detected with Cy3-coupled antibodies (red color; E–G, K–N, and P), while FITC-coupled secondary antibodies were used to visualize microtubules (Tub), (green color; G, J, L, O, and P). Additionally, DAPI was used to stain the nucleus (blue color; E–Q). In spermatocytes the whole cytoplasm is stained (E and F). In spermatids at early elongation phase TCTE3 is concentrated in the cytoplasm while weaker staining is observed in the nucleus region (I–L). During further differentiation of spermatids intensity of TCTE3 staining decreases. Some protein can be detected around the nucleus and in the cytoplasm surrounding the microtubular manchette (G, arrows). Figure H shows round spermatids incubated with secondary antibodies only. (M–Q) Immunostaining of mouse spermatozoa with anti-TCTE3 and anti-tubulin antibodies. The TCTE3 polypeptide is detectable along the whole flagella (M, N and P). Negligible staining was observed in sperm incubated only with secondary antibodies (Q).

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    Targeted disruption of the mouse Tcte3-3 gene. (A) Schematic representation of the Tcte3 gene locus on murine chromosome 17. Positions of Tcte3 exons are indicated by grey boxes. (B) Schematic representation of the targeting strategy. Wild-type Tcte3-3 locus (top), targeting construct (middle) and targeted allele (bottom). Exon 2 (black box) was replaced by the neomycin resistance gene cassette. Arrows indicate direction of transcription for both the Tcte3-3 and the neomycin resistance gene. (C) Southern blot analysis for genotyping the ES cells. Genomic DNA was restricted using KpnI restriction enzyme. An external probe (gray box, 3′-probe) gave rise to a 9.2 kb (mutant), and a 5.4 kb (wild-type) hybridization signal. (D) Hybridization with the neomycin fragment probe proved homologous recombination event in ES cell clone 1 and 3. (E) Long-range PCR was done using DNA from ES cells 1 and 3. With primers Neo3F and Tc-probe-r a 5.1 kb fragment was amplified from the targeted allele. Amplification using primer pair Tc-geno-F and Tc-probe-r resulted in a 4.8 kb fragment derived from the wild-type allele. (F) PCR genotyping of mice. Wild-type allele yields a PCR product of 900 bp using the Tc-fin-F and Tc-fin-R primers. The targeted allele is expected to generate an ∼600 bp fragment using the primers Neo3F and Tc-fin-R. (G) Quantitative real-time PCR analysis of Tcte3 mice. The average value calculated for the quantity of neomycin for each sample was plotted versus mice identification number. Putative homozygous mice have a value of ∼1 indicating the presence of two neomycin copies, while heterozygous mice containing one neomycin copy have quantities of ∼0.5. E, EcoRI; K, KpnI; S, SalI; N, NotI; X, XbaI; Xh, XhoI; * marks restriction sites deriving from cloning vector. Arrows () indicate positions of primers used for long range PCR.

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    Histology of Tcte3-3−/− testes. (A) shows a testis of a wild-type and a Tcte3-3−/− male of 3 months of age. (B–H) Light microscopically analyses of testicular and epididymal semi-thin sections stained with toluidine blue. (B) In wild-type testis different stages of spermatogenesis are visible and tails of elongated spermatids reach into the lumen of tubules. (C and D) Tcte3-3−/− seminiferous tubules show hypospermatogenesis or only focal spermatogenesis with release of premature germ cells in the lumen (arrowheads). Elongated spermatids frequently display head anomalities (arrows). Percentage of spermatids with head deformations varies in different tubule cross sections and was estimated at ∼15–20% compared to unaffected sperm. (E–G) In the epididymis, biflagellate or bend tail spermatozoa (arrowheads) and spermatozoa with head abnormalities (arrows) are also frequently found. Moreover, fewer spermatozoa are observed in cross sections compared to wild-type epididymis (H).

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    Transmission electron microscopy analyses of Tcte3-3−/− male germ cells. Testicular histology in Tcte3-3−/− males is normal until beginning of spermatid elongation. (A) shows a normal round spermatid (rSp), while elongating spermatid start to degenerate (deSp). In (B) spermatids with normal sperm head (eSp) are located beneath degenerating elongated spermatids (deSp). In spermatids and surrounding Sertoli cells, development of vacuoles (V) becomes obvious (C). The axonemal structure is not disturbed in Tcte3-3−/− sperm tails and outer dynein arms are detectable (arrows, D). In the epididymis often spermatozoa with two flagella or bend tails are observed (E, arrows indicate double tail formation; F, arrows indicate double tail formation or coiled flagella).

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    Apoptosis is increased in Tcte3-3−/− testes. (A and B) show testicular sections of a Tcte3-3−/− male after TUNEL reaction. TUNEL-positive staining is mostly evident in spermatogonia, however also primary spermatocytes and round spermatids are affected. Interstitial cells were not labeled. Only few cells are stained using the TUNEL assay in wild-type testis (C). (D) Quantification of the incidence of TUNEL-positive germ cells in wild-type (+/+) and Tcte3-3−/− males.

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    Results of the computer assisted analysis of sperm motility. Different parameters, (A–F) sperm velocities (μm/s), (G and H) lateral amplitude of the sperm tail beat (μm), (I) beat frequency of the sperm flagella (Hz) and (J) forward movement (%) of wild-type spermatozoa (+/+) and Tcte3-3−/− sperm (−/−) were measured after 1.5 and 3.5 h incubation time. Tcte3-deficient spermatozoa showed significantly reduced swim velocities and a decreased lateral amplitude in comparison to spermatozoa of wild-type and heterozygous mice (P<0.001 by Mann–Whitney U test). No significant difference is observed in the linearity of sperm movement. The beat frequency of Tcte3-3−/− sperm is slightly increased. For all parameters the medians (25–75%) and percentiles (5–95%) are shown.

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