Mol. Hum. Reprod. Advance Access originally published online on January 5, 2007
Molecular Human Reproduction 2007 13(3):155-163*; doi:10.1093/molehr/gal107
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Asthenoteratozoospermia in mice lacking testis expressed gene 18 (Tex18)
1 Institute of Human Genetics, University of Göttingen, Göttingen, Germany 2 Department of Anatomy and Cell Biology, University of Giessen, Giessen, Germany 3 Department of Hematology and Oncology, University of Göttingen, Göttingen, Germany 4 Department of Endocrinology, Utrecht University, Utrecht, The Netherlands
5 To whom correspondence should be addressed at: Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK. E-mail: karim.nayernia{at}ncl.ac.uk
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Testis expressed gene 18 (Tex18) is a small gene with one exon of 240 bp, which is specifically expressed in male germ cells. The gene encodes for a protein of 80 amino acids with unknown domain. To investigate the function of (Tex18) gene, we generated mice with targeted disruption of the (Tex18) gene by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J x 129/Sv) are fertile, while they are subfertile on the 129/Sv background, although mating is normal. We showed that Tex18(–/–) males are subfertile because of abnormal sperm morphology and reduced motility, which is called asthenoteratozoospermia, suggesting that (Tex18) affects sperm characteristics. Maturation of spermatids is unsynchronized and partially impaired in seminiferous tubules of Tex18(–/–) mice. Electron microscopical examination demonstrated abnormal structures of sperm head. In vivo experiments with sperm of Tex18(–/–) 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility, as determined by the computer-assisted semen analysis system, is significantly affected, compared to wild-type spermatozoa. Generation of transgenic mice containing Tex18-EGFP fusion construct revealed a high transcriptional activity of (Tex18) during spermiogenesis, a process with morphological changes of haploid germ cells and development to mature spermatozoa. These results indicate that (Tex18) is expressed predominantly during spermatid differentiation and subfertility of the male Tex18(–/–) mice on the 129/Sv background is due to the differentiation arrest, abnormal sperm morphology and reduced sperm motility.
Key words: asthenoteratozoospermia/Tex18, infertility/mouse model/gene targeting
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Introduction |
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The formation and organization of a mammalian sperm head occurs through diverse cellular and molecular processes during spermiogenesis. Such cellular events include sequential changes in the nucleus and the acrosome—which is derived from the Golgi apparatus—in concert with prominent bundles of microtubules, the manchette. However, these complex processes are readily impaired by a variety of intrinsic and extrinsic factors, eventually causing various types of male infertility—such as teratozoospermia—which include the deformation of sperm head (Szczygiel and Kurpisz, 1999). In some cases of male infertility, in addition, the motility of sperm is affected, which is called asthenoteratozoospermia. In recent years, some genes involved in asthenozoospermia and teratozoospermia have been discovered and characterized by generation and analysis of knock-out mice models (Adham et al., 2001; Mendoza-Lujambio et al., 2002; Nayernia et al., 2002; Neesen et al., 2001; Ogawa et al., 2004). However, all of these genes affect either morphology or motility of sperm. The only known genes, which cause sperm abnormality as well as disruption in sperm motility, are Cnot7 gene (Nakamura et al., 2004) and RXR beta gene (Kastner et al., 1996). In the present study, we describe a gene, designated as (Tex18), which affects both sperm morphology and sperm motility.
(Tex18) gene (testis expressed gene—accession number NM 031385) was identified by Page group together with 24 other testis specific genes (expressed in spermatogonia) through cDNA subtraction method (Wang et al., 2001). (Tex18) is a novel murine gene, localized on chromosome 10 and consisting of 1191 nucleotides, containing long 5' untranslated region (UTR) fragment and one exon of 240 bp, encoding an 80 amino acid protein. In order to determine the function of (Tex18) gene, we generated a knock-out mouse model, in which the whole gene was disrupted. We observed subfertility depending on the genetic background, increased percentage of abnormalities in the head shape of the sperm and reduced sperm motility. Testis sections of knock-out mice showed abnormalities, like arrest at the stage of round spermatid differentiation and lack of mature sperm. Furthermore, sperm lacking (Tex18) show reduced motility. These results prove that (Tex18) is involved in the regulation of the process of sperm formation in mice.
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Generation of (Tex18) knock-out mice
The mouse RZPD (The Resource Center and Primary Database, Berlin) genomic library 129/ola was screened using a (Tex18) 453-bp cDNA probe, generated by RT–PCR using primers Tex18F: 5'-GAT CAT TGC TTC AGG CTA CCA-3' and Tex18R: 5'-CTT CAC TTA AAA GGA GGC AAA-3'. Four positive clones were obtained: MPM Gc 121P06227Q2, MPM Gc 121P24194Q2, MPM Gc 121F12439Q2 and MPM Gc 121F07305Q2. For the determination of the restriction map of the (Tex18) locus, cosmid clones were examined by Southern blot analysis. The (Tex18)-targeting vector was constructed by using the vector pTK Neo, which contains a neomycin resistance gene driven by a TK promoter and a herpes simplex virus thymidine kinase gene (tk) cassette. A 5-kb XbaI/XbaI fragment containing the 5'-flanking region of the (Tex18) gene was isolated and ligated in pBS II KS vector. Fragment was then isolated using BamHI and SstII enzymes and cloned in pTK Neo vector, digested with the same enzymes. 3' wing of the construct was generated by RT–PCR, using primers containing restriction sites for SalI and ClaI enzymes. The resulting targeting construct was linearized with SalI and transfected into RI embryonic stem (ES) cells (Wurst and Joyner, 1993), and colonies resistant to G418 (400 µg/ml) and ganciclovir (GANC) (2 µM) were selected. Genomic DNA extracted from ES cell clones using standard methods (Laird et al., 1991) was digested with BstZ17I enzyme, electrophoresed, and blotted onto Hybond C membranes (Amersham, Braunschweig, Germany). Filters were hybridized with a 32P-labeled 5' external probe at 65°C overnight and washed at 65°C with 0.2 x SSC–0.1% SDS (1 x SSC contains 0.15 M NaCl and 0.015 M sodium citrate) for 30 min. A 5' external probe (747 bp) was generated by PCR using primers Text F2: 5'-TAG GCA GAG CTG TTT CCG CTC TGT GAT-3' and Text R2: 5'-GTT CCC CTA GCC TTC TAC CTT CTG AAC-3'. To confirm correct homologous recombination event of the targeted (Tex18) gene and the absence of additional random integration of the targeting construct, a neomycin fragment was used to reprobe Southern blots. One ES clone carrying the disrupted (Tex18) allele was injected into C57BL/6J blastocysts (Wurst and Joyner, 1993), and two male chimeric mice were generated. These males were mated to C57BL/6J and 129/Sv females, and the resulting F1 offspring were genotyped by PCR analyses. Heterozygous (Tex18) animals were crossed to obtain homozygous mice. Genomic DNA was extracted from mouse tails by using standard protocols (Hogan et al., 1986). PCR was carried out for 35 cycles using the following conditions: 30 sec at 94°C, 45 sec at 55°C and 2 min at 72°C. The following primers were used to discriminate wild-type and mutant allele: Tex18PCR F3 [(Tex18) sense], 5'-CCA TTG AAG ACA GTC TTC GGG -3'; Tex18PCR R3 [(Tex18) antisense], 5'-CTC TTA CCG TAC ATC GGC TAC-3'; Neo RI (Neo antisense), 5'-AGG AGC AAG GTG AGA TGA CAG-3'. The amplification products were analysed on 1% agarose gels. A 698-bp fragment of the mutant allele was amplified with primers Tex18PCR F3 and Neo RI whereas primers Tex18PCR F3 and Tex18PCR R3 amplified a 436 bp wild-type product with template DNA from both heterozygous and wild-type animals.
Generation of transgenic mice
Transgenic lines were generated by microinjection into FvB fertilized oocytes according to standard procedures (Hogan et al., 1986). For microinjection, the (Tex18)-enhanced green fluorescent protein (EGFP) transgene sequence was isolated and separated from vector sequences by digestion with SacI and purified after gel electrophoresis by binding to glass beads (BIO 101, Inc., La Jolla). The fragment was quantified and diluted to a concentration of 3 µg ml–1 in injection buffer consisting of 10 mmol l–1 Tris (pH 7.4) and 0.2 mmol l–1 EDTA as previously described (Hogan et al., 1986). The DNA was injected into the pronuclei of fertilized 1-cell mouse embryos. The injected embryos were transferred into FvB pseudopregnant hosts. Transgenic mice harbouring Tex18-EGFP sequence were identified by PCR as previously described (Hogan et al., 1986). Founders were bred with wild-type FvB mice. Transgenic progeny of such crosses were identified and bred together to produce homozygous animals.
RNA isolation and RT–PCR
Total RNA was isolated and cDNA synthesis was carried out with oligo-dT primers. One microgram of total RNA was mixed with 1 µl of oligo (dT) primer (10 pmol µl–1) and sterile water was added to a total volume of 12 µl. To avoid the possible secondary structure of the RNA, which might interfere with the synthesis, the mixture was heated to 70°C for 10 min, and then quickly chilled on ice. After a brief centrifugation, 4 µl of 5 x first strand buffer, 2 µl of 0.1 M dithiothreitol (DTT) and 1 µl of 10 mM dNTPs were added. The content of the tube was mixed gently and incubated at 42°C for 2 min. Then, 1 µl of reverse transcriptase enzyme (Superscript II) was added and further incubated at 42°C for 50 min for the first-strand cDNA synthesis. Then, the reaction was inactivated by heating at 70°C for 15 min. One microlitre of the first-strand reaction was used for the PCR reaction. RT–PCR amplification was performed using specific primers for EGFP and (Tex18). RT–PCR was achieved after 35 cycles of 94°C, 30 sec; 54°C (Tex18) or 58°C (EGFP), 30 sec; 72°C, 2 min (Tex18) or 45 sec (EGFP). Following primers were used for RT–PCR analysis: EGFP: 5'-CTG AAG TTC ATC TGC TGC ACC AAA-3', 5'-TTG AAG TCG ATG CCC TTC AGC-30; (Tex18): 5'-GAT CAT TGC TTC AGG CTA CCA-3', 5'-CTT CAC TTA AAA GGA GGC AAA-3'.
Northern blot analysis
Total RNA was extracted from mouse tissues. Twenty micrograms of RNA was size-fractionated by electrophoresis on a 1% agarose gel containing formaldehyde and transferred to a nylon membrane. The membrane was then hybridized with a 32P-labelled Neo probe.
Fertility test
To investigate the fertility of the (Tex18)-deficient males on a mixed background (C57BL/6J x 129/Sv) and on a 129/Sv genetic background, eight sexually mature male (Tex18) –/– mice from mixed background and 12 from 129/Sv genetic background were mated with both –/– and +/+ females for 3 months. Females were checked for the presence of vaginal plugs and/or pregnancy. Pregnant females were removed to holding cages to give birth. The number and size of litters sired by each group of males were determined in a 3-month mating period.
Sperm analysis
From eight (Tex18) (–/–) and three (Tex18) (+/–) male mice of the 129/Sv genetic background, the epididymides were collected and dissected in IVF medium. Sperm number in cauda epididymis was determined using the Neubauer cell chamber. To determine percentage of normal and abnormal sperm in cauda epididymis, 10 µl of sperm was smeared on superfrost slide, dried, fixed in paraformaldehyde (PFA) and stained with haematoxylin/eosin. At least 200 sperm were counted and the percentage of abnormal sperm was calculated. To investigate the acrosome reaction, spermatozoa were capacitated for 1.5 h in Tyrode's medium and then incubated for 5 min at 37°C in 5% CO2 with Tyrode's medium and 20 µM calcium ionophore A23187
[GenBank]
(Sigma-Aldrich Chemie). For the determination of the percentage of sperm that had undergone acrosome reaction, sperm were fixed and stained with Coomassie brilliant blue R250 (Thaler and Cardullo, 1995). At least 200 spermatozoa from each male were examined for the presence or absence of the characteristic dark blue acrosomal crescent. To investigate the sperm migration in the female reproductive tract, four (Tex18) (–/–) males of the 129/Sv genetic background were mated several times with mature CD1 females. The next day, if a vaginal plug was observed, uteri and oviducts from females were flushed with IVF medium and the sperm number determined.
Sperm motility analyses
Sperm motility analyses were performed as described previously (Nayernia et al., 2002). For statistical analysis, sperm motility measurements of each parameter were pooled for mouse type and for time of observation. Mann–Whitney U-tests were applied in order to define differences in all parameters comparing wild type and mutant (Tex18). Statistical analyses were performed by Statistica (StatSoft Inc., Tulsa, Okla).
Histological analysis
Freshly isolated testes were fixed in Bouin's solution. Tissues were embedded in paraffin and 5-µm sections were prepared. After deparaffination and rehydration, the sections were stained with periodic acid schiff and haematoxylin.
Immunocytochemistry and fluorescence microscopy
Sperm isolated from cauda epididymis were spread on superfrost slides, dried and fixed in 4% PFA in PBS (pH 7.4) for 1 h at ambient temperature. Fixed cells were rinsed in PBS (pH 7.4) and subsequently incubated overnight with primary antibody specific to outer acrosome membrane (OAM), midpiece (mpHGPx) and 
-tubulin. Cells were rinsed three times and incubated in the appropriate Cy3-conjugated secondary antibody (Sigma). All incubations were in PBS (pH 7.4), 5% BSA and 0.1% Triton-X100. For nuclear staining, slides were mounted with DAPI (40, 60-diamidino-2-phenylindole) (Vector) dye. Fluorescence microscopy was performed on a Zeiss fluorescence microscope.
Electron microscopy
Testes and epididymides were fixed with 5% glutaraldehyde in 0.2 M phosphate buffer, post-fixed with 2% osmium tetroxide, and embedded in epoxy (Epon) resin. Selected areas were sectioned and examined by electron microscopy.
FACS analysis of testis cell suspension
Freshly isolated testes were washed in PBS and incubated with collagenase in HBSS at 37°C for 30 min. Tubuli were washed 2–4 times in HBSS medium and centrifuged for 5 min at 150g each time. Cells were then incubated at 37°C in 1 M EDTA containing 0.25% trypsin for 5 min. Ten to twenty percentage of total volume of fetal bovine serum (FBS) was added to stop the reaction. Cells were then filtered through 70-µm pore Falcon filter. Filtrate was centrifuged for 5 min at 150g and the pellet was resuspended in Dulbecco's modified Eagle's medium (DMEM), containing 10% FBS and antibiotics. Cells were then sorted on FACStar Plus (Becton, Dickinson, USA).
Flow cytometric ploidy analysis
For determination of DNA content (1N, 2N and 4N), EGFP positive cells and wild-type cells (control) prepared as for FACS analysis were suspended in 100 µl of PBS. One millilitre of 98% ethanol was added, cells were vortexed and left at 4°C for 30 min. Cells were then centrifuged for 3 min at 150g, washed in PBS with 1% FBS, treated with 0.25% Triton-X100 in PBS for 5 min, washed again 3 times with PBS + FBS and finally stained for cell cycle measurement with propidium iodide (20 µg/ml) in PBS + FBS, with RNAse A (100 µg ml–1).
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Expression analysis of (Tex18) gene
(Tex18) contains a long 5' UTR fragment of 894 bp and one exon of 240 bp, encoding a 80 amino acid protein (Figure 1A). Expression of this gene was shown to be restricted to the testis (Wang et al., 2001). We have confirmed this data by RT–PCR using RNA extracted from nine different tissues and primers Tex18F and Tex18R amplifying 453-bp fragment of (Tex18) gene (Figure 1B).
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Expression analysis of (Tex18)-EGFP transgene
For the detailed studies of (Tex18) expression, transgenic mice lines were generated. For this purpose, a construct harbouring 1.6 kb promoter region of (Tex18) gene and EGFP was generated (Figure 2A). Two transgenic lines Tex18/4 and Tex18/16 were obtained, both expressing EGFP under (Tex18) promoter exclusively in testis, similar to endogenous gene. This specific expression was shown by northern blot analysis, using EGFP probe and by RT–PCR, using EGFP specific primers (Figure 2B and C). Histological sections of testis of (Tex18) transgenic mice demonstrated green signal emitted by sperm in the lumen of seminiferous tubules, not observed in the wild-type control. It suggests that the (Tex18) gene is expressed predominantly in post-meiotic stages of spermatogenesis (Figure 2D). Expression of green fluorescent protein gave us an opportunity to isolate EGFP-expressing cells of testis by fluorescence activated cell sorting (FACS). Whole testis cell suspension from transgenic male was applied for FACS analysis and a distinct population of EGFP positive cells was observed (Figure 2E). Interestingly, the percentage of EGFP positive cells increases with the age of the male. Only
3% of cells were EGFP positive in 5–20-day-old males, but 20% or more EGFP positive cells were found in the testes of adult males (Figure 2F). As post-meiotic germ cells occur in the male gonad at about 20 dpc, this observation supports the hypothesis that (Tex18) is expressed mainly during post-meiotic stages. To enforce the hypothesis, FACS analysis was applied for DNA content measurement. The percentage of cells in haploid (1N), diploid (2N) and tetraploid (4N) stage was determined among EGFP positive cells and total testis cell suspension from wild-type controls. This assay showed enrichment in number of haploid cells in the EGFP positive cells as compared with wild-type controls, as the ratio of haploid cells to all cell types (1N/1N + 2N + 4N) It was 1.39 in EGFP positive cells and 0.53 in wild-type controls (Figure 2G).
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Generation of (Tex18)-deficient mice
In order to disrupt the (Tex18) gene, a knock-out construct was generated by replacing whole gene with the neomycin gene (Neo) (Figure 3A). ES cells were transfected with the targeting construct and homologous recombinants were selected. DNA from positive clones was isolated and screened by Southern blot analysis using a 0.8-kb external probe generated by PCR. A fragment of 7 kb in the wild-type allele and a 12-kb fragment in the disrupted allele were recognized (Figure 3B). One recombinant positive ES cell clone was injected into C57BL/6J blastocyst and gave rise to two chimeric males that transmitted the mutant (Tex18) allele to their offspring. Males were bred with C57BL/6J and 129/Sv females to establish the (Tex18)-disrupted allele on a C57BL/6J x 129/Sv hybrid background and on a 129/Sv background, respectively. Heterozygous animals were crossed together and showed normal fertility with 29% of their homozygous offspring on the C57BL background and 25% on 129/Sv background. Examples of genotyping by PCR are shown (Figure 3C). Disruption of the (Tex18) gene resulted in the complete absence of the expression of gene, as it was shown by RT–PCR (Figure 3D).
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Analysis of fertility in (Tex18)-deficient mice
To evaluate consequences of (Tex18) disruption, we have tested fertility of homozygous males on both backgrounds, mating them with homozygous and wild-type females. All of the matings were performed for 3 months. Results showing total number of born mice and mean litter size with standard deviations are summarized in Table I, together with the results of breeding of heterozygous males. Mean litter size of offspring of HO x HO mating on both backgrounds was significantly different from litter size of HE x WT and HO x WT matings on the mixed background and from HE x WT mating on 129/Sv background, (P < 0.05, Mann–Whitney U-Test). Interestingly, among 12 homozygous males of 129/Sv background used for 3-month long fertility tests, 8 gave usually no more than 2 offspring, while another 4 were completely infertile. Therefore, we decided to investigate this line in detail.
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We observed no significant differences in sperm number in cauda epididymis of (Tex18)-deficient males compared to wild-type control (Mann–Whitney U-test), but a significant increase in number of sperm head abnormalities was observed (P < 0.05) (Table II). To evaluate sperm quality, its motility was measured after 1.5, 3.5 and 5.5 h. Essential differences in the sperm motility and progressive movement of (Tex18)-deficient mice were observed, in comparison to wild type. Proportions of motile and progressive movement showing spermatozoa of mutant mice were always 15–30% lower than that in controls (like 40% of motile mutant sperm versus 60.5% of motile sperm from control at 3.5 h or 19.6% of mutant sperm showing progressive movement versus 38.5% in wild type at 3.5 h). For further investigation of sperm motility, the following parameters were evaluated in detail: curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), beat frequency (BCF), straight forward movement (STR) and lateral head amplitude (ALH) (Figure 4). Statistically significant differences were observed for each parameter with P < 0.001, with three exceptions: for STR at 1.5 h, when P = 0.0017, BCF at 3.5 h, when P = 0.006 and STR at 5.5 h, when P = 0.024. Differences were especially high for all velocities and lateral head amplitude. This finding indicates that motility of (Tex18)-deficient sperm is greatly reduced. Testis of (Tex18)-deficient mice were of normal weight and size. Histological sections of homozygous male testis revealed abnormalities in seminiferous tubule structure. Efficiency of spermatogenesis was disturbed—it was very often arrested at the stage of round spermatid (they did not start the elongation process or did it too slowly), therefore reduced numbers (or sometimes not any) of elongated spermatids and mature sperm were observed in the lumen of seminiferous tubules. Vacuoles were observed in round spermatids, between nucleus and cytoplasm, on the other side of acrosome. They were observed also in Sertoli cells. Apoptotic spermatocytes were seen regularly in many seminiferous tubules; sometimes this leads to tubules where the spermatocytes were largely missing. Diploid spermatids, indicating problems in second meiotic division, were also often visible. Apoptotic round spermatids and rarely morphologically abnormal elongated spermatids were also seen (Figure 5). As it was mentioned before, an increased number of abnormal sperm were observed. Figure 6A shows typical sperm head abnormalities observed. Immunostaining of sperm fixed on the slides was applied in order to investigate abnormalities in detail. OAM antibody directed against OAM showed abnormal structure of acrosome in sperms of abnormal head shape (Figure 6B). Electron microscopy confirms abnormal acrosome shape in sperm, as well as formation of two tails in one spermatozoon, which was observed for some sperm (Figure 6C and D). Immunostaining using antibody against mitochondrial phospholipid hydroperoxide glutathione peroxidase (mpHGPx) localized in sperm midpiece and antibody directed against
-tubulin (localized in sperm tail) revealed no abnormalities of these structures (data not shown). To answer the question of whether the abnormal shape of the acrosome has a real effect, we examined the response of spermatozoa from mutant and wild-type mice to the calcium ionophore A23187.
[GenBank]
No significant difference in acrosome reaction between (Tex18) (–/–) and wild-type spermatozoa was observed, since 75.5% of sperm of homozygous mice and 82.5% sperm of wild-type control underwent normal acrosome reaction.
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In order to investigate if reduced motility has an effect on the migration of (Tex18)-deficient spermatozoa through the female genital tract, vaginal plug test was applied to determine the number of sperm in uterus and oviducts. Four (Tex18) (–/–) males were mated several times with mature CD1 females. The next day, if a vaginal plug was observed, uteri and oviducts from females were flushed with IVF medium and the sperm number was determined. We observed a 2-fold twice much reduction in the number of spermatozoa in uteri of females (0.4 x 106 in (Tex18) (–/–) versus 0.8 x 106 in wild-type control). A more marked decrease was observed in sperm number determined in oviducts (21 ± 58 in case of mutant and 301 ± 15 in case of control) (Figure 7).
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(Tex18) gene was described for the first time by Wang et al., (2001) in a systematic search for genes expressed in mouse spermatogonia, but not in somatic tissues. In the present study, we could confirmed testis specific expression and expression of (Tex18) in spermatogonia. However, we demonstrated that expression of (Tex18) gene is not restricted to spermatogonia, but occurs also in other stages of male germ cell development. We observed high expression levels of (Tex18) during spermatid differentiation. This expression could be detected only using RT–PCR analysis, but not with northern blot analysis (data not shown). However, we could detect expression of EGFP under control of (Tex18) promoter region using northern blot analysis (Figure 2B). One possibility for explanation of this discrepancy is that Tex18-EGFP transgene is integrated in the genome in higher copy number than endogenous (Tex18) gene, and therefore expression level of Tex18-EGFP transgene is higher than endogenous (Tex18) gene, and this expression can be detected by northern blot analysis. The segregation of Tex18-EGFP transgene during several generations indicates that Tex18-EGFP transgene is integrated in a single chromosomal locus. Another possible explanation is that (Tex18) acts at the RNA level and not at the protein level. At the protein level, no functional protein domain could be identified.
Breeding of the (Tex18) (–/–) mice revealed that the effect of the null mutation on male fertility depends on genetic background. The asthenoteratozoospermia in (Tex18) (–/–) males, which causes subfertility on the 129/Sv background and normal fertility in hybrid 129/Sv x C57Bl/6 background, indicates that the mutation in (Tex18) locus interacts with as yet unknown modifying genes. Interestingly, highly variable penetrance of male infertility on different genetic backgrounds has also been reported for mice carrying targeted null mutations of other genes affecting spermatogenesis, including sperm mitochondrion-associated cystein-rich protein (Smcp), dynein heavy chain and Tnp2 (Nayernia et al., 2002; Neesen et al., 2001; Adham et al., 2001).
The reduced number of spermatozoa recovered from the oviducts of females inseminated by (Tex18) (–/–) 129/Sv males pointed towards a defect in sperm motility. This hypothesis was confirmed by significant decrease in sperm velocities measured in vitro. It is possible that reduced motility is a secondary effect and is caused by abnormal sperm head morphology. Taken together, our data suggest that the subfertility of male (Tex18) (–/–) 129/Sv mice is caused by reduced sperm motility in the female reproductive tract. Our data also show that the failure to penetrate the zona pellucida is not caused by an impaired acrosome reaction. Conceivably, the impaired motility and abnormal morphology of the (Tex18)-deficient spermatozoa affects hypermotility, which is required for sperm migration and fertilization (Bedford, 1998, Nayernia et al., 2002).
The loss of (Tex18) function leads to higher percentage of immotile and abnormal spermatozoa. Loss or reduction in sperm motility together with sperm abnormality is known as asthenoteratozoospermia—one of the primary causes of untreatable infertility or subfertility in men (Acacio et al., 2000; Hristova et al., 2002). In about 70 to 80% of patients with asthenozoospermia, the impaired sperm movement is correlated with abnormalities in sperm structure (Courtade et al., 1998; Wilton et al., 1992) and is therefore similar to the phenotype of the (Tex18)-deficient mouse. Identification of human (Tex18) gene and screening for mutation could clarify some cases of asthenoteratozoospemia in men. Recently, we reported that targeted disruption of Smcp and dynein heavy chain genes causes asthenozoospermia (Nayernia et al., 2002; Neesen et al., 2001) and deletion of Tnp2 (transition protein 2) gene causes teratozoospermia in mice (Adham et al., 2001). In (Tex18)-deficient males, both asthenozoospermia and teratozoospermia were observed. Little is known about inherited defects in sperm morphology and sperm motility and, to our knowledge, (Tex18) and recently reported Cnot7 (Nakamura et al., 2004) and RXR beta (Hogan et al., 1986) deficient mice are the only mouse models for asthenoteratozoospermia. (Tex18)-deficient mice provide an experimental model to study the mechanisms of asthenoteratozoospermia and spermatid differentiation.
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We are grateful to M. Schindler and H. Riedesel for assistance with the generation of knockout and transgenic mice. We thank D. Meyer, B. Sadowski and A. Schneeberg for excellent technical assistance. This work was supported by the DFG grant (Graduiertenkolleg 242 to W.E.).
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* N.B. An error was made in the initial online pagination of Molecular Human Reproduction 13/3. The page span of this article was originally shown as 15–23. The publisher wishes to apologise for this error.
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Submitted on September 14, 2006; resubmitted on November 8, 2006; accepted on November 9, 2006.
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