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Previous Article | Next Article 
Molecular and Cellular Biology, January 2004, p. 629-637, Vol. 24, No. 2
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.2.629-637.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
INSERM U383 Génétique, Chromosome et Cancer, Clinique M. Lamy, Hôpital Necker Enfants Malades,1 Service de Tri Cellulaire, Institut de Recherche Necker Enfants Malades, Faculté de Médecine Necker, 75015 Paris, France,2 Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands3
Received 28 August 2003/ Returned for modification 26 September 2003/ Accepted 16 October 2003
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ABSTRACT |
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INTRODUCTION |
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DM1 is one of the growing group of diseases caused by dynamic mutations. This group currently comprises more than a dozen diseases, including Huntington's disease (HD), spinocerebellar ataxias, and fragile X syndrome, which are generally associated with CNG trinucleotide repeats (11, 46). The dynamics of the different trinucleotide repeats involved are very similar (intergenerational and somatic instability, bias towards expansions, CTG repeat number threshold for instability etc.), suggesting that the instability mechanisms are common to these diseases, especially those involving CTG or CAG tracts. Several hypotheses have been proposed to explain triplet repeat instability. These have been tested in bacteria, yeasts cell culture models, and transgenic mouse models. Expansions might occur during replication, through DNA polymerase pausing and/or slippage or FEN-1 endonuclease dysfunction in the CTG tract region (9, 20, 41-43, 45, 51). Furthermore, DNA repair mechanisms are clearly involved in instability, as the absence of MSH2, the main component of the mismatch repair pathway, leads in HD mice to the somatic stabilization of expanded repeats, showing that MSH2 is essential for the production of CAG expansions in tissues (29, 54a). Experiments using our DM300-328 transgenic mice, which carry >300 CTG repeats in their large (>45-kb) human genomic environment, revealed that the absence of MSH2 in this context does not stabilize the repeat but instead shifts the instability from expansions to contractions, not only in somatic tissues but also through generations (49). These results demonstrated that MSH2 is required for the formation of somatic and intergenerational CTG expansions. The CTG repeat is probably processed in a different way in the absence of MSH2, leading to intergenerational and somatic contractions. In a knock-in mouse model in which the 3' part of the DMPK murine homolog is replaced by the corresponding human 3' part of DMPK together with about 80 CTG repeats, van den Broek and collaborators observed a decrease of somatic CTG tract expansions in the absence of MSH3, one of the partners of MSH2 involved in mismatch repair and other mechanisms such as recombinational repair (54).
To determine the contribution of germinal mosaicism in the transmitting parent to intergenerational instability, several studies have been carried out on human patients with CAG repeat expansion diseases (HD, dentatorubropallidoluysian atrophy, spinocerebellar ataxia types 1 and 7, or fragile X syndrome) to look for correlations between the level of mosaicism in the sperm of the transmitting males and the size of the repeat inherited by their offspring. A high level of mosaicism was commonly observed in sperm, with this mosaicism overlapping the sizes inherited by the offspring, suggesting that intergenerational instability results mainly from germinal instability in the father (8, 12, 25, 34, 38, 52, 53). However, several studies, including one on fragile X fetuses and one on transmission in Friedreich ataxia families, suggested that intergenerational length changes could also involve a postzygotic instability event (13, 14, 37). In DM1 patients, a high level of CTG instability is observed in sperm from transmitting males, and this mosaicism more or less overlaps the CTG repeat sizes measured in blood from the offspring (22, 30).
A detailed analysis of CTG repeat-size variation between DM300-328 transgenic parents and their offspring carrying different MSH2 genotypes revealed that intergenerational instability probably results from the combination of two distinct events: germinal mosaicism in the transmitting parent and an MSH2-dependent instability event that takes place postzygotically, just after fertilization (49). Development analysis is required to elucidate the postzygotic instability event in our mice. Furthermore, the timing and dynamics of germinal and gametic instability in humans and in mouse models are still not fully understood. In HD mice carrying about 120 CAG repeats, male germinal expansions have been reported to be produced only in the latest stages of spermatogenesis (specifically during the transition from round spermatids to elongating spermatids) through a mechanism involving MSH2 (23, 28, 33). This is surprising, as the gene encoding MSH2 is not expressed during the final stages of mouse spermatogenesis and as no MSH2 protein can be detected in round spermatids in either men (5) or mice (47). In contrast, single-molecule DNA analysis of testicular germ cells from two HD patients suggested that expansions can occur before the end of the first meiotic division (55).
In several trinucleotide-repeat diseases and in our male transgenic mice, a positive correlation exists between the age of the transmitting parent and the repeat-length change in the offspring (48, 50). The evolution of parental gametic mosaicism throughout life may partly account for this association. A very small number of studies have measured repeat-size mosaicism in sperm from the same patient at different ages, but no clear increase was detected after 2 years in men with HD (25). However, the study periods may not be sufficiently long to detect changes in repeat-length mosaicism.
To elucidate the mechanism of germinal instability, we analyzed the status of CTG repeat instability in sperm at different ages to monitor the evolution of gametic mosaicism. By using the single-molecule PCR method, we demonstrated that a strong mosaicism towards expansions is present in spermatozoa, even very early during the mouse reproductive life. This mosaicism clearly increases with age, indicating that expansions are continuously produced through spermatogenesis in our mice. When assaying by normal or single-molecule PCR the levels of CTG repeat mosaicism in seven different germinal cell fractions sorted through a fluorescence-activated cell sorter (FACS), we found that mosaicism towards expansions was already present in spermatogonia. No differences were detected between mosaicisms in spermatogonia and spermatozoa. The presence of expansions in the first stages of spermatogenesis demonstrates that meiosis and postmeiotic mechanisms are probably not involved in germinal CTG repeat expansions. In Msh2-/- males, a majority of contractions were present in spermatogonia and no changes appeared in the following stages, indicating that MSH2 is necessary for the formation of germinal expansions in spermatogonia and that contraction events also occur before meiosis.
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MATERIALS AND METHODS |
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Mouse dissection and blood and sperm DNA extraction. Mice were killed and dissected at 7 weeks, 11 months, or 28 months of age, in accordance with the French veterinary laws. Blood was collected from the heart just after death, and DNA was extracted by the phenol-chloroform method (26). Semen was collected from both vas deferens, and DNA was carefully extracted from spermatozoa to avoid contamination by DNA from other cell types (49, 50).
FACS of different germinal cell types. After dissection at 7 weeks or 11 months of age, both testes were collected and cleaned in 1x phosphate-buffered saline (PBS). After removal of the tunica albuginea, the seminiferous cords were unwound and rinsed in 1x PBS and then carefully minced to obtain the maximum amount of individual germ cells. After filtration through a 70-µm-pore-size cell strainer (Falcon), which retains all cellular aggregates, cells were counted and the cell solution was centrifuged for 10 min at 2,500 rpm (Jouan GR 422 instrument) at room temperature. The cell pellet was resuspended in cold 70% ethanol overnight at a final concentration of 5 x 106 cells/ml. The cell suspension was then centrifuged for 10 min at 2,500 rpm at room temperature and rinsed twice with fresh RPMI 1640 medium (GibcoBRL). RNA was removed by incubating for 30 min at room temperature with 500 µg of RNase A (Boehringer) per ml. Digitonin (10 µg/ml; Sigma) was then added and left for 2 min at 4°C in the dark to permeabilize the cells. Digitonin was removed by centrifugation at 2,500 rpm at room temperature for 10 min. The cell pellet was resuspended at a final concentration of 1.5 x 106 cells/ml in fresh RPMI 1640 with 200 nM MitoTracker GreenFM (Molecular Probes) for 30 min at 4°C in the dark. Propidium iodide (10 µg/ml; Sigma) was added 2 min before analyzing cell labeling with a FACSVantage cell sorter (Becton Dickinson). Cell doublets, apoptotic cells, and necrotic cells were discarded. The combination of the two labels allowed us to sort seven different germinal cell types (44). Each cell fraction was collected in fresh RPMI 1640 before DNA extraction. Cells from each fraction were mounted on microscope slides and analyzed with a fluorescence microscope (Leica) to assay the purity.
Extraction of DNA from FACS-sorted cells. Cell fractions were centrifuged for 10 min at 10,000 rpm (Sigma 1K15 instrument) at room temperature, and the resulting pellet was resuspended in 0.2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1% sodium dodecyl sulfate with 1 M ß-mercaptoethanol for 1 h at room temperature. DNA was then extracted by the phenol-chloroform method.
Analysis of CTG repeat instability in the different germinal cell types. The CTG repeat region of each fraction was amplified with primers 101 and 102. Mosaicism was assayed by separating the amplification products on 3.5% acrylamide denaturing gels as previously described (49, 50).
PCR and single-molecule PCR. Blood, spermatozoa, and spermatogonia DNA was amplified by classical PCR and single-molecule PCR as previously described (35, 49).
Statistical analyses. Statistical analyses were performed with the StatView software by using the Mann-Whitney test (SAS Institute, Inc.).
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RESULTS |
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Figure 1A shows typical results obtained after amplification of the CTG repeat in blood and spermatozoa at the three studied ages by classical PCR with primers flanking the repeat. To ensure that the basal repeat size in each male had no effect on the level of somatic mosaicism, we studied only males that had equivalent numbers of CTG repeats in tail DNA upon weaning. Although no variation in the size of the CTG repeat was detectable in blood DNA, a distinct mosaicism that increased with age was observed in DNA from spermatozoa.
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Dynamics of CTG repeat mosaicism throughout spermatogenesis. The evolution of mosaicism throughout spermatogenesis may make it possible to measure the influence of particular steps of this process on instability, such as mitotic divisions, induced recombination, and meiosis. To determine the precise stage of spermatogenesis at which the germinal CTG repeat expansions are produced, we used two labels (one for DNA and one for mitochondria) and FACS to collect seven cellular fractions corresponding to the different stages of spermatogenesis. We then analyzed the CTG repeat-length mosaicism in each fraction, in mature spermatozoa, and also in blood as a control. We used the DNA intercalating agent propidium iodide to separate different germ cell types according to their DNA content: n (round and elongated spermatids), 2n (spermatogonia, preleptotene primary spermatocytes, and secondary spermatocytes), and 4n (leptotene/zygotene and pachytene primary spermatocytes). Figure 2A shows the DNA content labeling of germ cell samples from an 11-month-old transgenic male. Three major peaks were obtained, corresponding to the different DNA contents encountered during spermatogenesis. However, this labeling alone is not sufficient to separate germ cells from different types carrying the same DNA content, such as spermatogonia and secondary spermatocytes or round and elongated spermatids. We therefore also used a fluorescent label directed against a mitochondrial membrane protein to reflect the activity and repartition of mitochondria throughout spermatogenesis (44). According to the stage of spermatogenesis, germinal cells need more or less energy to perform specific processes and mitochondria have different activities. The combination of these two complementary labels enabled us to sort seven different germinal cell types (Fig. 2B): spermatogonia (R5), preleptotene (R6), leptotene/zygotene (R9) and pachytene (R8) primary spermatocytes, secondary spermatocytes (R7), and round (R3) and elongated (R4) spermatids (44). We ensured the purity of each cell type (estimated to be over 90%) by analyzing slides from each collected fraction by fluorescence microscopy (Fig. 3). After extraction of DNA from each cell fraction and also from blood and from mature spermatozoa, the level of CTG repeat-length mosaicism in each sample was measured. Interestingly, at 7 weeks of age, CTG repeat sizes appeared slightly larger in spermatogonia than in blood, with no detectable difference between spermatogonia and spermatozoa (Fig. 4). At 11 months of age, we observed marked mosaicism in spermatogonia, with larger CTG repeat sizes than in blood. In the following stages of spermatogenesis, including spermatozoa, mosaicism appeared to be equivalent to that observed in spermatogonia. The apparently faster migration products for round spermatids was not observed on repeated gels. The mosaicism was less pronounced at 7 weeks than at 11 months, showing that instability in spermatogonia increases with age in our transgenic mice. Although the separated seminiferous tubules were carefully washed in 1x PBS, some remaining testis interstitium cells such as Leydig cells (2n) may have been sorted together with spermatogonia (44). In adult mice, these cells represent 3.8% of the testicular volume, whereas the seminiferous tubules occupy 89.3% (36). Therefore, the proportion of Leydig cells remaining in the preparation and sorted with the spermatogonia is negligible. In addition, we observed a similar mosaicism pattern in preleptotene primary spermatocytes, the stage immediately after spermatogonia in spermatogenesis, which should not suffer from any contamination by somatic cells through these labels. These observations are consistent with mosaicism presented by the spermatogonia fraction being representative of instability in spermatogonia.
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DISCUSSION |
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We also studied the timing of CTG repeat instability throughout spermatogenesis, to determine the precise stage at which expansions are produced, especially with regard to meiosis, i.e., before, during, or after meiosis. This information should help us to understand the mechanism(s) involved in the production of expansions. We studied CTG repeat instability in spermatogonia, which are renewed and which proliferate during spermatogenesis in adult males; in primary spermatocytes (preleptotene, leptotene/zygotene, and pachytene), in which several molecular events successively occur (induced recombination, chromosome pairing, and DNA synthesis); in secondary spermatocytes, which are subject to the first meiotic division; and in round and elongated spermatids, which undergo major morphological changes and chromatin compaction before finally becoming spermatozoa. In yeast, the instability of CAG/CTG repeats increases during meiosis, mainly due to deletions (10, 21). Once cells enter meiosis, they may undergo molecular mechanisms such as repair associated with homologous recombination, taking place after self-induction of double-strand breaks in an SPO11-dependent manner (21). However, our data show that in our transgenic mice, germinal expansions are produced in spermatogonia and the length of the CTG repeat does not change between spermatogonia and mature spermatozoa. No meiosis-specific mechanisms appear to be necessary for expansions to occur during spermatogenesis in our transgenic mice. Our data suggest that germinal instability results from a mechanism that occurs during spermatogonia, therefore excluding the repair of self-induced double-strand breaks and homologous recombination during meiosis. The increase in mosaicism with age in the DM300-328 males probably results from the accumulation of expansions over lifetime, due to spermatogonia undergoing mitotic divisions and/or the repeated action of DNA repair mechanisms on these cells.
We recently studied the influence of DNA repair on CTG repeat instability by crossing our transgenic mice with mice having knockouts of genes belonging to the different DNA repair pathways, including the Msh2 mismatch repair pathway. We did not observe any stabilization of the repeat in transgenic Msh2-/- mice, unlike in another transgenic mice model (23, 29). In contrast, in the absence of MSH2, instability was shifted from expansions to contractions, both across generations and in tissues including spermatozoa (49). It is still unclear whether expansions in Msh2+/+ mice and contractions in Msh2-/- mice are produced by the same mechanism, with the direction of the instability depending on the presence of MSH2, or whether a totally different mechanism processes the CTG repeat in the absence of MSH2, leading to contractions instead of expansions. We decided to determine whether expansions in Msh2+/+ males and contractions in Msh2-/- males are observed at the same stage of spermatogenesis, in order to obtain further insight into the mechanism(s) involved in both types of change in CTG repeat length. We therefore monitored the CTG repeat contractions throughout spermatogenesis in Msh2-/- males and observed that contractions were already present in spermatogonia, with a length mosaicism similar to that observed in spermatozoa. The role of two partners of MSH2 in somatic instability was recently investigated in knock-in mice carrying the last exons of the human DMPK gene with 80 CTGs (54). The absence of MSH3 leads to the loss of somatic expansions, suggesting that MSH3, like MSH2, is involved in the somatic expansions of CTG repeats in mice. During mouse spermatogenesis, Msh2 is usually expressed to higher levels in spermatogonia and early primary spermatocytes than in other germinal cell types or tissues, whereas the expression of Msh3 is very weak in spermatogonia but peaks in early primary spermatocytes (preleptotene and leptotene) (47). MSH2 is crucial for expansions in spermatogonia, and MSH3 is probably important in germinal instability given its involvement in somatic mosaicism. However, as the level of expression of Msh3 is very low in spermatogonia, the stage where expansions occur in our mice, it is important to determine whether the absence of MSH3, a potential partner of MSH2 in the mechanisms generating expansions, modifies germinal instability. The formation of contractions observed in the absence of MSH2 remains limited to spermatogonia in our mice, as no further deletions were observed in the following stages of spermatogenesis. We showed that contractions and MSH2-dependent expansions are probably produced at the same stage of spermatogenesis in DM300-328 mice. However, this does not imply that expansions and contractions result from the same repair pathways, and the proteins involved in these events remain to be identified.
A role for MSH2 in the generation of germinal expansions via gap repair was previously proposed by Kovtun and McMurray (23). In mice carrying about 120 CAG repeats, expansions during spermatogenesis seem to be limited to the latest stages of spermatogenesis, with no mosaicism detectable in the earlier stages, including spermatogonia, whereas instability appears to occur before meiosis in human HD patients (55). When Msh2 is missing in these transgenic mice, no expansions occur in spermatozoa and the CAG repeat is stabilized, suggesting that germinal expansions are produced in spermatozoa by an Msh2-dependent mechanism. After the completion of meiosis, postmeiotic repair might occur in spermatids to ensure the DNA integrity of future gametes. However, at this step, the expression levels of many repair genes and genes encoding enzymes such as MSH2, MSH3, or PMS2 dramatically decrease, eventually becoming undetectable in elongated spermatids (47). In contrast, in our transgenic mice, germinal expansions appear to be generated in spermatogonia, closely matching what was reported for human HD patients (55). In addition, the absence of MSH2 shifts instability from expansions to contractions. One of the first hypotheses to explain these differences in the dynamics and timing of trinucleotide repeat instability is the different genomic contexts surrounding the repeats in these models, as it has already been suggested that they affect the direction and range of repeat-length changes (6, 9, 18, 26a, 50, 56). Furthermore, the frequency and size of intergenerational repeat expansions were very high in our mice. The different technical approaches could also partly explain the differences. We collected spermatozoa from the vas deferens and then extracted DNA from spermatozoa heads rather than from epididymis sperm. We have previously observed that DNA from epididymal cells (where instability of the repeat is very high, especially in the cauda epidydimis [data not shown]) can contaminate the DNA extracted from sperm without a specific protocol. By combining mitochondrial and DNA labelings, we were able to distinguish seven cell types found during spermatogenesis, whereas only three can be distinguished when only DNA content and cell size are analyzed. The range of trinucleotide repeat-length changes also differs, as does the method used to measure them in the two cases. We used single-molecule PCR on a large set of DNA samples containing single genomes. This is a very powerful tool for resolving the mosaic smears obtained after classical PCR amplification of trinucleotide repeats (35). We were able to detect a broad range of expansions in spermatogonia and spermatozoa, from +1 to +150 CTGs with a mean of about +30 CTGs. These expansions were clearly distinguishable from the PCR stutter classically observed during triplet-repeat amplification, due to polymerase slippage. This overcomes the problem of small expansions and contractions when the DNA input is too great, as discussed by Zhang et al. (56).
Our results imply that CTG repeat-length mosaicism is already present in germinal cells prior to meiosis and that the mechanism responsible for generating expansions is meiosis independent, occurs continuously throughout life, and involves MSH2. This limits the number of molecular mechanisms that can generate expansions. Interestingly, in addition to their role in mismatch repair, MSH2 and MSH3 are also involved in the repair of double-strand breaks by homologous recombination (15). MSH2 and MSH3 together ensure the homology of the sequence used as a template for this type of repair and scan this template for mismatches, small insertions, and deletions. Therefore, this process does not seem to be involved in the production of expansions in our mice, as shown by our previous results with Rad52 and Rad54, both of which are major actors in this repair pathway but which have no effect on the frequency of CTG repeat expansions (49). MSH2 also participates in single-strand annealing (SSA), which is able to repair double-strand breaks but does not involve classical double-strand break repair proteins like Rad51 or Rad54 (40). It would be interesting to study the effects of the ERRC1 and ERCC4 proteins (encoded by the mammalian homologs of the Saccharomyces cerevisiae Rad10 and Rad1 genes, respectively) on CTG repeat-size mosaicism, as these proteins, together with MSH2, are crucial for the ability of SSA to correct DNA damage (39). As trinucleotide repeat tracts are length-dependent breaking sites in yeast (16), the repeated action of the SSA pathway on these particular sequences may be the factor leading to repeat instability in some tissues. In all cases, the occurrence of germinal expansion events in spermatogonia, which are mitotically dividing 2n germ cells, suggests that the mechanisms responsible for instability in germ line and somatic tissues are potentially identical.
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ACKNOWLEDGMENTS |
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This work was supported by grants from INSERM, the Association Française contre les Myopathies (AFM), and the Université René-Descartes Paris V. C.S. was supported by a grant from the Ministère Français de la Recherche et de la Technologie.
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FOOTNOTES |
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