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a Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For recombination to be completed successfully, a number of events must occur in a precise sequence. According to the currently favored model [1], homologous chromosomes synapse and recombination is initiated by a double-strand break in one chromatid of the paired homologous chromosomes. The broken DNA strand is resected in a 5' to 3' direction, leaving a 3' single-stranded tail, which invades an adjacent homologous, nonsister chromatid. The ensuing processes of recombination result in the formation of a heteroduplex DNA molecule that potentially can have mismatched bases. These mismatched bases are corrected by the mismatch repair complex.
The bacterial MutHLS system first described in Escherichia coli is the prototypical mismatch repair complex responsible for correcting mispaired bases resulting from replication errors during mitosis or in recombinant heteroduplexes [3]. In this system, the MutS protein functions in the recognition of mispaired bases. Upon mismatch binding, the MutS protein forms a complex with the MutL protein, which in turn activates the MutH endonuclease, leading to the excision of the mismatched DNA and repair synthesis to replace the aberrant base. Eukaryotic homologues of the MutS and MutL proteins have been identified in yeast, mice, and humans. Although no MutH homologue has been found as yet, other nucleases recently have been suggested to fulfill the role of the MutH enzyme [4]. The MutS homologue MSH2 forms heterodimeric complexes with either MSH3 or MSH6, and these dimers function in the recognition of specific types of mismatches [3]. The mouse homologues of the MutL protein, MLH1 and PMS2, also form a heterodimer and are found in a multiprotein complex with the MutS homologues [3]. Two additional MutS homologues, MSH4 and MSH5, have been described in several species [5–8]. They are expressed only in meiotic cells in Saccharomyces cerevisiae, do not function in repair of mismatches, and are required specifically for the generation of crossovers [5, 8]. These proteins form heterodimers with each other but not with MSH2, MSH3, or MSH6 [9, 10].
Experimental evidence from Saccharomyces cerevisiae has implicated mismatch repair proteins in three facets of recombination that occur at different stages of meiotic prophase [11]. The first identified role for mismatch repair proteins in meiosis was the repair of mismatched bases in the heteroduplex intermediate present during pachynema [12, 13]. More recent data have shown that MSH2 binds in vitro to substrates containing structures resembling recombination intermediates [14] and, in conjunction with MSH6, facilitates their cleavage [15], suggesting a role in the resolution of crossovers near the end of meiotic prophase. Finally, mismatch repair proteins have been demonstrated to repress recombination between highly diverged sequences [16]. The requirement for the mismatch repair proteins in mammalian meiotic recombination is demonstrated by the phenotypes of the Mlh1, Pms2, and Msh5 knockout mice. Both male and female Mlh1-deficient mice are infertile, and the diplotene spermatocytes exhibit premature chromosome separation and meiotic arrest, suggesting a failure to form chiasmata [17, 18]. Of the Pms2-deficient mice, only the males are infertile [19]. Although some sperm were produced by these males, they were all abnormal, and the spermatocytes showed evidence of a defect in the synapsis of homologous chromosomes [19]. Male and female mice lacking Msh5 are also sterile and display aberrant patterns of chromosome synapsis similar to those in the Pms2 knockout mice [20, 21].
An understanding of the molecular mechanisms of recombination in mammals has lagged behind that in yeast, but recent data from knockout mice and other studies examining recombination and cell cycle proteins in mice are beginning to define the molecular requirements for meiotic recombination in higher eukaryotes. The mouse testis provides a readily accessible source of germ cells in all phases of meiosis in which to study recombination from its initiation to resolution. Here, we sought to expand our knowledge of the molecules involved in recombination in the mouse by examining the expression during spermatogenesis of three components of the mismatch repair complex: the MutL homologue, Pms2, and two MutS homologues, Msh2 and Msh3.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male ICR mice were obtained from Harlan Sprague Dawley Inc. (Indianapolis, IN). For studies of gene expression during development, whole testes were removed from mice at postpartum Days 7, 12, 17, 22, and 27 and from adults more than 8 wk old. The testes were decapsulated and immediately frozen in liquid nitrogen. Tissues were stored at -80°C until used for RNA extraction.
Cell Isolation
Enriched germ cell fractions were separated by velocity sedimentation at unit gravity [22]. Spermatogonia were obtained from 8-day-old mice. Leptotene, zygotene, and juvenile pachytene spermatocytes were isolated from 17-day-old mice. Adult mice at least 8 wk old were used to obtain pachytene spermatocytes, round spermatids, and residual bodies. Cells were frozen in liquid nitrogen and stored at -80°C until analyzed for RNA or protein.
Reverse Transcription (RT)-Polymerase Chain Reaction (PCR)
Levels of mismatch repair gene expression were analyzed by RT-PCR using commercially available primers (Stratagene, La Jolla, CA) according to the manufacturer's instructions with the modifications indicated below. The identity of the PCR products was confirmed by sequencing. RNA was extracted from the germ cell fractions or whole testes [23]. The RNA (1–5 µg) was reverse-transcribed using Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD). Ten percent of the first-strand synthesis reaction was used for the PCR. Actin template, separately reverse-transcribed, was included in the PCR reaction mix as an internal control. The PCR was run for 35–40 cycles depending on the primer set, and with 9 cycles remaining, the actin primers were added [24]. Preliminary experiments were performed to determine both the template concentration and the number of cycles required to produce PCR products within the linear range for each mismatch repair gene and the actin control (data not shown). The PCR products were analyzed by separation on a 1% agarose gel in single-strength TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA).
Western Blots
Isolated cell fractions were lysed in 1% NP-40-Tris-NaCl buffer according to a previously described protocol [25]. Protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA). Twenty micrograms of each sample was loaded onto a 7% SDS-polyacrylamide gel (SDS-PAGE). Kaleidoscope molecular weight markers (Bio-Rad) were used to assess molecular weight. After electrophoresis, the samples were transferred to nitrocellulose using a semi-dry transfer apparatus (Bio-Rad) for 45 min. The residual protein in the gel was stained with Coomassie blue to ascertain that an equal amount of protein was loaded in each lane. The blot was blocked with 5% powdered milk in TBS-Tween (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20) for 1 h. Primary antibodies against PMS2 and MSH2 (Calbiochem, San Diego, CA) were incubated with the blots for 1 h at room temperature. The blots were washed once in TBS-Tween at room temperature for 15 min and twice more for 5 min each. The secondary antibody, horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce, Rockford, IL), was reacted with the blots for 1 h at room temperature. The blots were washed once in TBS-Tween at room temperature for 15 min and 4 more times for 5 min each. The bands were visualized using the ECL reagent (Amersham Pharmacia Biotech, Piscataway, NJ).
Immunolocalization
Testes from 7-day-old and adult mice were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for immunostaining. After deparaffinization, the sections were boiled in a microwave oven twice for 5 min in 0.01 M sodium citrate, pH 6.0. The sections were then immunostained as previously described [26]. The antibodies used for the immunostaining of PMS2 and MSH2 were those used for the Western blots. The germ cell nuclear antigen (GCNA) antiserum was the kind gift of Dr. George Enders (University of Kansas Medical Center, Kansas City, KS). Goat anti-mouse rhodamine-conjugated and goat anti-rat fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Pierce) were used to visualize the antibody staining. The sections were counterstained with 4',6'-diamido-2-phenylindole (DAPI; Molecular Probes, Eugene, OR; 100 ng/ml) for 10 min, then washed twice for 5-min each with distilled water. Coverslips were mounted with Prolong Antifade (Molecular Probes). Immunofluorescent staining was observed with an epifluorescence microscope (Olympus America, Melville, NY). Images were captured to Adobe Photoshop (Adobe Systems, Inc., San Jose, CA) with a C5810 color chilled 3CCD camera (Hamamatsu, Hamamatsu City, Japan).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preliminary experiments were performed to establish the PCR conditions for amplifying the mismatch repair genes. Using total RNA isolated from adult testis, the expected bands of 757 base pairs (bp), 926 bp, and 870 bp were obtained for Pms2, Msh2, and Msh3, respectively. The primers used to amplify the actin control produced a band of 514 bp. The identity of each of these bands was confirmed by sequencing (data not shown). To determine the expression patterns for the mismatch repair genes in the germ cell lineage, an RT-PCR reaction was performed on testes at different developmental ages and enriched germ cell fractions.
Testis development The pattern of mismatch repair gene expression was first determined using RNA from testes of increasing developmental age for the RT-PCR reactions. Since the first spermatogenic cycle is synchronous, germ cells more advanced in their development appear sequentially with increasing age. Thus, in addition to somatic cells, testes at Day 7 contain spermatogonia. Leptotene/zygotene spermatocytes appear by 12 days, and pachytene spermatocytes are found by Day 17. At Day 22, more advanced pachytene spermatocytes and round spermatids are present, and by Day 27, elongating spermatids appear.
Expression of Pms2 in whole testes decreased gradually with increasing developmental age (Fig. 1A). The pattern of Msh2 expression showed a similar decline (Fig. 1B). The highest level of expression was found in testes from 7- and 12-day-old mice, in which at first spermatogonia and then leptotene/zygotene spermatocytes are present. With the appearance of later-stage germ cells at Days 17 through adult, the levels of Msh2 fell. By contrast, Msh3 expression peaked at ages 17, 22, and 27 days, coincident with the presence of spermatocytes and spermatids (Fig. 1C). These patterns of expression suggest that the mismatch repair genes are expressed in the developing germ cells.
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Germ cell fractions To ascertain whether germ cells express mismatch repair genes, RNA from enriched germ cell fractions was used for the RT-PCR assay. The levels of expression for Pms2 (Fig. 2A) were highest in spermatogonia and leptotene/zygotene spermatocytes whereas expression was extremely low in both juvenile and adult pachytene spermatocytes, round spermatids, and residual bodies. The expression of Msh2 (Fig. 2B), also was highest in spermatogonia and leptotene/zygotene spermatocytes. Reduced levels of Msh2 were observed in juvenile and adult pachytene spermatocytes and round spermatids. Msh2 expression was nearly undetectable in residual bodies. The pattern of Msh3 expression was entirely different from that of Pms2 and Msh2. Msh3 (Fig. 2C) expression was low in spermatogonia, increased in leptotene/zygotene and juvenile pachytene spermatocytes, and then declined. The germ cell-stage specificity of expression of the mismatch repair genes provides an explanation for the patterns observed during testis development.
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Protein Expression
The availability of suitable antibodies for PMS2 and MSH2 permitted the analysis by Western blot of whether the gene expression pattern reflected the presence of the proteins. The data indicate that the protein levels correlated well with the gene expression patterns for both proteins. The PMS2 antibody recognized a band of 96 kDa in adult testis (Fig. 3A) and in a control somatic cell line (data not shown). A strong band was present in spermatogonia and leptotene/zygotene spermatocytes. A faint band also was found in juvenile pachytene spermatocytes, but the very low levels of PMS2 in more mature pachytene spermatocytes or spermatids could only be detected upon prolonged exposure of the blots (data not shown). A minor band that reacted with the PMS2 antibody appeared in all spermatocyte and spermatid fractions, but the nature of this band is currently unknown. The MSH2 antibody recognized a band of 100 kDa in adult testis (Fig. 3B) and the control cell line (data not shown). MSH2 protein was detected in all germ cell fractions except residual bodies, with the highest levels in the spermatogonia and early spermatocytes and declining levels as germ cell development progressed.
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Protein Localization
Adult testes In adult testes, nuclei of spermatogonia were stained with the PMS2 antibody (Fig. 4A). Less intense staining was detected in spermatocytes and round spermatids. Nuclear localization of MSH2 (Fig. 4C) was observed in spermatogonia, spermatocytes, and round spermatids. Sertoli cells and elongating spermatids were not reactive for either PMS2 or MSH2.
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Immature testes Since mismatch repair proteins function to repair mispaired bases in newly replicated DNA in proliferating cells, it was conceivable that Sertoli cells, which continue to divide in immature testes up to Day 10 in the mouse [27], also would express PMS2 and MSH2. To examine this possibility, testes from 7-day-old mice were stained with antibodies to PMS2 or MSH2. An antibody to GCNA, which is specific for germ cells [28], was used to confirm that the positive cells were indeed germ cells. As can be seen in Figure 5, only the germ cells (arrows), with positive staining for GCNA (Fig. 5, B and D), expressed the mismatch repair proteins, PMS2 (Fig. 5A) and MSH2 (Fig. 5C). Sertoli cells were not stained by any of the antibodies (arrowheads). These data suggest that Sertoli cells did not contribute significantly to the expression of mismatch repair proteins in either the whole testis extracts or the isolated germ cell fractions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mitotic Expression
The mismatch repair system plays a vital role in protecting the genome during mitosis. In replicating cells. mismatch repair proteins monitor the newly synthesized DNA for errors in base incorporation or slippage of the DNA polymerase, which can introduce potentially deleterious mutations [29]. This has been demonstrated in Saccharomyces cerevisiae, in which deletion of mismatch repair proteins causes a mutator phenotype [30]. In humans, defects in the mismatch repair system have been associated with familial nonpolyposis colon cancer [31]. Patients with these tumors and their kindreds exhibit genomic instability and germline mutations in one of the mismatch repair proteins. In addition, transgenic mice with null mutations in Mlh1, Pms2, Msh2, and Msh6 all have a high incidence of somatic tumors and genomic instability [17–19, 32–34]. The high levels of both transcript and protein for Pms2 and Msh2 in spermatogonia are consistent with a role for these proteins in the accurate replication of the germline genome. Since immature Sertoli cells continue to divide mitotically for several days after birth, it might be expected that they also would require mismatch repair proteins for protection of their DNA. The fact that neither PMS2 nor MSH2 were detected in Sertoli cells is probably due to the low and decreasing mitotic index of the Sertoli cells at the age of the testes used for the immunolocalization studies [27]. The absence of detectable amounts of mismatch repair proteins in Sertoli cells further implies that the germ cells account for all of the expression observed in both the whole testes and the germ cell fractions.
Meiotic Expression
Most of our knowledge of the mechanisms of meiosis in general and of the role of mismatch repair in recombination comes from studies in Saccharomyces cerevisiae [1, 35]. Much less is known about the molecular events of recombination in mammals, although some of the major cytological events of meiotic prophase in mouse spermatocytes have been described, including the pairing of homologous chromosomes during early zygonema and completion of synapsis during the pachytene stage [36]. The high levels of both PMS2 and MSH2 in leptotene/zygotene spermatocytes are consistent with a role for a mismatch repair complex in homologue pairing in the mouse. Indeed, male Pms2 null mice are infertile and exhibit improper pairing, with extended segments of chromosomes incompletely synapsed and synapsis of regions of nonhomologous chromosomes [19]. Msh5 knockout mice have a similar phenotype that includes incomplete and nonhomologous chromosome pairing [20, 21]. These data suggest a critical requirement for PMS2 and MSH5 in homologue pairing, but there are some differences in phenotype between these two null mutations. For example, only the Pms2-deficient males, not the females, are infertile despite their production of some abnormal sperm. In contrast, both male and female Msh5 null mice are sterile, and the spermatocytes do not progress beyond the pachytene stage. Furthermore, the analysis of yeast msh5 mutants implies that pairing and heteroduplex formation is normal but that the resolution of recombination intermediates, which occurs later in meiosis, is impaired, leading to a reduction in crossing over [5]. Since the data from yeast suggest that PMS2 and MSH2 function together in a complex, it might be expected that Msh2-deficient mice also would exhibit defects in chromosome pairing, but apparently they do not as both males and females are fertile [33]. Taken together, these observations indicate both sex-specific and species-specific functional differences in the use of specific mismatch repair proteins during meiosis.
The model eukaryotic mismatch repair complex comprises a MutL heterodimer containing PMS2 and MLH1, and a MutS heterodimer containing MSH2 and either MSH3 or MSH6 [37]; thus, it might be predicted that the expression of these genes would be coordinated to facilitate the formation of the appropriate heterodimers. Here Pms2 and Msh2 expression peaked during leptonema/zygonema whereas Msh3 did not peak until the pachytene stage. A similar dissociation of the expression patterns for mismatch repair proteins has been noted previously in vegetative yeast, in which the levels of PMS1, the homologue of mouse Pms2, and MSH2 fluctuated during the cell cycle, with a peak of expression at the G1/S boundary, while MLH1 and MSH3 levels remained constant [38]. These differences in expression patterns could represent a means of regulating mismatch repair activity, or they could indicate that other MutL or MutS homologues participate in the complexes, providing more functional flexibility. Recent yeast data suggest that the MutL homologue, MLH3, interacts with MLH1, but not PMS1, and may function in an MSH3-dependent pathway [39]. Since PMS2 protein levels were quite low in pachytene spermatocytes, it is possible that a MutL homologue like MLH3 could replace PMS2 in complexes that participate in processes occurring late in meiotic prophase.
The MutL homologue, MLH1, and the MutS homologues, MSH4 and MSH5, are all required for the generation of crossovers in yeast [5, 8, 40]. Mice deficient for Mlh1 show a failure of crossing over and premature desynapsis of homologous chromosomes [17, 18]. MLH1 has been localized in discrete foci along the synaptonemal complex during pachynema in mouse spermatocytes, and the foci have been shown to correspond in frequency and distribution to the chiasmata [18, 41]. In yeast, MSH4 also has been localized in foci along the synaptonemal complex [8]. These localization data have supported the interpretation that these proteins are involved in either the formation or stabilization of crossovers. In the present study, the immunolocalization of PMS2 and MSH2 in tissue sections of adult mouse testis reflects the gene and protein expression patterns detected by both PCR and Western blotting. A similar localization of MSH2 was observed in human testis biopsy material [10]. Although several attempts were made to colocalize PMS2 or MSH2 with the synaptonemal complex in spread preparations of spermatocytes, no foci of either protein were detected. This could mean that PMS2 and MSH2 are too diffusely spread or too loosely associated with chromatin to be detected, whereas MLH1 and MSH4 could be anchored in complexes, which concentrates them at specific locations, facilitating their detection. Alternatively, PMS2 and MSH2 could be located within the complex such that the epitopes recognized by the antibodies are not readily accessible, and differences in the staining protocols could account for detection in tissue sections but not on spread chromosomes.
There are several possible explanations for the minor band recognized by the PMS2 antibody on the Western blot. One is that the band represents a posttranslational modification or processing of PMS2, but such changes have not been described previously. The band could represent an alternatively spliced variant, but there is no evidence that Pms2 transcripts are subject to alternative splicing. Only a single band of the expected size was found here, although a splicing event outside the region of the primers would not have been detected. The minor band could be a related protein that cross-reacts with the antiserum. Several human PMS2-related genes have been described, but these encode truncated proteins of much lower molecular size, ranging from 29 to 43 kDa, while the PMS1 protein is slightly larger [42]. The band could result from proteolytic degradation, but this also seems unlikely since all the protein lysates were prepared at the same time and in the presence of protease inhibitors. Further studies will be necessary to determine the nature of this protein.
The results presented here have established the expression pattern of several of the mismatch repair proteins during germ cell differentiation in the mouse. The mismatch repair proteins protect the genome from deleterious mutations and participate in recombination. Although the precise mechanisms of recombination and their timing in mammals have not yet been ascertained, the data emerging from studies of mismatch repair genes in the mouse, including the expression data reported here, indicate that there may be important functional differences between the mouse and yeast in the use of these proteins in meiotic recombination. It will be important to determine whether additional mismatch repair homologues participate in complexes that can account for the observed functional differences. The present study does suggest, however, that the mismatch repair proteins are expressed in mouse germ cells in a manner consistent with at least some of their known functions in both mitosis and meiosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the NIH, HD31376, to M.A.H. 
2 Correspondence: Laura L. Richardson, Department of Biochemistry and Cellular and Molecular Biology, Room M407 Walters Life Sciences Building, University of Tennessee, 1414 Cumberland Avenue, Knoxville, TN 37996-0840. FAX: 423 974 6306; lrichar5{at}utk.edu
Accepted: October 22, 1999.
Received: July 27, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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