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BOR - Papers in Press, published online ahead of print March 9, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.039123
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BIOLOGY OF REPRODUCTION 73, 123–130 (2005)
DOI: 10.1095/biolreprod.104.039123
© 2005 by the Society for the Study of Reproduction, Inc.

Nucleotide Excision Repair Activity Varies Among Murine Spermatogenic Cell Types1

Guogang Xu 3, Graciela Spivak 7, David L. Mitchell 8, Toshi Mori 9, John R. McCarrey 10, C. Alex McMahan 4, Ronald B. Walter 11, Philip C. Hanawalt 7, and Christi A. Walter 2 3,5,6,12   

Department of Cellular & Structural Biology,3 Department of Pathology,4 Barshop Institute for Longevity and Aging Studies,5 San Antonio Cancer Institute,6 The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900 Department of Biological Sciences,7 Stanford University, Stanford, California 94305-5020 Department of Carcinogenesis,8 The University of Texas M.D. Anderson Cancer Center, Science Park/Research Division, Smithville, Texas 78957 Radioisotope Center,9 Nara Medical University, Kashihara, Nara 634-0821, Japan Department of Biology,10 University of Texas at San Antonio, San Antonio, Texas 78249 Department of Chemistry and Biochemistry,11 Southwest Texas State University, San Marcos, Texas 78666 South Texas Veteran's Health Care System,12 San Antonio, Texas 78201


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Germ cells perform a unique and critical biological function: they propagate the DNA that will be used to direct development of the next generation. Genetic integrity of germ cell DNA is essential for producing healthy and reproductively fit offspring, and yet germ cell DNA is damaged by endogenous and exogenous agents. Nucleotide excision repair (NER) is an important mechanism for coping with a variety of DNA lesions. Little is known about NER activity in spermatogenic cells. We expected that germ cells would be more efficient at DNA repair than somatic cells, and that this efficiency may be reduced with age when the prevalence of spontaneous mutations increases. In the present study, NER was measured in defined spermatogenic cell types, including premeiotic cells (A and B type spermatogonia), meiotic cells (pachytene spermatocytes), and postmeiotic haploid cells (round spermatids) and compared with NER in keratinocytes. Global genome repair and transcription-coupled repair subpathways of NER were examined. All spermatogenic cell types from young mice displayed good repair of (6-4) pyrimidone photoproducts, although the repair rate was slower than in primary keratinocytes. In aged mice, repair of 6-4 pyrimidone photoproducts was depressed in postmeiotic cells. While repair of cyclobutane pyrimidine dimers was not detected in spermatogenic cells or in keratinocytes, the transcribed strands of active genes were repaired with greater efficiency than nontranscribed strands or inactive genes in keratinocytes and in meiotic and postmeiotic cells; spermatogonia displayed low to moderate ability to repair cyclobutane pyrimidine dimers on both DNA strands regardless of transcriptional status. Overall, the data suggest cell type-specific NER activity during murine spermatogenesis, and our results have possible implications for germ cell aging.

aging, gamete biology, gametogenesis, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is a differentiation process that encompasses mitotic, meiotic, and postmeiotic phases. In the mitotic phase, spermatogonia proliferate to expand or replenish the quantity of germ cells. In the meiotic phase, primary spermatocytes accomplish chromosomal synapsis and genetic recombination before undergoing a reductional division to produce secondary spermatocytes that divide to generate postmeiotic haploid cells. In the postmeiotic phase, the haploid spermatids are dramatically remodeled to form spermatozoa. It is essential for organisms to maintain the integrity of spermatogenic cell DNA to ensure that the continuation of the species is not compromised. Indeed, spermatogenic cells have been shown to have a lower spontaneous mutation frequency in a lacI transgene than in somatic tissues or cells [1–3]. Regardless, spermatogenic cells are constantly insulted by endogenous and exogenous agents such as reactive oxygen species (ROS), radiation, and chemicals, which produce a variety of DNA lesions. DNA damage in germ cells, if unrepaired, could interrupt successful reproduction by at least two mechanisms: 1) production of abnormal spermatozoa resulting in male infertility or miscarriage [4, 5]; and 2) generation of de novo germ line mutations resulting in genetic abnormalities and cancer, which may increase childhood mortality [6–8].

Fortunately, spermatogenic cells are equipped with defensive mechanisms to cope with DNA damage, including several DNA repair pathways. Among the DNA repair pathways, the nucleotide excision repair (NER) pathway is the most versatile, and involves at least 25 proteins [9]. NER repairs principally bulky DNA lesions, such as UV-induced photoproducts and benzo(a)pyrene diol epoxide DNA adducts. The NER pathway involves the following steps: 1) lesion recognition, 2) unwinding of the DNA helix around the damaged site, 3) dual incision at 5' and 3' sites flanking the lesion, 4) gap filling by DNA repair synthesis, and 5) ligation [10–12]. NER consists of two distinct subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). GGR is responsible for repairing DNA damage throughout the genome, whereas TCR is targeted to transcription-arresting lesions on the transcribed strand of transcriptionally active genes [13, 14]. It is well documented in somatic cells that UV-induced cyclobutane pyrimidine dimers (CPDs) are repaired at faster rates in the transcribed strands (TSs) of transcriptionally active genes than in the nontranscribed strands (NTSs) and the genome overall [15]. This preferential repair phenomenon has been documented for a number of genes and in a wide range of prokaryotic and eukaryotic cells [9, 12, 15–18].

In addition to the inherent genomic heterogeneity properties of NER, as mentioned above, it has been reported that NER activity in rats may be modified by aging or caloric restriction. For example, it was found that the overall repair rate of CPDs for the albumin gene was approximately 40% less in hepatocytes isolated from 24-mo-old (m/o) rats than in hepatocytes from 6 m/o rats, while dietary restriction could attenuate this decrease [18].

In contrast to the abundant knowledge of NER in somatic cells, little is known about NER in spermatogenic cells. Based on unscheduled DNA synthesis (UDS) as a measure of repair replication associated with NER, DNA repair was observed in spermatogenic cells after treatment with a variety of chemicals, UV irradiation, and x-rays [19– 21]. UDS was not detected in spermatids or spermatozoa [19, 21]. In one recent study, GGR and TCR were measured in rat spermatogenic cells after exposure to UVC irradiation, and limited NER activity was detected in intact meiotic and postmeiotic spermatogenic cells [22]. In contrast, high in vitro dual incision activity was found for the spermatogenic cells [22]. More interestingly, the in vitro incision activity was cell-type specific (i.e., highest in midpachytene spermatocytes and less in primary spermatocytes) in dipoletene of meiosis I, and haploid postmeiotic round spermatids [22]. Thus, although there are limited data regarding NER in spermatogenic cells, the available data indicate that NER activity is reduced compared with that found in somatic cells. These results are surprising in view of the presumed importance of maintaining germ line genetic integrity.

The present study is an initial attempt to determine the extent to which NER operates in mouse male germ cells to protect germ line DNA. Based on the essential need for genetic integrity in all genes for the development of healthy and reproductively fit offspring, we hypothesized that DNA repair activities, including NER, would be as proficient or greater in spermatogenic cells compared with that of somatic cells. Accordingly, we anticipated that all genes would be repaired as if they were transcriptionally active, regardless of transcriptional status, to ensure genetic integrity. To test the hypothesis, highly purified, defined spermatogenic cell types (i.e., premeiotic cells [type A and B spermatogonia], meiotic cells [pachytene spermatocytes], and postmeiotic cells [round spermatids]) were examined. After treatment with UVB irradiation, spermatogenic cells were allowed to repair DNA damage for various times. GGR of CPDs and pyrimidine (6-4) pyrimidone photoproducts (6-4-PPs) was studied in defined spermatogenic cell types obtained from young adult or middle-aged mice. In addition, TCR of CPDs was investigated in these spermatogenic cell types.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Preparations

Defined spermatogenic cell types (i.e., type A and B spermatogonia, pachytene spermatocytes, and round spermatids) were prepared using a STA-PUT apparatus (ProScience Inc., Toronto, Canada) as described previously [23]. Eight-day-old (d/o) CD1 male mice (Harlan) were used to obtain type A and B spermatogonia; and 2 m/o and 14 m/o CD1 male mice were used for preparations of pachytene spermatocytes and round spermatids, from young adult and middle-aged mice, respectively. Purities of recovered cells were based on morphological criteria and were >85% for type A and B spermatogonia and >90% for pachytene spermatocytes and round spermatids. Keratinocytes were prepared from the skin of 1–2 d/o newborn CD1 mice and cultured under low-calcium medium (0.02 mM) (Gibco-BRL Life Technology) according to established methods [24]. Before UVB treatment, keratinocytes were expanded in culture to 90% confluence. This experiment was approved by the Institutional Animal Care and Use Committee of University of Texas Health Science Center at San Antonio.

UVB Irradiation

UVB sources used in the present study were the same as those described previously [25]. A UVB dose-response was performed to identify the exposure that would yield approximately one CPD in the DNA fragments of interest, but with modest cell death. Cell viability was determined by trypan blue-exclusion (Sigma-Aldrich Co.). Three hundred J/m2 of UVB was the appropriate dose for type A and B spermatogonia, whereas 350 J/m2 was used for pachytene spermatocytes, round spermatids, and keratinocytes. Spermatogenic cells were suspended in 10 ml of PBS while on ice and treated with UVB after settling in a 100-mm culture dish. After irradiation, germ cells or keratinocytes were allowed to repair DNA damage for different times (i.e., 0, 12, and 24 h) in germ cell medium (modified Eagle medium, 15 mM Hepes, 1 mM sodium pyruvate, 6 mM sodium L-lactate, and 10% fetal bovine serum) at 33°C or in low-calcium medium (0.02 mM) at 37°C (Gibco-BRL Life Technology) before DNA isolation and subsequent analysis of repair.

Global Genome Repair

Global genome repair activity was measured using an immunoassay method as detailed previously [26–28]. Briefly, cellular DNA in TE buffer (10 mM Tris pH 7.5 and 1 mM EDTA) was denatured by boiling for 5 min, then placed on ice. One volume of 5x SSPE (0.9 M NaCl, 50 mM NaH2PO4, and 5 mM EDTA pH 7.7) was added, and samples were loaded in a slot blot apparatus (Schleicher & Schuell) with vacuum in triplicate for transfer onto a nitrocellulose membrane. Because CPDs are more abundantly induced than 6-4-PPs by UV irradiation, 1 µg of DNA was used for 6-4-PPs assays, whereas 0.1 µg of DNA was used for CPDs assays. The membrane was then baked at 80°C under vacuum for 2 h. Afterward, the membrane was incubated overnight at 4°C in PBS-0.2% Tween-20 (PBS-T) containing 5% skim milk. After multiple PBS-T washes at ambient temperature, the membrane was incubated for 2 h at ambient temperature with monoclonal antibodies specific for CPDs or 6-4-PPs, diluted 1/2000 in PBS. The monoclonal antibodies used were described previously [27, 28]. The membrane was washed as above and incubated for 1 h at ambient temperature with a peroxidase-labeled anti-mouse monoclonal antibody (Amersham) diluted 1/10 000 in PBS. After extensive washing, the membrane was visualized with an enhanced chemiluminescence kit following the manufacturer's recommendations (Pierce). The signal was quantified using a ChemiImager (Alpha Innotech Corp.).

Transcription-Coupled Repair

Transcription-coupled repair was assayed as described elsewhere [29]. Because primary spermatogenic cells do not replicate in vitro, and because no detectable replicated DNA was found for keratinocytes at 12 or 24 h after UVB irradiation, the cesium chloride centrifugation step to separate replicated DNA from parental DNA was not necessary. Three genes were studied: 1) dihydrofolate reductase (Dhfr), an endogenous housekeeping gene; 2) deleted in azoospermia-like gene (Dazl), expressed only in germ cells [30]; and 3) albumin (Alb), a liver-specific gene. Genomic DNA was digested with BamHI restriction endonuclease, which generated a ~17 kilobase (kb) fragment for the Dhfr gene, a ~14 kb fragment for Dazl, and a ~10 kb fragment for Alb. Strand-specific riboprobes were generated using an in vitro expression plasmid carrying a cDNA fragment of mouse Dhfr (generously provided by Dr. Isabel Mellon at Markey Cancer Center, University of Kentucky). A cDNA of the mouse Dazl gene (generously provided by Dr. Howard Cooke at MRC Human Genetics Unit, Edinburgh, U.K.) was subcloned into pGEM (Promega) and a cDNA of mouse Alb (generously provided by Dr. John Papaconstantinou at the University of Texas Medical Branch at Galveston, Galveston, TX) was subcloned into pBluescript II SK+ vector (Stratagene). T4 endonuclease V was generously provided by Dr. Stephen Lloyd (Oregon Health Sciences University, Portland, OR). TCR was measured at least three times for each gene and for each cell type using DNAs obtained from independently prepared cell cultures.

Statistical Analysis

Analysis of variance was used to analyze the difference of means between different spermatogenic cell types or different genes in terms of TCR or GGR activities. A Bonferroni adjustment was used to compare means. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
UVB Sensitivity of Different Cell Types

Seventy-two percent of type A or B spermatogonia remained viable 24 h after irradiation with 300 J/m2 of UVB, whereas ~100% of pachytene spermatocytes, round spermatids, and keratinocytes survived the same dose. After irradiation with 350 J/m2 57% of type A or B spermatogonia survived by 24 h, whereas 90% of pachytene spermatocytes, round spermatids, and keratinocytes survived. Therefore, 300 J/m2 of UVB was used for type A or B spermatogonia in DNA repair assays, whereas 350 J/m2 of UVB was used for other cell types.

Global Genome Repair in Defined Spermatogenic Cell Types

Approximately 60% of 6-4-PPs were removed in all tested spermatogenic cell types at 12 h after UV irradiation (Fig. 1). At 24 h, ~95% of 6-4-PPs were removed in type A spermatogonia, ~85% in type B spermatogonia, and ~80% in each pachytene spermatocytes and round spermatids (Fig. 1). All spermatogenic cell types investigated showed a lower repair efficiency compared with that of keratinocytes, which completely removed 6-4-PPs by 12 h after irradiation (P < 0.05; Fig. 1). In contrast, essentially no removal of CPDs was detected in spermatogenic cell types or keratinocytes at 12 or 24 h after UVB (Fig. 2).



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  		FIG. 1. Repair of 6-4-PPs in different spermatogenic cell types. The top panel is a representative immunoassay for different cell types. The bottom panel represents the quantitative data obtained from three independent experiments, each performed in triplicate. Data are presented as mean ± SEM. A, type A spermatogonia; B, type B spermatogonia; P, pachytene spermatocytes; RS, round spermatids; K, keratinocytes. aP < 0.05 compared to spermatogenic cell types at 12 h. Open bars represent 12 h after treatment and black bars represent 24 h after treatment



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  		FIG. 2. Repair of CPDs in different spermatogenic cell types. The top panel displays a representative immunoassay for different cell types. The bottom panel displays quantitative analysis of the immunoassays obtained from three independent experiments, each performed in triplicate. Data are presented as mean ± SEM. A, type A spermatogonia; B, type B spermatogonia; P, pachytene spermatocytes; RS, round spermatids; K, keratinocytes. The time of 0 h was designated as 100% of CPDs remaining. Open bars represent 12 h after treatment and black bars represent 24 h after treatment

Effect of Age on GGR in Different Spermatogenic Cells

To determine whether spermatogenic cell NER activity changed with age, removal of CPDs and 6-4-PPs were examined in pachytene spermatocytes and round spermatids obtained from middle-aged mice (14 m/o). No significant difference in the removal of CPDs was found at 12 and 24 h after UVB irradiation in spermatogenic cells obtained from middle-aged or young mice (data not shown). In contrast, a significant difference was observed in round spermatids obtained from middle-aged mice compared with that of the same cell type from young mice (Fig. 3). While 69% and 79% of 6-4-PPs were removed by 12 and 24 h, respectively, in round spermatids obtained from young mice, only 21% and 38% of 6-4-PPs were removed by 12 and 24 h, respectively, in round spermatids obtained from middle-aged mice (P < 0.01; Fig. 3). Pachytene spermatocytes from middle-aged mice also exhibited slightly decreased removal of 6-4-PPs compared with that of young mice, but the difference was not significant (P = 0.20 at 12 h and P = 0.18 at 24 h).



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  		FIG. 3. Global genome repair of 6-4-PPs in spermatogenic cell types obtained from young mice (2 m/o) or middle-aged mice (14 m/o). The top panel displays representative immunoassay images for P and RS. The bottom panel displays quantitative data from the immunoassays. Data are presented as mean ± SEM for three independent experiments. P, pachytene spermatocytes; RS, round spermatids. aSignificantly lower than young RS at the same time points, P < 0.05; bsignificantly lower than middle-aged P at same time points, P < 0.05. Open bars represent 12 h after treatment and black bars represent 24 h after treatment

TCR in Defined Spermatogenic Cell Types

Transcription-coupled repair activity was investigated in four spermatogenic cell types (i.e., type A and B spermatogonia [premeiotic cells], pachytene spermatocytes [meiotic cells], and round spermatids [postmeiotic cells]) and keratinocytes for a somatic comparison. Significantly faster repair of CPDs on the TS of Dhfr compared with that of the NTS was found in keratinocytes at 12 and 24 h after UV irradiation (P < 0.05; Fig. 4, Table 1). Type A and B spermatogonia displayed similar repair efficiencies of CPDs on the TS and the NTS at 12 and 24 h (Fig. 4, Table 1). The repair efficiency for type A and B spermatogonia on the TS and NTS was similar to the TS of keratinocytes. In contrast, pachytene spermatocytes repaired CPDs on the TS of the Dhfr gene significantly faster than those on NTS at 24 h after irradiation (P < 0.05; Fig. 4, Table 1). Round spermatids displayed faster repair rates of CPDs on the TS of the Dhfr gene than on the NTS at 12 h (P < 0.05), however, with slower efficiency at 24 h than those in type B spermatogonia and keratinocytes (P < 0.05; Fig. 4, Table 1).



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  		FIG. 4. TCR on different genes in different cell types. The top panel displays phosphorimages of TCR on TS (transcribed strand) and NTS (nontranscribed strand) of Dhfr, Dazl, and Alb genes in different cell types. The bottom panel displays quantitative data of TCR of three independent measurements. A, type A spermatogonia; B, type B spermatogonia; P, pachytene spermatocytes; RS, round spermatids; K, keratinocytes. Solid diamond represents Dhfr-TS, open diamond represents Dhfr-NTS, solid triangle represents Dazl-TS, open triangle represents Dazl-NTS, closed square represents Alb-TS, and open square represents Alb-NTS


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  		TABLE 1. Percentage (mean ± SEM) of CPDs removed in transcribed and nontranscribed genes in different cell types

Dazl is not expressed in keratinocytes [30] and repair of CPDs on the TS was less efficient than for Dhfr, but the differences were not statistically significant (Fig. 4, Table 1). Type A spermatogonia displayed approximately two times faster repair of CPDs on the TS than on the NTS by 24 h after irradiation; however, again, the difference did not reach statistical significance (P > 0.05). Type B spermatogonia removed CPDs with similar efficiency from both strands of Dazl by 24 h. Pachytene spermatocytes displayed significantly faster repair of CPDs on the TS of Dazl gene compared with that on the NTS at 24 h (P < 0.05), and round spermatids also indicated faster repair of CPDs on the TS of the Dazl by 24 h, but the differences were not statistically significant (Fig. 4, Table 1).

Alb is a nonexpressed gene in keratinocytes and spermatogenic cell types. Regardless, approximately 40% of the CPDs were removed from the TS by 24 h for type A spermatogonia and keratinocytes, while only approximately 25% were removed from the NTS in the same amount of time (P = 0.15; Fig. 4, Table 1). Type B spermatogonia displayed approximately 50% and 44% removal of CPDs from the TS and NTS, respectively, by 24 h. Pachytene spermatocytes and round spermatids displayed similar removal of CPDs in the TS and the NTS, however, the removal of CPDs in round spermatids was slower than for type B spermatogonia (P < 0.05; Fig. 4, Table 1).

Repair in Transcribed vs. Nontranscribed Genes

A comparison of TCR revealed that keratinocytes repaired CPDs on the TS of the transcriptionally active Dhfr gene faster than they did the transcriptionally inactive Dazl and Alb genes at 12 and 24 h after UVB irradiation (not statistically significant; Fig. 4, Table 1). A comparison of TCR in two transcriptionally active genes (i.e., Dhfr and Dazl) in spermatogenic cells revealed that all the spermatogenic cell types studied had similar repair efficiency of CPDs on the TS in the two genes (Fig. 4, Table 1). When repair was compared between transcriptionally active genes and inactive genes in spermatogenic cell types, pachytene spermatocytes and round spermatids were observed to repair CPDs on the TS in Dazl gene significantly faster than for the transcriptionally inactive Alb gene at 24 h post-UV irradiation (P < 0.05). However, type A and B spermatogonia repaired transcriptionally active and inactive genes at similar efficiencies at 24 h after irradiation (Fig. 4, Table 1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The successful completion of spermatogenesis depends on a highly orchestrated series of changes in gene expression [31]. Temporal separation of transcription and translation is not uncommon in spermatogenesis, such that RNA is transcribed at an earlier stage of spermatogenesis than translation [31, 32]. Dramatic changes in chromatin condensation transpire during gametogenesis so that the DNA is successfully compartmentalized in the mature spermatozoa. Genes not expressed in early spermatogenic cell types may be expressed in later stages to complete spermatogenesis [31, 32]. In addition, fusion of male and female gametes results in a cell—the zygote—which will give rise to an entire organism. Thus, in the germ line a recurring need exists for changes in the program of genes being transcribed to complete spermatogenesis and then again later, during development, when all genes are eventually needed. It is therefore important to maintain the genetic integrity in all genes in germ cells at different stages during spermatogenesis.

Although many agents create DNA damage repaired through the NER pathways, UVB-mediated DNA damage was selected as the model genotoxin for these initial studies for the following reasons: 1) UV-mediated DNA damage is, perhaps arguably, the best studied DNA damage repaired by NER; 2) because the lesions are well characterized; 3) methods for studying repair of photolesions in the genome and in specific sequences are well established; 4) the repair of photolesions is very similar to the repair of many chemically induced lesions; and 5) chemical half-lives vary substantially and they continue to exert their damaging effects long after they have been removed from the external medium while UV-mediated damage is limited to a very short time surrounding the actual treatment. Initially, GGR was examined following UVB-mediated DNA damage, a classical method of inflicting damage repaired largely by the NER pathway. Mouse primary spermatogenic cells and primary keratinocytes did not significantly repair CPDs in the genome overall. These findings are consistent with results reported for Chinese hamster ovary cell lines [33]. In marked contrast, more than 80% of 6-4-PPs in defined spermatogenic cell types and 100% of 6-4-PPs in keratinocytes were removed within 24 h (Fig. 1). This difference in repair between CPDs and 6-4-PPs in rodent cells can be explained by different damage recognition mechanisms. It is believed that 6-4-PPs are efficiently targeted for GGR by XPC/ hHR23, whereas the less helix-distorting CPDs require an additional factor, UV-damaged DNA binding protein (UV-DDB), before the action of XPC/hHR23 [13, 34]. UV-DDB consists of two subunits, p125 (DDB1) and p48 (DDB2; [35]). In human cells, p48 expression appears rate-limiting for UV-DDB activity [35]. However, p48 is not expressed in most rodent cells [36]; consequently, lesion recognition of CPDs by the GGR pathway is substantially reduced.

Although 6-4-PPs were repaired by spermatogenic cell types, keratinocytes were much more efficient in removing this lesion (Fig. 1). It was previously reported that rat pachytene spermatocytes treated with N-acetyoxy-2-acetylaminofluorene (NA-AAF), which induces C8-AAF adducts on guanine in DNA (C8-AAF adducts is another lesion that NER repairs), removed only 15% of dG-C8-AAF within 24 h [22]. In our study, ~80%–90% of 6-4-PPs were removed during 24 h by mouse spermatogenic cells, including pachytene spermatocytes. The different GGR efficiencies between the NA-AAF study [22] and our study could be due to one of the following: 1) different DNA lesions studied (6-4-PPs vs. dG-C8-AAF), 2) different species studied (i.e., mouse vs. rat), 3) differences in the purities of pachytene spermatocyte cell preparations (>85% in our study vs. 25%–75% in the NA-AAF study), or 4) a combination of these. Notably, in both studies, spermatogenic cell types displayed less efficient NER than somatic cells. A possible explanation for lower NER activity in germ cells compared with that in somatic cells is the lower temperature at which male germ cells are maintained in vivo and in vitro; 33°C for germ cells vs. 37°C for keratinocytes.

The lower NER activity observed in male germ cells initially appears to be counterintuitive. Why should DNA repair be less efficient in germ cells when the DNA is needed to direct development of the next generation? The answer may lie in the nature of the damage repaired by NER. Such damage can impede replication and transcription, and may interfere with the extensive chromatin condensation and remodeling that must occur in the male genome so that the DNA is contained in a streamlined cell, the spermatozoon. Because of the impressively large number of germ cells produced in the testis, it may be unnecessary to rescue germ cells containing damage repaired by the NER pathway. Apoptosis might be invoked to remove cells containing such damage. Apoptosis does occur, with peaks in the first wave of spermatogenesis, principally in spermatogonia and then a second peak occurring in pachytene spermatocytes [37]. In our study, type A and B spermatogonia were more sensitive to killing by UVB than were meiotic and postmeiotic cells. The high level of dual incision activity reported by Jansen et al. [22] could provide the means for signaling cells for removal via apoptosis because they would contain relatively high levels of strand breaks.

An increased risk of de novo germ-line mutations associated with increased paternal age is well recognized [38]. A lacI transgenic mouse model displays an 8- to 10-fold increased spontaneous mutant frequency in spermatogenic cell types obtained from old mice [2]. The increased spontaneous mutant frequency observed in mouse spermatogenic cells obtained from old mice corresponds to a decreased base excision repair activity in spermatogenic cell nuclear extracts and a corresponding reduction of Apex (apurinic/apyrimidinic endonuclease), an endonuclease involved in the base excision repair pathway [39]. The present study demonstrated a significant decline in NER activity directed toward removal of 6-4-PPs in postmeiotic round spermatids obtained from middle-aged mice. This result is consistent with a paternal age effect in which an increased risk for de novo germ-line mutations is realized [38]. At present, it is not known whether the abundance of NER proteins change with age in spermatogenic cell types. However, a decreased abundance of the NER proteins (e.g., XPA and RPA), has been reported to coincide with decreased NER activity in human dermal fibroblasts [40]. Thus it is possible that decreased DNA repair activity, including NER, may contribute to the paternal age effect, and decreased DNA repair may emanate from the decreased abundance of repair proteins.

Although GGR of CPDs is deficient in rodent cells, TCR of CPDs is intact because p48 is not involved in the TCR subpathway. Many studies have demonstrated preferential repair of CPDs on the transcribed strand of transcriptionally active genes in rodent somatic cells [9, 41, 42]. However, very little is known about TCR in spermatogenic cell types. In one study, rat primary spermatocytes removed 16.8% of CPDs on the transcribed strand of the transcriptionally active Scp1 gene by 16 h after UVC irradiation [22]. The authors concluded that male germ cells had a low level of TCR activity, but TCR activity was not studied in spermatogonia [22]. In our study, type A and B spermatogonia were found to be able to repair approximately 50% of CPDs in the TS of transcriptionally active genes (Dhfr or Dazl) by 24 h after UV irradiation (Fig. 4, Table 1). More interesting, the spermatogonial cell types removed CPDs on the TS of a transcriptionally inactive gene (i.e., Alb) as efficiently as in active genes (i.e., Dhfr or Dazl) by 24 h after UV irradiation. Type A and B spermatogonia efficiently repaired CPDs on both strands of a gene similarly regardless of transcriptional status. In contrast, pachytene spermatocytes, a meiotic cell type, demonstrated a preferential repair of CPDs in TS of transcriptionally active genes (i.e., Dhfr and Dazl). Notably, TCR activity in round spermatids decreased compared with that in type A and type B spermatogonia. These results suggest that TCR activity is spermatogenic cell type-specific.

Why would TCR activity vary among spermatogenic cell types? Unrepaired DNA damage in mitotically proliferating cells (e.g., type A spermatogonia) could lead to the production of a large number of mutant gametes. In contrast, unrepaired DNA damage in meiotic (e.g., pachytene spermatocytes) or postmeiotic cells (e.g., round spermatids) would result in only a few mutant gametes. Accordingly, mutations generated in premeiotic cells would have a larger impact on reproductive outcome than mutations in meiotic or postmeiotic cell types. Thus, it seems reasonable that type A and B spermatogonia repair DNA damage relatively efficiently whether it is in a transcribed or nontranscribed gene. In addition, meiotic and postmeiotic cell types in the testis are protected by a blood-testis barrier. Because mitotic type A and B spermatogonia are on the unprotected side of the barrier, they may be subject to more DNA damage from circulating genotoxins than are cells on the protected side. If true, premeiotic cells would have evolved with a greater need for the NER machinery than meiotic and postmeiotic cells. Unbiased and relatively efficient repair (i.e., non-gene-specific and non-strand-specific repair) in premeiotic spermatogenic cell types would also ensure the integrity of genes needed later during spermatogenesis and in development. Decreased NER in round spermatids may emanate from the extensive chromatin condensation that occurs during spermatogenesis [43]. Transition proteins replace histones in round spermatids [43] and the process is associated with additional chromatin condensation. Finally, protamines replace transition proteins in elongated spermatids and the chromatin is further compacted [43]. This high degree of condensation may preclude access of the NER proteins to the damaged DNA. Indeed, it is known that chromatin structure can affect NER activity [44]. For example, UV-mediated lesions in chromatin are less accessible to repair proteins than lesions in naked DNA [45].

TCR operates only on the transcribed strand of active genes, whereas the nontranscribed strand of active genes is dealt with by GGR [46]. As discussed above, GGR of CPDs is impaired in rodents. However, some of the cell types examined in the present study repaired CPDs on the nontranscribed strand at moderate efficiency (i.e., ≥30%) (Table 1, Fig. 4). Such a phenomenon was previously reported in rat myoblasts [47], rat PC12 pheochromocytoma cells [48], human neuron cells [26], and human HL60 promyelocytic leukemia cells [49] after differentiation. This phenomenon has been termed differentiation-associated repair (DAR), and seems to be confined to transcribed genes in differentiated cells [26]. In somatic cells, enhanced repair of the NTS strand occurred in differentiated cells for transcribed genes. In germ cells, the relatively undifferentiated spermatogonia displayed essentially equal repair activity on TS and NTS of transcribed and nontranscribed genes. Overall, the TCR activity of CPDs in spermatogenic cell types appears different from that in other differentiated cells.

In conclusion, the data indicate the following: 1) GGR of 6-4-PPs is more efficient in mouse primary keratinocytes than in spermatogenic cell types, although 6-4-PPs are largely repaired by 24 h post-UVB treatment in spermatogenic cell types, 2) GGR of 6-4-PPs decreases with age in postmeiotic spermatogenic cells, 3) TCR of CPDs in spermatogenic cells is cell type-specific, and 4) TCR efficiency declines in postmeiotic cell types.


   ACKNOWLEDGMENTS
 
We thank Drs. Isabel Mellon for generously providing mouse Dhfr clone, Howard Cooke for providing Dazl cDNA, John Papaconstantinou for providing albumin cDNA, and Stephen Lloyd for providing T4 endonuclease V.


   FOOTNOTES
 
1 Supported by grants ES09136 from the National Institute of Environmental Health Sciences; AG21163, AG19316, and AG24364 from the National Institute on Aging; a pilot project grant from the Children's Cancer Research Institute; the South Texas Environmental Hazards Center; and by the South Texas Veteran's Health Care System. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health or the Veteran's Health Care System. Back

2 Correspondence: Christi Walter, Department of Cellular & Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. FAX: 210 567 3803; walter{at}uthscsa.edu Back

Received: 15 December 2004.

First decision: 30 January 2005.

Accepted: 28 February 2005.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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