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Regular Article |
c Departments of Pharmacology and Therapeutics, and
d of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada H3G 1Y6
ABSTRACT
During germ cell development different spermatogenic cell types show remarkable variation in their susceptibility to stressful stimuli. Various cellular mechanisms are triggered in germ cells after exposure to stress, but the expression of only a few of the genes involved in such pathways has been studied during spermatogenesis. In the present study we determined the expression profiles of 216 stress response genes in isolated rat germ cells (pachytene spermatocytes, and round and elongating spermatids) using cDNA atlas arrays. Of the 216 genes studied, 86 were detected in pachytene spermatocytes, 82 in round spermatids, and 52 in elongating spermatids. Fifty percent (48) of the total number of genes detected during spermatogenesis were detected in all three cell types while nearly 25% (25) were expressed exclusively in pachytene spermatocytes and round spermatids; some cell specific transcripts were observed also. The use of the K means clustering method allowed us to group genes by their pattern of expression during spermatogenesis; five specific expression profiles were obtained and analyzed. To determine how stress response genes are regulated throughout spermatogenesis, we examined the expression of genes involved in stress response mechanisms such as heat shock proteins-chaperones, DNA repair, and oxidative stress. Genes belonging to these families were differentially expressed during germ cell development. We suggest that the differential expression of stress response genes during spermatogenesis contributes to the selectivity of the susceptibility of germ cells to stress.
apoptosis, gamete biology, gametogenesis, spermatid, spermatogenesis, toxicology
INTRODUCTION
Spermatogenesis is a highly regulated process that is initiated by the division of stem cells (spermatogonia) to form spermatocytes. Preleptotene spermatocytes undergo two meiotic divisions and give rise to round spermatids. Subsequently, these cells enter spermiogenesis and undergo major morphological changes to form mature spermatozoa [1]. The complex changes arising during spermatogenesis are orchestrated by the expression of genes encoding proteins that play essential roles during specific phases of germ cell development. Gene expression during spermatogenesis has been assessed for a limited number of genes [2, 3]. The expression of a large number of these genes is developmentally regulated during spermatogenesis. Both transcriptional and translational control mechanisms are responsible for temporal and stage-specific expression patterns [4].
During germ cell development, different spermatogenic cell types showed remarkable variation in their susceptibility to stressful stimuli. For example, spermatogonia were more sensitive to damage caused by x-ray irradiation [5], whereas round spermatids were highly sensitive to alkylating agents such as cyclophosphamide [6]. A differential response to heat stress was reported, with spermatogonia and spermatozoa being the most resistant, whereas spermatocytes and early spermatids showed the highest degree of sensitivity [7]. A variety of cellular mechanisms such as DNA repair [8], the heat shock response [9], apoptosis [10], and oxidative stress response [11] were triggered after exposure to different types of stressors. To date, the expression of only a limited number of genes involved in these mechanisms has been studied during spermatogenesis [2, 3]. The analysis of the expression of stress response genes during normal germ cell development is necessary to assess how these genes are regulated throughout spermatogenesis.
In the present study, we examined the expression of 216 stress response genes involved in different defense mechanisms. Gene expression was assessed in three isolated spermatogenic cell types: pachytene spermatocytes, round spermatids, and elongating spermatids, using the Atlas Rat Stress-Toxicology array (Clontech, Palo Alto, CA). Our results demonstrate that stress response genes are differentially expressed during spermatogenesis; we suggest that this contributes to the stage specificity of the susceptibility of germ cells to stress.
MATERIALS AND METHODS
Animals
Adult male Sprague-Dawley rats (425–450 g) were obtained from Charles River Canada (St. Constant, PQ, Canada). They were maintained in a 14L:10D cycle and provided with food and water ad libitum. Rats were injected with saline (1 mg/ml i.p.) and killed by decapitation 16 h later. Five animals were used in this study. All animal handling and care was done in accordance with the guidelines established by the Canadian Council on Animal Care.
Isolation of Spermatogenic Cell Types
The procedure for the isolation of spermatogenic cells was adapted from that previously described by Bellvé et al. [12]. Briefly, testes were removed, decapsulated, and incubated under 5% CO2 in air, with continuous agitation (120 cycles/min) at 33°C for 12 min in RPMI media (RPMI medium 160; Gibco BRL, Burlington, ON, Canada, supplemented with 0.5 mg/ml collagenase [C9891], Sigma, Oakville, ON, Canada). Cells were allowed to sediment and were washed three times with 24 mM NaHCO3, 0.066% C3H5O3Na (pH 7.2) to remove the dissociated interstitial tissue and remaining blood cells. Seminiferous tubules were incubated with 0.5 mg/ml trypsin (type 1, T8003; Sigma) and 1 µg/ml DNase I (type 1, DN-25; Sigma) under the same conditions as above for 16 min. After dissociation with a Pasteur pipette, cells were filtered through a 70-µm nylon mesh (Sefar Canada, Montréal, PQ, Canada) in the presence of 2 µg/ml DNase I (Sigma). The filtrate was washed three times with 0.5% BSA (Sigma) in RPMI and centrifuged (1000 x g; Beckman T-J-6 centrifuge; Beckman, Montreal, PQ, Canada). After each wash at 4°C, cells were resuspended in 10 ml of 0.5% BSA and filtered trough a 0.56-µm nylon mesh (Sefar Canada). A cell count was done using a hemocytometer, and a total of 5.6 x 108 cells in 25 ml of 0.5% BSA in RPMI were loaded in a velocity sedimentation cell separator apparatus (STA-PUT; Proscience, Don Mills, ON, Canada). Cells were introduced at a rate of 10 ml/min followed by a 2%–4% BSA gradient that allowed their separation by sedimentation velocity. The gradient was allowed to sediment for 1 h 45 min, then 100 fractions were collected at a rate of 13.3 ml/min. Pachytene spermatocytes, round spermatids, and elongating spermatids were distinguished by their chromatin packaging and morphological characteristics using light microscopy. Only fractions with high purity (>85%) of a specific spermatogenic cell type were pooled together. The average purity for each cell type was as follows: pachytene spermatocytes (89%), round spermatids (89%), and elongating spermatids (94%). Five separate cell separations were run, each using both testes from one animal.
RNA Extraction
Total RNA extraction was done with phenol/chloroform [13] followed by incubation with RNase-free DNase I (Sigma) for 1 h at 37°C to avoid contamination by genomic DNA. RNA quality was assessed by gel electrophoresis and spectrophotometric reading.
Probe Preparation and Hybridization to cDNA Atlas Arrays
Ribonucleic acid (5 µg) was reversed transcribed using Moloney-murine-leukemia virus (MMLV) reverse transcriptase and radiolabeled with [
-32P]dATP (Amersham Pharmacia Biotech, Baie d'Urfé, PQ, Canada; 10 µCi/µl). To purify labeled cDNAs from unincorporated 32P nucleotides, probes were filtered through CHROMA SPIN-200 DEPC-H2O columns (Clontech, Palo Alto, CA). The radioactive probes were added to nylon membranes (Rat Stress and Toxicology Atlas Arrays, Clontech) that contained 216 cDNAs spotted in duplicate, prehybridized with salmon testes DNA and ExpressHyb (Clontech) at 68°C; hybridization was allowed to occur overnight with continuous agitation at 68°C. After 18 h, the arrays were washed three times for 30 min with washing solution 1 (2x SSC, 1% SDS) and once for 30 min with washing solution 2 (0.1x SSC, 0.5% SDS) with continuous agitation at 68°C. After a final wash with 2x SSC at room temperature, the membranes were wrapped and exposed to a PhosphorImager plate for 24 h.
Analysis of Gene Expression
Arrays were visualized after scanning with a PhosphorImager (Storm; Molecular Dynamics, Sunnyvale, CA) and the image imported to AtlasImage software (version 1.5; Clontech) for quantification. The data generated by AtlasImage software were imported into GeneSpring (version 3.2.8; Silicon Genetics, Redwood, CA) for further analysis. The raw signal intensity obtained was the result of the subtraction of the background level on each membrane from that of each gene's intensity. The threshold for gene detection was set at a raw signal intensity for a given gene of two times the background intensity on that membrane for at least three out of five replicates. In order to minimize experimental variation and permit the comparison of different experiments to one another, we normalized our data with either of two different methods (GeneSpring). For experiment to experiment normalization, the raw value of a specific gene was divided by the median intensity of that single membrane. This was calculated for all five replicates and averaged to generate what is referred to as the relative intensity of a specific gene. In addition to the previous normalization, a gene-to-gene normalization was done exclusively for the cluster analysis. In this case, the signal strength of each gene was normalized relative to the median of all of the measurements taken for that gene in each experiment, defined as 1. This normalization was necessary to allow for better visualization of the profiles of expression, because all genes are on the same vertical scale rather than having a pattern of changes on widely differing vertical levels. Changes in gene expression were considered only when the difference in expression level was at least 1.5-fold and consistent in at least three out of five replicate experiments; a 1.5-fold change is equivalent to an increase by 50% or a decrease by 33%.
RESULTS
Overall Gene Expression During Spermatogenesis
The overall distribution of the genes detected in the different cell types is illustrated in Fig. 1. Of the 216 genes on the array, 96 were expressed during spermatogenesis in at least one of the three cell types. Their cellular distribution was as follows: 86 genes were detected in pachytene spermatocytes, 83 in round spermatids, and 52 in elongating spermatids. Several spermatogenic cell-specific genes were observed (Fig. 1, spotted circles), as well as two genes expressed uniquely in round and elongating spermatids and one uniquely in pachytene spermatocytes and elongating spermatids. Of the total number of genes expressed during spermatogenesis, interestingly, 50% (48) were detected in all three cell types, whereas nearly 25% (25) were expressed exclusively in pachytene spermatocytes and round spermatids; only one gene was expressed exclusively in elongating spermatids (Fig. 1, Table 1). These observations provide evidence that cellular contamination does not have an important effect on the gene expression profiles.
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Patterns of Gene Expression During Spermatogenesis
To determine the different expression profiles of the stress response genes detected during spermatogenesis, we used the K-means clustering method offered by GeneSpring (Fig. 2). K-means clustering allows for the partitioning of genes into groups based on similar expression patterns. The data are divided into K different clusters of greatest possible distinction by starting with K random clusters and moving the genes between clusters in order to minimize variability within clusters and maximize variability between clusters [14]. Prior to clustering by K means, the data were normalized (gene to gene), K was defined as 5, and distance as standard correlation.
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The 96 genes detected during spermatogenesis clustered into five specific expression profiles (Fig. 2). Strikingly, close to 50% (46) of the total number of genes decreased in expression level during germ cell development. A dramatic decrease from pachytene spermatocytes to round spermatids was observed for 33 genes (Fig. 2A); for example, the expression of genes involved in cell cycle regulation decreased by 98% (growth-related c-myc responsive gene; RCL); 95% (P55CDC); 90% (cyclin-dependent kinase 2 plus cyclin-dependent kinase 2ß, CDK2a + CDK2b); and 66.6% (cyclin-dependent kinase 4, CDK4). Of these, P55CDC was the only gene detected in elongating spermatids. Within the same cluster we found seven genes belonging to the family of DNA synthesis-recombination and repair pathways, and 10 involved in post-translational modification.
The remaining genes that decreased expression during spermatogenesis did so more gradually and thus partitioned into a different cluster (Fig. 2B). The expression of three genes (FKBP-rapamycin-associated protein, FRAP; cyclin-dependent kinase 7, CDK7; and ER60 protease, ER60) decreased by 38% from pachytene spermatocytes to the early spermatid stages. ER60 protease expression decreased further by 67% in elongating spermatids, whereas FRAP and CDK7 transcripts were undetectable by this stage. Members of the heat shock proteins (HSPs)-chaperone family (heat shock 90-kDa protein beta, HSP90-beta; testis-specific heat shock-related protein, HST; HSP70/HSP90-organizing protein, HOP; T-complex protein eta subunit, TCP1-eta; and mitochondrial stress-70 protein precursor, MTHSP70) partitioned also into this cluster.
Of the 21 genes present in Figure 2C, the expression of 17 peaked in the round spermatid stage. The genes that partitioned into this cluster were involved in multiple cellular processes, including protein turnover (polyubiquitin, 17-kDa ubiquitin conjugating enzyme E2; UBE2B), post-transcriptional modification (heat shock 47-kDa protein, HSP47; heat shock 27-kDa protein, HSP27; glucose-regulated 78-kDa protein, GRP78), DNA binding (murine double minute, MDM2), and intracellular transduction (ras associated with diabetes, RAD1; c-Jun N-terminal kinase 1, JNK1). In addition, the cell cycle genes, CDC-like kinase 3 (CLK3), G1/S-specific cyclin E (CCNE), G1/S-specific cyclin D2 (CCND2), and G2/M-specific cyclin B1 (CCNB1) were included in this cluster. Of these, CCND2 and CCNB1 were detected in round spermatids only. The high expression of some of the cell cycle genes in round spermatids suggests their involvement in functions other than cell proliferation, probably cell differentiation.
Surprisingly, 16 genes were expressed at higher levels in elongating spermatids (Fig. 2D) than in any other cell type. The highest level of expression in elongating spermatids was presented by cytoplasmic beta actin (ACTB) and heme oxygenase 2 (HO-2). This cluster included also extracellular regulated kinases ERK1 and ERK3; tubulin alpha-1 (TUBA1); ribosomal protein S30 (RPS30); four members of the DNA synthesis/recombination and repair family (apurinic/apyrimidinic endonuclease, APEX; 3-methyladenine DNA glycosylase, MPG; breast cancer and susceptibility gene, BRCA1; transcription factor 2H subunit p44, TFIIHp44); and the trafficking targeting protein nck, ash, and phospholipase C gamma-binding protein (NAP4). Of these, NAP4 had the largest increase in expression between round and elongating spermatids (12-fold), whereas TFIIHp44 increased the least (1.53-fold).
In contrast to the previous clusters, only nine genes were expressed at their lowest level in the round spermatid stage (Fig. 2E). Transcript levels were reduced by at least one third for ribosomal protein S29 40S subunit (RPS29) and ribosomal protein S19 40S subunit (RPS19), and the transcription genes cAMP-response element binding protein 1 (CREBP1) and DNA-binding protein inhibitor ID1.
Expression by Gene Family
To assess how the expression of stress response genes is regulated during spermatogenesis, we examined the expression of genes belonging to the HSP-chaperones, DNA repair, and oxidative stress families.
HSPs-chaperones The expression levels during spermatogenesis of eight HSP genes are shown in Figure 3A; of these eight detected transcripts, five decreased in expression from pachytene spermatocytes to elongating spermatids. HSP90-beta and the HSP70/HSP90-organizing protein (HOP) decreased gradually from pachytene spermatocytes to round spermatids (mean reduction of 37.5%), and from round to elongating spermatids (mean reduction of 54.5%). In contrast, HSP90-alpha and the HSCT-interacting protein (HIP) decreased more drastically (80%) during the early phases of spermatogenesis; their transcripts reached undetectable levels in the more mature germ cells. The testis-specific heat shock gene (HST) was the highest expressed HSP gene; expression levels were up to 40 times higher than the average expression on the arrays. Even though the degree of expression decreased strikingly (82%) from round to elongating spermatids, transcript levels in elongating spermatids remained higher than those of any other HSP in this cell type. In contrast, the expression of the transcript for heat shock 70-kDa protein (HSP70) increased during spermatogenesis, reaching levels four times higher in elongating spermatids than in the earlier haploid spermatids. For HSP27 and HSP47, transcript levels were the highest in round spermatids.
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For most of the other chaperones, the highest transcript levels were detected in pachytene spermatocytes (Figure 3B). The same genes decreased in expression from pachytene spermatocytes to round spermatids, with decreases ranging from 28% (calnexin, CANX) to 80% (t-complex protein 1 alpha, TCP1-a; and p23). The levels of expression in subsequent phases of spermatogenesis continued to decrease, except for calregulin (CALR), calnexin (CANX), and glucose-regulated 94-kDa protein (GRP94), which remained relatively unchanged in both haploid spermatogenic cell types.
DNA repair A total of 12 genes involved in nucleotide excision repair (NER), base excision repair (BER), and homologous recombination repair (HRR) were detected (Fig. 4). In the NER gene family, a particularly interesting pattern of expression was observed for proliferating cell nuclear antigen (PCNA); PCNA was the highest expressed DNA repair gene with levels increasing by 1.3-fold from pachytene spermatocytes to round spermatids and dropping dramatically thereafter (91.6%) to near undetectable levels in elongating spermatids. Among the other genes in the NER family, expression of the transcription factor subunit TFIIHp44 increased gradually during spermatogenesis, whereas the expression of the excision repair protein ERCC1 was at its lowest in round spermatids. Very low levels of the following genes were detected and these were only detected in specific cell types: Xeroderma pigmentosum group D protein (XPD) in round spermatids, replication protein A 70 kDa (RPA70) in pachytene spermatocytes, and replication protein A 32 kDa (RPA) in both pachytene spermatocytes and round spermatids. The BER genes, APEX and MPG, demonstrated a similar expression profile, with the highest transcript levels present in elongating spermatids. Expression of poly(ADP-ribose) polymerase (PARP) gradually decreased during spermatogenesis to reach near undetectable levels in elongating spermatids. Low and relatively constant expression up to the round spermatid stage was observed for ribosomal protein S3 (RPS3). The HRR gene, RAD51, was expressed exclusively in pachytene spermatocytes. On the other hand, BRCA1 was detected only in the haploid stages of spermatogenesis, and showed a 2.4-fold increase in expression level between round and elongating spermatids.
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Oxidative stress The expression of microsomal glutathione S-transferase (MGST1) and glutathione S-transferase Yb subunit (GSTM2) decreased by 32.4% and 47.3%, respectively, from pachytene spermatocytes to round spermatids (Fig. 5). Manganese-containing superoxide dismutase 2 (Mn SOD2) expression decreased gradually during spermatogenesis, whereas copper-zinc-containing superoxide dismutase 1 (Cu-Zn-SOD1) expression showed the exact opposite pattern. The most highly expressed gene in this family was heme oxygenase 2 (HMOX2, HO-2), levels of which increased 2.9-fold from pachytene spermatocytes to early haploid spermatids and 4.7-fold from round to elongating spermatids.
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DISCUSSION
The use of cDNA microarrays for large-scale analysis of gene expression has proven to be useful in providing information about tissue and cellular function [15, 16]. In the present study, we have used this technique to examine the expression of stress response genes during spermatogenesis.
Overall Expression of Stress Response Genes During Spermatogenesis
Analysis of the overall expression of stress response genes during spermatogenesis revealed a decrease in gene detection throughout germ cell development. A marked decrease (37%) in the number of genes detected was observed in the transition from the round to the elongating spermatid stage. This observation is supported by the previously reported transcription inactivation taking place during mid-spermiogenesis [17]. Despite the reduced ability of mature spermatids to transcribe stress response genes, elongating spermatids have been shown to be resistant to different types of stressful stimuli [18]. Male germ cells have developed posttranscriptional and posttranslational regulatory mechanisms to control gene expression throughout their development [19]. Activation of translationally repressed stress response transcripts during late stages of spermatogenesis could contribute to the reduced susceptibility of elongating spermatids to stress.
Selective gene expression throughout spermatogenesis was seen clearly, as illustrated in Figure 1. A number of genes were expressed in a cell-specific manner; interestingly, the differential expression of such genes may provide insight into which types of stress response mechanisms exist in each germ cell type. For example, the homologous recombination repair gene, RAD51, was expressed exclusively in pachytene spermatocytes; this correlates with chromosomal synapsis and recombination taking place at this stage of spermatogenesis, as well as with the previous localization of RAD51 in human and mouse spermatocytes [20]. The expression of topoisomerases was particularly interesting; topoisomerase I (TOP1) transcripts were restricted to elongating spermatids, whereas topoisomerase II alpha (TOP2A) transcripts were present up to the round spermatid stage. Replication, transcription, and recombination, as well as DNA damage recognition and repair, are common functions of topoisomerases [21, 22]. In addition, they are involved in regulating cellular sensitivity to a number of DNA damaging compounds [23]; thus, their differential expression during spermatogenesis may be indicative of a differential susceptibility of germ cells to such agents. On the other hand, genes involved in normal cellular functions, such as signal transduction, cytoskeletal motility, and translation, were detected in all germ cell types, as expected.
Patterns of Expression
The cluster analysis provided useful information about the behavior of stress response genes during germ cell development. The most prominent expression pattern observed was a decrease in transcript levels as spermatogenesis progressed. Apoptosis related genes (bcl-x, BAX, and BAD) presented a marked drop in expression from pachytene spermatocytes to round spermatids, with BAX reaching an undetectable level in early spermatids. This correlates with the recently reported immunohistochemical localization of BAX [24]. Even though the extent of apoptosis in spermatocytes in the adult testis remains unclarified, the presence of proapoptotic and antiapoptotic factors in germ cells during early spermatogenesis suggests that these cells have a greater ability to activate the cell death-survival signals that regulate apoptosis during germ cell development and after insult exposure [25–27]. The same profile of expression was seen for five cell cycle genes, supporting their important role in DNA-damage checkpoint mechanisms that take place during meiosis [28]. It is interesting that four members of this same family presented expression levels that peaked in round spermatids; however, a role in the stress response is less likely at this postmeiotic stage. Expression of cell cycle genes in nonproliferative germ cells has been reported previously [29, 30] but their function remains unknown. The previously described roles of cyclins and cdc2 serine/threonine kinases in chromosome condensation [31] as well as in sperm capacitation and the acrosome reaction [32] suggest that cell cycle genes may be involved in round spermatid differentiation.
Despite the observation that a large number of genes decreased in expression during spermatogenesis, the highest transcript levels for a number of genes were found in elongating spermatids. Elevated transcript levels in mature spermatids could be a consequence of a higher RNA stability, conferred by the formation of ribonucleoproteins. In fact, one of the mechanisms by which gene expression is post-transcriptionally regulated during spermatogenesis involves the association of mRNA, transcribed during early spermatogenesis, with RNA binding proteins, keeping the mRNA in a repressed state [33]. Later activation and translation of these transcripts takes place during transcriptionally inactive stages of spermatogenesis, providing the maturing cell with components necessary for its development [4]. Members of the DNA repair, oxidative stress, and signal transduction families presented this type of profile. As elongating spermatids are more resistant to insult, we suggest that "delayed" expression of components of the stress response machinery may contribute to part of this reduced susceptibility.
Expression by Gene Families
HSPs and correlated chaperones play an important role in stress responses. Aside from their functions in polypeptide transport and assembly, they act in protecting cellular proteins from misfolding and aggregation [34]. Their expression can be constitutive, developmentally regulated, or induced by elevated temperatures and other stressful stimuli [35]. Several members of this family have been previously studied during rodent spermatogenesis, mostly in mice [34, 36]; a large-scale analysis of their expression in rat testis has not been reported previously. The coexpression of HSPs and their partner proteins, such as DNAJ protein homolog 2 (DNAJ2), p23, HOP, and HIP, is indicative of the essential role this machinery plays during germ cell development, at least up to the round spermatid stage. The predominant reduction in expression levels during spermatogenesis may indicate that the ability of germ cells to cope with protein damage is decreased during the late stages of development. On the other hand, the contrasting increase in transcript levels of HSP70 is consistent with the expression pattern of HSC70t, a testis-spermatid specific HSP characterized in mouse [37]; however, no rat homolog for this protein has been reported yet. High levels of HSP70 in spermatids suggest an important role during spermatogenesis; HSP70 may act as a chaperone for the translationally regulated genes expressed in postmeiotic spermatids, or be involved in regulating the assembly of structures (acrosome, flagellum) taking place during spermiogenesis [38]. The expression of the majority of the other chaperones reported in this study has not been examined previously during spermatogenesis. The expression of members of the endoplasmic reticulum chaperone family (CALR, CANX, and GRP94) in all germ cell types supports the important role this family plays in proper assembly of nascent polypeptides during germ cell development.
The ability to cope with stress depends largely on the ability of the cell to undergo DNA repair. Unscheduled DNA synthesis, a measure of DNA repair, has been detected during spermatogenesis up to the early spermatid stages [39]. In this study we analyzed the expression of genes involved in NER, BER, and HRR. A more random pattern of expression of this family, compared with HSPs, points to a gene-specific developmental control of expression during spermatogenesis. The expression of PARP correlated with previous findings, where the highest levels of PARP mRNA were found in pachytene spermatocytes; these levels decreased to nearly undetectable in elongating spermatids [40]. The most abundant transcript observed for this family was PCNA, a gene involved in DNA replication during cellular proliferation and in DNA excision repair [41]. Elevated PCNA expression in germ cells in which no replication takes place, pachytene spermatocytes and round spermatids, indicates that PCNA may be involved in excision repair up to mid-spermatogenesis. Whether DNA repair can take place in elongating spermatids remains unknown. The expression of some components of the NER (TFIIHp44) and BER (MPG, APEX) pathways in mature spermatids may not be sufficient to suggest that DNA repair can take place at this stage of spermiogenesis. In fact, NER involves DNA damage recognition, excision, and DNA synthesis and ligation, requiring several components to achieve this process [42]; however, it is unknown whether some of the DNA repair proteins compensate for the role others play. The expression of DNA repair genes during spermatogenesis reported in this study gives insight into the DNA repair pathways present in the different germ cell types, however, functionality of these DNA repair mechanisms remains to be elucidated.
To assess whether or not germ cells are equipped with defense mechanisms against oxidative stress, we examined the expression of enzyme systems involved in antioxidant defense. Levels of GSTs were low and constant throughout spermatogenesis, in contrast to the increasing levels of SOD1 and HO-2 in later stages. The pattern of expression of SOD1 correlated with the highest mRNA levels reported in stages VI–VIII of rat testis [43]. Bilirubin, one of the products of heme degradation by heme oxygenases, has been shown to be a potent antioxidant in vitro [44]. HO-2 is the constitutive form of the stress inducible HO-1 gene and is highly expressed in brain and testis [45]. High susceptibility of sperm to lipid peroxidation [46], and the association between reactive oxygen species production and male infertility [47], support the importance of the antioxidant system in the testis. Indeed, loss of SOD1 activity results in higher susceptibility of human sperm to oxidative stress [48]. The abundant expression of HO-2 throughout spermatogenesis, consistent with the previously reported in situ localization of HO-2 [49], suggests that HO-2 plays an important role in germ cell development. Moreover, the similar profile of expression between HO-2 and SOD1, in addition to the recently reported evidence that HO-2 mediates neuroprotection against oxidative stress [50], suggest that HO-2 may have an important function in protecting germ cells from oxidative stress.
Male infertility and abnormal progeny outcome are some of the consequences resulting from the exposure of germ cells to stressors such as environmental chemicals and drugs [51]. Characterization of the protective mechanisms available in male germ cells during spermatogenesis is crucial to provide insight into the mechanisms leading to such consequences and to determine the susceptibility of germ cells to stress. In this study we found that stress response genes are differentially expressed during spermatogenesis; members of the HSPs, DNA repair, and oxidative stress families were present throughout spermatogenesis in a gene and stage-specific manner. Our results suggest that during male germ cell development cells have different abilities to cope with diverse types of stress such as oxidative stress, and protein and DNA damage. Selective expression of stress response genes during spermatogenesis may explain the differential susceptibility of germ cells to stress.
FOOTNOTES
First decision: 16 January 2000.
1 Supported by a grant from the Canadian Institutes of Health Research. 
2 Correspondence: B. Robaire, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montréal, PQ, Canada H3G 1Y6. FAX: 514 398 7120; brobaire{at}pharma.mcgill.ca
Accepted: February 14, 2001.
Received: December 31, 2000.
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Z. Yu, R. Guo, Y. Ge, J. Ma, J. Guan, S. Li, X. Sun, S. Xue, and D. Han Gene Expression Profiles in Different Stages of Mouse Spermatogenic Cells During Spermatogenesis Biol Reprod, July 1, 2003; 69(1): 37 - 47. [Abstract] [Full Text] [PDF]
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 A. Adeeko, D. Li, J. Doucet, G. M. Cooke, J. M. Trasler, B. Robaire, and B. F. Hales Gestational Exposure to Persistent Organic Pollutants: Maternal Liver Residues, Pregnancy Outcome, and Effects on Hepatic Gene Expression Profiles in the Dam and Fetus Toxicol. Sci., April 1, 2003; 72(2): 242 - 252. [Abstract] [Full Text] [PDF]
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 D. J. McLean, P. J. Friel, D. Pouchnik, and M. D. Griswold Oligonucleotide Microarray Analysis of Gene Expression in Follicle-Stimulating Hormone-Treated Rat Sertoli Cells Mol. Endocrinol., December 1, 2002; 16(12): 2780 - 2792. [Abstract] [Full Text] [PDF]
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A. Aguilar-Mahecha, B. F. Hales, and B. Robaire Chronic Cyclophosphamide Treatment Alters the Expression of Stress Response Genes in Rat Male Germ Cells Biol Reprod, April 1, 2002; 66(4): 1024 - 1032. [Abstract] [Full Text] [PDF]
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 P. Syntin, H. Chen, B. R. Zirkin, and B. Robaire Gene Expression in Brown Norway Rat Leydig Cells: Effects of Age and of Age-Related Germ Cell Loss Endocrinology, December 1, 2001; 142(12): 5277 - 5285. [Abstract] [Full Text] [PDF]
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