The complexity of the testis, including different interacting cell types as well as different stages of the cycle of the seminiferous epithelium, often proves to be a major hurdle when trying to understand toxicant responses and underlying mechanisms. In the current study, several different approaches are combined to dissect the structural complexity of the testis to examine gene expression patterns associated with germ cell apoptosis. Germ cell apoptosis induced by x-ray exposure is stage specific, with a higher incidence in stage II/III seminiferous tubules (Yamasaki et al., 2010
). A priming exposure to 2,5-hexanedione results in a marked reduction in x-ray–induced germ cell apoptosis in these affected stages (Yamasaki et al., 2010
). Because of the difficulty of physically separating seminiferous tubules by stage, the study of associated gene expression changes is often performed using RNA isolated from whole testis. However, examination of associated gene and/or protein expression in whole testis tissue is limited in its ability to distinguish responses at specific stages, revealing a muted picture of what is actually occurring. LCM is a novel technique for evaluating stage-specific gene expression in the testis, allowing for the assessment of gene expression from individual cells or cell groups when coupled with qRT-PCR (Espina et al., 2006
; Sluka et al., 2002
; Suarez-Quian et al., 2000
). In the present study, qRT-PCR of candidate genes hypothesized to be involved in the attenuated apoptotic response was conducted using RNA isolated from tissue captured from the basal compartment of SG1 and SG2 seminiferous tubules and whole testis tissue. The results reveal a role for Fas in HD-mediated attenuation of x-ray–induced germ cell apoptosis.
Fas, FasL, caspase 3, bcl-2, and p53 were investigated as candidate genes because previous studies have implicated them in the germ cell apoptotic response (Embree-Ku et al., 2002
; Hasegawa et al., 1997
; Van Houten et al., 1997
). Preliminary analyses revealed that the greatest changes in apoptotic gene expression occurred in seminiferous tubules of SG1, consistent with histological findings. These studies also confirmed the increased sensitivity of measuring stage-specific gene expression over whole testis (manual dissection). The results of Bcl-2 mRNA expression underscore the success of the LCM approach—Bcl-2, an intracellular antiapoptotic protein, is induced in late spermatocytes and spermatids following x-ray exposure (Beumer et al., 2000
; Van Houten et al., 1997
). The data showed that Bcl-2 mRNA expression from SGs 1 and 2, which did not include that of late spermatocytes or spermatids, was not changed in any group, whereas expression of manually dissected samples, which included all testicular epithelium, was slightly increased. These results suggest that the apoptosis-related genes were successfully quantified by the LCM approach. Follow-up studies focusing only on gene expression across the different treatment groups within SG1 seminiferous tubules collected by LCM revealed significant differences in Fas expression but no change in FasL or caspase 3. Fas expression was significantly increased with x-ray exposure and significantly attenuated with HD/5 Gy co-exposure. Extrinsic Fas/FasL-mediated signaling between Sertoli cells and germ cells plays a key role in mediating germ cell apoptosis (Richburg, 2000
). However, x-ray–induced apoptosis involves the upregulation of Fas, but not FasL, as seen here and in previous studies (Embree-Ku et al., 2002
). This may be because of direct activation of Fas, which has been demonstrated with ultraviolet radiation, or because of activation of Fas by tumor necrosis factor α in the absence of FasL (Aragane et al., 1998
; Suzuki et al., 1999
). Additional studies investigating these possible mechanisms of HD-mediated attenuated germ cell apoptosis during co-exposure are warranted. We originally hypothesized that the reduced apoptosis caused by HD pretreatment was a result of either decreased apoptotic signals (e.g., FasL) from the damaged Sertoli cells or decreased response of the germ cells because of an adaptation to the reduced Sertoli cell support. The results of the current study suggest that following co-exposure, the germ cells adapt to the lack of Sertoli cell support by reducing the Fas response to normal FasL signals ().
FIG. 8. Proposed mechanism of HD-mediated attenuation of germ cell apoptosis. (A) Testis after x-ray exposure; (B) testis after combined HD and x-ray exposure. Sertoli cells normally support and nurture the developing germ cells. After a direct toxic insult to (more ...)
These findings also demonstrate the utility of performing cell population and stage-specific analysis of gene expression in the testis. Although only a trend toward Fas induction with x-ray treatment, and slight attenuation with HD co-exposure, was observed in whole testis, more pronounced and statistically significant changes were observed for SG1 tissue. Selecting only for the targeted germ cells eliminated the noise produced by including somatic and unaffected germ cells. LCM extraction revealed an enhancement of the traces of signal that were buried in the noise of the whole testis samples. The only drawbacks to this technique are the technical difficulties—the required expertise to identify stages, the time and expense, and the low mRNA yields.
As another approach to identify potential mechanisms underlying the co-exposure response, microarray analysis performed in a separate study identified AEN and PUMA as genes whose protein products are potentially involved in the attenuated apoptotic response (Campion et al., 2010
). Microarray analysis proved to be challenging because of the large amounts of data generated and the lack of well-established methods for analyzing the gene-level effects of more than one chemical. Prior work by our laboratory has established an effective method for studying gene alterations in response to chemical mixtures (Campion et al., 2010
). Regardless of these advances, microarray data still have limitations; the expense often limits the number of samples analyzed, which may result in noisy variable data, and the dynamic range is relatively narrow, resulting in reduced sensitivity. Our microarray analysis revealed that Fas was significantly altered by x-ray exposure; however, the fold change detected by microarray was negligible, and it would not have been pursued had it not been for previous research implicating Fas in germ cell apoptosis. Fas is not a very abundant transcript in the testis, and it is likely that any changes in expression may have been lost in the limited dynamic range of the technology, as well as in the use of RNA extracted from whole testis.
The first step taken to pursue genes identified by the microarray analysis was to perform qRT-PCR with RNA isolated from whole testis. This analysis confirmed the x-ray–induced increases in both AEN and PUMA but did not confirm the HD-mediated attenuation of gene induction. Only a trend toward attenuated PUMA induction was observed at the 12-h time point. The greater fold change values detected by qRT-PCR as compared with the values detected by gene array demonstrate the increased sensitivity of qRT-PCR over array technology. These genes and their products are likely involved in x-ray–induced germ cell apoptosis but do not appear to be involved in the attenuated apoptosis effect seen with HD pretreatment.
To enhance the sensitivity of detection for AEN and PUMA, LCM was used to select out the sensitive populations. Although the AEN data obtained from analyzing whole testis versus LCM-collected tissue was very similar, we observed a discrepancy between the whole testis and LCM gene expression levels of PUMA. Localization of PUMA following x-ray exposure clarifies the reason for the discrepancy between whole testis and LCM. PUMA protein was found to be localized primarily to SG2 seminiferous tubules and not in the SG1 tissue collected by LCM. The trend toward attenuated PUMA in whole testis corresponded with the results of the microarray, which was also performed with whole testis. In contrast, SG1 tissue exhibited enhanced PUMA gene expression with co-exposure, which is likely different from the whole testis findings because of the localization of PUMA mainly to SG2. Large differences in PUMA staining between control, x-ray alone, and co-exposure treatment groups were not readily apparent, which may be because of the timing of analysis. The detected alterations at the gene level at 12 h may not manifest at the protein level until later time points. Additional studies are warranted to investigate the time-dependent changes and localization of PUMA protein following co-exposure. In light of these findings, microarray analysis might be best utilized in conjunction with LCM-captured, stage or cell-type specific tissue to better guide hypothesis generation in future studies.
Through our detailed analysis of apoptosis-related gene expression in the testis, Fas has emerged as a major factor in the attenuation of x-ray–induced apoptosis by HD co-exposure. These studies have also explored the differences and utility of using whole testis versus LCM-captured specific cell and stage populations, as well as comparing qRT-PCR to microarray results. These findings underscore the challenges of measuring gene expression in the testis and the importance of considering differences in specific cell populations and stages. Examination of gene expression in whole testis, which is technically easier and much faster, may be a good starting point for exploratory studies; however, the diluting effects of other cell types and stages in the testis that are unaffected by toxicant treatment could mask significant changes that may be physiologically relevant. These studies have shown that important considerations must be made to balance the technical difficulties with the sensitivity of the approach when investigating gene expression in the testis.