The present study investigated the response to combined exposure of unique testicular toxicants with dissimilar cellular targets in the testis. The significance of the coexposure model has long been an area of interest for this laboratory. A previous study that examined histopathological markers of exposure to two well-described testicular toxicants, HD and carbendazim, with the same cellular (Sertoli cell) and subcellular (microtubule) targets revealed a synergistic interaction. The coexposure model has begun to provide mechanistic insight into the complexity of real-life exposures to more than one testicular toxicant at a time (Markelewicz et al., 2004
). However, the cellular complexity of the testis and the importance of paracrine signaling make interpretation of toxicant coexposure challenging.
Consistent with previous findings, rats subjected to a subacute exposure of 1% HD in drinking water for 18 days exhibited decreased body and testis weights as well as an increased incidence of seminiferous tubules with greater than 3 RSH (Bryant et al., 2008
; Moffit et al., 2007
). Apoptosis was not increased by HD exposure alone after 18 days, as demonstrated in this study, while extended treatment with HD has been reported to induce an increase in apoptosis via the Sertoli cell-mediated pathway (Blanchard et al., 1996
). Simultaneous exposure to x-ray did not have an additional effect on RSH or other histopathological end points such as seminiferous tubule diameter, Sertoli cell vacuolization, or germ cell sloughing. The incidence of apoptosis as measured by TUNEL staining was significantly increased in a dose-dependent manner by x-ray exposure alone. Intriguingly, an 18-day priming period with HD resulted in an attenuation in the incidence of x-ray–induced germ cell apoptosis ().
It is well known that x-ray exposure induces apoptosis in germ cells undergoing DNA replication (Hasegawa et al., 1997
; Henriksen et al., 1996
). In this study, x-ray exposure resulted in an increased incidence of seminiferous tubules with greater than 3 TUNEL-positive cells. Apoptosis was primarily distributed in group 1 (stages I–VI) and peaked at stage II/III in both the 5 Gy and HD/5 Gy groups, suggesting that types A4 and intermediate spermatogonia, which are prevalent in this stage range, are most susceptible to x-ray–induced damage. These results are consistent with previous reports that A2 through B spermatogonia are most sensitive to x-ray exposure in rodents (Hasegawa et al., 1997
; Henriksen et al., 1996
). Interestingly, the coexposure model did not change the cell specificity of the x-ray killing effect seen here. This is a reasonable finding, as x-ray affects DNA replication of proliferating germ cells, but not the relatively quiescent Sertoli cells in the adult testis (Hasegawa et al., 1997
). The greatest percentage of TUNEL-positive cells observed in the HD group was found in stage group 4 (stages XII–XIV) at a distribution and rate comparable with that of the control group, indicating that at these doses, 18-day HD treatment does not affect the induction or distribution of apoptosis in any stage.
The stage-specific nature of seminiferous tubule sensitivity to toxic insult has been suggested previously. Wright et al. (1983)
demonstrated the presence of stage-specific proteins in the rat and more recently Johnston et al. (2008)
, using gene array technology, revealed that tubule stages are associated with unique transcriptomes. These data, combined with the histological distinctions between stages, support the hypothesis that the seminiferous tubule would exhibit a stage-related sensitivity to particular toxicants, a prospect that has been supported by numerous observations (Hikim et al., 1997
; Lue et al., 1999
; McClusky et al., 2007
The magnitude of x-ray–induced apoptosis in the HD/5 Gy group was attenuated by an 18-day priming exposure of HD (). However, the relative distribution of apoptosis across stages remained unchanged with coexposure, indicating that pretreatment with HD affected x-ray–induced apoptosis in a quantitative but not qualitative manner (). Interestingly, when examined as an average across all stages, the attenuative effect on apoptosis was stronger in the HD/5 Gy group than in the HD/2 Gy group.
Apoptosis of male germ cells can be induced in response to various internal or external cellular signals via the intrinsic or extrinsic pathways (Boekelheide et al., 2000
; Lee et al., 1997
). These pathways are differentially regulated depending on whether the toxicant targets are Sertoli or germ cells. Lee et al. (1999)
found that a toxicant-induced insult to the Sertoli cell will cause the secretion of proteins by the Sertoli cell that subsequently initiate the apoptosis-signaling cascade in germ cells. A direct insult to the germ cell, however, will bypass the Sertoli cell-signaling pathway and directly trigger apoptosis in the germ cell.
These phenomena combined with the increased complexity of the coexposure model demonstrated here have led us to posit two hypotheses explaining the attenuation of x-ray–induced germ cell apoptosis by HD pretreatment. Exposure to x-ray alone results in an increase in germ cell apoptosis (). Although this toxicant-cell interaction is direct, the germ cell remains under the influence of general support and paracrine signaling by the Sertoli cell. Conditioning by HD prior to x-ray exposure may have one of the following effects: HD may compromise the signaling ability of Sertoli cells, including that which is responsible for normal induction of apoptosis (); on the other hand, HD might hinder the general supportive ability of the Sertoli cell, encouraging the vulnerable germ cell to adapt characteristics that lend increased resistance to potential insults (). Both pathways allow for the same ultimate effect of attenuated apoptosis: the first by decreasing normal levels of apoptotic signaling by the Sertoli cell and the second by conferring increased toxicant resistance on germ cells by decreasing the Sertoli cell's supportive ability.
FIG. 7. Proposed mechanisms of HD-mediated attenuation of germ cell apoptosis. Sertoli cells normally support and nurture the developing germ cells. Following a direct toxic insult to germ cells by x-ray exposure, the susceptible germ cells undergo apoptosis (more ...)
To address these hypotheses and elucidate the pathway by which this coexposure model exerts its effect, we will have to move beyond simple histology. We have identified those tubule stages and cells most affected by exposure; now an analysis of the gene expression pathways is required to further elucidate the mechanistic pathways. This laboratory has conducted a simultaneous study using a gene array platform to examine a wide variety of gene expression changes allowing for an unbiased examination of potential alterations in genetic pathways and physiological functions affected by coexposure. In the companion studies, insights were drawn from both the gene array and histological analyses to identify candidate stages, cells and genes in the spermatogonial population that were most affected by exposure (Campion et al., 2010a
). To obtain a clear picture of gene changes by cell type, we utilized the laser capture microdissection technique which allowed for the capture of cell-specific RNA by histology (Campion et al., 2010b
). Candidate gene expression changes were analyzed by qRT-PCR, and conclusions were drawn based on the synthesis of histological and genetic data.
In summary, these experiments revealed an interesting and unexpected antagonistic effect on apoptosis following coexposure of two testicular toxicants with differing modes of action. Although a valuable observation in and of itself, investigation into the mechanisms of this attenuative effect will yield greater insight into the nature of real-world mixtures of exposures.