Superfund Research Program

Stage-specific Actions of Cadmium During Spermatogenesis

Project Leader: Gloria V. Callard
Grant Number: P42ES007381
Funding Period: 1995 - 2000

Project-Specific Links

Final Progress Reports

Year:   1999 

The overall objective of this project is to develop, validate and demonstrate the applicability of the shark testis model for stage-by stage analysis of spermatotoxicant actions and effects, using cadmium (Cd) as an illustrative spermatotoxicant. Previously, project investigators identified a powerful cadmium-accumulating mechanism in cell nuclei of germ cells in stem cell-spermatogonial stages of spermatogenesis and showed that Cd exposure in vivo and in vitro increased ongoing rates of apoptosis. Because ER are colocalized with the Cd-accumulating mechanism in stem cell-premeiotic stages, and previous studies showed effects of estrogen on apoptosis of spermatogonial clones, it was postulated that Cd's action as a spermatotoxicant could be due to interference with normal estrogen signaling mechanisms during spermatogenesis. Researchers tested this hypothesis by PCR cloning and characterizating cDNAs encoding cytochrome P450 aromatase (estrogen synthetase) and estrogen receptors in shark testis. RT-PCR analysis based on the isolated sequences showed that Cd exposure in vivo decreased P450arom mRNA to some extent in all stages of development, but preferentially reduced levels in meiotic stages (immediately upstream of major ER targets). Pretreatment with Cd also reduced ER (beta-subtype) mRNA levels in the testis. Results of this study indicate that perturbations of male reproduction (low sperm counts, feminization, testicular cancer) which have been ascribed to estrogen-like environmental chemicals (xenoestrogens) could, in fact, be due to Cd. Recently, it has been reported that Cd is able to bind to and activate ER. This suggests the possibility that Cd's effects on P450arom and ER expression in shark testis is as an estrogen mimic

Additionally, project investigators have used PCR-DD (differential display or mRNA fingerprinting) to identify genes that are specifically up- or down-regulated by Cd in Cd-sensitive stem cell/spermatogonial stages of spermatogenesis. Using 5 different primer sets, 49 stage-dependent and 39 Cd-responsive bands have been identified. One band, which was expressed specifically in stem cell/spermatogonial stages was upregulated 5- to 10-fold by pretreatment in vivo with Cd. Further analysis identified this band as a 450-bp sequence in the control region of the mitochondrial genome, and Northern analysis using this probe showed the same stage-and Cd-dependence as PCR-DD. Results suggest that a mechanism or a consequence of Cd's spermatotoxicity is enhanced transcription of mtDNA. Changes in mitochondrial structure and function are some of the earliest indicators of apoptosis. Of the 12 proteins encoded on the H-strand of mtDNA, the cytochrome oxidase subunits are especially interesting due to their involvement in caspase activation leading to apoptosis. Researchers are testing effects of Cd on expressed levels of each of the mitochondrial proteins, and screening a shark testis cDNA library to obtain full length sequences of the other identified Cd-responsive bands. These experiments illustrate the feasibility of PCR-DD as applied to the shark testis model for identification of stage-specific and toxicant sensitive genes. Future project work will apply PCR-DD to determine whether the banding patterns induced by Cd can be mimicked by ER agonists or antagonists.