The regulation and toxicology of spermatogenesis

Epidemiological, wildlife and laboratory studies have linked hormonally active agents (HAA) in the environment to declining sperm counts. The production and development of mature male gametes is essential for reproductive efficiency and survival of species. To understand normal hormonal control of spermatogenesis, and effects and mechanisms of HAA-mediated spermatotoxicity, three complementary animal models (shark Squalus acanthias; skate Leucoraja erinacea; mouse Mus musculus) were used to: (1) identify genetic markers of spermatogenic stage and condition; (2) identify biomarkers of HAA exposure and effect; (3) demonstrate applicability of these markers for detecting spermatotoxicity in laboratory and field studies; and (4) develop an in situ bioindicator species for detecting spermatotoxicants in polluted environments (skate). A homology cloning approach was employed to isolate cDNAs encoding androgen receptors (AR); progesterone receptors (PR); cytochrome P450 aromatase (P450arom); and zona pellucida protein 3 (ZP3)(shark, skate). Reverse transcription polymerase chain reaction analysis and real time quantitative PCR were used to measure expression of these and additional biomarkers (arylhydrocarbon receptors, AhR; cyp1A) (skate, shark). Histomorphometric and PCNA- and TUNEL-labeling techniques were applied to assess spermatogenic condition and proliferative and apoptotic activities of germ cells (shark, skate, mouse). Intratesticular levels of AR, PR, P450arom, and AhR were stage-related during spermatogenesis, but each had a unique expression profile and was differentially affected by administered toxicants (estrogen, dioxin, cadmium). The testes of AhR null mice, compared to wild type, revealed no differences in seminiferous tubule morphology or PCNA- or TUNEL-staining implying that AhR has no essential physiological role in spermatogenesis. Skates collected at different distances from a polluted site (New Bedford Harbor Superfund Site), in comparison to reference controls, displayed a gradient of effects on testicular expression of AR and P450arom. Biomarker effects correlated with histomorphometric changes indicative of spermatotoxicity, namely, a block in progression of spermatogonial clones into meiosis. Induction of cyp1A and ZP3 in affected skates suggests exposure to dioxin- and estrogen-like chemicals. This thesis project provides researchers with new information and reagents to apply to the problem of spermatotoxicity, and demonstrates how environmentally relevant wild-caught animals can complement a conventional laboratory model for understanding male reproduction.