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5α-Dihydrotestosterone (DHT) is a potent androgen in mammals with multiple roles; however the physiological actions of DHT in male fishes are not well known. To address this knowledge gap, male mummichog ( Fundulus heteroclitus ) were continuously exposed to 0, 5, and 50 μg/L DHT for 21 days. Following exposure, testes were separated for histology, ex vivo incubation to measure steroidogenic capacity, and gene expression analyses (real-time PCR and microarray). DHT significantly decreased ex vivo 11-ketotestosterone (11KT) production in males exposed to 50 μg/L DHT but not 5 μg/L DHT, and DHT exposure did not affect ex vivo testosterone production. Histological examination revealed that the amount of interlobular and connective tissue present in the testes was increased in the 50 μg/L DHT treatment. Despite reductions in the production of 11KT, DHT did not affect the expression of targeted genes in the steroidogenic pathway such as steroidogenic acute regulatory protein ( star ), P450 side chain cleavage ( cyp11a1 ) and 11β-hydroxysteroid dehydrogenase ( hsd11b3 ). Microarray analysis in the testes of individuals from control and 50 μg/L DHT revealed that males exposed to 50 μg/L DHT showed regulated transcriptional sub-networks that were related to immunity, regulation of blood flow, lipids and xenobiotic clearance, suggesting that DHT may be involved in the physiological regulation of these processes in the fish testes. A second objective of this study was to determine the feasibility of measuring mRNA levels in tissues used for ex vivo steroid production by comparing RNA integrity and transcript levels in testes of both immediately flash frozen tissue and incubated tissue. There was no significant difference in RNA quality between the two time points, indicating RNA integrity can remain intact for at least 18 h in ex vivo assays, thereby providing a viable option for researchers assessing multi-level biological reproductive endpoints when limited tissue is available. While the gene expression levels of actb , efla , rps12 , rps18 , star , and hsd11b3 remained unchanged, esr2a ( esrba ), esr2b ( esrbb ) and cyp11a1 were significantly lower in incubated tissue compared to flash frozen tissue. Therefore caution must be used as the steady-state levels of select genes may change over time. This study improves our understanding of DHT action in the teleostean testis and generates new hypotheses regarding cell processes that are regulated by this underexplored and potent androgen. [ABSTRACT FROM AUTHOR]