Feswick A, Ings J S, Doyle M A, Bosker T, Munkittrick K R, Martyniuk C J
Canadian Rivers Institute and Department of Biology, University of New Brunswick, Saint John, New Brunswick E2L 4L5, Canada.
Canadian Rivers Institute and Department of Biology, University of New Brunswick, Saint John, New Brunswick E2L 4L5, Canada.
Gen Comp Endocrinol. 2014 Jul 1;203:106-19. doi: 10.1016/j.ygcen.2014.01.003. Epub 2014 Jan 19.
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.
5α-双氢睾酮（DHT）是一种在哺乳动物中具有多种作用的强效雄激素；然而，DHT在雄性鱼类中的生理作用尚不清楚。为了填补这一知识空白，将雄性食蚊鱼（Fundulus heteroclitus）连续暴露于0、5和50μg/L的DHT中21天。暴露后，分离睾丸进行组织学检查、离体孵育以测量类固醇生成能力以及基因表达分析（实时PCR和微阵列）。DHT显著降低了暴露于50μg/L DHT而非5μg/L DHT的雄性个体的离体11-酮睾酮（11KT）产量，且DHT暴露不影响离体睾酮产量。组织学检查显示，在50μg/L DHT处理组中，睾丸中的小叶间和结缔组织量增加。尽管11KT产量降低，但DHT不影响类固醇生成途径中靶向基因的表达，如类固醇生成急性调节蛋白（star）、细胞色素P450侧链裂解酶（cyp11a1）和11β-羟基类固醇脱氢酶（hsd11b3）。对对照组和50μg/L DHT处理组个体的睾丸进行微阵列分析发现，暴露于50μg/L DHT的雄性个体显示出与免疫、血流调节、脂质和外源性物质清除相关的转录子网受到调节，这表明DHT可能参与了鱼类睾丸中这些过程的生理调节。本研究的第二个目的是通过比较立即速冻组织和孵育组织的睾丸中的RNA完整性和转录水平，确定测量用于离体类固醇生成的组织中mRNA水平的可行性。两个时间点之间的RNA质量没有显著差异，表明在离体试验中RNA完整性至少可在18小时内保持完整，从而为研究人员在组织有限时评估多层次生物生殖终点提供了一个可行的选择。虽然actb、efla、rps12、rps18、star和hsd11b3的基因表达水平保持不变，但与速冻组织相比，孵育组织中的esr2a（esrba）、esr2b（esrbb）和cyp11a1显著降低。因此，必须谨慎使用，因为某些基因的稳态水平可能随时间变化。本研究增进了我们对硬骨鱼睾丸中DHT作用的理解，并就这种未充分研究的强效雄激素所调节的细胞过程产生了新的假设。
