Sperm mRNA transcripts are indicators of sub-chronic low dose testicular injury in the Fischer 344 rat

Current human reproductive risk assessment methods rely on semen and serum hormone analyses, which are not easily comparable to the histopathological endpoints and mating studies used in animal testing. Because of these limitations, there is a need to develop universal evaluations that reliably reflect male reproductive function. We hypothesized that toxicant-induced testicular injury can be detected in sperm using mRNA transcripts as indicators of insult. To test this, we exposed adult male Fischer 344 rats to low doses of model testicular toxicants and classically characterized the testicular injury while simultaneously evaluating sperm mRNA transcripts from the same animals. Overall, this study aimed to: 1) identify sperm transcripts altered after exposure to the model testicular toxicant, 2,5-hexanedione (HD) using microarrays; 2) expand on the HD-induced transcript changes in a comprehensive time course experiment using qRT-PCR arrays; and 3) test these injury indicators after exposure to another model testicular toxicant, carbendazim (CBZ). Microarray analysis of HD-treated adult Fischer 344 rats identified 128 altered sperm mRNA transcripts when compared to control using linear models of microarray analysis (q < 0.05). All transcript alterations disappeared after 3 months of post-exposure recovery. In the time course experiment, time-dependent alterations were observed for 12 candidate transcripts selected from the microarray data based upon fold change and biological relevance, and 8 of these transcripts remained significantly altered after the 3-month recovery period (p < 0.05). In the last experiment, 8 candidate transcripts changed after exposure to CBZ (p < 0.05). The two testicular toxicants produced distinct molecular signatures with only 4 overlapping transcripts between them, each occurring in opposite directions. Overall, these results suggest that sperm mRNA transcripts are indicators of low dose toxicant-induced testicular injury in the rat.当前人类生殖风险评估方法主要依赖精液与血清激素分析，但此类方法难以与动物试验中采用的组织病理学终点及交配研究结果进行有效对标。鉴于上述局限，亟需开发能够可靠反映雄性生殖功能的通用评估手段。我们提出假说：可通过mRNA转录本作为损伤标志物，在精子中检测到毒物诱导的睾丸损伤。为验证该假说，本研究将成年雄性费希尔344（Fischer 344）大鼠暴露于低剂量的经典睾丸毒物，采用经典方法表征睾丸损伤的同时，同步分析同一动物个体的精子mRNA转录本水平。 本研究整体目标如下：1）利用微阵列（microarray）技术筛选暴露于经典睾丸毒物2,5-己二酮（2,5-hexanedione，HD）后发生表达变化的精子转录本；2）通过qRT-PCR阵列（qRT-PCR array）开展全面的时间进程实验，深入分析HD诱导的转录本表达变化；3）在暴露于另一种经典睾丸毒物多菌灵（carbendazim，CBZ）后，对上述损伤标志物进行验证。 对HD处理的成年费希尔344大鼠进行微阵列分析，采用微阵列线性模型与对照组进行比较后，共筛选得到128个差异表达的精子mRNA转录本（q<0.05）。所有转录本的表达变化均在暴露后3个月的恢复期内完全消失。在时间进程实验中，根据表达倍数变化与生物学相关性，从微阵列数据中筛选得到12个候选转录本，其表达变化呈现时间依赖性；其中8个转录本在3个月恢复期后仍存在显著表达差异（p<0.05）。在最后一项实验中，暴露于CBZ后共有8个候选转录本发生表达变化（p<0.05）。两种睾丸毒物诱导产生了截然不同的分子特征，二者仅存在4个重叠的差异转录本，且这些转录本的表达变化方向完全相反。综上，本研究结果表明，精子mRNA转录本可作为大鼠体内低剂量毒物诱导睾丸损伤的标志物。