D'Andrea M R, Lawrence D, Nagele R G, Wang C Y, Damiano B P
Johnson & Johnson Pharmaceutical Research and Development, Welsh & McKean Roads, Spring House, Pennsylvania, USA.
Biotech Histochem. 2008 Oct;83(5):211-20. doi: 10.1080/10520290802521804.
It is well known that toxicants such as cyclophosphamide and ethanol can have deleterious effects on normal spermatogenesis. End points such as testis weight and sperm counts have been used widely to assess gross structural and functional changes in testes resulting from toxicant exposure. Histopathological assessments are more sensitive measures of testicular health, but generally they are neither quantitative nor sensitive enough to detect early toxicity. Recently, immunolabeling cells with proliferating cell nuclear antigen (PCNA) has been used to identify proliferating spermatogonia; however, there have been no systematic attempts to quantify these changes. We have developed a sensitive, reliable and quantitative assay using immunohistochemistry on formalin fixed, paraffin embedded rat testes to assess the degree of proliferation-related toxicity. An indexing scheme was derived based on the determination of radially positioned PCNA-positive cells within similarly staged seminiferous tubules presenting a single layer of PCNA-positive cells along the basement membrane of the basal tubular compartment. An average of 60 tubules in the testes were counted per animal. Our results show significant decreases in the PCNA index in rats treated with an experimental compound that has been shown to produce testicular histopathology. The analysis provides a quick, reliable, sensitive, and quantitative means for assessing early testicular toxicity. The assay has potential utility as an in vivo biomarker for detecting early testicular toxicity of experimental compounds in preclinical development as well as for refining follow-up compounds with reduced testicular toxicity.
众所周知，环磷酰胺和乙醇等毒物会对正常精子发生产生有害影响。睾丸重量和精子计数等终点指标已被广泛用于评估毒物暴露导致的睾丸总体结构和功能变化。组织病理学评估是衡量睾丸健康状况更敏感的指标，但通常既不具有定量性，也不够敏感，无法检测早期毒性。最近，用增殖细胞核抗原（PCNA）对细胞进行免疫标记已被用于识别增殖的精原细胞；然而，尚未有系统地尝试对这些变化进行量化。我们开发了一种灵敏、可靠且定量的检测方法，利用免疫组织化学技术对福尔马林固定、石蜡包埋的大鼠睾丸进行检测，以评估增殖相关毒性的程度。基于对处于相似阶段的生精小管内径向定位的PCNA阳性细胞的测定得出了一种索引方案，这些生精小管在基底管状隔室的基底膜处呈现单层PCNA阳性细胞。每只动物的睾丸平均计数60个小管。我们的结果显示，用已被证明会导致睾丸组织病理学变化的实验性化合物处理的大鼠，其PCNA指数显著降低。该分析为评估早期睾丸毒性提供了一种快速、可靠、灵敏且定量的方法。该检测方法有潜力作为一种体内生物标志物，用于检测临床前开发中实验性化合物的早期睾丸毒性，以及用于优化睾丸毒性降低的后续化合物。
