Proteomic analysis for busulfan-induced spermatogenesis disorder

Busulfan is the most commonly used drug for the treatment of chronic myelogenous leukemia and pretreatment for hematopoietic stem cell transplantation, which can damage the reproductive and immune system. However, little is known about the protein expression profiling in busulfan treated testis. This research studies the proteomics for busulfan-induced spermatogenesis disorder. The model of busulfan-induced mouse spermatogenesis disorder was subjected to label-free quantification proteomics analysis. Clustering heatmap, gene ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein interaction analyses were performed and validated by molecular experiments. The busulfan-treated mouse model showed abnormal testis morphology and reduced sperm number and testis weight. Testicular and sperm damage was most severe at 30 days after busulfan treatment. The busulfan-treated mouse testes were subjected to label-free quantification proteomics, which revealed 190 significantly downregulated proteins including lactate dehydrogenase A like 6B (LDHAL6B) and ubiquitin-specific protease 7 (USP7). In addition, the testis and spermatozoa in the epididymis progressively improved from 70 to 80 days after busulfan treatment, and that the testis weight and spermatozoa number gradually increased from 40 to 80 days after busulfan treatment. Western blotting revealed that LDHAL6B protein significantly increased at 10 days, decreased from 20 to 60 days, and then gradually elevated from 70 to 80 days after busulfan treatment. We revealed 190 significantly downregulated proteins in busulfan-treated mouse testes at 30 days and indicated that 70 days is the cut-off point of spermatogenic recovery for busulfan-treated mouse testis, increasing our understanding of this reproductive disorder model. An increased understanding of busulfan’s toxic effect will help to prevent and treat reproductive diseases.

白消安（Busulfan）是治疗慢性髓系白血病（chronic myelogenous leukemia）及造血干细胞移植（hematopoietic stem cell transplantation）预处理的最常用药物，其可损伤生殖系统与免疫系统。然而，目前针对白消安处理后睾丸的蛋白质表达谱研究仍较为匮乏。本研究围绕白消安诱导的生精障碍开展蛋白质组学研究，对构建的白消安诱导小鼠生精障碍模型开展无标记定量蛋白质组学（label-free quantification proteomics）分析，依次完成聚类热图、基因本体论（Gene Ontology, GO）、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路及蛋白质相互作用分析，并通过分子实验对结果进行验证。
白消安处理的小鼠模型呈现睾丸形态异常、精子数量减少及睾丸重量降低的表型；在白消安处理后30天，睾丸与精子损伤最为严重。对该模型小鼠睾丸开展无标记定量蛋白质组学分析后，共鉴定出190个显著下调的蛋白质，其中包括乳酸脱氢酶A类似蛋白6B（LDHAL6B）与泛素特异性蛋白酶7（USP7）。此外，在白消安处理后70至80天，小鼠睾丸与附睾内精子的损伤状态逐步得到改善；同时睾丸重量与精子数量自处理后40至80天呈逐步上升趋势。蛋白质免疫印迹（Western Blotting）结果显示，LDHAL6B蛋白在白消安处理后10天显著上调，20至60天表达量下降，随后在70至80天再次逐步升高。
本研究明确了白消安处理后30天的小鼠睾丸内存在190个显著下调的蛋白质，并指出70天是白消安处理小鼠睾丸生精功能恢复的临界时间点，加深了我们对该生殖障碍模型的认知。加深对白消安毒性作用的认知，将有助于生殖系统疾病的预防与治疗。