Proteomic analysis for busulfan-induced spermatogenesis disorder

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Proteomic analysis; busulfan; spermatogenesis; testis.

Figure 1.

Obvious damage of testis and spermatozoa in epididymis appeared at 30 days after busulfan treatment. Mice were injected intraperitoneally once with busulfan, and then testes and epididymes were separated for H&E staining (A) and measurement of testis weight (B) and spermatozoa number (D) after 10, 20 and 30 days treatment. Arrow heads indicate abnormal germ cells (A). Body weight was also recorded before and after busulfan treatment (C). NC, negative control; *p < 0.05.

Figure 2.

Proteomic analysis of busulfan-treated mouse testis. 190 significantly downregulated proteins were identified in busulfan-treated mouse testis using volcano plot analysis (A). Partially downregulated proteins were shown in the clustering heatmap (B). Mice were injected intraperitoneally once with busulfan, and then testes were separated for label-free quantification proteomics after 30 days. NC, negative control.

Figure 3.

The differentially expressed proteins in busulfan-treated mouse testis were analyzed by bioinformatics. 190 significantly downregulated proteins were analyzed by gene ontology (A) and KEGG pathway analysis (B).

Figure 4.

Protein interactions in busulfan-treated testis were analyzed by bioinformatics. Partially downregulated proteins were analyzed using the STRING database. The colored lines show known or predicted interactions of protein. The color for the outer edge of the circle represents difference, and the lighter of the color means the greater of the difference.

Figure 5.

LDHAL6B protein was significantly reduced after 30 days for busulfan-treated mouse testis. Mice were injected intraperitoneally once with busulfan, and then testes were analyzed by immunofluorescence for detection of LDHAL6B protein after 30 days. NC, negative control.

Figure 6.

Spermatogenic recovery in busulfan-induced spermatogenesis disorder mouse testis. Recovery of testis and spermatozoa in epididymis progressively improved from 70 days to 80 days after busulfan treatment. Mice were injected intraperitoneally once with busulfan, and then testes and epididymes were separated for morphological imaging and staining with H&E (A), and measurement of testis weight (B) and spermatozoa number (C) after 40, 50, 60, 70 and 80 days. NC, negative control; *p < 0.05.

Figure 7.

LDHAL6B significantly decreased from 20 to 60 days and increased from 70 to 80 days after busulfan treatment. Mice were injected intraperitoneally once with busulfan, and then testes were separated for detection of LDHAL6B after the specified number of days (A). Densitometry of protein band was analyzed (B). NC, negative control; *p < 0.05; ***p < 0.001