Busulfan impairs blood-testis barrier and spermatogenesis by increasing noncollagenous 1 domain peptide via matrix metalloproteinase 9

Backgrounds: Sterility induced by anti-cancer treatments has caused significant concern, yet the mechanism and treatment exploration are little for male infertility after Cancer therapy. Busulfan, the antineoplastic that was widely applied before bone marrow transplantation, was known to induce male reproductive disorder.

Objectives: To investigate the effect of busulfan on blood-testis barrier function in adult rats and determine whether noncollagenous 1 domain peptide, the biologically active fragment proteolyzed from the collagen α3 chain (IV) by matrix metalloproteinase 9, was involved during this process.

Materials and methods: Adult male rats were treated with one-dose or double-dose of busulfan (10 mg/kg) before euthanized at day 35. Blood-testis barrier integrity assay, HE staining, immunofluorescence, and Western blot were used to validate the effect of busulfan on blood-testis barrier permeability and spermatogenesis. JNJ0966 was applied to specifically inhibit the matrix metalloproteinase 9 activity. The polymerization activity of F-actin/G-actin and microtubule/tubulin in the testis were assessed by using commercial kits.

Results: A noteworthy blood-testis barrier injury and significant up-regulation of matrix metalloproteinase 9 activity and noncollagenous 1 level after a single-dose busulfan (10 mg/kg) treatment in adult rat testis were revealed. The application of JNJ0966 was found to decrease noncollagenous 1 level and rescue the busulfan-induced blood-testis barrier injury including the mis-localization of junction proteins across the seminiferous epithelium, by recovering the organization and polymerization of both F-actin and microtubule. The busulfan-induced spermatogenesis impairment was also improved by JNJ0966.

Conclusion: These findings thus demonstrate that the elevation in matrix metalloproteinase 9 and noncollagenous 1 might participate in busulfan-induced blood-testis barrier disruption in adult male rats. As such, busulfan-induced male infertility could possibly be managed through interventions on noncollagenous 1 production.