Interaction of Insulin-like Growth Factor-binding Protein-3 and BAX in Mitochondria Promotes Male Germ Cell Apoptosis

摘要

Germ cell apoptosis is crucial for spermatogenesis and can be triggered by various stimuli, including intratesticular hormone deprivation. This study proposes a role for insulin-like growth factor binding protein-3 (IGFBP-3) in male germ cell apoptosis. Groups of adult Sprague-Dawley male rats received one of the following treatments for 5 days: (i) daily intratesticular (IT) injections with saline (control); (ii) a single subcutaneous injection of the gonadotropin-releasing hormone antagonist (GnRH-A), acyline, on day 1 and a daily IT injection of saline; (iii) daily IT injection of IGFBP-3; and (iv) a GnRH-A injection on day 1 and a daily IT injection of IGFBP-3. Germ cell apoptosis increased significantly after IGFBP-3 or GnRH-A treatment which was further enhanced by the combined treatment. After co-immunoprecipitation with BAX antibody, IGFBP-3 association with BAX was demonstrated in total and mitochondrial fractions but not in the cytosol of testis extracts. BAX-associated IGFBP-3 expression was increased in mitochondria after treatment compared with control, which was confirmed by an IGFBP-3 enzyme-linked immunosorbent assay. Dot blot studies further validated the BAX-IGFBP-3 binding in vitro. IGFBP-3 as well as BAX induced release of cytochrome c and DIABLO from isolated testicular mitochondria in vitro. IGFBP-3, when combined with an ineffective dose of BAX, triggered release of these proteins from isolated mitochondria at a 4-fold lower dose than IGFBP-3 alone. Our data demonstrate that the IGFBP-3 and BAX interaction activates germ cell apoptosis via the mitochondria-dependent pathway. This represents a novel pathway regulating germ call homeostasis that may have significance for male fertility and testicular disease.