Humanin Reduces Cyclophosphamide Induced Male Germ Cell Apoptosis in Mice

Many young men after cancer chemotherapy develop long term and often irreversible infertility. Chemotherapeutic drugs adversely affect fertility because they kill germ cells in the testes.  Cyclophosphamide is one of the alkylating anti-cancer medications commonly used for patients with lymphomas, leukemia, some forms of brain cancer and testicular cancer. Humanin is a 24 amino-acid mitochondrial derived peptide that has cytoprotective action on neurons, endothelial cells and germ cells.  We hypothesized that humanin may prevent cyclophosphamide induced germ cell apoptosis in testes.  To test this hypothesis, groups of 4 young adult mice (12-14 week-old, C57BL/6J) received the single intraperitoneal injections of: 1) control (saline), cyclophosphamide group (200mg/kg), humanin group (40mg/kg), and cyclophosphamide + humanin group (the same doses as above). All mice were sacrificed 24 hours after treatment. Bouin’s fixed paraffin embedded testicular sections were used for TUNEL assay to detect apoptosis. The rate of germ cell apoptosis was quantified by using the % of seminiferous tubules with TUNEL positive cells.  The results showed that cyclophosphamide significantly increased the number of germ cell apoptosis compared to control (p< 0.05).  Humanin significantly reduced cyclophosphamide induced germ cell apoptosis (p<0.05), while the humanin alone group had no change compared to control animals.  Thus, we conclude that Humanin reduces cyclophosphamide induced germ cell apoptosis in mice. The study of the cytoprotective action of humanin on germ cells may have implications in prevention of infertility in men treated by cancer chemotherapy.