Multi-cycle cisplatin treatment alters spermatogonial functional stem cell behavior and niche

Harman, James Gregory
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A typical clinical cis-diamminedichloroplatinum (II) (cisplatin) dosing regimen consists of repeated cycles of five to seven daily low dose treatments followed by a one to two week recovery period. While effective, this dosing structure results in a prolonged, and sometimes permanent, infertility in men. Undifferentiated spermatogonia, including spermatogonial stem cells (SSCs), are theoretically capable of repopulating the seminiferous tubules after exposure has ceased. It is proposed that an altered spermatogonial environment during recovery from the initial treatment cycle may drive an increase in SSC mitotic cell activity, rendering the SSC pool increasingly susceptible to cisplatin-induced cell death from subsequent cycles. The undifferentiated spermatogonia population and niche of the adult mouse (C57/BL/6J) were examined during the recovery period of a clinically-relevant course of one and two cycles of 2.5 mg/kg/d of intraperitoneal cisplatin and were compared to mice receiving an equivalent cumulative dose in a single cycle (5.0 mg/kg/d) and vehicle treated controls. Histological examination of the testicular epithelium revealed an increase in the disorganization of spermatogenesis correlating with the number of exposure cycles. Quantification of TUNEL positive cells showed an increase in apoptotic germ cells early in the recovery period in mice exposed to cisplatin compared to control animals. Immunohistochemical (IHC) examination of Foxo1 (undifferentiated spermatogonia marker) showed an increase in the undifferentiated spermatogonia population late in the recovery period in mice exposed to one cycle of 2.5 mg/kg/d, but not following two cycles of 2.5 mg/kg/d. Analysis of BrdU incorporation after dosing indicated a decrease in mitotic activity of early germ cells immediately after cisplatin exposure followed by a return to basal levels by the conclusion of the initial recovery period. No such rebound was observed during the second recovery period. IHC investigation of glial cell line-derived neurotrophic factor (GDNF), a recognized SSC niche factor, revealed an increase in production along the basal Sertoli cell membrane throughout the recovery period in all treatment groups. Taken together, these data establish that the impact of cisplatin exposure on the functional stem cell pool and niche correlates with: (1) the number of dosing cycles; (2) mitotic activity of early germ cells; and (3) alterations in the basal Sertoli cell GDNF expression levels after cisplatin-induced testicular injury.