Reproduction Abstracts

The past few decades have seen marked improvements in life expectancy following childhood cancer due, in part, to advances in chemotherapy. While these drugs are effective in treating malignant disease, one of the main adverse outcomes can be infertility. This is of particular concern for prepubertal boys, for whom there are not yet any established methods of fertility preservation. The objective of this study was to gain a better understanding of the effects of the widely-used chemotherapy drug, cyclophosphamide, on the prepubertal testis. This study was carried out in a primary tissue culture model. Immature mouse testes from pnd5 were dissected, cut into fragments, and each fragment was cultured individually on a membrane floating on 1 ml of medium. After 24 h, half of the medium was replaced with medium spiked with phosphoramide mustard (PM), the active metabolite of cyclophosphamide, to give final concentrations within the range of reported patient plasma concentrations (0.01–10 μM). Twenty-four hours after the drug was added, membranes were transferred into fresh, drug-free medium and cultured for a further 48 h. Testis fragments not exposed to PM throughout the culture period were used as a control. Fragments were then fixed, sectioned and analysed using immunofluorescence for expression of testis cell-type-specific and apoptotic markers. Concentrations of 1 and 10 μM PM markedly decreased germ cells, which fell to 10.29 and 7.18% of control levels respectively. Proliferation in the testis and numbers of Sertoli cells and interstitial cell types showed no significant changes. Expression of the apoptotic marker, cleaved caspase 3, only increased after exposure to the highest, 10 μM, concentration. In conclusion, this study found that in vitro exposure of the prepubertal mouse testis to concentrations equivalent to mid-high therapeutic concentrations of PM caused significant cell death, with specific and marked loss of germ cells.