Granulocyte-colony stimulating factor does not prevent in vitro cisplatin-induced germ cell reduction in immature human and mouse testis

Abstract

Background Currently there are no established fertility preservation options for pre-pubertal boys facing cancer treatment. Granulocyte-colony stimulating factor (G-CSF) treatment has been proposed to be chemoprotective against spermatogonial cell loss in an alkylating chemotherapy model of busulfan treated adult mice. Having previously shown that exposure to the alkylating-like chemotherapy cisplatin resulted in a reduction in germ cell numbers in immature human testicular tissues, we here investigate whether G-CSF would prevent cisplatin-induced germ cell loss in immature human and mouse (fetal and pre-pubertal) testicular tissues. Methods Organotypic in vitro culture systems were utilised to determine the effects of clinically-relevant concentrations of G-CSF in cisplatin-exposed immature testicular tissues. Human fetal (n = 14 fetuses) and mouse pre-pubertal (n = 4 litters) testicular tissue pieces were cultured and exposed to cisplatin or vehicle control for 24 hrs and analysed at 72 and 240 hrs post-exposure. Combined G-CSF and cisplatin exposure groups explored varying concentrations and duration of G-CSF supplementation to the culture medium (including groups receiving G-CSF before, during and after cisplatin exposure). In addition, effects of G-CSF supplementation alone were investigated. Survival of total germ cell and sub-populations were identified by expression of AP2γ and MAGE-A4 for human gonocytes and (pre)spermatogonia, respectively, and MVH and PLZF, for mouse germ cells and putative spermatogonial stem cells (SSCs) respectively, were quantified. Results Exposure to cisplatin resulted in a reduced germ cell number in human fetal and mouse pre-pubertal testicular tissues at 240 hrs post-exposure. Germ cell number was not preserved by combined exposure with G-CSF using any of the exposure regimens (prior to, during or after cisplatin exposure). Continuous supplementation with G-CSF alone for 14 days did not change the germ cell composition in either human or mouse immature testicular tissues. Conclusions This study demonstrates that exposure to G-CSF does not prevent cisplatin-induced germ cell loss in immature human and mouse testicular tissues in an in vitro system.

Alkylating agents‐induced gonadotoxicity in prepubertal males: Insights on the clinical and preclinical front

“…So far, only cisplatin and carboplatin have been tested in such models. [89][90][91][92] Possibly due to easier accessibility, research was carried out on fetal tissue after it was found to exhibit a similar pattern and timing of damage to child-derived tissue following treatment. 89 As expected, this group found some differences when comparing the response of mice and human-derived tissue.…”

“…88 This led another group to rely on this model to investigate the effects of cisplatin and carboplatin treatment on the prepubertal human testis. [89][90][91][92] To the best of our knowledge, these are pioneering experiments concerning the use of prepubertal-aged human tissues in chemotherapy-induced gonadotoxicity studies and will be further discussed in more detail. Despite their novel usage of child-derived tissue, no constraints regarding tissue viability were mentioned.…”