P-428 Impact of chemotherapy exposure using 4-hydroperoxycyclophosphamide on a male germ cell line (GC1-spg) and testes from prepubertal mouse
Authors: Klees C , Manzano N , Demeestere I , Lybaert P 
Published: June 2023

Abstract

Study question
What are the dynamics of the toxicity from 4-hydroperoxycyclophosphamide (4-HC) on male germ cell line and prepubertal mouse testes?

Summary answer
Chemotherapy has dose- and time- dependent harmful effects on genes regulating apoptosis and survival in the two different models.

What is known already
Fertility preservation strategies offered to prepubertal boys during oncological treatment are limited. The development of a noninvasive pharmacological protection of spermatogenic cells during oncological treatments would be a major advancement in the field. Studies in the female gonad have shown that chemotherapy exposure alters the expression profile of miRNAs and that miRNA-mimics administration could be used as an innovative tool to protect germ cells during gonadotoxic treatment. This project aims to assess the acute effect of 4-HC exposure on apoptosis and proliferation in spermatogonia in order to evaluate the differential miRNAs expression profile in male germ cells following gonadotoxic insult.

Study design, size, duration
GC-1 spermatogonial (GC1-spg) cells were cultured for 48h and exposed during the last 24h to different doses of 4-HC (20, 30 and 50µM) before assessing apoptosis and proliferation to evaluate the gonadotoxic dose of this agent. Cells were then exposed to 4-HC-50µM and apoptosis/proliferation were evaluated after 1,4,8,12,16,20 and 24h to establish the acute timepoint of gonadotoxicity. Similar experiments were then conducted on post-natal day 3 mouse testes during a culture of 24h. (N = 3)

Participants/materials, setting, methods
Histological analyses were performed to detect early apoptosis within spermatogonia cells using DDX4 and Cleaved Caspase 3 (CC3) co-immunostaining. DNA damages were evaluated on testes using TUNEL assay. A large screening of apoptosis gene expression was performed on cells with Taqman array (RT-qPCR). Apoptosis and cell proliferation were further assessed in both models through gene expression analyses of CC3 and Ki-67, respectively.

Main results and the role of chance
After 24 hours of in vitro exposure, a significant increase of apoptosis was observed in the cells treated with 30 µM of 4-HC and 50 µM of 4-HC, reflected by a higher level of CC3 expression in those two conditions compared to the control and the 4-HC-20 µM conditions. The panel screening of apoptosis gene expression performed on cells exposed to 20µM 4-HC during 24h showed no significant difference of expression between the control and the treated condition, sustaining the absence of harmful effect of 4-HC at this concentration in our model. The different timepoint analyses on cells using 50µM 4-HC have shown that chemotherapy-induced CC3 expression occurs approximatively 20 hours after the initiation of the treatment (p = 0.014). Similarly, there is a significant increase of apoptosis in prepubertal mouse testes following exposure to 50 µM of 4-HC compared to control, while no significant effect was observed at 20 µM 4-HC exposure. In both models, no change in proliferation through Ki-67 gene expression analyses was observed between non-treated and treated conditions. Finally, histological analyses performed on newborn mouse testes previously exposed or not to 4-HC revealed an increase of DNA fragmentation in the treated testes compared to control.

Limitations, reasons for caution
The use of a mouse cell line and in vitro cultured mouse testes is a limitation because of the species barrier. The direct exposure to chemotherapy (compared to in vivo models) can influence the level of toxic damage.

Wider implications of the findings
Those data characterize the dynamics of the toxic effect of 4-HC on spermatogonia. They will precise the optimal temporal window to detect the modifications in miRNA profiles by sequencing, to address the expression alterations and provide new targets to reduce cyclophosphamide gonadotoxic effects on the testicular germ cells.

Trial registration number
not applicable