Vitamin E but Not GSH Decreases Reactive Oxygen Species Accumulation and Enhances Sperm Production during In Vitro Maturation of Frozen-Thawed Prepubertal Mouse Testicular Tissue.

Brahim Arkoun,Christine Rondanino,Ludovic Galas,Justine Saulnier,Nathalie Rives,Marion Delessard,Ludovic Dumont,Aurélie Rives

doi:10.3390/ijms20215380

Abstract

Freezing–thawing procedures and in vitro culture conditions are considered as a source of stress associated with increased reactive oxygen species (ROS) generation, leading to a damaged cell aerobic metabolism and consequently to oxidative stress. In the present study, we sought to investigate whether vitamin E (Vit E) or reduced glutathione (GSH) enhances sperm production by decreasing ROS accumulation during in vitro maturation of prepubertal mice testes. Testes of prepubertal mice were cryopreserved using a freezing medium supplemented or not supplemented with Vit E and were cultured after thawing. In presence of Rol alone in culture medium, frozen-thawed (F-T) testicular tissues exhibited a higher ROS accumulation than fresh tissue during in vitro culture. However, Vit E supplementation in freezing, thawing, and culture media significantly decreased cytoplasmic ROS accumulation in F-T testicular tissue during in vitro maturation when compared with F-T testicular tissue cultured in the presence of Rol alone, whereas GSH supplementation in culture medium significantly increased ROS accumulation associated with cytolysis and tissue disintegration. Vit E but not GSH promoted a better in vitro sperm production and was a suitable ROS scavenger and effective molecule to improve the yield of in vitro spermatogenesis from F-T prepubertal mice testes. The prevention of oxidative stress in the cytoplasmic compartment should be regarded as a potential strategy for improving testicular tissue viability and functionality during the freeze–thaw procedure and in vitro maturation.

Highlights

Testicular tissue cryopreservation is a prerequisite and the only potential strategy for fertility preservation in prepubertal boys affected by cancer [1]
Detection in Testicular Cell Suspension The signal emitted by the CellROX orange probe was localized into the cytoplasm of the cells named “cytoplasmic” reactive oxygen species (ROS) in the current study (Figure 1a2,b2)
We showed that cytoplasmic ROS accumulation was significantly decreased in fresh testicular tissue cultured with Rol when compared with basal medium (BM) alone at D45 and D60 of culture, whereas nuclear ROS were not detected under these two conditions

Summary

Introduction

Testicular tissue cryopreservation is a prerequisite and the only potential strategy for fertility preservation in prepubertal boys affected by cancer [1]. Despite the use of cryoprotectants and optimized protocols that seem to maintain post-thaw structural and functional integrity of testicular tissue, several other parameters should be improved, notably concerning the deleterious effects of freezing and thawing steps [3]. These chemical and physical conditions could damage cell aerobic metabolism and induce oxidative stress associated with high levels of reactive oxygen species (ROS) generation [6]. ROS comprise both free radical and non-free radical oxygen-derived reactive molecules that are constantly generated, as part of the normal aerobic life, during the intermediate steps of oxygen reduction along the mitochondrial electron transport chain [6]. ROS are involved in both the proliferation and differentiation of different stem cells including spermatogonial stem cells (SSCs) and play a critical and positive role on self-renewal of SSCs [8]

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Discussion

Conclusion