Establishment, maintenance and functional integrity of the blood–testis barrier in organotypic cultures of fresh and frozen/thawed prepubertal mouse testes

Can the spatio-temporal formation of an intact blood–testis barrier (BTB), which is essential for the progression of spermatogenesis, be reproduced in cultures of fresh or frozen/thawed prepubertal mouse testes? Organotypic cultures allow the establishment and maintenance of major BTB components and the formation of a functional BTB in mouse testicular tissues. In vitro maturation of prepubertal testicular tissues is a promising approach to restore fertility in adult survivors of childhood cancer. Although gametes can be successfully obtained from prepubertal mouse testes in organotypic cultures, the spermatogenic yield remains low compared to in vivo controls. Mouse testicular tissues were frozen using controlled slow freezing (CSF) or solid surface vitrification (SSV) procedures. A total of 158 testes (fresh n = 58, CSF n = 58 or SSV n = 42) from 6 to 7 days postpartum (dpp) mice were cultured at 34°C in basal medium (α-MEM, 10% KnockOut Serum Replacement, 5 μg/ml gentamicin) at a gas–liquid interphase (under 20% O2), with or without 10−6 M retinol, for 9, 16 and 30 days. In addition, 32 testes from 6–7, 15–16, 22–23 and 36–37 dpp mice were used as in vivo controls. The mRNA levels of BTB genes (Claudin 3, Claudin 11, Zonula occludens 1 and Connexin-43), germ cell-specific genes (Sal-like protein 4, Kit oncogene, Stimulated by retinoic acid gene 8, Synaptonemal complex protein 3, Transition protein 1 and Protamine 2), markers of Sertoli cell immaturity/maturity (anti-Mullerian hormone, androgen receptor, cyclin-dependent kinase inhibitor 1b) and the androgen-regulated gene Reproductive homeobox 5 (Rhox5) were measured by quantitative RT-PCR (RT-qPCR). The localization of BTB proteins in seminiferous tubules was studied by immunohistochemistry and spermatogenic progression was evaluated histologically. The integrity of the BTB was assessed using a biotin tracer. Modest differences in Claudin 11 (Cldn11), Zonula occludens 1 (Zo-1), Connexin-43 (Cx43) transcript levels and in the localization of the corresponding proteins were found between in vitro cultures of fresh or frozen/thawed testes and in vivo controls (P < 0.05). However, a 32–77-fold decrease in Claudin 3 (Cldn3) mRNA levels and a lack of CLDN3 immunolabelling in 36–44% of seminiferous tubules were observed in 30-day organotypic cultures (P < 0.05). Although Sertoli cell maturation and the completion of a full spermatogenic cycle were achieved after 30 days of culture, meiotic and postmeiotic progression was altered in cultured testicular tissues (P < 0.05). Moreover, an increased BTB permeability and a decreased expression of Rhox5 were observed at the end of the culture period in comparison with in vivo controls (P < 0.05). Completion of spermatogenesis occurred in vitro in seminiferous tubules with an intact BTB, and in those expressing or lacking CLDN3. None. Further studies will be needed to determine whether the expression of other BTB components is altered and to decipher the reason for lower Cldn3 and Rhox5 mRNA levels in organotypic cultures. This work contributes to a better understanding of the molecular mechanisms occurring in in vitro matured prepubertal testes. The organotypic culture system will have to be developed further and optimized for human tissue, before potential clinical applications can be envisaged. This work was supported by Rouen University Hospital, Ligue contre le Cancer (to L.D.), and co-supported by European Union and Région Normandie (to A.O.). Europe gets involved in Normandie with European Régional Development Fund (ERDF). The authors declare that they have no conflict of interest.