Effects of Vitamin A on In Vitro Maturation of Pre-Pubertal Mouse Spermatogonial Stem Cells

Abstract

Testicular tissue cryopreservation is the only potential option for fertility preservation in pre-pubertal boys exposed to gonadotoxic treatment. Completion of spermatogenesis after in vitro maturation is one of the future uses of harvested testicular tissue. The purpose of the current study was to evaluate the effects of vitamin A on in vitro maturation of fresh and frozen-thawed mouse pre-pubertal spermatogonial stem cells in an organ culture system. Pre-pubertal CD1 mouse fresh testes were cultured for 7 (D7), 9 (D9) and 11 (D11) days using an organ culture system. Basal medium was supplemented with different concentrations of retinol (Re) or retinoic acid (RA) alone or in combination. Seminiferous tubule morphology (tubule diameter, intra-tubular cell type), intra-tubular cell death and proliferation (PCNA antibody) and testosterone level were assessed at D7, D9 and D11. Pre-pubertal mouse testicular tissue were frozen after a soaking temperature performed at -7°C, -8°C or -9°C and after thawing, were cultured for 9 days, using the culture medium preserving the best fresh tissue functionality. Retinoic acid at 10-6M and retinol at 3.3.10-7M, as well as retinol 10-6M are favourable for seminiferous tubule growth, maintenance of intra-tubular cell proliferation and germ cell differentiation of fresh pre-pubertal mouse spermatogonia. Structural and functional integrity of frozen-thawed testicular tissue appeared to be well-preserved after soaking temperature at -8°C, after 9 days of organotypic culture using 10-6M retinol. RA and Re can control in vitro germ cell proliferation and differentiation. Re at a concentration of 10-6M maintains intra-tubular cell proliferation and the ability of spermatogonia to initiate spermatogenesis in fresh and frozen pre-pubertal mouse testicular tissue using a soaking temperature at -8°C. Our data suggested a possible human application for in vitro maturation of cryopreserved pre-pubertal testicular tissue.

Retinol Improves In Vitro Differentiation of Pre-Pubertal Mouse Spermatogonial Stem Cells into Sperm during the First Wave of Spermatogenesis

“…Recently, our group demonstrated the beneficial effect of RE on the in vitro maturation of fresh and thawed frozen mouse pre-pubertal SSCs in an organ culture system using polycarbonate membrane inserts [ 18 ]. Indeed, RE at a concentration of 10 -6 M, rather than RA, maintained intra-tubular cell proliferation, the ability of spermatogonia to enter meiosis and Leydig cells functionality [ 18 ]. However, this system of culture did not allow pachytene spermatocytes to reach the post-meiotic stage and induced significant testicular tissue necrosis after 11 days of culture [ 18 ].…”

“…Indeed, RE at a concentration of 10 -6 M, rather than RA, maintained intra-tubular cell proliferation, the ability of spermatogonia to enter meiosis and Leydig cells functionality [ 18 ]. However, this system of culture did not allow pachytene spermatocytes to reach the post-meiotic stage and induced significant testicular tissue necrosis after 11 days of culture [ 18 ]. Therefore, we aimed to improve the production yield of spermatozoa from pre-pubertal mice SSCs by using the agarose gel system at a gas-liquid interphase.…”

Mammalian gonocyte and spermatogonia differentiation: recent advances and remaining challenges

“…The role of RA in spermatogonial differentiation was further clarified using Rbp4 null mice, in order to prevent the formation of RA from endogenous retinol pools (Ghyselinck et al 2006). RA and retinol were reported to induce the in vitro differentiation of spermatogonia, using an organotypic culture system of fresh and frozen PND6 and PND7 mouse testes (Travers et al 2013). Spermatogonia and primary spermatocytes were visualized using Tra98 immunostaining, which detects germ cell nuclear antigen (GCNA), while undifferentiated and differentiated spermatogonia were identified using Plzf C and Kit C immunostaining respectively.…”

Assessment of the optimal vitrification protocol for pre-pubertal mice testes leading to successful in vitro production of flagellated spermatozoa

“…Cellular death via necrosis was evaluated according to the methodology recently described by our team (Travers et al, 2013) and slightly modified using pyknotic nuclei quantification in seminiferous tubules. Briefly, seminiferous tubules were classified in four categories: no pyknotic if the seminiferous tubule was pyknotic-free, slightly pyknotic (<10% of pyknotic nuclei per seminiferous tubule), partially pyknotic (10-90% of pyknotic nuclei) or totally pyknotic (>90% of pyknotic nuclei).…”