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.
Highlights
Spermatogenesis is a highly organized process of cell proliferation and terminal differentiation that leads to the formation of mature spermatozoa
RE3 appeared to be deleterious for seminiferous tubule growth as well as RE4 and RERA5; these conditions impaired significantly the seminiferous tubule growth after 11 days of organotypic culture compared with the other conditions tested (p
After exclusion of the above mentioned conditions, seminiferous tubules area was significantly higher after 7 days of culture, for RE6 compared to RARE6 and
Summary
Spermatogenesis is a highly organized process of cell proliferation and terminal differentiation that leads to the formation of mature spermatozoa. Sperm cryopreservation is proposed for young adult males and adolescents prior to gonadotoxic treatment [4]. For pre-pubertal boys exposed to gonadotoxic treatment, testicular tissue freezing appears to be the only potential option to preserve their future fertility, even if this procedure remains not currently proposed. Open testicular biopsy is generally carried out under general anaesthesia in combination with another clinical care of the patient (tumor ablation, central line placement) [5,6,7]. The parents of young boys consented to testicular biopsy in 76% [5] or 93.5% [7] of cases and few sequelae occurred during intraor post-operative procedure [5,7]
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