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Improving Freezing Protocols and Organotypic Culture: A Histological Study on Rat Prepubertal Testicular Tissue

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Abstract

Testicular tissue freezing before gonadotoxic treatments allows the preservation of fertility for children suffering from cancer. Recently, the testis organ culture method was presented as a relevant method to restore the fertility of these patients. However, the yield of spermatozoa production is low in the mouse model and no gamete has been obtained in vitro in the rat model. Here, we assess different cryopreservation protocols and culture conditions to improve the efficiency of in vitro maturation of rat prepubertal testes. Testes from male rats aged 5 or 8 days post-partum were cultured onto agarose gels of different percentages. After determining the best culture conditions, different cryopreservation protocols were assessed. Finally, testicular tissues were cultured with media of various compositions and analyzed at different time points. Our results show that the cryopreservation protocols allow the preservation of tissue architecture, cell proliferation, with no or moderate increase of cell death. In vitro spermatogenesis did not proceed beyond the pachytene spermatocyte stage. Only 2 of the 6 tested media allowed the survival of differentiated germ cells over the 45-day culture period. In conclusion, this study highlights the necessity to further improve the organ culture method before applying it into the clinics.

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Abbreviations

SSV:

Solid surface vitrification

CSF:

Controlled slow freezing

Dpp :

Days post-partum

Dx:

Day x of culture

FT:

Fresh tissues

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Acknowledgments

The authors are grateful to Shirley Troche and Dimitri Hamel for excellent technical assistance, Amandine Bironneau and Véronique Sétif for their technical advices. This work was supported by a PhD Grant from University of Rouen Normandy (awarded to Justine Saulnier) and funding from Fondation de l’Avenir under Grant AP-RM-18-031, 2020.

Conflict of interest

The authors have nothing to disclose.

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Authors and Affiliations

  1. Department of Reproductive Biology–CECOS, Rouen University Hospital, EA 4308 “Gametogenesis and Gamete Quality”, UNIROUEN, Normandie Univ, 22 Boulevard Gambetta, 76000, Rouen, France

    Justine Saulnier, Antoine Oblette, Marion Delessard, Ludovic Dumont, Aurélie Rives, Nathalie Rives & Christine Rondanino

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  1. Justine Saulnier
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  2. Antoine Oblette
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Correspondence to Christine Rondanino.

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Saulnier, J., Oblette, A., Delessard, M. et al. Improving Freezing Protocols and Organotypic Culture: A Histological Study on Rat Prepubertal Testicular Tissue. Ann Biomed Eng 49, 203–218 (2021). https://doi.org/10.1007/s10439-020-02535-8

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