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Does soaking temperature during controlled slow freezing of pre-pubertal mouse testes influence course of in vitro spermatogenesis?

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Abstract

The banking of testicular tissue before highly gonadotoxic treatment is a prerequisite for the preservation of fertility in pre-pubertal boys not yet producing sperm. The aim of the current study is to evaluate the impact of a soaking temperature performed at −7 °C, −8 °C or −9 °C on the ability of frozen-thawed mouse spermatogonial stem cells (SSCs) to generate haploid germ cells after in vitro maturation. Testes of 6.5-day-old post-partum CD-1 mice were cryopreserved by using a controlled slow freezing protocol with soaking at −7 °C, −8 °C or −9 °C. Frozen-thawed pre-pubertal testicular tissues were cultured in vitro on agarose gel for 30 days. Histological evaluations were performed and flagellated late spermatids were counted after mechanical dissection of the cultured tissues. The differentiation of frozen SSCs into elongated spermatids was more efficient after treatment at −9 °C than at −7 °C and −8 °C. After dissection, flagellated late spermatids were observed by using Shorr staining. The number of flagellated late spermatids was significantly decreased after slow freezing when compared with a fresh tissue control. Therefore, the soaking temperature during slow freezing of pre-pubertal mouse testicular tissue might positively influence the course of in vitro spermatogenesis. Our slow freezing protocol with a soaking temperature at −9 °C was the optimal condition in terms of the achievement of in vitro spermatogenesis with a higher production of elongated spermatids, although the effectiveness of the maturation process was reduced compared with the fresh tissue control.

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Acknowledgments

The authors are grateful to Agathe Way for her assistance with the immunohistochemistry.

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Authors and Affiliations

  1. EA 4308 “Gametogenesis and Gamete Quality”, Reproductive Biology Laboratory - CECOS, Rouen University Hospital, Institute for Biomedical Research, University of Rouen, 76031, Rouen Cedex, France

    Brahim Arkoun, Ludovic Dumont, Jean-Pierre Milazzo, Christine Rondanino, Amandine Bironneau & Nathalie Rives

  2. Biochemistry Laboratory, Rouen University Hospital, Institute for Biomedical Research, University of Rouen, 76031, Rouen Cedex, France

    Julien Wils

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  1. Brahim Arkoun
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  2. Ludovic Dumont
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  3. Jean-Pierre Milazzo
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  4. Christine Rondanino
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  5. Amandine Bironneau
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  6. Julien Wils
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  7. Nathalie Rives
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Correspondence to Nathalie Rives.

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The authors declare no competing interests.

Additional information

This study was supported by a PhD grant from ”La Région Haute-Normandie” (awarded to Brahim Arkoun) and funding from Rouen University Hospital, Institute for Research and Innovation in Biomedicine, Agence de la Biomédecine and “la Ligue nationale contre le cancer”.

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Fig. S1

Representation of experimental procedures performed with pre-pubertal (6.5 dpp) mouse testes. a Controlled slow freezing of pre-pubertal mouse testes with various soaking temperatures (−7 °C, −8 °C or −9 °C) before their storage in liquid nitrogen at −196 °C. b Thawing procedure of immature mouse testes. c In vitro culture of frozen-thawed pre-pubertal mouse testes on agarose gels at a gas-liquid interphase, according to the various soaking temperatures used above. d Set of experiments performed after culture of the frozen-thawed pre-pubertal mouse testicular tissues (D DMSO, dpp day post-partum, FCS fetal calf serum, KSR knock-out serum replacement, L-15 Leibovitz medium, LN 2 liquid nitrogen, α-MEM alpha-minimum essential medium, RE retinol, RIA radioimmunoassay, S sucrose). (PDF 363 kb)

Fig. S2

Assessment of Leydig cell functional integrity during in vitro culture of frozen-thawed pre-pubertal (6.5 dpp) mouse testes using the −9 °C condition. Results indicate the mean of testosterone concentration in the culture medium under the various conditions tested. Data are expressed as mean ± SEM; n = 4 mouse testes for each condition. *Statistically significant difference between −9 °C and fresh tissue control; P < 0.05 (D day, NS not significant). (PDF 246 kb)

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Arkoun, B., Dumont, L., Milazzo, JP. et al. Does soaking temperature during controlled slow freezing of pre-pubertal mouse testes influence course of in vitro spermatogenesis?. Cell Tissue Res 364, 661–674 (2016). https://doi.org/10.1007/s00441-015-2341-2

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