Arkoun Brahim, Dumont Ludovic, Milazzo Jean-Pierre, Rondanino Christine, Bironneau Amandine, Wils Julien, Rives Nathalie
EA 4308 "Gametogenesis and Gamete Quality", Reproductive Biology Laboratory - CECOS, Rouen University Hospital, Institute for Biomedical Research, University of Rouen, 76031, Rouen Cedex, France.
Biochemistry Laboratory, Rouen University Hospital, Institute for Biomedical Research, University of Rouen, 76031, Rouen Cedex, France.
Cell Tissue Res. 2016 Jun;364(3):661-674. doi: 10.1007/s00441-015-2341-2. Epub 2015 Dec 29.
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.
在进行高性腺毒性治疗之前储存睾丸组织，是尚未产生精子的青春期前男孩保留生育能力的前提条件。本研究的目的是评估在-7℃、-8℃或-9℃下进行浸泡温度处理，对冻融后的小鼠精原干细胞（SSCs）在体外成熟后产生单倍体生殖细胞能力的影响。产后6.5天大的CD-1小鼠的睾丸，采用控制慢速冷冻方案，并在-7℃、-8℃或-9℃下浸泡进行冷冻保存。将冻融后的青春期前睾丸组织在琼脂糖凝胶上进行30天的体外培养。进行组织学评估，并在对培养组织进行机械解剖后计数有鞭毛的晚期精子细胞。在-9℃处理后，冷冻的SSCs分化为细长型精子细胞比在-7℃和-8℃处理时更有效。解剖后，使用Shorr染色观察有鞭毛的晚期精子细胞。与新鲜组织对照相比，慢速冷冻后有鞭毛的晚期精子细胞数量显著减少。因此，青春期前小鼠睾丸组织慢速冷冻过程中的浸泡温度，可能对体外精子发生过程产生积极影响。就实现体外精子发生并产生更多细长型精子细胞而言，我们在-9℃浸泡温度下的慢速冷冻方案是最佳条件，尽管与新鲜组织对照相比，成熟过程的有效性有所降低。
