Univ Rouen Normandie, INSERM, NORDIC UMR 1239 - Team Adrenal and Gonadal Pathophysiology (AGoPath), Rouen, France.
Normandie, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France.
Front Endocrinol (Lausanne). 2023 Mar 17;14:1112834. doi: 10.3389/fendo.2023.1112834. eCollection 2023.
Suitable cryopreservation procedures of pre-pubertal testicular tissue associated with efficient culture conditions are crucial in the fields of fertility preservation and restoration. spermatogenesis remains a challenging technical procedure to undergo a complete spermatogenesis.The number of haploid cells and more specifically the spermatic yield produced in mice is still extremely low compared to age-matched controls and this procedure has never yet been successfully transferred to humans.
To evaluate the impact of in vitro culture and freezing procedure, pre-pubertal testicular mice testes were directly cultured until day 4 (D4), D16 and D30 or cryopreserved by controlled slow freezing then cultured until D30. Testes composed of a panel of 6.5 dpp (days postpartum), 10.5 dpp, 22.5 dpp, and 36.5 dpp mice were used as controls. Testicular tissues were assessed by histological (HES) and immunofluorescence (stimulated by retinoic acid gene 8, STRA8) analyses. Moreover, a detailed transcriptome evaluation study has been carried out to study the gene expression patterns throughout the first spermatogenic wave.
Transcriptomic analyses reveal that cultured tissues expression profiles are almost comparable between D16 and D30; highlighting an abnormal kinetic throughout the second half of the first spermatogenesis during cultures. In addition, testicular explants have shown dysregulation of their transcriptomic profile compared to controls with genes related to inflammation response, insulin-like growth factor and genes involved in steroidogenesis.
The present work first shows that cryopreservation had very little impact on gene expression in testicular tissue, either directly after thawing or after 30 days in culture. Transcriptomic analysis of testis tissue samples is highly informative due to the large number of expressed genes and identified isoforms. This study provides a very valuable basis for future studies concerning spermatogenesis in mice.
适合的未成年睾丸组织冷冻保存程序与有效的培养条件在生育力保存和恢复领域至关重要。精子发生仍然是一个具有挑战性的技术过程，无法完成完整的精子发生。与年龄匹配的对照相比， 单倍体细胞的数量，特别是精子产量，在小鼠中仍然极低，而且这一过程从未成功地应用于人类。
为了评估体外培养和冷冻程序的影响，直接培养未成年小鼠睾丸组织直至第 4 天（D4）、第 16 天（D16）和第 30 天（D30），或通过控制缓慢冷冻进行冷冻保存，然后培养至第 30 天。使用 6.5 dpp（产后天数）、10.5 dpp、22.5 dpp 和 36.5 dpp 的小鼠睾丸组织作为对照。通过组织学（HES）和免疫荧光分析（视黄酸基因 8 刺激，STRA8）评估睾丸组织。此外，还进行了详细的转录组评估研究，以研究整个第一次精子发生波中的基因表达模式。
转录组分析表明，培养组织的表达谱在 D16 和 D30 之间几乎相同；突出显示在培养过程中第二次精子发生的后半段存在异常动力学。此外，与对照相比，睾丸外植体的转录组谱失调，与炎症反应、胰岛素样生长因子和类固醇生成相关的基因有关。
本工作首次表明，冷冻保存对睾丸组织的基因表达几乎没有影响，无论是在解冻后直接进行，还是在培养 30 天后进行。由于表达基因和鉴定的同工型数量众多，对睾丸组织样本的转录组分析提供了非常有价值的基础，可用于未来对小鼠精子发生的研究。
