Dumont Ludovic, Rives-Feraille Aurélie, Delessard Marion, Saulnier Justine, Rondanino Christine, Rives Nathalie
Department of Reproductive Biology - CECOS, Normandie Univ, UNIROUEN, EA 4308 "Gametogenesis and Gamete Quality", Rouen University Hospital, Rouen, France.
Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France.
Andrology. 2021 Mar;9(2):673-688. doi: 10.1111/andr.12928. Epub 2020 Nov 16.
Oncological procedures have irreversible side effects on germ cells for childhood cancer survival boys. In vitro culture of prepubertal testicular tissue has been proposed to restore fertility; however, recent data on animal models showed that meiotic and post-meiotic progression was impaired.
As potential key inducers of the mitosis-meiosis switch, type 2 cannabinoid receptor (CB ) has been proposed to play a central role in the meiotic entry of male germ cells. Herein, the in vitro first spermatogenesis wave in mice was used to understand the impact of CB activation on the differentiation of spermatogonia until elongated spermatids.
A first set of cultured testicular explants of 6.5 days post-partum (dpp) mice was performed to assess the impact of a range of JWH133 supplementation (10 nm, 100 nm, 1 µm, 10 µm). Then, the progressive development of germ cells at key timepoints of spermatogenesis was evaluated throughout (i) in vitro culture (day 2 [D2], D3, D6, D10, D18, and D30) coupled with (ii) in vivo counterparts (8.5, 9.5, 12.5, 16.5, 24.5, and 36.5 dpp).
CB was detected at the plasma membrane of cells, and a successful completion of spermatogenesis was obtained in vitro. One day after the activation of CB by 1 μm of the agonist JWH133, percentage of zygotene spermatocyte I increased.
After 30 days of culture, (i) an enrichment of haploid germ cells detected by flow cytometry, (ii) a reduced necrotic area, and (iii) an increase in the density of post-meiotic germ cells were observed. We showed that the activation of CB improves in vitro entry into meiosis and differentiation of spermatogonia, mimicking physiological meiotic transition.
肿瘤治疗程序会对童年癌症存活男孩的生殖细胞产生不可逆的副作用。有人提出对青春期前睾丸组织进行体外培养以恢复生育能力；然而，近期动物模型数据显示减数分裂及减数分裂后进程受损。
作为有丝分裂-减数分裂转换的潜在关键诱导因子，有人提出2型大麻素受体（CB）在雄性生殖细胞减数分裂起始过程中起核心作用。在此，利用小鼠体外首次精子发生波来了解CB激活对精原细胞直至延长型精子细胞分化的影响。
对出生后6.5天（dpp）小鼠的第一组培养睾丸外植体进行实验，以评估一系列JWH133添加量（10 nM、100 nM、1 μM、10 μM）的影响。然后，在精子发生的关键时间点，通过（i）体外培养（第2天[D2]、D3、D6、D10、D18和D30）并结合（ii）体内对应阶段（8.5、9.5、12.5、16.5、24.5和36.5 dpp）来评估生殖细胞的逐步发育。
在细胞膜上检测到CB，且体外成功完成了精子发生。用1 μM激动剂JWH133激活CB一天后，偶线期精母细胞I的百分比增加。
培养30天后，观察到（i）通过流式细胞术检测到单倍体生殖细胞富集，（ii）坏死面积减小，以及（iii）减数分裂后生殖细胞密度增加。我们表明，CB的激活改善了体外减数分裂起始及精原细胞分化，模拟了生理性减数分裂转变。
