P017 The maintenance of testicular architecture and germ cell in adult testis tissue under organ culture condition based on the gas-liquid interface method
P017 The maintenance of testicular architecture and germ cell in adult testis tissue under organ culture condition based on the gas-liquid interface method
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P017 基于气液界面法的器官培养条件下成人睾丸组织睾丸结构和生殖细胞的维持
DOI：
10.1093/humrep/deab130.016
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发表时间：
2021
影响因子：
6.1
通讯作者：
Ogawa T
中科院分区：
文献类型：
--
作者：
Komeya M;Odaka H;Matsumura T;Yamanaka H;Sato T;Yao M;Masumori N;Ogawa T
气液界面器官培养系统在小鼠体内实现了体外精子发生，是否也能支持人睾丸的体外精子发生？概要答案新鲜和冷冻保存的睾丸组织中，虽然没有观察到精子发生的进程，但生殖细胞在结构没有退化的情况下得以维持。已知的情况尽管体外精子发生的研究已经进行了100年，但只有采用气液界面法的器官培养系统实现了小鼠体外精子发生。目前还没有证实这种培养系统是否可以应用于包括人类在内的其他哺乳动物并诱导精子发生。研究设计、大小、持续时间睾丸组织取自接受性别重置手术的变性患者。睾丸标本立即处理培养或冷冻保存，使用玻璃化冷冻方案。在三种不同的培养基中进行睾丸碎片的器官培养，最长时间为3周，以评估培养组织的短期变化（生殖细胞和体细胞的活力、增殖和维持）。参与者/材料、设置、方法新鲜和冷冻保存-解冻的睾丸碎片（1-2 mm 3）使用器官培养系统在具有敲除血清替代物的α-MEM中培养（K组），A组为α-MEM+高脂BSA，D组为DMEM + FBS。补充黄体生成素、卵泡刺激素和睾酮。每周用免疫组化染色法检测睾丸生精细胞的数量（DDX 4法）、增殖活性（EdU法）和小管内细胞凋亡（TdT介导的dUTP缺口末端标记法）。主要结果和机会的作用新鲜培养组和冻存培养组的生精小管结构一直保持到培养第2周。D、K、A组生精小管DDX 4阳性细胞数分别为49 ± 24、55 ± 21、50 ± 26个/曲细精管，1周32 ± 13、42 ± 7、36 ± 21个/曲细精管。2周后逐渐减少至26 ± 8、24 ± 6和27 ± 18个细胞/小管，各组间无差异。D、K、A组肾小管内EdU阳性细胞数分别为0.2 ± 0.2、2.8 ± 2.1、1.1 ± 0.8个/小管，1周时分别为0.1 ± 0.2、0.5 ± 0.6、0.3 ± 0.6个/小管。2周时的值分别为0.01、0.05和0.03。因此，EdU阳性细胞从培养的第一周开始急剧减少。冻存培养组DDX 4阳性生殖细胞和小管内EdU阳性细胞数与新鲜培养组无差异。局限性、注意事项目前的器官培养体系不完善，无法诱导人类体外精子发生。本研究的意义在于：我们的器官培养系统能够维持睾丸结构和生殖细胞的完整性。本研究通过使用变性患者的睾丸组织，促进生殖细胞增殖和分化所需的培养条件的研究。试验注册号创新领域科学研究资助金18 H 05546、青年科学家资助金（A）17 H 05098、武田科学基金会
Study questionCan the gas-liquid interface organ culture system that achieved in vitro spermatogenesis in mice also support in vitro spermatogenesis in human adult testis?Summary answerAlthough the progression of spermatogenesis was not observed, germ cells were maintained without the degeneration of the architecture in both fresh and cryopreserved testicular tissues.What is known alreadyAlthough the research on in vitro spermatogenesis have been conducted for 100 years, only the organ culture system using gas-liquid interface method achieved in vitro spermatogenesis in mice. It has not been verified whether this culture system can be applied to other mammals including humans and induce spermatogenesis.Study design, size, durationTesticular tissue was obtained from the transgender patients receiving sex reassignment surgery. Testicular specimens were either immediately processed for cultivation or cryopreserved, using a vitrification freezing protocol. Organ culture of testicular fragments was performed in three different media for a maximum period of 3 weeks to evaluate the short-term changes in the cultured tissues (viability, proliferation and maintenance of germ and somatic cells).Participants/materials, setting, methodsFresh and cryopreserved-thawed testis fragments (1–2 mm3) were cultured using the organ culture system in alpha-MEM with knock-out serum replacement (K group), alpha-MEM with lipid-rich BSA (A group) and DMEM with FBS (D group). Luteinizing hormone, follicle stimulating hormone and testosterone were supplemented. The number of germ cells (using DDX4), proliferative activity of germ cells (using EdU assay) and intratubular cell apoptosis (by TdT-mediated dUTP Nick End Labeling) were evaluated by immunohistochemical staining weekly.Main results and the role of chanceThe architecture of the seminiferous tubules was maintained until the second week of culture in both the fresh and the cryopreserved culture group. The number of DDX4-positive germ cells per seminiferous tubule in groups D, K, and A was 49 ± 24, 55 ± 21, 50 ± 26 cells/tubule in 1 day, 32 ± 13, 42 ± 7, 36 ± 21 cells/tubule in 1week, respectively. The numbers gradually decreased to 26 ± 8, 24 ± 6 and 27 ± 18 cells/tubule, in 2 weeks, respectively, with no difference among the groups. The number of intratubular EdU-positive cells of groups D, K, and A was 0.2 ± 0.2, 2.8 ± 2.1, 1.1 ± 0.8 cells/tubule at 1 day, 0.1 ± 0.2, 0.5 ± 0.6, 0.3 ± 0.6 cells/tubule at 1 week, respectively. The values were 0.01, 0.05, and 0.03 at 2 weeks. Thus, EdU-positive cells drastically decreased from the first week of culture. The number of DDX4-positive germ cells and the intratubular EdU-positive cells in the cryopreserved culture group was not different from that in the fresh culture group.Limitations, reasons for cautionCurrent organ culture systems are incomplete, being unable to induce human in vitro spermatogenesis. Further research is needed to improve culture condition with the aim of producing fertile sperm of infertile adult male patients.Wider implications of the findings: Our organ culture system could maintain testis structure and germ cells. By using the testis tissues of the transgender patients, which are available with their consent, we will promote the investigation of the culture condition necessary for germ cell proliferation and differentiation.Trial registration numberGrant-in-Aid for Scientific Research on Innovative Areas 18H05546, Grant-in-Aid for Young Scientists (A) 17H05098 and Takeda Science Foundation