男性不育的核心机制是睾丸生精及微环境功能障碍，但缺乏有效模型直观分析生精及微环境的相互作用。类器官是研究细胞与微环境间相互作用的理想模型，而前期研究只能构建具部份功能的睾丸3D细胞球，是由于所用原代细胞难完成细胞自组装。已知mTORC2/PKC通路是促进细胞自组装关键，FOXO1可激活该通路，且FOXO1/3/4是调控生精及微环境的重要因子。同时预实验发现人尿源诱导多能干细胞(u-iPSCs)分化的睾丸功能细胞具干性利于自组装。据此，我们提出“通过FOXO1激活mTORC2/PKC通路促进u-iPSCs来源功能细胞自组装形成人睾丸类器官”的假说。本项目拟采用u-iPSCs结合3D打印构建具有仿生结构和功能的人睾丸类器官，并结合基因芯片等分析FOXO1/3/4调控生精与生精微环境间的相互作用机制，旨在获得FOXO1/3/4调控睾丸生精及微环境的可靠证据，为寻找男性不育新疗法提供科学依据。

The core pathologic mechanism of male infertility is spermatogenesis dysfunction and spermatogenic microenvironment disorder, but there is no effective model to visually analyze the interaction between spermatogenesis and spermatogenic microenvironment. Organoids is an ideal model for studying the interaction between cells and microenvironment. However, our and others' previous studies only constructed testicular 3D spheroids with partial functions, due to that it is difficult to complete cell self-assembly for the primary cells used. Studies have shown that mTORC2/PKC pathway is the key to promote cell self-assembly, which can be activated by FOXO1. Meanwhile, FOXO1/3/4 are important factors to regulate spermatogenesis and spermatogenic microenvironment. At the same time, our pilot experiments found that testicular functional cells, which are differentiated from human urine derived-induced pluripotent stem cells (u-iPSCs), possess stemness and better self-assembly ability. Based on the above basis, we propose a hypothesis that " FOXO1 can promote the self-assembly of testicular functional cells from u-iPSCs to form human testicular organoids via activating the mTORC2/PKC pathway." We are intending to construct human testicular organoids that have both bionic structure and spermatogenesis and testosterone secretion function by combining u-iPSCs and 3D bio-printing. Based on these human testicular organoids, the interaction mechanism between spermatogenesis and spermatogenic microenvironment regulated by FOXO1/3/4 will be analyzed by using gene array, in order to obtain reliable evidence that FOXO1/3/4 can regulate spermatogenesis and spermatogenic microenvironment, providing a scientific basis for new therapies target on male infertility.