Objective: To investigate the role of Rictor/mTORC2 in the formation of blood testis barrier (BTB), testicular development, and spermatogenesis.
Methods: Amh Cre positive mice homozygous for rictor loxP with Sertoli cell specific deletion of rictor were obtained by cross breeding Amh Cre mice with rictor loxP mice. The histology of the reproductive organs, seminiferous tubules and epididymis of the transgenic mice was observed with HE staining. The cell subgroups of the germ cells in the seminiferous tubule were detected by flow cytometry with propidium iodide labeling. The expression levels of Ki 67 and separase were detected with immunofluorescence assay, and the expression levels of BTB associated proteins were detected with immunofluorescence and Western blotting.
Results: Compared with the control (Amh Cre-, rictorloxP/loxP or rictorloxP/-) mice, the mice with Sertoli cell specific rictor deletion showed significantly decreased testicular weight and epididymis weight (P<0.05), significantly increased diploid cells (P<0.01), and decreased haploid cells (P<0.01) but comparable tetraploid cells and similar expression levels of Ki 67 and separase. The mice with rictor knockout also showed aberrant localization of BTB associated proteins, which were scattered over the whole seminiferous epithelium, but the expression levels of the protein remained stable.
Conclusion: Rictor in testicular Sertoli cells is essential for maintaining BTB integrity and function and ensuring normal spermatogenesis in mice.
目的: 研究Rictor/mTORC2在睾丸血睾屏障形成、睾丸发育和精子发生中的作用及相关机制。
方法: 采用Cre-loxP重组酶系统，构建睾丸支持细胞中rictor基因敲除小鼠。比较敲除鼠和对照鼠的生殖器官外观和质量。HE染色观察睾丸和附睾的组织形态。采用流式细胞技术检测生精小管的细胞周期。采用免疫荧光技术检测增殖蛋白（Ki-67）和分离酶蛋白的表达。采用免疫组化和Western blot技术检测血睾屏障相关蛋白表达。
结果: 相比对照鼠，敲除鼠的睾丸和附睾体积和重量都明显变小（P < 0.01）；敲除鼠的生精细胞中二倍体细胞显著上升（P < 0.01），单倍体细胞显著下降（P < 0.01），而四倍体细胞、Ki-67和分离酶蛋白无显著差异；血睾屏障相关蛋白出现严重的位置错乱，而蛋白表达量不受影响。
结论: 睾丸支持细胞中rictor基因的正常表达，在血睾屏障结构完整性维持和功能发挥以及精子正常发生中起着至关重要作用。