Autophagy core protein ATG5 is required for elongating spermatid development, sperm individualization and normal fertility in male mice
Autophagy core protein ATG5 is required for elongating spermatid development, sperm individualization and normal fertility in male mice
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自噬核心蛋白 ATG5 是延长雄性小鼠精子细胞发育、精子个体化和正常生育能力所必需的
DOI：
10.1080/15548627.2020.1783822
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发表时间：
2020-07-18
期刊：
影响因子：
13.3
通讯作者：
Zhang, Zhibing
中科院分区：
文献类型：
--
作者：
Huang, Qian;Liu, Yunhao;Zhang, Zhibing
摘要精子发生是精子发生过程中最长的阶段，其形态发生剧烈变化，是精子形成的最后一步，它涉及蛋白质的降解和多余细胞质的去除，因此我们推测大自噬/自噬可能参与了这一过程。为了验证这一假设，我们研究了ATG5的功能，ATG5是男性生殖细胞发育中的一种核心自噬蛋白。将ATG5和Stra8−iCre小鼠杂交，有条件地灭活雄性生殖细胞中的ATG5。在ATG5FLOX/FLOX；Stra8−iCre突变小鼠中，睾丸自噬标记LC3A/B-II的表达显著降低，而自噬受体SQSTM1/P62的表达显著增加，表明睾丸自噬活性降低。突变小鼠的生育力大幅下降，约70%是不育的。与对照组相比，精子数量和活动率也显著降低。组织学检查发现，突变的睾丸在生精上皮内正常吸收后，其管腔内有许多大的残留体。附睾尾部腔内充满松弛的生殖细胞、大的细胞质小体和头部和尾部杂乱的精子。附睾尾精子的电子显微镜检查显示，精子头部畸形，中段有不连续的附属物结构，顶体形成异常，精子个性化丧失。精子免疫荧光染色显示突变小鼠精子线粒体和顶体分布异常。ATG5被证明通过调节多种信号来诱导自噬来维持正常的发育过程。我们的研究表明，ATG5对男性生育能力是必不可少的，并参与精子发生的各个方面。缩写：AKAP4：A-激酶锚定蛋白4；ATG5：自噬相关5；ATG7：自噬相关7；ATG10：自噬相关10；ATG12：自噬相关12；CKO：条件性基因敲除；DDX4：DEAD-box解旋酶4；MAP1LC3/LC3/TG8：微管相关蛋白1轻链3；PBS：磷酸盐缓冲盐水；PIWIL2/MILI：Piwi样RNA介导的基因沉默2；RT-PCR：逆转录聚合酶反应；SQSTM1/p62：序列1；TBC：小管球蛋白复合体；WT：野生型。
ABSTRACT Spermiogenesis is the longest phase of spermatogenesis, with dramatic morphological changes and a final step of spermiation, which involves protein degradation and the removal of excess cytoplasm; therefore, we hypothesized that macroautophagy/autophagy might be involved in the process. To test this hypothesis, we examined the function of ATG5, a core autophagy protein in male germ cell development. Floxed Atg5 and Stra8− iCre mice were crossed to conditionally inactivate Atg5 in male germ cells. In Atg5flox/flox; Stra8− iCre mutant mice, testicular expression of the autophagosome marker LC3A/B-II was significantly reduced, and expression of autophagy receptor SQSTM1/p62 was significantly increased, indicating a decrease in testicular autophagy activity. The fertility of mutant mice was dramatically reduced with about 70% being infertile. Sperm counts and motility were also significantly reduced compared to controls. Histological examination of the mutant testes revealed numerous, large residual bodies in the lumen of stages after their normal resorption within the seminiferous epithelium. The cauda epididymal lumen was filled with sloughed germ cells, large cytoplasmic bodies, and spermatozoa with disorganized heads and tails. Examination of cauda epididymal sperm by electron microscopy revealed misshapen sperm heads, a discontinuous accessory structure in the mid-piece and abnormal acrosome formation and loss of sperm individualization. Immunofluorescence staining of epididymal sperm showed abnormal mitochondria and acrosome distribution in the mutant mice. ATG5 was shown to induce autophagy by mediating multiple signals to maintain normal developmental processes. Our study demonstrated ATG5 is essential for male fertility and is involved in various aspects of spermiogenesis. Abbreviations: AKAP4: a-kinase anchoring protein 4; ATG5: autophagy-related 5; ATG7: autophagy-related 7; ATG10: autophagy-related 10; ATG12: autophagy-related 12; cKO: conditional knockout; DDX4: DEAD-box helicase 4; MAP1LC3/LC3/tg8: microtubule-associated protein 1 light chain 3; PBS: phosphate-buffered saline; PIWIL2/MILI: piwi like RNA-mediated gene silencing 2; RT-PCR: reverse transcription-polymerase chain reaction; SQSTM1/p62: sequestosome 1; TBC: tubulobulbar complexes; WT: wild type.