Tet1/Tet2缺陷影响生殖衰老及其分子机制

Tet (ten-eleven translocation) family proteins include Tet1, Tet2 and Tet3. Tets regulate DNA demethylation by epigenetic modification and play important roles in many biological processes, including embryonic development, stem cell pluripotency and differentiation, and tumorigenesis. Telomeres consist of repeated DNA repeats and associated proteins located at the end of chromosomes and maintain chromosomal stability. Telomere maintenance and function are involved in proliferation of pluripotent stem cells and pluripotency, tumorigenesis and aging including germ cells and ovarian aging. Recently we and others found that Tet1 and Tet2 deficiency leads to telomere shortening and reduced differentiation competency of pluripotent embryonic stem cells. Moreover, our preliminary observations indicate that Tet1 and Tet2 double knockout mice exhibit premature aging symptoms with age. This proposed study is to investigate whether Tets deficiency leads to gonad premature aging via telomere shortening of germ cells and somatic cells and the underlying molecular mechanisms using Tet1 and Tet2 double knockout mice. We propose the following four aims to be tested: 1) Tet1 and Tet2 deficient females exhibit premature ovarian aging; 2) Tet1 and Tet2 deficiency reduces fertility of males with age; 3) Tet1 and Tet2 deficiency impairs meiotic pairing and recombination, leading to aneuploidy and reduced embryo development; and 4) Tet1 and Tet2 mediated telomere shortening affect genome-wide gene expression which could be implicated in ovarian and testis dysfunction with age. We anticipate that the data obtained from this study will provide new mechanisms of reproductive aging and potential strategies for anti-gonad aging, benefitting human reproductive health.

Tet (ten-eleven translocation) 家族蛋白包括Tet1, Tet2 和Tet3，调控DNA去甲基化，在胚胎发育、干细胞多能性及肿瘤发生中起重要作用。端粒影响干细胞增殖和多能性维持、肿瘤发生以及衰老。Tet缺陷是否会导致早衰尚不清楚。我们发现Tet1/Tet2缺陷会导致端粒缩短，影响多能性干细胞分化发育能力，并且Tet1/Tet2缺陷小鼠随年龄增长表现出提早衰老症状。本项目试图研究Tets是否通过DNA去甲基化改变，使细胞端粒变短从而引起卵巢和睾丸早衰并揭示其分子机制。提出并验证四个方面的实验假设：1）Tet缺陷引起卵巢早衰；2）Tet缺陷加快雄性生殖能力随年龄增长而下降；3）Tet缺陷引起减数分裂异常、非整倍体增多及胚胎发育能力下降；4）Tet缺陷导致端粒变短和功能异常从而引起性腺早衰。期望为生殖衰老的机制探寻和医疗策略提供新思路，使人类生殖健康受益。

衰老涉及细胞和分子诸多方面的变化，例如基因组不稳定性，端粒损耗，表观遗传改变，干细胞衰竭等。DNA甲基化伴随着年龄增长或者体细胞表观衰老时钟而改变，表明DNA甲基化与衰老之间存在潜在的相关性。但是，异常的DNA去甲基化是否影响生殖衰老仍然是未知的。TET酶（ten-eleven translocation enzymes）可以促使DNA去甲基化，在胚胎干细胞的自我更新、肿瘤发生中起重要作用。Tet1和Tet2敲除小鼠的产生表明Tet1和Tet2在发育上是非必需的。TET酶是否参与调控成年小鼠生殖衰老尚待阐明。我们的研究显示，Tet1缺陷的成年小鼠随年龄增长，包括未分化精原细胞在内的生殖细胞逐渐丧失，精子发生减少，生殖衰老加速。Tet1缺失会降低精原细胞中5hmC的水平，并下调细胞周期，生殖细胞分化，减数分裂相关基因的表达，从而导致生殖早衰。同时，我们发现Tet2缺失也会影响雄性生殖衰老，但并不显著。我们还研究了Tet缺失对卵巢衰老的影响，发现Tet1或Tet2缺失均会降低生殖能力并导致卵巢过早衰老。但是，Tet1和Tet2缺失导致早衰的机制有所不同。值得注意的是，Tet1缺陷小鼠随着年龄的增长，其卵泡数量显著降低，卵泡储备减少，这与卵巢早衰症状一致。Tet2缺失不会影响卵泡数量，但会显著延迟第一次减数分裂前期进程和极体排出，卵母细胞质量下降，进而导致生育能力下降。通过单细胞转录组分析，Tet1缺失会导致减数分裂缺陷，从而降低生殖细胞发育和卵泡发生。Tet2缺失会损伤生殖细胞的减数分裂细胞周期，DNA修复，纺锤体装配检验点和表观遗传调控，并显著地诱导肌动蛋白细胞骨架通路的异常激活，这些因素共同导致第一次减数分裂延迟或停滞，进而导致极体排出异常。Tet1缺失会降低卵母细胞和卵泡储备，导致卵巢早衰，而Tet2缺失会损伤卵母细胞的质量。我们的研究揭示了TET酶在雄性和雌性生殖衰老中的不同作用。