FBXW24 通过调控 SYCP3 的泛素化水平来控制雌性减数分裂前期的进程。
FBXW24 controls female meiotic prophase progression by regulating SYCP3 ubiquitination.
机构信息
State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China.
Analysis and Test Center, Nanjing Medical University, Nanjing, China.
出版信息
Clin Transl Med. 2022 Jul;12(7):e891. doi: 10.1002/ctm2.891.
BACKGROUND
An impeccable female meiotic prophase is critical for producing a high-quality oocyte and, ultimately, a healthy newborn. SYCP3 is a key component of the synaptonemal complex regulating meiotic homologous recombination. However, what regulates SYCP3 stability is unknown.
METHODS
Fertility assays, follicle counting, meiotic prophase stage (leptotene, zygotene, pachytene and diplotene) analysis and live imaging were employed to examine how FBXW24 knockout (KO) affect female fertility, follicle reserve, oocyte quality, meiotic prophase progression of female germ cells, and meiosis of oocytes. Western blot and immunostaining were used to examined the levels & signals (intensity, foci) of SYCP3 and multiple key DSB indicators & repair proteins (γH2AX, RPA2, p-CHK2, RAD51, MLH1, HORMAD1, TRIP13) after FBXW24 KO. Co-IP and immuno-EM were used to examined the interaction between FBXW24 and SYCP3; Mass spec was used to characterize the ubiquitination sites in SYCP3; In vivo & in vitro ubiquitination assays were utilized to determine the key sites in SYCP3 & FBXW24 for ubiquitination.
RESULTS
Fbxw24-knockout (KO) female mice were infertile due to massive oocyte death upon meiosis entry. Fbxw24-KO oocytes were defective due to elevated DNA double-strand breaks (DSBs) and inseparable homologous chromosomes. Fbxw24-KO germ cells showed increased SYCP3 levels, delayed prophase progression, increased DSBs, and decreased crossover foci. Next, we found that FBXW24 directly binds and ubiquitinates SYCP3 to regulate its stability. In addition, several key residues important for SYCP3 ubiquitination and FBXW24 ubiquitinating activity were characterized.
CONCLUSIONS
We proposed that FBXW24 regulates the timely degradation of SYCP3 to ensure normal crossover and DSB repair during pachytene. FBXW24-KO delayed SYCP3 degradation and DSB repair from pachytene until metaphase II (MII), ultimately causing failure in oocyte maturation, oocyte death, and infertility.
背景
完美的减数分裂前期对于产生高质量的卵子至关重要，而卵子的质量又直接决定了新生儿的健康。SYCP3 是联会复合体的关键组成部分，可调节减数分裂同源重组。然而，SYCP3 稳定性的调节机制尚不清楚。
方法
利用生育能力检测、卵泡计数、减数分裂前期阶段（细线期、偶线期、粗线期和双线期）分析和活细胞成像技术，研究 FBXW24 敲除（KO）如何影响雌性生育能力、卵泡储备、卵子质量、雌性生殖细胞减数分裂前期进展以及卵母细胞减数分裂。Western blot 和免疫染色用于检测 FBXW24 KO 后 SYCP3 及其多个关键双链断裂（DSB）指标和修复蛋白（γH2AX、RPA2、p-CHK2、RAD51、MLH1、HORMAD1、TRIP13）的水平和信号（强度、焦点）。免疫共沉淀和免疫电镜用于检测 FBXW24 和 SYCP3 之间的相互作用；质谱用于鉴定 SYCP3 中的泛素化位点；体内和体外泛素化实验用于确定 SYCP3 和 FBXW24 中的关键泛素化位点。
结果
Fbxw24-KO 雌性小鼠由于减数分裂进入时大量卵母细胞死亡而不育。由于 DNA 双链断裂（DSBs）增加和同源染色体分离不完全，Fbxw24-KO 卵母细胞存在缺陷。Fbxw24-KO 生殖细胞显示 SYCP3 水平升高、减数分裂前期进展延迟、DSB 增加和交叉焦点减少。接下来，我们发现 FBXW24 直接结合并泛素化 SYCP3 以调节其稳定性。此外，还鉴定了几个对 SYCP3 泛素化和 FBXW24 泛素化活性很重要的关键残基。
结论
我们提出，FBXW24 调节 SYCP3 的适时降解，以确保在粗线期的正常交叉和 DSB 修复。FBXW24-KO 延迟了 SYCP3 的降解和从粗线期到中期 II（MII）的 DSB 修复，最终导致卵母细胞成熟失败、卵母细胞死亡和不育。
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