Hypoxanthine phosphoribosyltransferase (HPRT)-deficiency is associated with impaired fertility in the female rat.
Hypoxanthine phosphoribosyltransferase (HPRT)-deficiency is associated with impaired fertility in the female rat.
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次黄嘌呤磷酸核糖转移酶（HPRT）缺乏与雌性大鼠的生育能力受损有关。
DOI：
10.1002/mrd.23413
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发表时间：
2020
影响因子：
2.5
通讯作者：
Meek S
中科院分区：
文献类型：
--
作者：
Meek S
嘌呤代谢产物在哺乳动物早期胚胎发育中起着重要的调控作用。环AMP、环GMP和次黄嘌呤的水平在体内调节小鼠卵母细胞的减数分裂停滞，而次黄嘌呤、腺嘌呤或肌苷的水平升高可在体外胚胎发育期间破坏第一卵裂阶段（Dienhart &唐斯，1996; Wigglesworth等人，2013年）。次黄嘌呤磷酸核糖基转移酶（HPRT）是嘌呤补救途径的重要组成部分，参与次黄嘌呤和鸟嘌呤的再循环，为核酸和关键代谢物（包括第二信使）的合成提供底物。HPRT基因位于X染色体上，当在人类中发生突变时，会导致男性的衰弱性神经系统疾病Lesch-Nyhan病（Lesch & Nyhan，1964）。在这里，我们报告了HPRT的缺乏破坏了早期胚胎发育，导致雌性Hprt敲除（KO）大鼠的生育能力受损。我们先前描述了使用靶向大鼠DA胚胎干细胞产生Hprt KO大鼠（Meek等人，2016年）。Hprt突变大鼠缺乏Hprt基因的外显子7和8，并且不表达HPRT蛋白。尽管Hprt KO大鼠看起来总体上健康，但它们在中脑中表现出降低的多巴胺水平，这与先前在Hprt KO小鼠和人类Lesch-Nyhan患者中进行的观察一致（Meek等人，2016年）。为了检查大鼠胚胎发育期间对HPRT功能的需求，我们将Hprt KO大鼠杂交，但重复交配未能产生任何后代（图1a）。尽管从KO× KO交配中回收了受精的1细胞胚胎，但在E4天仅回收了碎片胚胎。5，当野生型胚胎正常达到囊胚阶段时（图1b，c）。Hprt KO雄性是可生育的，并且可以产生具有类似DA/Sprague道利混合遗传背景的大鼠典型的正常大小的窝仔（图1a; Meek等人，2020年）。相比之下，与野生型雄性交配的Hprt KO雌性大鼠在E4天产生了许多碎片胚胎和< 50%的扩张囊胚。5，这与足月时窝仔数减少相对应（图1b和1 e）。有趣的是，这些窝仔中有雄性幼仔，尽管数量略有减少，表明“拯救”并不依赖于来自携带X染色体精子的完整Hprt等位基因的贡献（图1a）。在与来自携带Rex 1-EGFP敲入报告基因的转基因系的野生型雄性交配中，Rex 1-EGFP敲入报告基因首先在4-8细胞阶段表达（Meek et al.，2020），Rex 1-EGFP荧光在几乎所有碎片胚胎中都很明显（图1d，e）。这证实了大多数HPRT缺陷卵母细胞的受精已经发生，并且合子基因激活已经在大多数退化胚胎中开始。未能从Hprt KO大鼠之间的杂交中恢复完整的囊胚证明，HPRT活性对于早期胚胎发育的初始卵裂的适当进展是必需的。
Purine metabolites play critical roles in regulating early embryonic development in mammals. The levels of cyclic AMP, cyclic GMP and hypoxanthine regulate meiotic arrest of mouse oocytes in vivo, whilst elevated levels of hypoxanthine, adenine, or inosine can disrupt the first cleavage stages during embryonic development in vitro (Dienhart & Downs, 1996; Wigglesworth et al., 2013). The enzyme hypoxanthine phosphoribosyltransferase (HPRT) is an essential component of the purine salvage pathway, involved in recycling hypoxanthine and guanine to provide substrates for the synthesis of nucleic acids and key metabolites including second messengers. The HPRT gene is located on the X chromosome and when mutated in humans causes the debilitating neurological disorder Lesch–Nyhan disease in males (Lesch & Nyhan, 1964). Here, we report that absence of HPRT disrupts early embryonic development leading to impaired fertility in female Hprt knock‐out (KO) rats. We previously described the generation of Hprt KO rats using targeted rat DA embryonic stem cells (Meek et al., 2016). The Hprt mutant rats lack exons 7 and 8 of the Hprt gene and do not express HPRT protein. Although the Hprt KO rats appeared generally healthy, they exhibited reduced levels of dopamine in the midbrain, in line with previous observations made in Hprt KO mice and in human Lesch–Nyhan patients (Meek et al., 2016). To examine the requirement for HPRT function during rat embryonic development we crossed Hprt KO rats, but repeated matings failed to produce any offspring (Figure 1a). Although fertilized 1‐cell embryos were recovered from KO× KO matings, only fragmented embryos were recovered at day E4. 5, when wild‐type embryos would normally reach the blastocyst stage (Figure 1b, c). Hprt KO males were fertile and could produce normal sized litters typical of rats with a similar DA/Sprague Dawley mixed genetic background (Figure 1a; Meek et al., 2020). In contrast, Hprt KO female rats mated with wild‐type males produced many fragmented embryos and< 50% expanded blastocysts at day E4. 5, which corresponded with reduced litter sizes at term (Figures 1b and 1e). Interestingly, male pups were represented in these litters, albeit at slightly reduced numbers, indicating that “rescue” did not rely on the contribution of an intact Hprt allele from X‐chromosome‐bearing sperm (Figure 1a). In matings with wild‐type males from a transgenic line carrying a Rex1‐EGFP knock‐in reporter gene that is first expressed at the 4–8‐cell stage (Meek et al., 2020), Rex1‐EGFP fluorescence was evident in almost all fragmented embryos (Figure 1d, e). This confirmed that fertilization of most HPRT‐deficient oocytes had taken place, and zygotic gene activation had begun in the majority of the degenerating embryos. The failure to recover intact blastocysts from crosses between Hprt KO rats demonstrated that HPRT activity is essential for proper progression through the initial cleavages of early embryonic