Division of Animal Sciences, University of Missouri, Columbia, MO, USA.
Department of Obstetrics, Gynecology and Women's Health, University of Missouri, Columbia, MO, USA.
Biol Reprod. 2021 Jan 4;104(1):117-129. doi: 10.1093/biolre/ioaa186.
The ubiquitin-proteasome system plays diverse regulatory and homeostatic roles in mammalian reproduction. Ubiquitin ligases are the substrate-specific mediators of ubiquitin-binding to its substrate proteins. The NEDD4-like ubiquitin ligase 2 (aliases NEDL2, HECW2) is a HECT-type ubiquitin ligase that contains one N-terminal HECW ubiquitin ligase domain, one C-terminal HECT ubiquitin ligase domain, one C2 domain, and two WW protein-protein interaction modules. Beyond its predicted ubiquitin-ligase activity, its cellular functions are largely unknown. Current studies were designed to investigate the content and distribution of NEDL2 in porcine spermatozoa, oocytes, zygotes, and early preimplantation embryos, and in cumulus cells before and after in vitro maturation with oocytes, and fibroblast cells as positive control by western blot and immunocytochemistry, and to examine its roles during oocyte fertilization. Multiple isoforms of NEDL2 were identified by WB. One at approximately 52 kDa was detected only in the germinal vesicle (GV) stage and metaphase II oocytes, and in early preimplantation embryos. Other isoforms were high mass bands at 91, 136, and 155 kDa, which were only detected in somatic cells. Interestingly, ejaculated spermatozoa prominently displayed the same 52 kDa band as oocytes; they also had two minor bands of 74 and 129 kDa, which were not detected in somatic cells or oocytes. By immunofluorescence, NEDL2 showed a diffused cytoplasmic localization in all cell types and accumulated in distinct foci in the germinal vesicles (GVs) of immature oocytes, in maternal and paternal pronuclei of zygotes and nuclei of embryo blastomeres and somatic cells. In blastocysts, the labeling intensity of NEDL2 was stronger in the inner cell mass than in trophoblast, indicating higher NEDL2 content in the ICM cells than in trophectoderm. NEDL2 abundance was 10 times higher in post-maturation oocyte-surrounding cumulus cells than that of cumulus cells before in vitro maturation with hormones, indicating that NEDL2 may have a unique role in cumulus cells after ovulation. Microinjection of anti-NEDL2 antibody into oocyte before IVF did not affect the percentage of oocytes fertilized, percentage of oocytes cleaved, or blastocyst formation. However, the anti-NEDL2 antibody decreased the number of pronuclei, accelerated the formation of nuclear precursor bodies at 6 h postfertilization, inhibited sperm DNA decondensation, and resulted in more fertilized oocytes without male pronuclear formation. In summary, NEDL2 may play a key role during fertilization, especially during sperm DNA decondensation.
泛素-蛋白酶体系统在哺乳动物生殖中发挥着多样化的调节和内稳态作用。泛素连接酶是泛素结合到其底物蛋白的底物特异性介质。NEDD4 样泛素连接酶 2（别名 NEDL2、HECW2）是一种 HECT 型泛素连接酶，包含一个 N 端 HECW 泛素连接酶结构域、一个 C 端 HECT 泛素连接酶结构域、一个 C2 结构域和两个 WW 蛋白-蛋白相互作用模块。除了其预测的泛素连接酶活性外，其细胞功能在很大程度上尚不清楚。目前的研究旨在通过 Western blot 和免疫细胞化学法，研究 NEDL2 在猪精子、卵母细胞、受精卵和早期胚胎以及体外成熟卵母细胞前后的卵丘细胞中的含量和分布，并作为阳性对照检查其在卵母细胞受精过程中的作用。通过 WB 鉴定出 NEDL2 的多种同工型。一种约 52 kDa 的同工型仅在生发泡 (GV) 期和中期 II 期卵母细胞以及早期胚胎中检测到。其他同工型是 91、136 和 155 kDa 的高分子量条带，仅在体细胞中检测到。有趣的是，射出的精子与卵母细胞一样明显显示出相同的 52 kDa 条带；它们还具有 74 和 129 kDa 的两个较小条带，在体细胞或卵母细胞中均未检测到。通过免疫荧光，NEDL2 在所有细胞类型中均显示弥散的细胞质定位，并在未成熟卵母细胞的生发泡 (GV) 中、受精卵的母本和父本原核以及胚胎卵裂球和体细胞的核中积累在不同的焦点中。在囊胚中，内细胞团中 NEDL2 的标记强度强于滋养外胚层，表明内细胞团中的 NEDL2 含量高于滋养外胚层。与激素体外成熟前的卵丘细胞相比，成熟后卵母细胞周围卵丘细胞中的 NEDL2 丰度高 10 倍，表明 NEDL2 在排卵后卵丘细胞中可能具有独特的作用。在 IVF 前将抗 NEDL2 抗体注入卵母细胞中不会影响卵母细胞受精的百分比、卵母细胞分裂的百分比或囊胚形成。然而，抗 NEDL2 抗体减少了原核的数量，加速了受精后 6 小时核前体的形成，抑制了精子 DNA 去浓缩，并导致更多的受精卵母细胞没有雄性原核形成。总之，NEDL2 可能在受精过程中发挥关键作用，尤其是在精子 DNA 去浓缩过程中。
