Division of Animal Science, University of Missouri, Columbia, MO 65211, USA.
Department of Agricultural Education, College of Education, Sunchon National University, Suncheon 57922, Korea.
Cells. 2021 Sep 17;10(9):2450. doi: 10.3390/cells10092450.
Propagation of paternal sperm-contributed mitochondrial genes, resulting in heteroplasmy, is seldom observed in mammals due to post-fertilization degradation of sperm mitochondria, referred to as sperm mitophagy. Whole organelle sperm mitochondrion degradation is thought to be mediated by the interplay between the ubiquitin-proteasome system (UPS) and the autophagic pathway (Song et al., Proc. Natl. Acad. Sci. USA, 2016). Both porcine and primate post-fertilization sperm mitophagy rely on the ubiquitin-binding autophagy receptor, sequestosome 1 (SQSTM1), and the proteasome-interacting ubiquitinated protein dislocase, valosin-containing protein (VCP). Consequently, we anticipated that sperm mitophagy could be reconstituted in a cell-free system consisting of permeabilized mammalian spermatozoa co-incubated with porcine oocyte extracts. We found that SQSTM1 was detected in the midpiece/mitochondrial sheath of the sperm tail after, but not before, co-incubation with oocyte extracts. VCP was prominent in the sperm mitochondrial sheath both before and after the extract co-incubation and was also detected in the acrosome and postacrosomal sheath and the subacrosomal layer of the spermatozoa co-incubated with extraction buffer as control. Such patterns are consistent with our previous observation of SQSTM1 and VCP associating with sperm mitochondria inside the porcine zygote. In addition, it was observed that sperm head expansion mimicked the early stages of paternal pronucleus development in a zygote during prolonged sperm-oocyte extract co-incubation. Treatment with anti-SQSTM1 antibody during extract co-incubation prevented ooplasmic SQSTM1 binding to sperm mitochondria. Even in an interspecific cellular environment encompassing bull spermatozoa and porcine oocyte extract, ooplasmic SQSTM1 was recruited to heterospecific sperm mitochondria. Complementary with the binding of SQSTM1 and VCP to sperm mitochondria, two sperm-borne pro-mitophagy proteins, parkin co-regulated gene product (PACRG) and spermatogenesis associated 18 (SPATA18), underwent localization changes after extract coincubation, which were consistent with their degradation observed inside fertilized porcine oocytes. These results demonstrate that the early developmental events of post-fertilization sperm mitophagy observed in porcine zygote can be reconstituted in a cell-free system, which could become a useful tool for identifying additional molecules that regulate mitochondrial inheritance in mammals.
父系精子来源的线粒体基因的传播，导致异质性，在哺乳动物中很少观察到，这是由于精子线粒体在受精后的降解，即精子自噬。整个细胞器精子线粒体的降解被认为是由泛素-蛋白酶体系统（UPS）和自噬途径之间的相互作用介导的（Song 等人，Proc. Natl. Acad. Sci. USA，2016）。猪和灵长类动物受精后的精子自噬都依赖于泛素结合自噬受体，即自噬相关蛋白 1（SQSTM1）和包含 valosin 的蛋白（VCP）。因此，我们预计精子自噬可以在一个由透化的哺乳动物精子与猪卵母细胞提取物共孵育组成的无细胞系统中重新构成。我们发现，SQSTM1 在与卵母细胞提取物共孵育后而不是之前，在精子尾部的中段/线粒体鞘中被检测到。VCP 在精子线粒体鞘中在提取物共孵育前后都很突出，并且在与提取缓冲液共孵育的精子的顶体和顶体后鞘以及亚顶体层中也被检测到。这种模式与我们之前观察到的 SQSTM1 和 VCP 与猪受精卵内的精子线粒体结合的模式一致。此外，在延长的精子-卵母细胞提取物共孵育过程中，观察到精子头部的扩张模拟了合子中父本原核发育的早期阶段。在提取物共孵育期间用抗 SQSTM1 抗体处理可防止卵质 SQSTM1 与精子线粒体结合。即使在包含公牛精子和猪卵母细胞提取物的种间细胞环境中，卵质 SQSTM1 也被招募到异源精子线粒体上。与 SQSTM1 和 VCP 与精子线粒体的结合互补的是，两种精子携带的促自噬蛋白，即 parkin 共调节基因产物（PACRG）和精子发生相关 18（SPATA18），在提取物共孵育后发生了定位变化，这与它们在受精的猪卵母细胞内的降解一致。这些结果表明，在猪受精卵中观察到的受精后精子自噬的早期发育事件可以在无细胞系统中重新构成，这可能成为鉴定调节哺乳动物线粒体遗传的其他分子的有用工具。
