Olli Kristine E, Li Kun, Galileo Deni S, Martin-DeLeon Patricia A
Department of Biological Sciences, University of Delaware, Newark, Delaware.
J Cell Physiol. 2018 Jan;233(1):11-22. doi: 10.1002/jcp.25882. Epub 2017 Mar 28.
Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca -ATPase 4 (Pmca4) gene which encodes the highly conserved Ca efflux pump, PMCA4. This is the major Ca clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co-immunoprecipitation (Co-IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca ] in capacitated and Ca ionophore-treated sperm and with neuronal (nNOS) at basal [Ca ] (ucapacitated sperm). FRET efficiencies for PMCA4-eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4-nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co-immunoprecipitated with eNOS in a Ca -dependent manner. In Pmca4 sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild-type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co-ordinates Ca and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4 males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance.
精子活力降低（弱精子症）及由此导致的不育是由于编码高度保守的钙外流泵PMCA4的质膜钙ATP酶4（Pmca4）基因缺失所致。PMCA4是小鼠精子中的主要钙清除蛋白。由于PMCA4缺失或活性降低导致弱精子症的潜在机制尚不清楚，我们研究了精子PMCA4是否对一氧化氮合酶（NOSs）产生负调控作用，以及在其缺失时一氧化氮、过氧亚硝酸盐和氧化应激水平是否会升高。通过免疫共沉淀（Co-IP）和荧光共振能量转移（FRET），我们发现，在获能精子和经钙离子载体处理的精子中，胞质钙离子浓度升高时，PMCA4与NOSs存在关联；在基础钙离子浓度（未获能精子）时，PMCA4与神经元型一氧化氮合酶（nNOS）存在关联。在获能精子和未获能精子中，PMCA4与内皮型一氧化氮合酶（eNOS）的FRET效率分别为35%和23%，差异显著（p < 0.01），分子间距小于10 nm。对于PMCA4-nNOS，这种相互作用仅在获能精子中可见，其FRET效率为24%，显著高于未获能精子（6%）（p < 0.05）。PMCA4和NOSs被确定为一种四聚体复合物中的相互作用伙伴，该复合物包括小窝蛋白1，小窝蛋白1以钙离子依赖的方式与eNOS进行免疫共沉淀。在Pmca4基因缺失的精子中，获能/未获能精子的NOS活性比野生型精子升高了两倍，同时过氧亚硝酸盐水平增加了两倍，凋亡生殖细胞数量显著增加（p < 0.001）。这些数据支持一种四聚体复合物模型，即PMCA4协调钙离子和一氧化氮信号以维持精子活力，Pmca4基因缺失的雄性个体中一氧化氮水平升高会导致弱精子症。研究结果提示PMCA4突变与人类弱精子症有关，具有诊断意义。
