Modelski Mark J, Menlah Gladys, Wang Yipei, Dash Soma, Wu Kathie, Galileo Deni S, Martin-DeLeon Patricia A
Department of Biological Sciences, University of Delaware, Newark, Delaware.
Department of Biological Sciences, University of Delaware, Newark, Delaware
Biol Reprod. 2014 Nov;91(5):109. doi: 10.1095/biolreprod.113.115857. Epub 2014 Sep 17.
To initiate the crucial cell adhesion events necessary for fertilization, sperm must penetrate extracellular matrix barriers containing hyaluronic acid (HA), a task thought to be accomplished by neutral-active hyaluronidases. Here we report that the ~57 kDa hyaluronidase 2 (HYAL2) that in somatic tissues has been highly characterized to be acid-active is present in mouse and human sperm, as detected by Western blot, flow cytometric, and immunoprecipitation assays. Immunofluorescence revealed its presence on the plasma membrane over the acrosome, the midpiece, and proximal principal piece in mice where protein fractionation demonstrated a differential distribution in subcellular compartments. It is significantly more abundant in the acrosome-reacted (P = 0.04) and soluble acrosomal fractions (P = 0.006) (microenvironments where acid-active hyaluronidases function) compared to that of the plasma membrane where neutral hyaluronidases mediate cumulus penetration. Using HA substrate gel electrophoresis, immunoprecipitated HYAL 2 was shown to have catalytic activity at pH 4.0. Colocalization and coimmunoprecipitation assays reveal that HYAL2 is associated with its cofactor, CD44, consistent with CD44-dependent HYAL2 activity. HYAL2 is also present throughout the epididymis, where Hyal2 transcripts were detected, and in the epididymal luminal fluids. In vitro assays demonstrated that HYAL2 can be acquired on the sperm membrane from epididymal luminal fluids, suggesting that it plays a role in epididymal maturation. Because similar biphasic kinetics are seen for HYAL2 and SPAM1 (Sperm adhesion molecule 1), it is likely that HYAL2 plays a redundant role in the catalysis of megadalton HA to its 20 kDa intermediate during fertilization.
为启动受精所需的关键细胞黏附事件，精子必须穿透含有透明质酸（HA）的细胞外基质屏障，这一任务被认为是由中性活性透明质酸酶完成的。在此我们报告，通过蛋白质印迹、流式细胞术和免疫沉淀分析检测发现，在体细胞组织中已被高度表征为酸活性的约57 kDa透明质酸酶2（HYAL2）存在于小鼠和人类精子中。免疫荧光显示其存在于小鼠顶体、中段和近端主段的质膜上，蛋白质分级分离表明其在亚细胞区室中呈差异分布。与中性透明质酸酶介导卵丘穿透的质膜相比，它在顶体反应的部分（P = 0.04）和可溶性顶体部分（P = 0.006）（酸活性透明质酸酶发挥作用的微环境）中含量显著更高。使用HA底物凝胶电泳，显示免疫沉淀的HYAL 2在pH 4.0时有催化活性。共定位和共免疫沉淀分析表明，HYAL2与其辅因子CD44相关，这与CD44依赖性HYAL2活性一致。HYAL2也存在于整个附睾中，在那里检测到了Hyal2转录本，并且存在于附睾管腔液中。体外分析表明，HYAL2可以从附睾管腔液中获取到精子膜上，这表明它在附睾成熟过程中发挥作用。由于HYAL2和SPAM1（精子黏附分子1）表现出相似的双相动力学，因此HYAL2可能在受精过程中催化百万道尔顿的HA转化为其20 kDa中间体时发挥冗余作用。
