Intracellular activation of ovastacin mediates pre-fertilization hardening of the zona pellucida
Intracellular activation of ovastacin mediates pre-fertilization hardening of the zona pellucida
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DOI：
10.1093/molehr/gax040
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发表时间：
2017-09-01
影响因子：
4
通讯作者：
Stoecker, Walter
中科院分区：
文献类型：
--
作者：
Koerschgen, Hagen;Kuske, Michael;Stoecker, Walter
研究问题：如何以及在哪里激活原ovastacin，以及活性ovastacin如何调节透明质酸硬化（ZPH）和成功受精？研究结果：已知：金属蛋白酶ovastacin储存在皮质颗粒中，它在受精时切割透明带蛋白2（ZP 2），从而破坏ZP精子配体并触发ZPH。研究设计，样品/材料，方法：我们分离卵母细胞从野生型和卵蛋白缺乏（Astl（null））FVB小鼠受精前和受精后（体外和体内）和定量的卵蛋白活性和切割的ZP 2通过免疫印迹。我们通过使用α-糜蛋白酶测量ZP消化时间和通过测定ZP 2裂解来评估ZPH。我们使用域特异性奥伐他汀抗体通过免疫荧光测定奥伐他汀的细胞分布。实验至少进行三次，最少20个卵母细胞。使用Shapiro-Wilk检验对数据进行预分析。在正态分布的情况下，通过双侧Student's t检验确定显著性，而在非正态分布的情况下，通过Mann-Whitney U检验确定显著性。主要结果和机会的作用：中期II（MII）卵母细胞中含有无活性的原ovastacin和活化的ovastacin。免疫印迹和ZP消化试验显示，在野生型小鼠受精前，ZP 2就已部分裂解。部分卵裂与生殖囊破裂和MII相吻合，尽管存在胎球蛋白-B蛋白，内源性ovastacin抑制剂，在卵泡和输卵管液。胞吐后，奥司他辛的部分C末端结构域仍然附着在质膜上，而N末端活性奥司他辛结构域则被分泌。这一发现可能会解决以前相互矛盾的数据表明，ovastacin作为一个卵膜受体称为SAS 1B（精子顶体SLLP 1结合蛋白; SLLP，精子溶菌酶样蛋白）和一个分泌的蛋白酶介导ZP 2 cleavage.Limitations，理由解释：对于这项研究，只有从野生型和ovastacin缺陷的FVB小鼠分离的卵母细胞进行了研究。一些实验涉及卵母细胞激活的Ca 2+离子载体A23187触发ZPH.WIDER的影响的发现：这项研究提供了一个详细的空间和时间视图提前卵裂ZP 2的奥伐他汀，这是已知的不利影响IVF率在小鼠和human.LARGE规模的数据：无。
STUDY QUESTION: How and where is pro-ovastacin activated and how does active ovastacin regulate zona pellucida hardening (ZPH) and successful fertilization?STUDY FINDING: Ovastacin is partially active before exocytosis and pre-hardens the zona pellucida (ZP) before fertilization.WHAT IS KNOWN ALREADY: The metalloproteinase ovastacin is stored in cortical granules, it cleaves zona pellucida protein 2 (ZP2) upon fertilization and thereby destroys the ZP sperm ligand and triggers ZPH. Female mice deficient in the extracellular circulating ovastacin-inhibitor fetuin-B are infertile due to pre-mature ZPH.STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We isolated oocytes from wild-type and ovastacin-deficient (Astl(null)) FVB mice before and after fertilization (in vitro and in vivo) and quantified ovastacin activity and cleavage of ZP2 by immunoblot. We assessed ZPH by measuring ZP digestion time using alpha-chymotrypsin and by determining ZP2 cleavage. We determined cellular distribution of ovastacin by immunofluorescence using domain-specific ovastacin antibodies. Experiments were performed at least in triplicate with a minimum of 20 oocytes. Data were pre-analyzed using Shapiro-Wilk test. In case of normal distribution, significance was determined via two-sided Student's t-test, whereas in case of non-normal distribution via Mann-Whitney U-test.MAIN RESULTS AND THE ROLE OF CHANCE: Metaphase II (MII) oocytes contained both inactive pro-ovastacin and activated ovastacin. Immunoblot and ZP digestion assays revealed a partial cleavage of ZP2 even before fertilization in wild-type mice. Partial cleavage coincided with germinal-vesicle breakdown and MII, despite the presence of fetuin-B protein, an endogenous ovastacin inhibitor, in the follicular and oviductal fluid. Upon exocytosis, part of the C-terminal domain of ovastacin remained attached to the plasmalemma, while the N-terminal active ovastacin domain was secreted. This finding may resolve previously conflicting data showing that ovastacin acts both as an oolemmal receptor termed SAS1B (sperm acrosomal SLLP1 binding protein; SLLP, sperm lysozyme like protein) and a secreted protease mediating ZP2 cleavage.LIMITATIONS, REASONS FOR CAUTION: For this study, only oocytes isolated from wild-type and ovastacin-deficient FVB mice were investigated. Some experiments involved oocyte activation by the Ca2+ ionophore A23187 to trigger ZPH.WIDER IMPLICATIONS OF THE FINDINGS: This study provides a detailed spatial and temporal view of pre-mature cleavage of ZP2 by ovastacin, which is known to adversely affect IVF rate in mice and humans.LARGE SCALE DATA: None.