Studies on the Regulation of Fertilization and Activation of Development

为了阐明受精和激活发育的调控，我们研究了小鼠精子在精子成熟、精子从子宫转移到输卵管、受精和卵子激活等方面的可能功能。实验结果如下：1。附睾精子质膜上的tritonx -100不溶性微结构域，即脂筏，在获能过程中逐渐从精子赤道区向顶区移动。缺乏PH-20精子的雄性小鼠仍然具有生育能力，尽管从积云团中分散积云细胞的能力降低。精子提取物的Western blot分析显示，除PH-20外，其他透明质酸酶(s)可能存在于精子顶体中。这些数据提供了至少在小鼠中PH-20不是受精所必需的证据，并表明其他透明质酸酶可能在精子穿透积云细胞层和/或卵细胞透明带中发挥重要作用，可能与PH-20.3合作。小鼠基因组克隆编码42 kda的精子丝氨酸蛋白酶TESP5。免疫化学分析显示42-和4l-kDa形式的TESP5定位于附睾尾部精子的头部、细胞质液滴和尾部，可能是糖基磷脂酰肌醇锚定的膜蛋白。此外，这两种形式的TESP5被选择性地包含在精子膜的Triton x -100不溶性微结构域，即脂筏中。这些结果表明，TESP5可能参与精子/卵子的相互作用。在小鼠睾丸中存在两种不同的ADAMI亚型，ADAMla和ADAMlb，而不是ADAMl。ADAMlb有限地存在于生殖细胞的内质网中，而附睾精子仅在细胞表面含有ADAMlb。ADAMla和ADAMlb的前体在生殖细胞内质网中与ADAM2的前体形成异二聚体复合物。成熟形态的ADAMlb和ADAM2之间的异二聚体复合体也存在于精子表面。这些数据暗示了ADAMla和ADAMlb分别在精子发生和受精中的潜在作用。当具有编码磷脂酶ζ的聚(a)尾的合成mRNA被注入未受精卵时，观察到原核形成。少

To elucidate the regulation of fertilization and activation of development, we have examined possible functions of mouse sperm in sperm maturation, sperm transit from uterus to oviduct, fertilization, and egg activation. The following experimental results have been obtained :1. The TritonX-100-insoluble microdomains, lipid rafts, on the plasma membranes of epididymal sperm gradually move to the apical region from the equatorial region of sperm during capacitation.2. Male mice lacking sperm PH-20 are still fertile, in spite of a reduced ability to disperse cuwnulus cells from the cumulus mass. Western blot analysis of sperm extracts reveals the presence of other hyaluronidase(s), except PH-20, presumably within the sperm acrosome. These data provide evidence that PH-20 is not essential for fertilization at least in the mouse, and suggest that the other hyaluronidase(s) may play an important role in the sperm penetration through the cumulus cell layer and/or the egg zona pellucida, possi … More bly in cooperation with PH-20.3. Mouse genomic clones encoding a 42-kDa sperm serine protease, TESP5, have been identified. Immunochemical analysis reveals that 42- and 4l-kDa forms of TESP5 are localized in the head, cytoplasmic droplet, and tail of cauda epididymal sperm probably as a glycosylphosphatidylinositol-anchored membranous protein. Moreover, these two forms of TESP5 are selectively included into Triton X-100-insoluble microdomains, Iipid rafts, of the sperm membranes. These results suggest a possible involvement of TESP5 in the sperm/egg interactions.4. Two different ADAMI isoforms, ADAMla and ADAMlb, instead of ADAMl are present in the mouse testis. ADAMla is resttrictedly present within the endoplasmic reticulum of germ cells, whereas epididymal sperm contain only ADAMlb on the cell surface. The precursors of ADAMla and ADAMlb form a heterodimeric complex with that of ADAM2 in the endoplasmic reticulum of germ cells. The heterodimeric complex between the mature forms of ADAMlb and ADAM2 is also found on the sperm surface. These data imply the potential roles of ADAMla and ADAMlb in spermatogenesis and fertilization, respectively.5. When a synthesized mRNA with a poly(A) tail encoding phospholipase ζ is injected into unfertilized eggs, pronuclear formation is observed. Less