Molecular basis of spermatogenesis and fertilization in mammals

哺乳动物精子发生和受精的分子基础

基本信息

批准号：
15208033
负责人：
BABA Tadashi
金额：
$ 27.96万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15208033/
关键词：
Sperm Egg Fertilization Spermatogenesis Egg activation Cell fusion Oviduct Knockout mouse 精子成熟輪卵管

项目摘要

To elucidate the molecular basis of mammalian spermatogenesis and fertilization, we have examined the synthesis, processing, and functions of sperm proteins involved in fertilization. The following experimental results have been obtained :1. In mouse, two different isoforms of ADAM1, ADAM1a and ADAM1b, are produced in the testis. ADAM1a is localized within the endoplasmic reticulum of testicular germ cells (TGC), whereas epididymal sperm contain only ADAM1b on the plasma membrane. We show that the loss of ADAM1a results in the male infertility because of the severely impaired ability of sperm to migrate from the uterus into the oviduct through the uterotubal junction. Among testis (sperm)-specific proteins examined, only the level of ADAM3 was strongly reduced in ADAM1a-deficient mouse sperm. Moreover, the appearance of ADAM3 on the sperm surface was dependent on the formation of a fertilin protein complex between ADAM1a and ADAM2 in TGC. These results suggest that ADAM1a/ADAM2 fertili … More n may be implicated in the selective transport of specific sperm proteins including ADAM3 from the endoplasmic reticulum of testicular germ cells onto the cell surface.We also show that mutant male mice lacking ADAM1b are fertile, and the loss of ADAM1b results in no significant defect in the sperm functions. ADAM1b-deficient epididymal sperm showed a severe reduction of ADAM2 on the cell surface, despite the normal presence of ADAM2 in TGC. The appearance of ADAM1b and ADAM2 on the sperm surface depended on formation and abundance of ADAM1b/ADAM2 fertilin in TGC. Thus, mouse ADAM1b/ADAM2 fertilin may play the crucial role not in the sperm/egg fusion, but in the appearance of these two ADAMs on the sperm surface.2. A glycosylphosphatidylinositol (GPI)-anchored hyaluronidase, PH-20, on the sperm surface has long been believed to assist sperm penetration through the cumulus mass surrounding the eggs. However, mouse sperm lacking PH-20 were still capable of penetrating the cumulus mass despite a delayed dispersal of cumulus cells. Intriguingly, a 55-kDa hyaluronan-hydrolyzing protein was abundantly present in wild-type and PH-20-deficient mouse sperm. In this study, we have purified the 55-kDa mouse protein from soluble protein extracts released from epididymal sperm by acrosome reaction, and have identified as a novel hyaluronidase, Hyal5. Hyal5 was exclusively expressed in the testis, and formed a 160-kbp gene cluster together with Hyalp1, Hyal4, and Ph-20 on mouse chromosome 6. Hyal5 was a single-chain hyaluronidase present on the plasma and acrosomal membranes of sperm presumably as a GPI-anchored protein. Moreover, hyaluronan zymography revealed that Hyal5 is enzymatically active in the pH range 5-7, and inactive at pH 3 and 4. Both Hyal5-enriched, PH-20-free soluble protein extracts, and PH-20-deficient mouse sperm were capable of dispersing cumulus cells from the cumulus mass. The cumulus cell dispersal was strongly inhibited by the presence of a hyaluronidase inhibitor, apigenin. These results suggest that in the mouse, Hyal5 may function principally as a "cumulus matrix depolymerase" in the sperm penetration through the cumulus mass, and in the local hyaluronan hydrolysis near or on the surface of the egg zona pellucida to enable the proximal region of sperm tail to move freely. PH-20 may compensate in part for the functional roles of Hyal5.3. We have previously identified a 42-kDa serine protease, TESP5, identical to testisin, esp-1, or tryptase 4 as a candidate enzyme involved in the sperm penetration of egg ZP. Mouse TESP5 is glycosylphosphatidylinositol (GPI)-anchored on the cell surface of cauda epididymal sperm. To elucidate the role(s) of TESP5 in fertilization, we have produced mutant mice carrying a targeted mutation in the TESP5 gene by homologous recombination in embryonic stem cells. The TESP5-deficient male and female mice showed a normal fertility. However, in vitro fertilization assay revealed that epididymal sperm lacking TESP5 barely fertilize metaphase II-arrested oocytes. The loss of TESP5 on sperm resulted in the reduced ability to bind the ZP in vitro. Even when TESP5-deficient sperm bound to the ZP, the rate of ZP-induced acrosome reaction was very low. Moreover, the ability of sperm to fuse with egg in vitro was severely impaired by the TESP5 loss. These results suggest that TESP5 may act in the acrosome reaction, and imply that the impaired function of TESP5-deficient sperm may be compensated for during the transit through the uterus and/or oviduct. Less

为了阐明哺乳动物精子发生和受精的分子基础，我们研究了与受精相关的精子蛋白的合成，加工和功能。已经获得以下实验结果：1。在小鼠中，在睾丸中产生了ADAM1，ADAM1A和ADAM1B的两种不同的同工型。 ADAM1A位于测试生殖细胞的内质网中（TGC），而附域精子仅在质膜上仅包含ADAM1B。我们表明，ADAM1A的丧失导致雄性不育症，因为精子通过子宫内蛋白蛋白蛋白蛋白蛋白酶结的能力严重受损地从子宫迁移到卵形。在检查的睾丸（精子）特异性蛋白中，仅在ADAM1A缺陷小鼠精子中，ADAM3的水平大大降低。此外，ADAM3在精子表面的外观取决于TGC中ADAM1A和ADAM2之间的肥料蛋白复合物的形成。这些结果表明，在选择性运输特定的精子蛋白（包括来自ADAM3）的ADAM1A/ADAM2肥料中，可能隐含更多的N，包括ADAM3的aDAM3从内胞质网状网状网状网络表面上。我们还表明，缺少ADAM1B的突变雄性小鼠是aDAM1B的，并且没有大量ADAM1B的损失导致精子功能的大量缺陷。尽管TGC中ADAM2正常存在，但ADAM1B缺陷的附睾精子显示了细胞表面ADAM2的严重降低。 ADAM1B和ADAM2在精子表面上的出现取决于TGC中ADAM1B/ADAM2肥料的形成和抽象。这是小鼠ADAM1B/ADAM2肥料可能在精子融合中起着至关重要的作用，而是在精子表面上的这两个Adams出现。2。长期以来，人们一直认为，精子表面上的糖基磷脂酰肌醇（GPI）锚定的透明质酸酶，PH-20，可以帮助精子穿过卵周围的积积质量。然而，尽管积云细胞的散布延迟，但缺乏PH-20的小鼠精子仍能够穿透积云质量。有趣的是，在野生型和pH-20缺陷的小鼠精子中绝对存在55 kDA水解蛋白。在这项研究中，我们通过反应从附睾精子释放出的固体蛋白提取物中纯化了55 kDa小鼠的蛋白质，并确定为一种新型的透明质酸酶Hyal5。 Hyal5在睾丸中专门表达，并在小鼠6染色体上形成了160-KBP基因簇，以及Hyalp1，Hyal4和PH-20。Hyal5是一种单链透明质酸酶，存在于Plasma和Acrosome Membranes上的单链透明质酸酶。此外，透明质酸酶学表明，透明度5在pH范围5-7中具有酶活性，在pH 3和4处无活性。透明5含有透明的pH-20无pH-20无pH-20固体蛋白提取物，pH-20缺陷小鼠的精子能够从累积细胞中分散累积细胞。透明质酸酶抑制剂阿apegenin的存在强烈抑制积云细胞的分散。这些结果表明，在小鼠中，Hyal5可能主要用作精子穿过积云质量的精子穿透的“积积基质去聚合酶”，以及在卵子Zona Pellucida附近或表面的局部水解中，以使精子近端区域的近端区域自由移动。 pH-20可以部分补偿透明5.3的功能作用。我们先前已经将与睾丸，ESP-1或胰蛋白酶4相同的42 kDa丝氨酸蛋白酶（TESP5）鉴定为参与EGG ZP精子渗透的候选酶。小鼠TESP5是在Cauda附睾精子的细胞表面上锚定的糖基磷脂酰肌醇（GPI）。为了阐明Tesp5在受精中的作用，我们通过胚胎干细胞中的同源重组产生了在TESP5基因中携带靶向突变的突变小鼠。 TESP5缺乏雄性和雌性小鼠表现出正常的生育能力。然而，体外受精测定法表明，缺乏TESP5的附子精子几乎没有施肥化生II捕获的卵母细胞。对精子的TESP5损失导致体外结合ZP的能力降低。即使tesp5缺陷精子与ZP结合，跨反应引起的ZP速率也很低。此外，TESP5损失严重损害了精子与卵子体外融合的能力。这些结果表明，tesp5可能在跨反应中起作用，这意味着在通过子宫和/或卵形的过渡期间，可以完成tesp5缺陷精子的功能受损。较少的