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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，我们研究了参与受精的精子蛋白的合成、加工和功能。得到如下实验结果： 1．在小鼠中，睾丸中产生两种不同的 ADAM1 亚型：ADAM1a 和 ADAM1b。 ADAM1a 位于睾丸生殖细胞 (TGC) 的内质网内，而附睾精子的质膜上仅含有 ADAM1b。我们发现 ADAM1a 的缺失会导致男性不育，因为精子从子宫通过子宫输卵管连接处迁移到输卵管的能力严重受损。在检测的睾丸（精子）特异性蛋白质中，只有 ADAM1a 缺陷小鼠精子中 ADAM3 的水平大幅降低。此外，ADAM3 在精子表面的出现取决于 TGC 中 ADAM1a 和 ADAM2 之间生育素蛋白复合物的形成。这些结果表明，ADAM1a/ADAM2 的生育能力可能与包括 ADAM3 在内的特定精子蛋白从睾丸生殖细胞内质网选择性转运到细胞表面有关。我们还表明，缺乏 ADAM1b 的突变雄性小鼠具有生育能力，并且 ADAM1b 的缺失不会导致精子功能出现明显缺陷。 ADAM1b 缺陷的附睾精子显示细胞表面 ADAM2 严重减少，尽管 TGC 中 ADAM2 正常存在。 ADAM1b 和 ADAM2 在精子表面的出现取决于 TGC 中 ADAM1b/ADAM2 肥力素的形成和丰度。因此，小鼠ADAM1b/ADAM2生育素可能不是在精子/卵子融合中发挥关键作用，而是在这两种ADAM在精子表面的出现中发挥关键作用。 2．长期以来，人们一直认为精子表面的糖基磷脂酰肌醇 (GPI) 锚定透明质酸酶 PH-20 有助于精子穿透卵子周围的卵丘团。然而，尽管卵丘细胞的扩散延迟，但缺乏 PH-20 的小鼠精子仍然能够穿透卵丘团。有趣的是，野生型和 PH-20 缺陷型小鼠精子中大量存在 55 kDa 透明质酸水解蛋白。在本研究中，我们通过顶体反应从附睾精子释放的可溶性蛋白提取物中纯化了55-kDa的小鼠蛋白，并鉴定为一种新型透明质酸酶Hyal5。 Hyal5 仅在睾丸中表达，并与小鼠 6 号染色体上的 Hyalp1、Hyal4 和 Ph-20 一起形成 160 kbp 的基因簇。Hyal5 是一种单链透明质酸酶，存在于精子的质膜和顶体膜上，可能作为 GPI 锚定蛋白。此外，透明质酸酶谱分析显示，Hyal5 在 pH 范围 5-7 内具有酶活性，在 pH 3 和 4 时无活性。富含 Hyal5、不含 PH-20 的可溶性蛋白质提取物和缺乏 PH-20 的小鼠精子都能够从卵丘团中分散卵丘细胞。透明质酸酶抑制剂芹黄素的存在强烈抑制卵丘细胞的扩散。这些结果表明，在小鼠中，Hyal5可能主要作为精子穿透卵丘团的“卵丘基质解聚酶”，并在卵透明带附近或表面的局部透明质酸水解中发挥作用，从而使精子尾部的近端区域能够自由移动。 PH-20 可以部分补偿 Hyal5.3 的功能作用。我们之前已经鉴定出一种 42 kDa 丝氨酸蛋白酶 TESP5，与睾丸素 esp-1 或类胰蛋白酶 4 相同，作为参与精子穿透卵子 ZP 的候选酶。小鼠 TESP5 是糖基磷脂酰肌醇 (GPI)，锚定在附睾精子尾部的细胞表面。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携带 TESP5 基因靶向突变的突变小鼠。 TESP5缺陷的雄性和雌性小鼠表现出正常的生育能力。然而，体外受精试验显示，缺乏 TESP5 的附睾精子几乎无法与中期 II 停滞的卵母细胞受精。精子上 TESP5 的缺失导致体外结合 ZP 的能力降低。即使当TESP5缺陷的精子与ZP结合时，ZP诱导的顶体反应的速率也非常低。此外，TESP5 缺失严重损害了精子在体外与卵子融合的能力。这些结果表明TESP5可能在顶体反应中发挥作用，并意味着TESP5缺陷的精子受损的功能可能在通过子宫和/或输卵管的过程中得到补偿。较少的