Molecular basis of spermatogenesis and fertilization in mammals

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。实验结果如下：1。在小鼠中，ADAM1的两种不同亚型ADAM1a和ADAM1b在睾丸中产生。ADAM1a定位于睾丸生殖细胞（TGC）的内质网内，而附睾精子仅在质膜上含有ADAM1b。我们发现ADAM1a的缺失导致男性不育，因为精子从子宫通过子宫输卵管交界处迁移到输卵管的能力严重受损。在检测的睾丸（精子）特异性蛋白中，只有ADAM3的水平在adam1a缺乏的小鼠精子中强烈降低。此外，ADAM3在精子表面的出现依赖于TGC中ADAM1a和ADAM2之间的受精蛋白复合物的形成。这些结果表明，ADAM1a/ADAM2可能参与了包括ADAM3在内的特定精子蛋白从睾丸生殖细胞内质网到细胞表面的选择性运输。我们还发现，缺乏ADAM1b的突变雄性小鼠具有生育能力，ADAM1b的缺失不会导致精子功能的显著缺陷。尽管ADAM2在TGC中正常存在，但缺乏adam1b的附睾精子显示细胞表面ADAM2严重减少。ADAM1b和ADAM2在精子表面的出现取决于TGC中ADAM1b/ADAM2受精蛋白的形成和丰度。因此，小鼠ADAM1b/ADAM2受精卵可能不是在精子/卵子融合中起关键作用，而是在这两个ADAMs在精子表面的出现中起关键作用。长期以来，精子表面的糖基磷脂酰肌醇（GPI）锚定的透明质酸酶（PH-20）被认为有助于精子穿透卵子周围的积云团。然而，缺乏PH-20的小鼠精子仍然能够穿透积云团，尽管积云细胞的扩散延迟。有趣的是，一种55-kDa的透明质酸水解蛋白在野生型和ph -20缺陷小鼠精子中大量存在。在这项研究中，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，并鉴定了一种新的透明质酸酶，Hyal5。Hyal5仅在睾丸中表达，与Hyalp1、Hyal4、Ph-20在小鼠6号染色体上形成160 kbp的基因簇。Hyal5是一种单链透明质酸酶，存在于精子的血浆和顶体膜上，可能是一种gpi锚定蛋白。此外，透明质酸酶谱分析显示，Hyal5在pH值5-7范围内具有酶活性，在pH值3和4范围内无活性。富含hyal5、不含ph -20的可溶性蛋白提取物和缺乏ph -20的小鼠精子都能使积云细胞从积云团中分散出来。透明质酸酶抑制剂芹菜素的存在强烈抑制了积云细胞的扩散。这些结果表明，在小鼠中，Hyal5可能主要作为“积云基质解聚合酶”在精子穿透积云团中起作用，并在卵细胞透明带附近或表面局部水解透明质酸，使精子尾部近端区域自由移动。PH-20可能部分补偿Hyal5.3的功能作用。我们之前已经确定了一种42 kda的丝氨酸蛋白酶TESP5，与睾丸素，sp-1或胰蛋白酶4相同，作为参与精子穿透卵子ZP的候选酶。小鼠TESP5是固定在附睾尾部精子细胞表面的糖基磷脂酰肌醇（GPI）。为了阐明TESP5在受精中的作用，我们在胚胎干细胞中通过同源重组产生了携带TESP5基因靶向突变的突变小鼠。缺乏tesp5的雄性和雌性小鼠的生育能力正常。然而，体外受精实验显示，缺乏TESP5的附睾精子几乎不能使中期ii期阻滞的卵母细胞受精。精子上的TESP5缺失导致体外结合ZP的能力降低。即使当缺乏tesp5的精子与ZP结合时，ZP诱导的顶体反应率也很低。此外，精子在体外与卵子融合的能力因TESP5的缺失而严重受损。这些结果表明，TESP5可能在顶体反应中起作用，并暗示缺乏TESP5的精子功能受损可能在通过子宫和/或输卵管的运输过程中得到补偿。少

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