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，我们研究了精子蛋白的合成、加工和功能。取得了如下实验结果：1.在小鼠中，在睾丸中产生两种不同的ADAM 1亚型，ADAM 1a和ADAM 1b。ADAM 1a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ADAM 1b。我们发现，ADAM 1a的缺失导致男性不育，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ADAM 1a缺陷小鼠精子中的ADAM 3水平显著降低。此外，在TGC中，精子表面的ADAM 3的出现依赖于ADAM 1a和ADAM 2之间的受精素蛋白复合物的形成。这些结果表明，ADAM 1a/ADAM 2可育， ...更多信息 n可能参与特异性精子蛋白包括ADAM 3从睾丸生殖细胞内质网到细胞表面的选择性转运，我们还表明，缺乏ADAM 1b的突变雄性小鼠是有生育能力的，并且ADAM 1b的缺失不会导致精子功能的显著缺陷。ADAM 1b缺陷的附睾精子表现出细胞表面的ADAM 2严重减少，尽管TGC中的ADAM 2正常存在。TGC中精子表面的ADAM 1b和ADAM 2的出现依赖于ADAM 1b/ADAM 2受精素的形成和丰度。因此，小鼠ADAM 1b/ADAM 2受精蛋白可能不是在精卵融合中起关键作用，而是在精子表面这两种亚当斯的出现中起关键作用.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。然而，缺乏PH-20的小鼠精子仍然能够穿透卵丘质量，尽管卵丘细胞的分散延迟。有趣的是，一个55 kDa的透明质酸水解蛋白大量存在于野生型和PH-20缺陷小鼠精子。在这项研究中，我们已经纯化了55 kDa的小鼠蛋白质从附睾精子顶体反应释放的可溶性蛋白提取物，并已确定为一种新的透明质酸酶，Hyal 5。Hyal 5仅在睾丸中表达，并与Hyalp 1、Hyal 4和Ph-20一起在小鼠6号染色体上形成160-kbp的基因簇。Hyal 5是存在于精子的质膜和顶体膜上的单链透明质酸酶，推测为GPI锚定蛋白。此外，透明质酸酶谱显示Hyal 5在pH 5-7范围内具有酶活性，而在pH 3和4下无活性。Hyal 5富集，PH-20自由可溶性蛋白提取物，PH-20缺乏小鼠精子能够分散卵丘细胞从卵丘质量。的卵丘细胞分散强烈抑制透明质酸酶抑制剂，芹菜素的存在。这些结果表明，在小鼠中，Hyal 5可能主要作为“卵丘基质解聚酶”在精子穿透卵丘团中起作用，并且在卵透明质膜表面附近或表面上的局部透明质酸水解中起作用，以使精子尾部的近端区域自由移动。PH-20可以部分补偿Hyal5.3的功能作用。我们以前已经确定了一个42 kDa的丝氨酸蛋白酶，TESP 5，相同的testisin，ESP-1，或类胰蛋白酶4作为候选酶参与精子穿透卵ZP。小鼠TESP 5是锚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TESP 5在受精中的作用，我们通过在胚胎干细胞中同源重组产生了携带TESP 5基因靶向突变的突变小鼠。TESP 5缺陷的雄性和雌性小鼠显示出正常的生育能力。然而，体外受精试验表明，缺乏TESP 5的附睾精子几乎不能使中期II停滞的卵母细胞受精。精子上TESP 5的缺失导致体外结合ZP的能力降低。即使TESP 5缺陷精子与ZP结合，ZP诱导的顶体反应率也非常低。此外，TESP 5的缺失严重损害了精子与卵子在体外融合的能力。这些结果表明，TESP 5可能在顶体反应中起作用，并暗示TESP 5缺陷精子的受损功能可能在通过子宫和/或输卵管的过程中得到补偿。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