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.在小鼠睾丸中产生了ADAM1a和ADAM1b两种不同的亚型。ADAM1a定位于睾丸生殖细胞(TGC)的内质网，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，因为精子通过子宫输卵管连接处从子宫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ADAM1a缺陷小鼠精子中的ADAM3水平显著降低。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体的形成。这些结果表明，ADAM1a/ADAM2可育…更多的n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。我们还表明，缺乏ADAM1b的突变雄性小鼠是可育的，ADAM1b的丢失不会导致精子功能的显著缺陷。ADAM1b缺陷的附睾精子显示细胞表面的ADAM2严重减少，尽管TGC中正常存在ADAM2。ADAM1b和ADAM2在精子表面的出现取决于TGC中ADAM1b/ADAM2受精素的形成和丰度。因此，小鼠ADAM1b/ADAM2受精素可能不是在精子/卵子融合中起关键作用，而是在精子表面出现这两种ADAM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-20一直被认为有助于精子穿过卵子周围的堆积物。然而，缺乏PH-20的小鼠精子仍然能够穿透卵丘，尽管卵丘细胞的扩散延迟。有趣的是，一种55 kDa的透明质酸水解蛋白在野生型和PH-20缺乏的小鼠精子中大量存在。在本研究中，我们从附睾精子通过顶体反应释放的可溶性蛋白提取物中纯化了55 kDa的小鼠蛋白，并鉴定为一种新的透明质酸酶，即Hyal5。Hyal5仅在睾丸中表达，并与小鼠6号染色体上的Hyalp1、Hyal4和Ph-20一起形成一个160kbp的基因簇。Hyal5是一种存在于精子浆和顶体膜上的单链透明质酸酶，可能是一种GPI锚定蛋白。此外，透明质酸酶谱显示，在pH为5-7的范围内，透明质酸具有酶活性，而在pH为3和4的范围内，透明质酸不具有活性。富含透明质酸的、不含PH-20的可溶性蛋白提取物和缺乏PH-20的小鼠精子都能将卵丘细胞从卵丘团中分散开来。透明质酸酶抑制剂芹菜素的存在强烈抑制了卵丘细胞的扩散。这些结果表明，在小鼠中，Hyal5可能主要作为“卵丘基质解聚酶”在精子穿透卵丘团中发挥作用，并在卵子透明带附近或表面的局部透明质酸水解中使精子尾巴的近端区域能够自由移动。PH-20可能部分补偿了Hyal5.3的功能作用。我们先前已经确定42 kDa的丝氨酸蛋白酶TESP5与睾丸蛋白ESP-1或类胰蛋白酶4相同，是参与精子穿透卵子ZP的候选酶。小鼠TESP5是一种糖基化磷脂酰肌醇(GPI)，定位于附睾尾精子的细胞表面。为了阐明TESP5在受精中的作用(S)，我们通过胚胎干细胞中的同源重组产生了TESP5基因的定向突变突变小鼠。TESP5基因缺陷的雄性和雌性小鼠表现出正常的生育能力。然而，体外受精试验表明，缺乏TESP5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诱导的顶体反应的发生率也很低。此外，TESP5基因的缺失严重削弱了精子与卵子在体外融合的能力。这些结果表明，TESP5可能参与了顶体反应，提示TESP5缺陷精子在通过子宫和/或输卵管的过程中，其受损的功能可能得到补偿。较少

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