Oterino J, Sánchez Toranzo G, Zelarayán L, Ajmat M T, Bonilla F, Bühler M I
Departamento de Biología del Desarrollo, Chacabuco 461, 4000 San Miguel de Tucumán, Argentina.
Zygote. 2006 May;14(2):97-106. doi: 10.1017/S0967199406003662.
During activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.
在两栖类卵激活过程中，皮质颗粒胞吐作用会导致卵黄膜发生精细的超微结构变化。这些变化包括卵黄膜结构的改变以及受精膜（FE）的形成，受精膜不再能被精子穿透。在布氏蟾蜍中，当卵穿过输卵管时，卵黄膜会被输卵管分泌的一种类胰蛋白酶改变，这会使卵黄膜对精子溶解素的敏感性增加。经孕酮体外成熟的布氏蟾蜍完全成熟的卵母细胞是多精受精的，尽管皮质颗粒胞吐作用似乎按正常时间顺序发生。这些卵母细胞无论是否经过胰蛋白酶处理都能受精，这表明卵黄膜对精子是完全可渗透的。未经胰蛋白酶处理的卵黄膜既不能保留gp90也不能保留gp96。这表明这些糖蛋白参与了对多精受精的阻断，并且在授精前对体外成熟的卵母细胞进行胰蛋白酶处理是使卵黄膜能够阻断多精受精所必需的。从经胰蛋白酶处理并通过电刺激激活的体外成熟的布氏蟾蜍卵母细胞中分离得到的卵黄膜，其结合能力丧失，这表明先前的胰蛋白酶处理是精子阻断发生的必要步骤。当体外成熟的卵母细胞与从体外成熟的卵母细胞中获得的皮质颗粒产物（vCGP）一起孵育时，经胰蛋白酶处理的卵黄膜能够阻断精子进入。这些卵母细胞表现出激活的特征性迹象。这些结果支持了布氏蟾蜍卵母细胞可被外部刺激激活的观点，并表明存在与受精激活机制相关的未知卵母细胞表面受体。通过SDS - PAGE对体外成熟卵母细胞的可溶性卵黄膜进行电泳分析，结果显示gp40（未经胰蛋白酶处理的体外成熟卵母细胞）转化为gp38（归因于胰蛋白酶活性或皮质颗粒产物活性，CGP），以及gp70转化为gp68（归因于胰蛋白酶活性加CGP活性）。考虑到只有经胰蛋白酶处理并激活的体外成熟卵母细胞的卵黄膜才能阻断精子进入，我们可以推测gp70向gp68的转化与精子结合相关的变化有关。
