哺乳动物精子顶体反应的位点 - 山东大学课程中心30

哺乳动物精子顶体反应的位点在哺乳动物和其他许多动物中，受精过程包括精子头顶端的顶体（一个被膜包裹的拥有各种酶的细胞器）建立能够渗透穿过卵黄膜的通道。

受精发生前，精子必须首先发生顶体反应（AR），其中涉及顶体膜和其上覆盖的膜（精子细胞膜）之间的点融合。

顶体反应释放了顶体内的溶解性酶，促进了在穿透卵黄膜过程中通道的建立（虽然各种证据现在质疑卵黄膜溶解是否为兽类哺乳动物所使用的机制）。

在美国国家科学院院刊（PNAS）中，利用体外受精，Jin等人报告了来自顶体和精子尾部中段被不同荧光标签标记的转基因小鼠的精子顶体反应的位点及时间。

这种采用视频成像的方法，使他们能够记录到，与在裸卵表面的精子相比，精子顶体反应更容易在围绕在新排出的小鼠卵周围的卵丘细胞团的空隙中发生。

此外，比起在裸卵表面的精子，不仅顶体反应更大程度上和更快速地在精子穿透卵丘的过程中发生，在卵丘细胞覆盖裸卵表面的情况下，精子也会与裸卵相互作用。

最重要的是，Jin等人通过对所记录影像的追溯检查发现了一些与卵丘细胞反应后的精子能够继续使卵受精。

鉴于过去的几年中，顶体反应已经被用不同的手段，在各种各样的哺乳动物中，包括人，进行了研究。

那么为什么目前的研究在现有尝试的背景下去理解兽类哺乳动物受精的机制中如此重要呢？顶体反应的起始非哺乳动物受精的模型已经十分明确，精子必须创造一个能穿透卵黄膜的通道，根据物种的不同，顶体反应被卵黄膜或通常被卵黄膜外的胶状物（卵胶膜）诱导。

然而，在兽类哺乳动物中，由于卵黄膜，或称透明带在排卵后被物种特异性的卵丘细胞（一种经常嵌在含有丰富透明质酸基质中的多层细胞团）包围数小时，建立顶体反应开始的位点很困难。

在早期利用豚鼠和几种啮齿动物的研究中，Austin 和Bishop报告了精子在卵丘中发生顶体反应，并且他们错误地认为该反应是精子获能（特别是在兽类哺乳动物中，精子在女性阴道中经历膜相关的变化，最终使精子具有受精能力）的形态学表现。

后来，在电子显微镜下观察到，兔精子在卵丘的边缘通常是具有完整顶体法，而在卵丘最里面的精子此时已经发生了部分顶体反应并且大部分与透明带结合（3）。

此后，在叙利亚和中国仓鼠甚至人的精子中都观察到了顶体反应发生在卵丘中的现象。

然而，不久前，在分子层面上定义受精的研究有了重要的进展，小鼠透明带中含有三种糖蛋白——ZP1，ZP2和ZP3（8）。

因为大多数哺乳动物没有卵丘的卵可以在体内或体外受精，那么观察到小鼠的顶体反应可以被ZP3（9）诱导就可以表明透明带诱导了顶体反应。

虽然这看起来给之前在其他物种中观察到的顶体反应发生在卵丘中的结论大打折扣，虽然也偶有例外，但是大部分关于小鼠的研究是利用没有卵丘的卵进行的，透明带是哺乳动物顶体反应生理上的诱导者的这种观点已经成为其他报道的焦点（11，12），并且被普遍接受。

因此，Jin等人（1）的研究的特殊优点不仅在于观察活体小鼠精子的事件，他们法观察代表着在这方面回归生物体内的现实情况。

这表明这些精子通常在到达透明带之前，在卵丘中发生顶体反应，使用荧光尾标签这也表明了这样的精子可以继续参与受精的关键事实。

发生了顶体反应的小鼠精子可以继续穿透透明带的发现对于描述精子头部表面的包括参与受精时精卵结合在内的成分有重要的影响。

然而，考虑这项工作的其他影响，它需要最终显示出其结果能够代表在体内受精的情况。

此外，尽管在卵丘中荧光顶体标记消失，据推测反应的顶体壳一直保留到当精子头部与透明带表明结合时。

虽然这些在文献中没有强调，但广泛的跨物种的顶体反应研究表明，兽类哺乳动物已发生反应的顶体有不寻常的稳定性，因此，顶体的寿命可能是由不溶性的顶体基质成分带来的。

这种稳定似乎是对允许它坚持系在精子头部直到精子穿透遇到相对强大的密度较高的透明带基质的一个适应。

卵丘的重要性？最后，Jin等人的文章进一步关注了两个问题。

虽然在小鼠卵丘中顶体反应的诱导剂是什么还不知道，但作者们在最后一点指出，在多种哺乳动物，包括人类中，卵丘细胞可以产生孕酮，孕酮可在获能精子中诱导顶体反应（13）。

这又带来一个重要的问题：卵丘中和透明带表面对与顶体反应刺激包含的分子机制，是相同还是非常不同？第二，文章强调了一个更广泛的问题：关于兽类哺乳动物中卵丘适应性的重要性（在卵相对较小的有袋类哺乳动物排卵前滤泡细胞也会脱落）。

事实上，没有一个单一的或简单的答案，因为卵丘就其排卵后的寿命，形式及其功能显示出综合性。

例如，在鼩的两个非常不同的物种中稠密的透明带，通过缝隙连接稳定，无细胞间基质，这样的透明带似乎无法刺激顶体反应，但其确实发挥了受精中作为顶体反应诱导剂的重要作用（14）。

顶体在精子到达透明带前脱落，在这种情况下，反应后的精子在到达透明带表面前穿透并杀死一列卵丘细胞（15）。

因为鼩通常被认为是类似于兽类的祖先，所以卵丘在受精中的重要作用可能代表了进化的情况，基于精子头部解剖，可至少将其应用于表明其他食虫动物的进化情况。

然而，尽管在大部分哺乳动物的研究中，受精可以在没有卵丘的情况下发生，但其形式和排卵后的生活有很大的不同。

卵丘在一些物种中排列紧密并且只含有少量的基质，但丰富的透明质酸基质可以使卵丘明显扩大，使卵在宽敞的输卵管壶腹部受精，这在兽类哺乳动物中是很常见的。

从有蹄类和灵长类动物到啮齿目和兔类，卵丘的扩大可提供一个更大的与卵相关的靶点来隔绝宽松的壶腹部中出现的少量精子（16），促进卵的早期受精（17）。

Site of the mammalian sperm physiological acrosome reactionIn mammals and the many other animals in which fertilization involves the establishment of a penetration path through the egg coat, at the apex of the sperm head lies the acrosome—a membrane-limited organelle that houses a complex of enzymes. For fertilization to occur, spermatozoa must first undergo the acrosome reaction (AR), which involves point fusions between the outer acrosomal and overlying cell membrane. This reaction releases the acrosome’s soluble lysins, which facilitate the creation of a path in the egg coat during penetration (although a variety of evidence now calls into question whether egg-coat lysis is the mechanism used by eutherian mammals). In PNAS, using in vitro fertilization, Jin et al.(1) report their tracking of the site and timing of the AR in spermatozoa from transgenic mice whose acrosome and sperm tail midpiece were tagged with different fluorescent markers. This approach, which used video imaging, allowed them to record that, compared with sperm on a naked zona surface, the AR was generally initiated more readily in the interstices of the cell mass of the cumulus oophorus that surrounds the newly ovulated mouse egg. Moreover, not only did the AR generally occur to a greater degree and often more rapidly in sperm penetrating the cumulus than on a naked zona surface, but this was also true for sperm interacting with naked eggs in the presence of cumulus-invested oocytes. Most important, Jin et al. (1) establish by retroactiveexamination of recorded images that some spermatozoa reacting within the cumulus were then able to go on to fertilize the egg. Given that the AR has been examined in different ways over the years and in a variety of mammals, including man, why then is this current study so important in the context of present efforts to understand the mechanisms of fertilization in eutherian mammals?Initiation of the ARIt has long been clear for the nonmammalian models of fertilization in which spermatozoa must create a penetration path that, according to species, the AR is induced by the egg coat or often, by jelly that surrounds that coat. In eutherian mammals, however, establishing the site where the AR begins is made difficult by the fact that the egg coat—the zona pellucida—is surrounded for a species-variable number of hours after ovulation by the cumulus oophorus, a multilayered mass of cells often embedded in a hyaluronic acid-rich matrix. In early studies in the guinea pig and several rodents, Austin and Bishop (2) reported spermatozoa undergoing the AR within the cumulus and, mistakenly, they then suggested that the reaction was a morphological manifestation of sperm capacitation—a sperm membrane-related change undergone in the female tract, peculiar to therian mammals, that finally confers on spermatozoa the ability to fertilize. Later, as seen in the electron microscope, rabbit spermatozoa at the fringe of the cumulus oophorus generally had intact acrosomes, whereas most caught within the cumulus had undergone some phase of the AR by that time, as had most spermatozoa bound to the zona pellucida (3). Subsequently, similar observations of the AR within the cumulus were made for spermatozoa of the Syrian (4, 5) and Chinese hamster (6) and even man (7). Not long before that, however, inroads into the problem of defining fertilization in molecular terms had begun with the important finding that the (mouse) zona pellucida comprises three glycoproteins—ZP1, ZP2, and ZP3 (8). Because cumulus-free eggs of most mammals can be fertilized in vivo or in vitro, the equally exciting observation a few years later that the mouse AR can be induced by ZP3 (9) led to the view that the zona pellucida induces the AR. Although seeming to discount previous observations of the AR within the cumulus in other species and although, with occasional exceptions(10), most mouse studies were performed using cumulus-free eggs, this view of the zona pellucida as the physiological inducer of the mammalian AR has been a focus of other reports (11, 12), and is now widespread. Thus, the observations of Jin et al. (1) represent a return to biological reality in this regard. The particular merit of their study lies not only in the observation of events in living mouse spermatozoa. To reiterate, it shows that these spermatozoa frequently undergo the AR within the cumulus before reaching the zona pellucida and, using a fluorescent tail tag, it also shows the key fact that such spermatozoa can then go on to fertilize.The observation that mouse spermatozoa with reacted acrosomes can then go on to adhere to and penetrate the zona pellucida has important implications for efforts to characterize the sperm head surface component(s) involved in sperm/ egg binding at fertilization. However, in considering other implications of this work, it needs to be shown eventually that its findings represent the situation during fertilization in vivo. Moreover, despite the disappearance of the fluorescent acrosomal tag within thecumulus, it is presumed that the reacted acrosomal carapace is retained until and while the sperm head binds to the zona surface. Although this has not been stressed in the literature, a broad cross-species survey of the AR suggests that the reacted acrosome in eutherian mammals has an unusual stability and therefore, lifespan, possibly brought by an insoluble acrosomal matrix component. This stability seems to be an adaptation that then allows it to persist and tether the sperm head until penetration of the relatively formidable, dense zona matrix begins.Significance of the Cumulus Oophorus?Finally, the paper of Jin et al. (1) serves to focus two further issues. Although the identity of the AR induction agent in the mouse cumulus oophorus is not known, as a last point these authors note that in several mammals, including man, progesterone, which cumulus cells may produce, can induce the AR in capacitated spermatozoa (13). This brings an important question as to whether the stimulus for the AR in the cumulus oophorus and that on the zona surface, and the molecular steps involved, are the same or very different. Second, the study serves to highlight a broader question as to the adaptive significance of the cumulus oophorus, which appears to be unique to eutherian mammals, the follicular cellsbeing shed before ovulation even in marsupial mammals that have comparably small eggs. In fact, there is no single or simple answer, because the cumulus oophorus displays a variety of radiations in regard to its post-ovulatory life, its form, and its function. For instance, in two very different species of shrew, the dense cumulus is stabilized by gap junctions and has no intercellular matrix, but does have an essential role in fertilization as the inducer of the AR, which the zona pellucida seems unable to stimulate (14). The acrosome is shed before spermatozoa reach the zona pellucida, and in one case, the reacted spermatozoa penetrate and kill a line of cumulus cells in reaching the zona surface (15). Because shrews are often considered to be akin to the eutherian progenitors, this essential role of the cumulus in fertilization may represent the evolutionary situation, and based on sperm head anatomy may apply at least to other insectivores. However, although fertilization can occur in the absence of a cumulus in most mammals studied, its form and the postovulatory life can vary widely. The cumulus oophorus may be compact and have little matrix in some species, but a hyaluronic acid-rich matrix expands the ovulated cumulus oophorus significantly where the eggs are fertilized in a spacious tubal ampulla, which is very common in eutherian mammals. Seen in a range of species from ungulates and primates to rodents and rabbits, this expanded state of the cumulus may act to provide a larger egg-associated target for and sequester the very few spermatozoa present in a voluminous ampulla (16), and consistent with the present study, it may foster early fertilization of the eggs (17).。