Tulsiani D R, Skudlarek M D, Holland M K, Orgebin-Crist M C
Center for Reproductive Biology Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2633.
Biol Reprod. 1993 Feb;48(2):417-28. doi: 10.1095/biolreprod48.2.417.
Spermatozoa acquire fertilizing ability during passage through the epididymis. Modification of oligosaccharide moieties on sperm surface glycoproteins are some of the biochemical changes believed to be important in the production of functionally mature spermatozoa during passage through the epididymis. In an attempt to understand the mechanism underlying these modifications, we quantified four glycosyltransferase activities (the enzymes that catalyze the transfer of sugar residues from nucleotide sugar donor to the sugar chains on glycoproteins and glycolipids) of spermatozoa and fluid from various regions of the epididymis. Our results are as follows. (1) Only 10-20% of the total glycosyltransferase activities (sialyltransferase, fucosyltransferase, galactosyltransferase, and N-acetyl glucosaminyltransferase) sedimented with the spermatozoa; the remaining 80-90% of the four enzymes were present in soluble form in the epididymal fluid. (2) When the four transferase activities were expressed per 10(6) spermatozoa, only sialyltransferase and fucosyltransferase activities showed maturation-dependent changes. The former enzyme was significantly higher on the proximal caput spermatozoa and the latter on the distal caput spermatozoa. The higher levels of the two enzymes on caput spermatozoa could be due to their binding to the endogenous sugar acceptor molecules on the sperm surface, and subsequent release following sequential sialylation and fucosylation of the molecules in the proximal and distal caput spermatozoa, respectively. (3) When spermatozoa from the proximal and distal caput, corpus, and proximal and distal cauda were incubated with fucose-labeled nucleotide sugar (GDP[14C]fucose), higher levels of radioactivity were routinely incorporated into the spermatozoa from the distal caput. (4) The [14C]fucose-labeled spermatozoa or sperm plasma membranes, when solubilized, resolved on SDS-PAGE, and visualized by autoradiography, showed that the radioactivity had been incorporated into an endogenous acceptor of 86 kDa (major component) and several minor components. Treatment of the solubilized spermatozoa with N-glycanase suggested that the [14C]fucose is mainly present on N-linked oligosaccharide units. These studies demonstrate that some of the sperm surface components are fucosylated during sperm maturation. The potential significance of the in vitro fucosylation of sperm surface components in the production of functionally mature spermatozoa is discussed.
精子在通过附睾的过程中获得受精能力。精子表面糖蛋白上寡糖部分的修饰是一些被认为在精子通过附睾过程中产生功能成熟精子方面很重要的生化变化。为了试图理解这些修饰背后的机制，我们对来自附睾不同区域的精子和液体中的四种糖基转移酶活性（催化糖残基从核苷酸糖供体转移到糖蛋白和糖脂上的糖链的酶）进行了定量。我们的结果如下。（1）总糖基转移酶活性（唾液酸转移酶、岩藻糖基转移酶、半乳糖基转移酶和N-乙酰葡糖胺基转移酶）中只有10%-20%与精子一起沉淀；其余80%-90%的这四种酶以可溶形式存在于附睾液中。（2）当以每10^6个精子来表示这四种转移酶活性时，只有唾液酸转移酶和岩藻糖基转移酶活性显示出成熟依赖性变化。前一种酶在附睾头近端精子上显著更高，后一种酶在附睾头远端精子上更高。附睾头精子上这两种酶的较高水平可能是由于它们与精子表面的内源性糖受体分子结合，随后分别在附睾头近端和远端精子中分子依次进行唾液酸化和岩藻糖基化后释放。（3）当将来自附睾头近端和远端、附睾体以及附睾尾近端和远端的精子与岩藻糖标记的核苷酸糖（GDP[14C]岩藻糖）一起孵育时，通常会有更高水平的放射性掺入到附睾头远端的精子中。（4）[14C]岩藻糖标记的精子或精子质膜，在溶解后，在SDS-PAGE上分离，并通过放射自显影进行可视化，结果显示放射性已掺入到一个86 kDa的内源性受体（主要成分）和几个次要成分中。用N-糖苷酶处理溶解后的精子表明，[14C]岩藻糖主要存在于N-连接的寡糖单元上。这些研究表明，精子表面的一些成分在精子成熟过程中会发生岩藻糖基化。本文讨论了精子表面成分的体外岩藻糖基化在产生功能成熟精子方面的潜在意义。
