Pereira B M, Abou-Haila A, Tulsiani D R
a Center for Reproductive Biology Research and Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2633, USA.
Biol Reprod. 1998 Dec;59(6):1288-95. doi: 10.1095/biolreprod59.6.1288.
In previous publications (Tulsiani et al., Biochem J 1993; 290:427-436 and Tulsiani et al., Dev Biol 1995; 167:584-595), we reported that sperm surface mannosidase is present in rat testis and is modified during spermatogenesis and sperm maturation. The present studies were directed towards examining the origin of alpha-D-mannosidase activity present on fertile spermatozoa. Mixed germ cells prepared after sequential enzymatic digestions of rat testis were separated by unit gravity sedimentation using 2-4% linear bovine serum albumin gradient. Fractions enriched in spermatocytes, round spermatids, and condensed/elongated spermatids (> 95% pure cells) were separately pooled and assayed for [3H]Man9-mannosidase activity before (intact) and after lysis with Triton X-100. Interestingly, the cells contained a significant level of alpha-D-mannosidase activity. Approximately 70% of the total [3H]Man9-mannosidase activity present in the detergent-solubilized germ cell extract cross-reacted with anti-rat sperm mannosidase, and 25% of the activity cross-reacted with anti-Golgi mannosidase I. This result indicates that most of the mannosidase activity present in the germ cell extract is antigenically similar to the enzyme present on the cauda spermatozoa. Using cell fractionation techniques, we obtained evidence suggesting that the germ cell-associated mannosidase activity is an integral component of the plasma membranes. Taken together, these results indicate that sperm surface mannosidase is first expressed on the testicular germ cells.
在之前的出版物中（图尔西亚尼等人，《生物化学杂志》1993年；290:427 - 436以及图尔西亚尼等人，《发育生物学》1995年；167:584 - 595），我们报道了精子表面甘露糖苷酶存在于大鼠睾丸中，并且在精子发生和精子成熟过程中会发生修饰。目前的研究旨在探究可育精子上存在的α - D - 甘露糖苷酶活性的来源。通过用2 - 4%线性牛血清白蛋白梯度进行单位重力沉降，分离经连续酶消化大鼠睾丸后制备的混合生殖细胞。分别收集富含精母细胞、圆形精子细胞以及浓缩/伸长精子细胞（> 95%纯细胞）的组分，并在使用Triton X - 100裂解之前（完整状态）和之后检测其[3H]Man9 - 甘露糖苷酶活性。有趣的是，这些细胞含有显著水平的α - D - 甘露糖苷酶活性。去污剂溶解的生殖细胞提取物中存在的总[3H]Man9 - 甘露糖苷酶活性中，约70%与抗大鼠精子甘露糖苷酶发生交叉反应，25%的活性与抗高尔基体甘露糖苷酶I发生交叉反应。这一结果表明，生殖细胞提取物中存在的大部分甘露糖苷酶活性在抗原性上与附睾精子上存在的酶相似。使用细胞分级分离技术，我们获得了证据表明与生殖细胞相关的甘露糖苷酶活性是质膜的一个组成部分。综上所述，这些结果表明精子表面甘露糖苷酶首先在睾丸生殖细胞上表达。
