Reyes-Moreno Carlos, Boilard Mathieu, Sullivan Robert, Sirard Marc-André
Centre de Recherche en Biologie de la Reproduction, Départment des Sciences Animales, Université Laval, Sainte-Foy, Québec, Canada.
Mol Reprod Dev. 2002 Dec;63(4):500-9. doi: 10.1002/mrd.10192.
Epididymis provides a safe environment in which stored-spermatozoa could survive for days before ejaculation. In vitro studies suggested that epididymal proteins seem to be implicated in sperm survival during coincubation with cultured epididymal cells. This study was basically designed to confirm if secretory proteins from bovine epididymal cell cultures provide sperm protection against rapid loss of sperm motility in vitro. Bovine spermatozoa were incubated in conditioned media (CM), which were prepared from cultured cauda epididymal cell (CEC). Motion parameters were recorded using a computer-assisted sperm analyzer. Sperm-free protein extracts from CM were fractionated by ultrafiltration through a 10-kDa cut off membrane. A significantly positive effect on sperm motility was observed when spermatozoa were incubated in CM (54 +/- 4%) and CM > 10 kDa (57 +/- 4%) compared to CM < 10-kDa fraction (30 +/- 3%) or fresh media (34 +/- 3%), after a 6-hr incubation period. This beneficial effect on sperm motility was abolished when the CM > 10-kDa fraction was heat-treated at 100 degrees C for 10 min. The CM > 10 kDa fraction provides factors that remained active even though spermatozoa were washed twice after a 2-hr preincubation period. To identify potential beneficial factors, bovine spermatozoa were incubated with radiolabeled proteins obtained using (35)S-methionine in culture medium. SDS-PAGE analysis of proteins extracted from CM-preincubated spermatozoa revealed the presence of a 42-kDa protein strongly associated to the sperm surface. This 42-kDa spot was trypsin-digested and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as a protein homologue to a 35-kDa bovine estrogen-sulfotransferase. This protein can play a role in epididymal biology and sperm function. Taken together, these results suggest that specific epididymal proteins can be implicated in the sperm protection in vitro, and can be characterized in our cell culture system.
附睾提供了一个安全的环境，储存的精子在射精前可在其中存活数天。体外研究表明，附睾蛋白似乎与培养的附睾细胞共孵育期间精子的存活有关。本研究主要旨在确认牛附睾细胞培养物中的分泌蛋白是否能在体外为精子提供保护，防止精子活力快速丧失。将牛精子在条件培养基（CM）中孵育，该条件培养基由培养的附睾尾细胞（CEC）制备。使用计算机辅助精子分析仪记录运动参数。通过10 kDa截留膜超滤对CM中的无精子蛋白提取物进行分级分离。在6小时孵育期后，与CM < 10 kDa级分（30 ± 3%）或新鲜培养基（34 ± 3%）相比，精子在CM（54 ± 4%）和CM > 10 kDa（57 ± 4%）中孵育时，观察到对精子活力有显著的积极影响。当CM > 10 kDa级分在100℃热处理10分钟时，对精子活力的这种有益影响被消除。CM > 10 kDa级分提供的因子即使在2小时预孵育期后精子被洗涤两次仍保持活性。为了鉴定潜在的有益因子，将牛精子与在培养基中使用（35）S-甲硫氨酸获得的放射性标记蛋白一起孵育。对从CM预孵育的精子中提取的蛋白质进行SDS-PAGE分析，发现存在一种与精子表面强烈相关的42 kDa蛋白质。对这个42 kDa的斑点进行胰蛋白酶消化，并通过基质辅助激光解吸电离飞行时间（MALDI-TOF）鉴定为与35 kDa牛雌激素磺基转移酶同源的蛋白质。这种蛋白质可能在附睾生物学和精子功能中发挥作用。综上所述，这些结果表明特定的附睾蛋白可能参与体外精子保护，并可在我们
