Akhondi M A, Chapple C, Moore H D
Department of Obstetrics and Gynaecology, University of Sheffield, UK.
Hum Reprod. 1997 Mar;12(3):514-22. doi: 10.1093/humrep/12.3.514.
Human epididymal tissue was recovered from 11 patients undergoing orchidectomy without anti-androgen treatment. Everted epithelial fragments from the caput and corpus epididymis of six patients were successfully cultured in a modified RPMI 1640 medium supplemented with HEPES and androgens for up to 110 days (mean 56 +/- 28) in 5% CO(2) in air at 37 degrees C. Epithelial cells from human oviduct and non-reproductive tract cells (breast epithelial cells, fibroblasts) were also cultured for comparison. The proportion of epididymal epithelial cells in primary cultures assessed by immunofluorescent localization using a cytokeratin monoclonal antibody was shown to be >70% for the first 6-8 weeks of culture. Light and electron microscopy indicated that epithelial cells maintained polarity and some normal morphology during the culture period. Washed epididymal or ejaculated spermatozoa prepared by a 'swim-up' procedure were co-incubated (i) directly with epididymal cells in culture wells, (ii) in 12 mm Millicell inserts within culture wells, thereby preventing contact of spermatozoa with culture cells; and (iii) in culture medium alone. A significant proportion of spermatozoa in direct contact with culture cells or in Millicell inserts were viable after 6 days of co-incubation (30-45%) and exhibited progressive motility, while all spermatozoa in medium alone were non-motile by 3 days. Using computer-assisted sperm analysis it was shown that the progressive motility of viable spermatozoa decreased gradually for the first 5 days in culture and then remained constant (approximately 30 microm/s, average path velocity). After 12 days of co-incubation, 15 +/- 4% of spermatozoa in direct contact with epithelial cells remained motile; in one experiment, a few spermatozoa (<1%) were motile at 17 days. Light and electron microscope observations indicated that prolonged sperm survival was associated with close apposition of spermatozoa (by equatorial segment) to the apical membrane of epithelial cells. Oviductal epithelial cells were also beneficial for sperm survival, but other cell types had no effect.
从11例接受睾丸切除术且未接受抗雄激素治疗的患者身上获取了人类附睾组织。将6例患者附睾头和附睾体的外翻上皮片段成功培养于添加了HEPES和雄激素的改良RPMI 1640培养基中，在37℃、含5%二氧化碳的空气中培养长达110天（平均56±28天）。还培养了人类输卵管上皮细胞和非生殖道细胞（乳腺上皮细胞、成纤维细胞）用于比较。使用细胞角蛋白单克隆抗体通过免疫荧光定位评估，原代培养中附睾上皮细胞的比例在培养的前6 - 8周显示大于70%。光镜和电镜观察表明，上皮细胞在培养期间保持极性和一些正常形态。通过“上游”程序制备的洗涤过的附睾精子或射出精子，分别进行如下共孵育：(i) 直接与培养孔中的附睾细胞共孵育；(ii) 在培养孔内的12毫米Millicell插入物中，从而防止精子与培养细胞接触；(iii) 仅在培养基中。共孵育6天后，与培养细胞直接接触或在Millicell插入物中的精子有很大比例仍存活（30 - 45%）并表现出进行性运动，而仅在培养基中的所有精子在3天时均无运动能力。使用计算机辅助精子分析表明，存活精子的进行性运动在培养的前5天逐渐下降，然后保持恒定（约30微米/秒，平均路径速度）。共孵育12天后，与上皮细胞直接接触的精子中有15±4%仍有运动能力；在一项实验中，少数精子（<1%）在17天时仍有运动能力。光镜和电镜观察表明，精子的长期存活与精子（通过赤道段）与上皮细胞顶端膜的紧密贴附有关。输卵管上皮细胞对精子存活也有益，但其他细胞类型则无影响。
