Yunaini Luluk, Pujianto Dwi Ari
Doctoral Program for Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta 10430, Indonesia.
Department of Medical Biology, Faculty of Medicine, Universitas Indonesia, Jakarta 10430, Indonesia.
Vet World. 2023 Jun;16(6):1325-1332. doi: 10.14202/vetworld.2023.1325-1332. Epub 2023 Jun 13.
Sperm maturation occurs in the epididymis through interactions with existing molecules inside the lumen. However, the mechanism of epididymis molecular transfer is currently unclear. This study was aimed to determine the necessity of the epididymal epithelial cells (EECs) in the process of sperm maturation in terms of sperm kinetics and tyrosine phosphorylation.
A true experimental research design was used in this study. The medium tested was a primary culture of mice caput epididymal cells (cells and culture medium), conditioned medium (CM) (supernatant of EECs), and secretome (CM filtered at 0.22 μm). Sperm was cocultured in EEC culture, CM, and secretome for 1, 2, 3, or 4 h. The original culture medium was used as the control. Sperm kinetic analysis was performed after the indicated times using computer-assisted sperm analysis, and tyrosine phosphorylation was detected using the Western blot technique.
A primary culture of caput EECs was successfully generated. The results showed increased sperm motility and progressive movement after 3 h of incubation (p < 0.05). There was a significant decrease in the average path velocity (VAP) values after 4 h of incubation (p < 0.05), but there was no significant change in the 1, 2, and 3 h incubation groups. The EEC culture-CM and secretome groups showed a significant increased progressivity and VAP percentage values compared with the control medium (p < 0.05). In terms of percentage motility, the culture and CM groups were significantly different from the control medium, but the secretome group was not.
The sperm kinetics of sperm cultured in CM, secretome, and EEC were significantly increased after 3 h of incubation, suggesting that CM and secretome can be used to replace EECs, especially when analyzing molecules secreted by the epididymal epithelium during sperm maturation. The results of this study highlight the potential of CM and secretome as therapy mediums for sperm kinetic abnormalities.
精子在附睾中通过与管腔内现有分子相互作用而成熟。然而，附睾分子转运的机制目前尚不清楚。本研究旨在从精子动力学和酪氨酸磷酸化方面确定附睾上皮细胞（EECs）在精子成熟过程中的必要性。
本研究采用真实验研究设计。测试的培养基为小鼠附睾头细胞原代培养物（细胞和培养基）、条件培养基（CM）（EECs的上清液）和分泌蛋白质组（经0.22μm过滤的CM）。精子在EEC培养物、CM和分泌蛋白质组中共同培养1、2、3或4小时。使用原始培养基作为对照。在指定时间后，使用计算机辅助精子分析进行精子动力学分析，并使用蛋白质免疫印迹技术检测酪氨酸磷酸化。
成功建立了附睾头EECs原代培养物。结果显示，孵育3小时后精子活力和前向运动增加（p<0.05）。孵育4小时后平均路径速度（VAP）值显著降低（p<0.05），但在1、2和3小时孵育组中无显著变化。与对照培养基相比，EEC培养物-CM和分泌蛋白质组组的前向性和VAP百分比值显著增加（p<0.05）。在活力百分比方面，培养物和CM组与对照培养基有显著差异，但分泌蛋白质组组无差异。
在CM、分泌蛋白质组和EEC中培养的精子在孵育3小时后精子动力学显著增加，表明CM和分泌蛋白质组可用于替代EECs，特别是在分析精子成熟过程中附睾上皮分泌的分子时。本研究结果突出了CM和分泌蛋白质组作为精子动力学异常治疗介质的潜力。
