Kunkitti Panisara, Chatdarong Kaywalee, Suwimonteerabutr Junpen, Nedumpun Teerawut, Johannisson Anders, Bergqvist Ann-Sofi, Sjunnesson Ylva, Axnér Eva
Department of Surgery and Theriogenology, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen, Thailand.
Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
Anim Reprod Sci. 2017 Oct;185:148-153. doi: 10.1016/j.anireprosci.2017.08.014. Epub 2017 Aug 23.
During the cryopreservation process, spermatozoa are exposed to hypertonic solutions contributed by the high concentration of cryoprotectant. During addition and removal of cryoprotectant the spermatozoa are subjected to a substantial osmotic stress. Spermatozoa of different species and different stages of maturation may have different susceptibility to osmotic stress depending on the biology of the cell membrane and this will affect their tolerance to the freezing-thawing stress. The aims of this study were to determine the osmotic tolerance limits for motility, membrane integrity and mitochondrial membrane potential of feline epididymal spermatozoa and to study the effect of osmotic stress on the feline spermatozoa of different epididymal regions. Epididymal spermatozoa from three regions (caput, corpus and cauda) were pre-exposed to various osmolalities (75, 300, 600, 900, 1200 mOsm) in a single step for 10min and returned to 300 mOsm afterward. Percentage of motile spermatozoa was measured subjectively and membrane integrity (SYBR-14 positive cells) was evaluated prior to and after exposure to different osmolalities. The mitochondrial membrane potential (JC1) of spermatozoa were evaluated using flow cytometer and compared between epididymal regions (caput, corpus and cauda). All the parameters were compared using a mixed procedure. The percentage of motile epididymal spermatozoa decreased significantly when spermatozoa were exposed to 75 mOsm and 600 mOsm. Epididymal spermatozoa showed signs of damage when pre-exposed to 900 and 1200 mOsm and returned to isotonic condition as significant reduction of membrane integrity and mitochondrial membrane potential were observed (P<0.05). The plasma membrane of spermatozoa from the cauda epididymal region showed higher susceptibility to osmotic stress than the other regions as demonstrated by a significant difference between regions after return to isotonicity from 900 mOsm (P>0.01) and a difference between caput and corpus after return from 1200 mOsm (P<0.05). The corpus and cauda epididymal spermatozoa had higher percentage of spermatozoa with high mitochondrial membrane potential than those from caput when exposed to 75, 300 and 600 mOsm (P<0.05). In conclusion, a single step exposure to hypertonic solution of greater than 600 mOsm prior to return to isotonic condition can cause severe damage to sperm membrane and mitochondrial membrane potential compared to non-returning (exposure to various osmolality but not returned to isotonic condition). Changes in osmolality impacted mostly on sperm motility. Spermatozoa from cauda epididymis were more susceptible to osmotic stress compared to those from corpus and caput indicating that the maturation changes in the sperm membrane during passage through the epididymis increase susceptibility to the osmotic changes that may occur during sperm cryopreservation.
在冷冻保存过程中，精子会暴露于由高浓度冷冻保护剂造成的高渗溶液中。在添加和去除冷冻保护剂的过程中，精子会受到相当大的渗透应激。不同物种以及处于不同成熟阶段的精子，可能因其细胞膜生物学特性的不同而对渗透应激具有不同的敏感性，这会影响它们对冻融应激的耐受性。本研究的目的是确定猫附睾精子活力、膜完整性和线粒体膜电位的渗透耐受极限，并研究渗透应激对猫不同附睾区域精子的影响。来自三个区域（头部、体部和尾部）的附睾精子在一步操作中预先暴露于各种渗透压（75、300、600、900、1200 mOsm）下10分钟，之后再恢复到300 mOsm。主观测量活动精子的百分比，并在暴露于不同渗透压之前和之后评估膜完整性（SYBR-14阳性细胞）。使用流式细胞仪评估精子的线粒体膜电位（JC1），并在附睾区域（头部、体部和尾部）之间进行比较。所有参数均使用混合程序进行比较。当精子暴露于75 mOsm和600 mOsm时，附睾活动精子的百分比显著下降。当预先暴露于900和1200 mOsm并恢复到等渗状态时，附睾精子出现损伤迹象，因为观察到膜完整性和线粒体膜电位显著降低（P<0.05）。附睾尾部区域精子的质膜对渗透应激的敏感性高于其他区域，从900 mOsm恢复到等渗状态后各区域之间存在显著差异（P>0.01），从1200 mOsm恢复后头部和体部之间存在差异（P<0.05）。当暴露于75、300和600 mOsm时，附睾体部和尾部精子中具有高线粒体膜电位的精子百分比高于头部精子（P<0.05）。总之，与不恢复（暴露于各种渗透压但不恢复到等渗状态）相比，在恢复到等渗状态之前一步暴露于大于600 mOsm的高渗溶液会对精子膜和线粒体膜电位造成严重损伤。渗透压的变化主要影响精子活力。附睾尾部的精子比体部和头部的精子对渗透应激更敏感，这表明精子在通过附睾的过程中细胞膜的成熟变化增加了其对精子冷冻保存期间可能发生的渗透变化的敏感性。
