Steckler D, Stout T A E, Durandt C, Nöthling J O
Section of Reproduction, Faculty of Veterinary Science, Department of Production Animal Studies, Onderstepoort, South Africa.
Section of Reproduction, Faculty of Veterinary Science, Department of Production Animal Studies, Onderstepoort, South Africa; Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Theriogenology. 2015 Jun;83(9):1451-60. doi: 10.1016/j.theriogenology.2015.01.019. Epub 2015 Jan 21.
The aim of this study was to determine whether flow cytometric evaluation of combined merocyanine 540 and Yo-Pro 1 (M540-YP) staining would identify viable dog sperm that had undergone membrane stabilization known to be associated with capacitation in other species, and whether such destabilization is detected earlier than when using the tyrosine phosphorylation and ethidium homodimer (TP-EH) stain combination with epifluorescence microscopy. Semen from nine dogs was collected and incubated in parallel in bicarbonate-free modified Tyrode's medium (-BIC), medium containing 15 mM bicarbonate (+BIC), dog prostatic fluid, and in PBS. Aliquots for staining were removed at various time points during incubation of up to 6 hours. Staining with M540-YP allowed the classification of dog sperm as viable without destabilized membranes, viable with destabilized membranes, nonviable without destabilized membranes, or nonviable with destabilized membranes. The percentage of viable sperm detected using EH (83.5 ± 1.37%; mean ± SEM) was higher than when using YP (66.7 ± 1.37%: P < 0.05; n = 54 semen samples). On the other hand, M540-YP identified a higher percentage of viable sperm with destabilized membranes than TP-EH (75 ± 1.76% vs. 35 ± 1.70%: P < 0.05; n = 54 semen samples). Staining with M540-YP indicated a rapid increase in the percentage of viable sperm with destabilized membranes, reaching a maximum during the first 30 minutes of incubation in +BIC. For all other treatments (i.e., -BIC, prostatic fluid, and PBS), the peak in the percentage of viable sperm with destabilized membranes was reached as much as 90 to 210 minutes later than incubation in +BIC. The lowest percentage of viable sperm showing signs of capacitation was recorded during incubation in PBS. We conclude that YP identifies sperm committed to cell death earlier than EH, and that the M540-YP stain combination identifies membrane destabilization known to be associated with capacitation in other species earlier than the TP-EH stain combination.
本研究的目的是确定用流式细胞术评估部花青540和Yo-Pro 1（M540-YP）联合染色能否识别经历了膜稳定化的存活犬精子，这种膜稳定化已知与其他物种的获能相关，以及这种膜去稳定化是否比使用酪氨酸磷酸化和溴化乙锭同二聚体（TP-EH）染色组合并结合落射荧光显微镜检测得更早。收集9只犬的精液，并分别在不含碳酸氢盐的改良Tyrode氏培养基（-BIC）、含15 mM碳酸氢盐的培养基（+BIC）、犬前列腺液和磷酸盐缓冲液（PBS）中平行孵育。在长达6小时的孵育过程中的不同时间点取出用于染色的等分试样。用M540-YP染色可将犬精子分类为膜未去稳定化的存活精子、膜去稳定化的存活精子、膜未去稳定化的非存活精子或膜去稳定化的非存活精子。使用EH检测到的存活精子百分比（83.5±1.37%；平均值±标准误）高于使用YP时（66.7±1.37%：P<0.05；n = 54份精液样本）。另一方面，M540-YP识别出的膜去稳定化的存活精子百分比高于TP-EH（75±1.76%对35±1.70%：P<0.05；n = 54份精液样本）。用M540-YP染色表明，膜去稳定化的存活精子百分比迅速增加，在+BIC中孵育的最初30分钟内达到最大值。对于所有其他处理（即-BIC、前列腺液和PBS），膜去稳定化的存活精子百分比达到峰值的时间比在+BIC中孵育晚90至210分钟。在PBS中孵育期间，显示获能迹象的存活精子百分比最低。我们得出结论，YP比EH更早识别出走向细胞死亡的精子，并且M540-YP染色组合比TP-EH染色组合更早识别出已知与其他物种获能相关的膜去稳定化。
