Caballero Ignacio, Vazquez Juan M, Mayor Gloria M, Almiñana Carmen, Calvete Juan J, Sanz Libia, Roca Jordi, Martinez Emilio A
Department of Medicine and Surgery, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain.
Int J Androl. 2009 Oct;32(5):505-13. doi: 10.1111/j.1365-2605.2008.00887.x. Epub 2008 Apr 9.
PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma that is able to preserve, in vitro, the viability, motility and mitochondrial activity of highly-extended boar spermatozoa. However, a relationship between the protective effects of the heterodimer and sperm capacitation is still unclear. The present study investigated the effect of the PSP-I/PSP-II (1.5 mg/mL) on membrane stability, intracellular calcium concentration (Ca(2+)) and plasma membrane and acrosome integrity of highly extended boar spermatozoa. Boar spermatozoa were diluted to 1 x 10(6) spermatozoa/mL and incubated at 38 degrees C in Phosphate-buffered saline (PBS) for 10, 30, 60, 120 and 300 min or in modified Tris-buffered medium (mTBM) for 10, 20, 30, 60 and 120 min. After each incubation time, the membrane stability (using Merocyanine-540/Yo-Pro-1), elevation of Ca(2+) (using Fluo-3-AM/PI) and the sperm plasma membrane and acrosome integrity (using SYBR-14/PI/PE-PNA) were evaluated by flow cytometry. As expected, exposure of the spermatozoa to the PSP-I/PSP-II preserved the plasma membrane and acrosome integrity compared to non-exposed spermatozoa in both media PBS and mTBM (p < .01). The evaluation of membrane stability showed no differences in the percentages of viable sperm with instable plasma membrane in the presence of the PSP-I/PSP-II compared to controls irrespective of the dilution media. The evaluation of the Ca(2+) levels showed that while spermatozoa incubated in mTBM and exposed to PSP-I/PSP-II had lower Ca(2+) than controls (39.08% vs. 47.97%, respectively; p < .05), no differences were observed in those samples incubated in PBS. However, a temporal evaluation of the samples showed that a similar proportion of live spermatozoa were able to achieve high levels of Ca(2+) and membrane instability independent of the presence of PSP-I/PSP-II. In conclusion, PSP-I/PSP-II exert a non-permanent decapacitation effect on highly extended boar spermatozoa that is related with a delay in the increase of Ca(2+) levels.
PSP-I/PSP-II异二聚体是公猪精浆中的一种主要蛋白质，能够在体外维持高度稀释的公猪精子的活力、运动能力和线粒体活性。然而，该异二聚体的保护作用与精子获能之间的关系仍不清楚。本研究调查了PSP-I/PSP-II（1.5毫克/毫升）对高度稀释的公猪精子膜稳定性、细胞内钙浓度（Ca(2+)）以及质膜和顶体完整性的影响。将公猪精子稀释至1×10(6)个精子/毫升，并在38℃下于磷酸盐缓冲盐水（PBS）中孵育10、30、60、120和300分钟，或在改良的Tris缓冲培养基（mTBM）中孵育10、20、30、60和120分钟。在每个孵育时间后，通过流式细胞术评估膜稳定性（使用部花青-540/Yo-Pro-1）、Ca(2+)的升高（使用Fluo-3-AM/PI）以及精子质膜和顶体完整性（使用SYBR-14/PI/PE-PNA）。正如预期的那样，与在PBS和mTBM这两种培养基中未暴露的精子相比，精子暴露于PSP-I/PSP-II可保持质膜和顶体完整性（p <.01）。膜稳定性评估显示，无论稀释培养基如何，与对照组相比，在存在PSP-I/PSP-II的情况下，质膜不稳定的活精子百分比没有差异。Ca(2+)水平评估显示，虽然在mTBM中孵育并暴露于PSP-I/PSP-II的精子的Ca(2+)低于对照组（分别为39.08%对47.97%；p <.05），但在PBS中孵育的样品中未观察到差异。然而，对样品的时间评估显示，无论是否存在PSP-I/PSP-II，相似比例的活精子能够达到高水平的Ca(2+)和膜不稳定性。总之，PSP-I/PSP-II对高度稀释的公猪精子发挥非永久性的去能作用，这与Ca(2+)水平升高的延迟有关。
