de Andrade A F C, de Arruda R P, Celeghini E C C, Nascimento J, Martins S M M K, Raphael C F, Moretti A S
Laboratory of Semen Biotechnology and Andrology, University of Sao Paulo (USP), Pirassununga, SP, Brazil.
Reprod Domest Anim. 2007 Apr;42(2):190-4. doi: 10.1111/j.1439-0531.2006.00751.x.
The purpose of this study was to validate a technique for simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa, using an association of fluorescent probes: Propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and JC-1. Three ejaculates from each of four different boars, all showing motility >or=80% and abnormal morphology <or=10%, were diluted in TALP medium and split into two aliquots. One of the aliquots was flash frozen in liquid nitrogen and thawed in three continuous cycles, to induce damage in cellular membranes and to disturb mitochondrial function. Three treatments were prepared with the following fixed ratios of fresh semen : flash frozen semen; 100 : 0 (T100), 50 : 50 (T50), and 0 : 100 (T0). The samples were then submitted to a stain technique. To a 150-microl aliquot of diluted semen it was added 3 microl of PI (0.5 mg/ml), 2 microl of JC-1 (153 microm) and 50 microl of FITC-PSA (100 microg/ml). Samples were incubated at 38.5 degrees C for 8 min, in the dark. An 8-microl sample was put on a slide, coverslipped and immediately evaluated by epifluorescent microscopy. The association of fluorescent probes was divided into eight cell classes, according to plasma membrane integrity, intact acrosome and mitochondrial function. For plasma membrane integrity, detected by PI probe, the equation: (p < 0.0001) and R(2) = 0.97 was obtained. The intact acrosome, verified by the FITC-PSA probe, produced the equation: (p < 0.0001) and R(2) = 0.98. The mitochondrial potential, marked by JC-1, was estimated by the equation: (p < 0.001) and R(2) = 0.99. The group of spermatozoa with combined intact plasma membrane, intact acrosome and high mitochondrial potential (IPIAH), was estimated by the equation: (p < 0.0001) and R(2) = 0.97. The resulting linear equations demonstrate that this technique is efficient and practical for the simultaneous evaluations of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa.
本研究的目的是验证一种使用荧光探针组合同时评估公猪精子中质膜、顶体膜和线粒体膜的技术：碘化丙啶（PI）、异硫氰酸荧光素偶联的豌豆凝集素（FITC-PSA）和JC-1。从四头不同公猪中各采集三份精液，所有精液的活力均≥80%，异常形态均≤10%，将其在TALP培养基中稀释并分成两份。其中一份在液氮中速冻，并连续解冻三个循环，以诱导细胞膜损伤并扰乱线粒体功能。用以下新鲜精液与速冻精液的固定比例制备三种处理：100:0（T100）、50:50（T50）和0:100（T0）。然后将样品进行染色技术处理。向150微升稀释精液中加入3微升PI（0.5毫克/毫升）、2微升JC-1（153微摩尔）和50微升FITC-PSA（100微克/毫升）。样品在38.5℃下于黑暗中孵育8分钟。取8微升样品置于载玻片上，盖上盖玻片，立即用落射荧光显微镜进行评估。根据质膜完整性、完整顶体和线粒体功能，将荧光探针组合分为八类细胞。对于通过PI探针检测的质膜完整性，得到方程：（p<0.0001）且R² = 0.97。通过FITC-PSA探针验证的完整顶体，得到方程：（p<0.0001）且R² = 0.98。由JC-1标记的线粒体电位，通过方程估计：（p<0.001）且R² = 0.99。具有完整质膜、完整顶体和高线粒体电位（IPIAH）的精子组，通过方程估计：（p<0.0001）且R² = 0.97。所得线性方程表明，该技术对于同时评估公猪精子中的质膜、顶体膜和线粒体膜是有效且实用的。
