کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2096523 1082170 2007 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium
موضوعات مرتبط
علوم زیستی و بیوفناوری علوم کشاورزی و بیولوژیک علوم دامی و جانورشناسی
پیش نمایش صفحه اول مقاله
Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium
چکیده انگلیسی

Low-level production of the superoxide anion (O2−) is an important signal transduction event in sperm function including capacitation; however, excessive production of O2− can be detrimental to sperm function. The objective of this study was to assess dihydroethidium (DHE) as a probe for O2− in equine spermatozoa. Ejaculated spermatozoa were separated by centrifugation over a Percoll gradient (40:80), and loaded with DHE (2.0 μM) as well as with calcein-acetoxymethylester (CAM, 7.8 nM) to determine cell viability. In Experiment 1, cells were incubated with the xanthine–xanthine oxidase (X, 0.1 mM; XO, 0.01 U/mL) generating system for the production of O2−, with or without the addition of superoxide dismutase (SOD, 150 U/mL) or the SOD mimetic, Tiron (0.1, 1.0 or 5.0 mM) for 1 h. Changes in fluorescence of DHE were determined for the live cell population (calcein-positive cells) by flow cytometry. The DHE fluorescence increased with the X–XO incubation; this increase was inhibited by SOD or Tiron, indicating that DHE is specific for O2− detection. In Experiment 2, spermatozoa were loaded with DHE/CAM, treated with calcium ionophore A23187 (0, 0.8, or 8.0 μM), and incubated for 15 min. Cell fluorescence was again determined by flow cytometry. Calcium ionophore A23187 increased O2− production in a dose-dependent manner. In Experiment 3, cells were loaded with DHE/CAM, treated with NADPH (0.0, 0.25, 0.5, or 1 mM) with or without 0.5% Triton X-100, and incubated for 15 min prior to flow cytometry. Cells treated with NADPH with or without 0.5% Triton X-100 did not have O2− levels that were significantly different from the control. In Experiment 4, spermatozoa loaded with DHE/CAM were incubated under capacitating conditions (1.2 mM dibutryl-cAMP + 1.0 mM caffeine) or in control media for 3 h. Although O2− generation increased over time in control and capacitated treatments, spermatozoa incubated under capacitating conditions had higher O2− production than those incubated in control media. Therefore, DHE was a useful probe for the detection of O2− in equine spermatozoa and elevation in intracellular calcium as well as capacitation in vitro were associated with increased generation of O2−.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Theriogenology - Volume 67, Issue 3, February 2007, Pages 580–589
نویسندگان
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