Breininger E, Dubois D, Pereyra V E, Rodriguez P C, Satorre M M, Cetica P D
Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Universidad de Buenos Aires, Buenos Aires, Argentina.
Investigaciones en Producción Animal (INPA), CONICET - Universidad de Buenos Aires, Buenos Aires, Argentina.
Reprod Domest Anim. 2017 Oct;52(5):731-740. doi: 10.1111/rda.12973. Epub 2017 Apr 10.
The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential-interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/10 spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/10 and 2.22 ± 0.14 U/10 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/10 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.
这项工作的目的是测定公猪精子中磷酸果糖激酶（PFK）、苹果酸脱氢酶（MDH）和异柠檬酸脱氢酶（IDH）的酶活性，并研究它们在碳酸氢盐诱导的获能和卵泡液诱导的顶体反应中的作用。通过分光光度法测定公猪精子提取物中这些酶的酶活性。精子悬液在碳酸氢盐（40 mM，一种众所周知的获能诱导剂）或卵泡液（30%，作为顶体反应诱导剂）以及分别为PFK、IDH和MDH抑制剂的不同浓度的草酰戊二酸、草酰苹果酸和羟基苹果酸存在下孵育。通过金霉素（CTC）荧光技术测定获能百分比，通过台盼蓝和微分干涉对比光学显微镜测定真正的顶体反应。公猪精子酶提取物中PFK的活性为1.70±0.19 U/10个精子，NAD和NADP依赖性IDH的活性分别为0.111±0.005 U/10和2.22±0.14 U/10个精子，MDH的活性为4.24±0.38 U/10个精子。添加这些酶的特异性抑制剂可阻止精子获能，并在获能过程中降低精子活力，抑制顶体反应（AR），而在此过程中不影响精子活力。我们的结果证明了PFK、IDH和MDH参与公猪精子中碳酸氢盐诱导的获能和卵泡液诱导的顶体反应，有助于阐明在猪精子中产生这些过程所需能量的机制。
