C18 The hunt for biomarkers in presymptomatic large animal model: mitochondrial metabolism in spermatozoa of transgenic minipigs modelling huntington’s disease

Background Large-animal minipig transgenic model of Huntington’s disease (HD), carrying N-terminal part of human mutated huntingtin, was developed in Libechov and is expected to resemble to clinical picture of HD patients. Phenotyping of this model started immediately in presymptomatic stage. Despite of ongoing discussion, whether the glycolytic or mitochondrial metabolism (MM) is crucial for sperm ATP production; we decided to use spermatozoa as a promising biological material to assess MM parameters in HD. Aims The aims of this study were: to optimise methods for MM measurement in minipig spermatozoa; to assess its MM and to establish possible biomarkers of HD. Methods Semen samples were obtained repeatedly (12 boars, two generations, aged 12–65 months). Mitochondrial energy generating system (MEGS) capacity was measured by radiolabeled substrates oxidation, respiration by polarography and citrate synthase (CS) by spectrophotometry. Results Three possibly interfering factors were statistically analysed: the effects of HD; generation and ageing. Eight analysed parameters were incident to distinct generation. These parameters were influenced also by the effect of ageing. MEGS incubation with [1–14C] pyruvate+carnitine+ADP was affected significantly by all analysed effects. Four MM parameters were significantly diminished in HD spermatozoa: [U-14C]malate+pyruvate+malonate+ADP (I2) and [1–14C] pyruvate+ADP incubations, I2/CS ratio and complex I-dependent respiration might be considered as biomarkers of HD. Conclusions Used methods are suitable for MM analysis in minipig spermatozoa. Interestingly, spermatozoa can preferentially utilise pyruvate by sperm-specific isoform of lactate dehydrogenase. Our results in pyruvate-containing MEGS-incubations might indicate both MM and glycolytic defect in porcine HD spermatozoa, but further analyses are needed. Support The Project Contract No. MSMT-28477/2014 “HUNTINGTON” 7F14308 and NPU 1609 (MSMT).