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Background: Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. Objective: We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. Methods: In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. Results: Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. Conclusions: The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt.

Background: Huntington disease (HD) is a fatal neurodegenerative disorder involving reduced muscle coordination, mental and behavioral changes, and testicular degeneration. In order to further clarify the decreased fertility and penetration ability of the spermatozoa of transgenic HD minipig boars (TgHD), we applied a set of mitochondrial metabolism (MM) parameter measurements to this promising biological material, which can be collected noninvasively in longitudinal studies. Objective: We aimed to optimize methods for MM measurements in spermatozoa and to establish possible biomarkers of HD in TgHD spermatozoa expressing the N-terminal part of mutated human huntingtin. Methods: Semen samples from 12 TgHD and wild-type animals, aged 12-65 months, were obtained repeatedly during the study. Respiration was measured by polarography, MM was assessed by the detection of oxidation of radiolabeled substrates (mitochondrial energy-generating system; MEGS), and the content of the oxidative phosphorylation system subunits was detected by Western blot. Three possibly interfering factors were statistically analyzed: the effect of HD, generation and aging. Results: We found 5 MM parameters which were significantly diminished in TgHD spermatozoa and propose 3 specific MEGS incubations and complex I-dependent respiration as potential biomarkers of HD in TgHD spermatozoa. Conclusions: Our results suggest a link between the gain of toxic function of mutated huntingtin in TgHD spermatozoa and the observed MM and/or glycolytic impairment. We determined 4 biomarkers useful for HD phenotyping and experimental therapy monitoring studies in TgHD minipigs.

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).