Neurodegener Dis. 2016;16(3-4):245-59. doi: 10.1159/000443665.
Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars.
Monika Macakovaa,b, Bozena Bohuslavovaa,b, Petra Vochozkovaa,b, Antonin Pavloka, Miroslava Sedlackovac, Daniela Vidinskaa,b, Klara Vochyanovaa,b, Irena Liskovaa,d, Ivona Valekovaa,b, Monika Baxaa,b, Zdenka Ellederovaa, Jiri Klimaa, Stefan Juhasa, Jana Juhasovaa, Jana Klouckovae, Martin Haluzike, Jiri Klempird, Hana Hansikovaf, Jana Spacilovaf, Ryan Collinsg, Ian Blumenthalg, Michael Talkowskig, James F. Gusellag, David S. Howlandh, Marian DiFigliai and Jan Motlika,∗
a) Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and Genetics, Czech Academy of Science, Libechov, Czech Republic
b) Department of Cell Biology, Faculty of Science, Charles University in Prague, Prague, Czech Republic
c) Department of Histology and Embryology, Masaryk University in Brno, Faculty of Medicine, Brno, Czech Republic
d) Department of Neurology and Centre of Clinical Neuroscience, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic
e) 3rd Department of Medicine, Department of Endocrinology and Metabolism, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
f) Laboratory for Study of Mitochondrial Disorders, Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
g) Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA
h) CHDI Foundation, Princeton, NY, USA
i) Department of Neurology, Massachusetts General Hospital, Boston, MA, USA
Background: Huntington’s disease is induced by CAG expansion, in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) causes primarily 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 months 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.
KEYWORDS: Huntington´s Disease, pig model, mutant huntingtin, spermatozoa, testes, degeneration
A large animal model for Huntington’s disease (HD), a minipig transgenic for N terminal part of human huntingtin (548aa) with the mutation of 124 CAG repeats, was generated by us in 2009. At the moment we breed four generations of TgHD minipigs and their WT siblings. Even though HD is linked with neurodegeneration, the first phenotype observed in F1 generation of this model was a male fertility failure at 13 months (Baxa et al. 2013). Testicular pathology was further described in featured manuscript (Macakova et al.2016). Mutant huntingtin causing Huntington’s disease (HD) is expressed in all tissues of the body. Interestingly, the highest levels have been found in the brain and testes where there is a highest similarity in gene expression patterns. Also in R6/2 mice the testicular degeneration was revealed before the neurodegenerative phenotype, at 4 weeks of age (Sathasivam K,et al 1999). Furthermore, mtHtt causes testicular atrophy and male infertility before neurodegeneration also in YAC128 mouse HD model (Rath D et al.1997). Testes from HD patients were analyzed only after post-mortem ( O’Donnell L et al. 2011). This analysis revealed a decreased number of spermatocytes and spermatids, and thicker seminal tubules of testis from the HD patients compared to the healthy controls.
In our TgHD minipig model we detected reduced number of spermatozoa , and also their mal function. In vitro penetration assay revealed inability of TgHD spermatozoa to penetrate oocytes with intact zona pellucida, and lower number of sperms entering zona free oocytes compared to spermatozoa from WT siblings (Figure 1). Moreover, the sperm motility, and progressivity tests indicated a motility defect in TgHD spermatozoa. Electron microscopy (EM) revealed anamorphic nucleus associated with incomplete chromatin condensation and abnormal acrosome, and the absence of residual bodies in TgHD spermatozoa. Proximal cytoplasmic droplets were often linked with disorganized mitochondrial sheaths. The high expression of mtHtt in the tail of TgHD spermatozoa was detected by immunocytochemistry (ICC) and Western blot (WB).
Figure 1. In vitro penetration assay. (A)Unfertilized oocyte with intact zona pellucida after fertilization with TgHD spermatozoa. (B) Fertilized oocyte with intact zona pellucida after fertilization with WT spermatozoa. (C) Lower number of TgHD spermatozoa compared to the WT spermatozoa (D) entering a zona pellucida free oocyte during the in vitro penetration test.
Furthermore, detailed examination of TgHD testes showed degenerative changes, the testes as well as epididydimis was smaller compared to the Wt siblings (Figure 2). High expression and localization of mtHtt was detected by immunohistochemistry (IHC) (Figure 3). Apoptotic spermatogonia and Sertoli cells were revealed.
Figure 2. Atrophic testes of TgHD (A) boar compared to his WT siblings (B) at the age of 36 months. TgHD testes: length 5.7cm; width 4 cm; circuit 10.5cm; Epididymis is smaller, the circuit of sperms is almost invisible WT testes – length 7cm; width 5 cm; circuit 14.8 cm
Figure 3. Localization of mtHtt in the Tg HD bors testis. Immunofluorescence for 3B5H10 (anti N-termnal fragment of 171 aa with 65Q in red) show localization of mtHtt in apical part of Sertoli cells of Tg HD boars (n=2) and cytoplasm of Sertoli cells in case of progresive apoptosis germ cells in seminiferous tubules of TgHD boar (n=1).
EM of 24 and 36 months old testicular tissues revealed degenerative changes. An increased density of cytoplasm of Sertoli cells linked with its vacuolization, swollen mitochondria, stretched endoplasmic reticulum and clumps of heterochromatin in the nucleus were found in TgHD tissue compared to WT. Lamina basalis was thicker and undulated. The diameter of TgHD tubules was reduced, not containing any spermatogenic elements. An immunohistochemical cell proliferation assay of TgHD testicular tissue measured by Ki-67 protein (Ki-67) and proliferating cell nuclear antigen (PCNA) immunoreactivity proved the impaired spermatogenesis in 90% of seminiferous tubules. The significant decrease in proliferation of spermatogonia in human seminiferous epithelium was detected also by monoclonal antibodies against Ki-67 and PCNA in testis biopsies of men with spermatogenic defects (Stegler et al, 1998).
In addition, we performed 31P magnetic resonance spectroscopy, a non-invasive method allowing an evaluation of in vivo metabolism at the molecular level, of testes from 24 months old TgHD and WT boars ( Jozefovicova M et al. 2015). The analysis showed a significant reduction of relative phosphodiester (PDE) concentration, PDE/ γ‑ ATP ratio, in testicular parenchyma of TgHD boars compared to WT siblings (Figure 4).We assume that the significant reductionof relative PDE concentration may be related to sperm motility or reduced concentration of seminal fluid. This hypothesis agrees with the observed testicular pathology in featured manuscript.
Figure 4. Distribution of the ratios PDE/γ-ATP, PME/γ-ATP and Pi/γ-ATP in the group of TgHD minipigs (°) and control wild type (nontransgenic) siblings (•).
Horizontal lines show average values and standard deviations. Asterix marks signifi cance level p < 0.05. Reprinted with permission of Ambit media adopted from CeskSlovNeurol N2015; 78/111 (Suppl 2)
The therapeutic potential of hormone therapy in the HD R6 ⁄ 1 mouse model (HD mice) was tested by testosterone treatment that significantly raised serum testosterone in both wild-type and HD mice. However, this treatment had a limited effect on motoric deficit and no effect on cognition and neurogenesis. In contrast, it significantly improved testicular atrophy and spermatogenesis (Hannan and Ransome, 2011). Our EM and hormonal studies unequivocally confirmed that the testicular interstitium and Leydig cells function in TgHD boars is comparable with WT controls. It means that testicular phenotype in the tubuli seminiferi and the ejaculated spermatozoa represents the gain of toxic effect of mHtt and its proteolytic fragments.
The noninvasive approach to boar ejaculates offer a longitudinal follow up of pharmacological treatment of HD on the biomedical model.
This testicular phenotype can be easily monitored in the TgHD minipigs and therefore represents a biomarker that can be suitable for therapeutics.
The research leading to these results has received funding from the CHDI foundation (A-5378), the National Sustainability Programme, project no. LO1609 (Czech Ministry of Education, Youth and Sports), the Norwegian Financial Mechanism 2009-2014 and the Ministry of Education, Youth and Sports under the Project Contract no. MSMT-28477/2014 and the Charles University in Prague, project GA UK no. 378215.
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