Macakova Monika, Bohuslavova Bozena, Vochozkova Petra, Pavlok Antonin, Sedlackova Miroslava, Vidinska Daniela, Vochyanova Klara, Liskova Irena, Valekova Ivona, Baxa Monika, Ellederova Zdenka, Klima Jiri, Juhas Stefan, Juhasova Jana, Klouckova Jana, Haluzik Martin, Klempir Jiri, Hansikova Hana, Spacilova Jana, Collins Ryan, Blumenthal Ian, Talkowski Michael, Gusella James F, Howland David S, DiFiglia Marian, Motlik Jan
Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and Genetics, Czech Academy of Science, Libechov, Czech Republic.
Neurodegener Dis. 2016;16(3-4):245-59. doi: 10.1159/000443665. Epub 2016 Mar 10.
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.
We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt.
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.
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.
The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt.
亨廷顿舞蹈症由编码亨廷顿蛋白的单基因中的CAG重复序列扩增所致。突变的亨廷顿蛋白（mtHtt）主要导致脑内神经元变性，但也会影响包括睾丸在内的外周组织。
我们研究了表达人mtHtt N端区域的转基因公猪的精子和睾丸。
本研究比较了F1代（24 - 48月龄）和F2代（12 - 36月龄）转基因（TgHD）和野生型（WT）公猪的生殖参数测量结果以及精子和睾丸的电子显微镜（EM）图像。此外，进行了免疫荧光、免疫组织化学、蛋白质免疫印迹、激素分析和全基因组测序，以阐明mtHtt的作用。
在13月龄时观察到两代TgHD公猪均存在生育力下降的证据。生殖参数随年龄下降并逐渐恶化。电子显微镜显示，24月龄和36月龄的TgHD公猪的精子尾部和睾丸上皮存在许多病理特征。此外，免疫组织化学证实转基因睾丸中生精细胞的增殖活性显著降低。mtHtt在TgHD公猪的精子和睾丸中高表达，并定位于生精小管的所有细胞中。TgHD和WT同胞的生育相关激素水平没有差异。基因组分析证实慢病毒构建体的插入未中断猪基因组中的任何编码序列。
TgHD公猪的精子和睾丸变性是由高表达的mtHtt的功能获得所致。
