College of Veterinary Medicine, Jilin University, Changchun, China.
First Bethune Hospital, Jilin University, Changchun, China.
Andrologia. 2021 Jul;53(6):e14056. doi: 10.1111/and.14056. Epub 2021 Mar 24.
Germplasm cryopreservation and expansion of gonocytes/prospermatogonia or spermatogonial stem cells (SSCs) are important; however, it's difficult in cattle. Since inhibitors of Mek1/2 and Gsk3β (2i) can enhance pluripotency maintenance, effects of 2i-based medium on the cultivation of bovine prospermatogonia from the cryopreserved tissues were examined. The testicular tissues of newborn bulls were well cryopreserved. High mRNA levels of prospermatogonium/SSC markers (PLZF, GFRα-1) and pluripotency markers (Oct4/Pouf5, Sox2, Nanog) were detected and the PLZF /GFRα-1 prospermatogonia were consistently identified immunohistochemically in the seminiferous cords. Using differential plating and Percoll-based centrifugation, 41.59% prospermatogonia were enriched and they proliferated robustly in 2i medium. The 2i medium boosted mRNA abundances of Pouf5, Sox2, Nanog, GFRα-1, PLZF, anti-apoptosis gene Bcl2, LIF receptor gene LIFR and enhanced PLZF protein expression, but suppressed mRNA expressions of spermatogonial differentiation marker c-kit and pro-apoptotic gene Bax, in the cultured prospermatogonia. It also alleviated H O -induced apoptosis of the enriched cells and decreased histone H3 lysine (K9) trimethylation (H3K9me3) and its methylase Suv39h1/2 mRNA level in the cultured seminiferous cords. Overall, 2i medium improves the cultivation of bovine prospermatogonia isolated from the cryopreserved testes, by inhibiting Suv39h1/2-mediated H3K9me3 through Mek1/2 and Gsk3β signalling, evidencing successful cryopreservation and expansion of bovine germplasm.
种质的冷冻保存和生殖细胞/精原干细胞（SSC）的扩增很重要；然而，在牛中却很困难。由于 Mek1/2 和 Gsk3β 的抑制剂（2i）可以增强多能性的维持，因此研究了基于 2i 的培养基对冷冻保存组织中牛精原前体细胞的培养效果。新生公牛的睾丸组织得到了很好的冷冻保存。检测到精原细胞/SSC 标志物（PLZF、GFRα-1）和多能性标志物（Oct4/Pouf5、Sox2、Nanog）的高 mRNA 水平，在曲细精管中始终通过免疫组织化学鉴定 PLZF/GFRα-1 精原前体细胞。通过差异 plating 和基于 Percoll 的离心，富集了 41.59%的精原前体细胞，它们在 2i 培养基中大量增殖。2i 培养基提高了 Pouf5、Sox2、Nanog、GFRα-1、PLZF、抗凋亡基因 Bcl2、LIF 受体基因 LIFR 的 mRNA 丰度，并增强了 PLZF 蛋白的表达，但抑制了培养精原前体细胞中精原细胞分化标志物 c-kit 和促凋亡基因 Bax 的 mRNA 表达。它还减轻了富集细胞中 H2O2 诱导的凋亡，并降低了培养曲细精管中组蛋白 H3 赖氨酸（K9）三甲基化（H3K9me3）及其甲基转移酶 Suv39h1/2 的 mRNA 水平。总之，2i 培养基通过 Mek1/2 和 Gsk3β 信号通路抑制 Suv39h1/2 介导的 H3K9me3，改善了从冷冻保存的睾丸中分离出的牛精原前体细胞的培养，证明了牛种质的成功冷冻保存和扩增。
