Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Haidian District, Beijing, People's Republic of China.
CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Chaoyang District, Beijing, People's Republic of China.
Mol Hum Reprod. 2019 Sep 1;25(9):507-518. doi: 10.1093/molehr/gaz043.
Spermatogonial stem cells (SSCs) self-renew and contribute genetic information to the next generation. Pig is wildly used as a model animal for understanding reproduction mechanisms of human being. Inducing directional differentiation of porcine SSCs may be an important strategy in exploring the mechanisms of spermatogenesis and developing better treatment methods for male infertility. Here, we established an in-vitro culture model for porcine small seminiferous tubule segments, to induce SSCs to differentiate into single-tail haploid spermatozoa. The culture model subsequently enabled spermatozoa to express the sperm-specific protein acrosin and oocytes to develop to blastocyst stage after round spermatid injection. The addition of retinoic acid (RA) to the differentiation media promoted the efficiency of haploid differentiation. RT-PCR analysis indicated that RA stimulated the expression of Stra8 but reduced the expression of NANOS2 in spermatogonia. Genes involved in post-meiotic development, transition protein 1 (Tnp1) and protamine 1 (Prm1) were upregulated in the presence of RA. The addition of an RA receptor (RAR) inhibitor, BMS439, showed that RA enhanced the expression of cAMP responsive-element binding protein through RAR and promoted the formation of round spermatids. We established an efficient culture system for in-vitro differentiation of pig SSCs. Our study represents a model for human testis disease and toxicology screening. Molecular regulators of SSC differentiation revealed in this study might provide a therapeutic strategy for male infertility.
精原干细胞 (SSC) 自我更新，并将遗传信息传递给下一代。猪被广泛用作研究人类生殖机制的模式动物。诱导猪 SSC 的定向分化可能是探索精子发生机制和开发更好的男性不育治疗方法的重要策略。在这里，我们建立了一个体外培养猪小精直小管段的模型，以诱导 SSC 分化为单尾单倍体精子。该培养模型随后使精子表达精子特异性蛋白顶体酶，并在圆形精子注射后使卵母细胞发育到囊胚阶段。在分化培养基中添加视黄酸 (RA) 可提高单倍体分化效率。RT-PCR 分析表明，RA 刺激 Stra8 的表达，但降低精原细胞中 NANOS2 的表达。在存在 RA 的情况下，涉及减数后发育的基因，过渡蛋白 1 (Tnp1) 和精蛋白 1 (Prm1) 的表达上调。添加视黄酸受体 (RAR) 抑制剂 BMS439 表明，RA 通过 RAR 增强 cAMP 反应元件结合蛋白的表达，并促进圆形精子的形成。我们建立了一个有效的猪 SSC 体外分化培养系统。我们的研究代表了人类睾丸疾病和毒理学筛选的模型。本研究中揭示的 SSC 分化的分子调节剂可能为男性不育提供一种治疗策略。
