犏牛与其亲本牛精原细胞分化为初级精母细胞差异表达miRNAs功能研究

Some studies indicated that the differentiational obstruction from spermatogonia to primary spermatocytes is the key step of spermatogenic arrest of cattleyak, during which post-transcriptional depression ocurred on the expression of a large number of genes. As the important post-transcriptional regulatory factor, miRNA plays extremely important roles in maintaining defferentiaional potential of spermatogenic cells, inducing cellular differentiation and maintaining cellular superficial characteristics. Therefore, the aims of this project are as followings: .(1) to build miRNA libraries of type A spermatogonia and pachytene spermatocytes for cattleyak, the patrilineal cattle and the matrilineal yak using the new generation of high-throughput sequencing technology and screen significantly expressed miRNAs during the differentiation from spermatogonia to primary spermatocytes between cattleyak and its parental species; (2) to analyze the expression patterns of significantly expressed miRNAs in testicular tissues and spermatogenic cells of different Bovidae species; (3) to explore the biological regulation mechanisms of key miRNAs correlated with cattleyak spermatogenesis on predicted target genes during the proliferation and differentiation of type A spermatogonia of cattleyak by performing miRNA over-expression, interference strategies and cellular function ‘saving’ experiments; (4) to clarify the correlations of key miRSNPs with the male infertility of cattle-yak and the effects of key miRSNPs on differentiation of type A spermatogonia of cattleyak. All these are expected to provide new theoretical evidences for clarifying the mechanisms of cattleyak spermatogenic arrest and exhibiting new means to overcome the male infertility of cattle-yak.

精原细胞到初级精母细胞分化的障碍是犏牛精子发生阻滞的关键环节且有大量基因表达在此环节受到转录后抑制，而miRNA作为转录后水平重要调控因子，对动物生精细胞分化潜能和细胞表征的维持以及细胞分化的诱导等均起极其重要作用。本项目利用新一代高通量测序技术构建犏牛与其亲本牛（普通牛和牦牛）A型精原细胞和粗线期初级精母细胞miRNA文库并筛选犏牛与其亲本牛精原细胞到初级精母细胞分化过程差异表达miRNA，然后分析其在不同牛种生精细胞表达变化规律。通过双荧光报告试验揭示与犏牛精子发生相关关键miRNA对其靶基因的调控作用，采用miRNA的过表达、抑制表达和细胞的功能“拯救”等技术探究关键miRNA调控犏牛未分化精原细胞增殖分化的作用机制，阐明关键miRSNPs与犏牛雄性不育的关联性以及对犏牛精原细胞增殖分化的影响，为犏牛精子发生阻滞机制的解析提供新理论依据并为雄性不育的克服展现新思路和方法。

犏牛是雄性普通牛和雌性牦牛的种间F1 代杂种，具有优异的杂种优势，但雄性犏牛因精子发生阻滞而表现不育，使其优秀基因型组合无法遗传给下一代，成为牦牛杂交育种的最大障碍。因此，探明犏牛精子发生阻滞机制，从根本上解决其雄性不育问题具有重要意义；精原细胞到初级精母细胞分化的障碍是犏牛精子发生阻滞的关键环节，而miRNA作为转录后水平重要调控因子，对生精细胞分化潜能和细胞表征的维持以及细胞分化的诱导等均起极其重要作用。本项目对犏牛及其亲本牛睾丸组织学和全转录组差异表达基因和miRNA、犏牛及其亲本牛精原细胞和初级精母细胞的特征以及精原细胞分化为初级精母细胞差异表达关键miRNA及其功能进行了深入研究。通过犏牛与牦牛转录组差异表达基因分析发现犏牛精子发生阻滞可能始于精原干细胞阶段，而后在精原细胞分化和精母细胞减数分裂期间阻滞程度加重，最终导致犏牛睾丸中出现数量极其稀少且无受精能力的精子；犏牛与牦牛相比其睾丸组织部分下调表达miRNA可能与精原干细胞自我更新的失败、精原细胞分化的抑制以及有丝分裂向减数分裂转换的失败有关，由此进一步证实犏牛精子发生阻滞时期可能始于精原干细胞阶段。在精原细胞分化为初级精母细胞过程中犏牛中下调表达的部分miRNAs可能与犏牛精原干细胞的维持和精原细胞的分化、大量生精细胞的凋亡以及曲细精管中初级精母细胞数量的骤减有关；关键bta-miR-34c靶向CDK4和CDC25A基因通过促进CELL CYCLE和p53 SIGALING通路中细胞周期蛋白/周期蛋白依赖性激酶复合体，即CycA/CDK2和CyclinD/CDK4的表达，从而导致精原细胞分化中细胞周期G1期的阻滞以及S期向G2期的转换的抑制，因此关键bta-miR-34c及其靶基因在犏牛精子发生阻滞中的发挥重要作用，为犏牛精子发生阻滞提供了新的依据，将为犏牛雄性不育的克服提供了新的思路。