CryomiRs调控大熊猫精子冷冻保存效果的作用机制研究

Spermatozoa cryopreservation plays an important role in the long-term storage of giant panda genetic resources and artificial breeding. However, the unidentified action and cryoinjury mechanism result in low birth rate and poor utilization efficiency using artificial insemination with frozen-thawed semen of giant panda. microRNA (miRNA) can regulate spermatogenesis, maturation process of spermatozoa, and also effect the early embryonic development after fertilization. CryomiRs, a cold-associated family of miRNAs, are mainly involved in cold or freezing tolerance in some insects and hibernating mammals under extreme environmental stresses through the depression of metabolic rate and other physiological and biochemical processes. Our previous study indicated that cryopreservation can induce miRNA expression changes and probably modulate metabolic-related physiological and biochemical processes of spermatozoa. It was hypothesized that miRNAs and their target genes may participate in the regulation of important physiological and biochemical process of spermatozoa, and then affect post-thawed semen quality and its fertilization effects of giant panda. To identify this hypothesis, this project aims to detect the cryomiRs expression and the signal pathways of their target genes using high-throughput miRNA sequencing, flow cytometry and RNA interference techniques, and to explore the mechanism of miRNAs modulating the cryopreservation effects of giant panda frozen semen, and try to explain, from a new point of view, the reason of low efficiency of artificial insemination with frozen-thawed semen and provide a new idea for the optimization and improvement of the freezing extenders of giant panda spermatozoa.

精子冷冻保存在大熊猫种质资源保护和人工繁育中具有重要的作用。但是大熊猫冷冻精液人工授精的产仔率低下，导致冷冻精液利用效率极差，原因在于冷冻对大熊猫精子的损伤及作用机制尚不明确。miRNA在精子发生和成熟过程中具有重要的调控作用，并能影响受精后早期胚胎的发育。CryomiRs是耐寒性昆虫和冬眠动物中一类与抗冷/抗冻相关的miRNAs。我们前期研究发现，冷冻能够诱导精子miRNA表达的变化，推测miRNA及靶基因可能参与调控精子重要的生理生化过程，影响精子的冻后质量和受精效果。因此，本项目拟采用miRNA高通量测序、流式细胞分析和miRNA干扰等技术，探讨冷冻过程中大熊猫精子CryomiRs表达规律及其靶基因作用的信号途径，揭示miRNA调控大熊猫精子冷冻保存效果的作用机制，从新的角度阐明大熊猫冷冻精液人工授精效率低下的原因，为优化和改善大熊猫精子冷冻稀释液提供新的思路。

人工授精技术在大熊猫的保护与繁育工作中扮演着至关重要的角色。但绝大多数大熊猫人工授精采用新鲜精液进行，采用大熊猫冷冻精液人工授精也取得了一定的成功，但是产仔率低下、解冻后精子活力也较低，导致大熊猫冷冻精液人工授精推广和使用受到极大限制。因此，揭示大熊猫精子在冷冻保存过程中的冷冻损伤及其作用机制，为进一步改善和优化大熊猫精液冷冻稀释液、提高大熊猫繁殖率和人工授精产仔率等具有重要的科学和实践价值。同时，对家养动物精子冷冻保存相关研究也具有重要的参考意义。本研究主要通过检测大熊猫精子在冷冻保存过程中与冷冻损伤和抗冻性按的转录组mRNA和非编码RNA，如miRNA、lncRNA和piRNA的表达变化，揭示CryomiRs与精子冷冻损伤、抗冻性、活力和受精力相关的分子信号途径。主要结果如下：.（1）冷冻前后大熊猫精子中转录组mRNA、非编码的miRNA、lncRNA和piRNA均发生了显著的差异表达；.（2）进一步分析发现，与精子功能相关的信号通路如嗅觉传导、Ca2+信号途径与精子抗冻性相关；与精子凋亡相关的P53、PI3K-AKT等信号通路中相关miRNAs及其靶基因以及与精子代谢相关的miRNA及其靶基因的差异表达，从而影响精子的活力、受精能力及抗冻性等功能。.（3）发现一个与精子糖代谢相关的miR-26a及其靶基因PDHX。进一步在猪精子中的验证表明，miR-26a介导PDHX调控丙酮酸向乙酰辅酶A的转化，揭示其调控精子的存活和活力的分子机制。.（4）比较分析揭示了与精子抗冻性密切相关的嗅觉受体-嗅觉传导-CNG离子通道及其调控途径，相关后续研究正在进行之中。. 综上所述，通过对大熊猫精子冷冻前后差异表达的CryomiRs及其靶基因的分析和验证，对揭示冷冻保存过程中精子的冷冻损伤和抗冻机制，改善和优化精子冷冻稀释液，提高大熊猫和猪冷冻精液人工授精推广与应用等具有重要的科学和实践价值。