低压低氧对小鼠精子发生及其小RNA表达的影响

doi:10.16829/j.slxb.150649

兽类学报 ›› 2022, Vol. 42 ›› Issue (5): 579-589.DOI: 10.16829/j.slxb.150649

低压低氧对小鼠精子发生及其小RNA表达的影响

李双¹^,²^,³, 贾功雪¹^,²^,³, 陶海萍¹^,²^,³, 王玉军¹^,²^,³, 李斌业⁴, 杨其恩¹^,²^,³()

^1.中国科学院西北高原生物研究所，中国科学院高原生物适应与进化重点实验室，西宁 810001
^2.中国科学院大学，北京 100049
^3.中国科学院西北高原生物研究所，青海省动物生态基因组学重点实验室，西宁 810001
^4.青海省人民医院，生殖医学中心，西宁 810007

收稿日期:2021-12-22 接受日期:2022-07-05 出版日期:2022-09-30 发布日期:2022-09-21
通讯作者: 杨其恩
作者简介:李双 (1990- )，女，博士研究生，主要从事动物生殖与发育研究.
基金资助:
国家自然科学基金(81960287);中国科学院青年创新促进会(2021432);青海省重大专项“三江源区代表性动物基因资源保护与应用”

Effects of hypobaric hypoxia on spermatogenesis and the expression of small RNA in mice

Shuang LI¹^,²^,³, Gongxue JIA¹^,²^,³, Haiping TAO¹^,²^,³, Yujun WANG¹^,²^,³, Binye LI⁴, Qien YANG¹^,²^,³()

^1.Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China
^2.University of Chinese Academy of Sciences, Beijing 100049, China
^3.Qinghai Key Laboratory of Animal Ecological Genomics, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China
^4.Center for Reproductive Medicine, Qinghai People’s Hospital, Xining 810007, China

Received:2021-12-22 Accepted:2022-07-05 Online:2022-09-30 Published:2022-09-21
Contact: Qien YANG

摘要/Abstract

摘要：

作为青藏高原最为关键的环境因子，低压低氧对高原非习服动物繁殖和生殖系统功能有不利影响。已有的研究表明，低氧环境会导致雄性生殖细胞凋亡、精子畸形率升高、精子质量下降，进而影响受精和早期胚胎发育，但目前缺乏低氧损伤精子功能的机理研究。小RNA (small RNA) 是在转录后及翻译水平上调控基因表达的重要功能分子，不同类型的small RNA通过诱导基因沉默或调控翻译等方式参与调节精子发生。本研究通过低压氧舱模拟海拔5 000 m处理4周建立缺氧小鼠模型，发现低氧处理导致小鼠曲精细管中生精细胞排列紊乱，精子数量未发生显著变化但畸形率增加17.5倍 (P < 0.001)。通过small RNA测序发现，低氧组小鼠精子中的small RNA碱基偏好性与对照组一致，第一碱基对尿嘧啶 (U) 有很强的偏好性。低氧组小鼠精子中21 nt长度的small RNA比例显著减少4.4% (P < 0.05)。低氧组小鼠精子中piRNA、tsRNA表达无差异，但miRNA表达上调21个，下调58个。对差异miRNAs靶基因与相同低氧处理小鼠睾丸组织差异基因比对，共比对到831个差异表达基因，其中上调miRNAs的靶基因中有429个差异表达基因；下调miRNAs的靶基因中有813个差异表达基因。此外，上调miRNAs的靶基因富集在FoxO信号通路、甲状腺激素信号通路、类固醇生物合成和HIF?1信号等通路，而下调miRNAs的靶基因富集在脂肪酸代谢等通路。本研究获得了缺氧小鼠精子small RNA变化图谱，对人类和其他动物在缺氧环境下精子表观遗传修饰变化的研究具有参考价值。

关键词: 低压低氧, 精子, 小RNA, 靶基因

Abstract:

As the most crucial environmental factors, low atmospheric pressure and low oxygen content have adverse effects on the reproductive system of non-adapted animals living in the Qinghai?Tibet Plateau. Compelling evidence has shown that hypoxia exposure caused increased germ cell apoptosis and lower sperm quality, leading to defects in fertilization and preimplantation embryo development. Currently, how hypoxia affects testicular gene expression and sperm function is not well-understood. small RNA regulates gene expression at the post-transcriptional and translational level and participates in spermatogenesis by inducing gene silencing or transcription. In this study, a hypoxia mouse model was established by simulating the environment at an altitude of 5 000 m in a hypobaric oxygen chamber for 4 weeks. The results showed that the germ cells within seminiferous tubules of hypoxic mice were arranged disorderly. Although the total number of sperm did not change significantly, the proportion of abnormal sperm had a 17.5 fold increase in Hypo-4W animals (P < 0.001). small RNA sequencing revealed that the number of small RNAs with a length of 21 nt in hypoxic mouse sperm was reduced by 4.4% (P < 0.05), and the first base had a strong preference for Uracil (U). There were no significant differences in piRNA and tsRNA in sperm of control and hypoxia exposed animals, however, 21 miRNAs were up-regulated and 58 were down-regulated. Furthermore, we analyzed the target genes of these miRNAs and differentially expressed genes in the hypoxia-treated testicular tissues of mice. The results showed that 429 target genes of up-regulated miRNAs and 813 target genes of down-regulated miRNAs were differentially expressed. Finally, enrichment analysis revealed that the target genes of up-regulated miRNAs were enriched in FoxO signaling pathway, thyroid hormone signaling pathway, steroid biosynthesis and HIF-1 signaling pathway, while the targets of down-regulated miRNAs were enriched in fatty acid metabolism. In summary, the miRNA expression dynamics of hypoxic mouse sperm obtained in the present study provides an important reference value for further dissecting changes in sperm epigenome in humans and other animals under hypoxic environment.

Key words: Hypobaric hypoxia, Sperm, small RNA, Target genes

中图分类号:

Q522

李双, 贾功雪, 陶海萍, 王玉军, 李斌业, 杨其恩. 低压低氧对小鼠精子发生及其小RNA表达的影响[J]. 兽类学报, 2022, 42(5): 579-589.

Shuang LI, Gongxue JIA, Haiping TAO, Yujun WANG, Binye LI, Qien YANG. Effects of hypobaric hypoxia on spermatogenesis and the expression of small RNA in mice[J]. ACTA THERIOLOGICA SINICA, 2022, 42(5): 579-589.

图/表 5

图1 低氧组小鼠精子形态异常.a：实验设计示意图；b：对照组与低氧组小鼠睾丸组织苏木素―伊红染色，星号表示生殖细胞紊乱曲精细管，箭头指向核浓缩的生殖细胞，标尺：100 μm；c：对照组与低氧组小鼠精子苏木素―伊红染色代表图，标尺：10 μm；d：对照组与低氧组小鼠精子总数及畸形精子比例统计，每个点代表一个生物重复.数据以平均值 ± 标准误统计. *** P < 0.001

Fig. 1 Abnormal morphology of sperm in Hypo-4W animals. a: Schematic diagram of experimental design. b: HE staining of testicular tissues of Control and Hypo-4W animals, the asterisks indicate germ cell disorder convoluted tubules, the arrows point to germ cells with concentrated nucleus, Scale bars = 100 μm. c: Representative images of HE staining of sperm of mice in Control and Hypo-4W animals, Scale bars = 10 μm. d: Total number of sperms and proportion of abnormal sperms of mice in Control and Hypo-4W animals, each dot represents a biological repeat. Data were presented as the mean ± SE. *** P < 0.001

图2 低氧组小鼠精子small RNA 测序分析. a：small RNA序列长度分布统计，* P < 0.05；b：miRNAs及其他不同种类的RNA分子所占比例统计；c：小鼠精子miRNAs前6个碱基序列的碱基偏好性；d：小鼠精子不同片段长度miRNAs第1个碱基序列的碱基偏好

Fig. 2 small RNA sequencing analysis of sperm for Hypo-4W animals. a: Statistics of small RNA sequence length distribution, * P<0.05; b: Statistics on the proportion of miRNAs and another different type of small RNA molecules; c: Base bias of the first 6 base sequences of miRNAs in mouse sperm; d: Base bias of the 1st base sequence of miRNAs of different fragment lengths in mouse sperm

图3 低氧组小鼠精子miRNA差异表达. a：低氧组小鼠精子miRNA表达量的样本相关性热图；b：低氧组小鼠精子miRNA差异表达数量火山图 (P < 0.05, |log2(Fold Change)| > 1)

Fig. 3 Differential expression of miRNA in mouse sperm for Hypo-4W animals. a: Sample correlation heat map of expression quantity; b: Volcano plot of miRNA differential expression in sperm of mice for Hypo-4W animals (P < 0.05, |log2(Fold Change)| > 1)

图4 低氧组小鼠精子差异miRNA靶基因分析. a：下调miRNAs的靶基因比对到同样低氧处理的小鼠睾丸组织差异基因火山图；b：上调miRNAs的靶基因比对到同样低氧处理的小鼠睾丸组织差异基因火山图

Fig. 4 Analysis of differential miRNA target genes in mice sperm of Hypo-4W animals. a: The volcanic map of target genes of down-regulated miRNAs were compared to the differential genes in testicular tissues of mice with the same treatment; b: The volcanic map of target genes of up-regulated miRNAs were compared to the differential genes in testicular tissues of mice with the same treatment

图5 低氧组小鼠精子差异miRNA靶基因富集分析. a：下调miRNAs的靶基因KEGG气泡图；b：下调miRNAs的靶基因GO生物过程分析柱状图；c：上调miRNAs的靶基因KEGG气泡图；d：上调miRNAs的靶基因GO生物过程分析柱状图

Fig. 5 Enrichment analysis of differential miRNA target genes in mice sperm of Hypo-4W animals. a: KEGG bubble map of down-regulated miRNAs target genes; b: Histogram of GO biological process analysis of target genes of down-regulated miRNAs; c: KEGG bubble map of up-regulated miRNAs target genes; d: Histogram of GO bioprocess analysis of target genes of up-regulated miRNAs

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低压低氧对小鼠精子发生及其小RNA表达的影响

Effects of hypobaric hypoxia on spermatogenesis and the expression of small RNA in mice

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