体细胞核移植（Somatic cell nuclear transfer，SCNT）技术的实现离不开健康成熟的卵母细胞以及合适的核供体细胞。哺乳卵母细胞的成熟需要经历四个重要的时期，依次是生发泡（Germinal vesicle，GV）时期、生发泡破裂（Germinal vesicle breakdown，GVBD）时期、第一次减数分裂中期（Metaphase Ⅰ，MI）、第二次减数分裂中期（Metaphase ⅠI，MII），成熟的卵母细胞停滞于MII期，在显微镜下可清晰看到第一极体。期间大量DNA、RNA、蛋白质及其他物质的合成储备，对后续的受精过程、胚胎的早期发育至关重要。供体细胞核的DNA甲基化状态会影响SCNT胚胎的重编程，影响克隆效率。目前，在山羊上关于表观遗传修饰参与调控卵母细胞成熟以及体细胞核移植的研究较少，因此，本研究以山羊GV期、MII期卵母细胞以及核移植供体细胞为材料，探究组蛋白甲基化相关基因及其相关LncRNA对卵母细胞成熟过程以及体细胞核移植的影响。本研究主要分为以下四个部分： 试验一：山羊GV期和MII期卵母细胞差异LncRNA和mRNA表达分析 本试验旨在研究山羊卵母细胞成熟过程中LncRNA和mRNA的表达模式，通过使用单细胞测序技术对GV期和MII期的山羊卵母细胞进行转录组分析，共鉴定出27,033个新LncRNA，并筛选出4,516个差异mRNA和3,019个差异LncRNA，涉及RNA剪接、线粒体翻译、氧化磷酸化、胞质多腺苷酸化以及组蛋白甲基化等多个生物学过程，说明山羊卵母细胞的成熟受到诸多因素的共同调节。本试验对进一步研究LncRNA和mRNA在山羊卵母细胞成熟过程中的调控机理具有重要的参考价值，为构建山羊卵母细胞成熟过程中LncRNA和mRNA表达调控网络打下了坚实的基础。 试验二：LSD1对山羊卵母细胞体外成熟影响的研究 本试验旨在研究组蛋白去甲基化酶LSD1参与调控山羊卵母细胞成熟的作用机理，通过使用LSD1抑制剂GSK-LSD1探究LSD1对山羊卵母细胞体外成熟的影响。结果显示，随着GSK-LSD1浓度的增加，山羊卵母细胞的极体排出率显著降低（Control：58.84 ± 0.95%；2.5 μM：52.14 ± 0.51%，P < 0.01；50 μM：41.22 ± 0.42%，P < 0.001；100 μM：29.78 ± 1.78%，P < 0.001）。使用50 μM抑制剂处理山羊卵母细胞12 h结果显示，与对照组（CTL）相比，处理组（GSK-LSD1）卵母细胞胞内H3K4me2的甲基化水平显著上升（P < 0.001），纺锤体组装异常率（CTL：25.94 ± 1.02%；GSK-LSD1：71.15 ± 3.32%；P < 0.01）、染色体排列异常率（CTL：22.93 ± 1.11%；GSK-LSD1：76.03 ± 3.25%；P < 0.01）和骨架蛋白分布异常率（CTL：15.31 ± 1.60%；GSK-LSD1：67.50 ± 3.09%；P < 0.001）均显著上升，胞内p-H2AX表达水平也显著上升（P < 0.01）。与正常MII期卵母细胞相比，在使用50 μM抑制剂处理后仍能排出第一极体的卵母细胞中，BCL2/BAX的比值显著下降（P < 0.01），CNOT6L（P < 0.001）和CNOT7（P < 0.001）的mRNA表达水平也显著下降。以上结果表明LSD1在山羊卵母细胞体外成熟过程中具有重要作用。 试验三：LncRNA LUZP1与LSD1靶标关系初步验证 本试验旨在研究LncRNA LUZP1及其靶基因LSD1的靶标关系，揭示在山羊颗粒细胞以及成纤维细胞中LUZP1和LSD1的调控方式，从侧面验证测序结果的可靠性。首先运用生物信息学技术对LUZP1和LSD1的位置关系、表达模式进行分析，而后通过在颗粒细胞和成纤维细胞中转染siRNA探究二者的作用方式。结果显示，LUZP1与LSD1相距1,394 bp，在山羊卵母细胞GV期向MII期发育过程中RNA表达水平均显著上升（P < 0.001）；在颗粒细胞以及成纤维细胞中干扰LUZP1均会显著降低LSD1的mRNA水平（P < 0.001），而干扰LSD1同样也会显著降低LUZP1的RNA水平（P < 0.001）。因此本试验结果表明，LncRNA LUZP1能够和LSD1相互影响。 试验四：LSD1对克隆山羊成纤维细胞DNA甲基化的影响 本试验旨在研究LSD1、DNA甲基化和SCNT之间的相互联系，探究再克隆效率逐渐降低的原因，为SCNT技术的发展提供参考。首先对两种核移植供体细胞（普通山羊成纤维细胞，Goat fibroblast cells，GFC；克隆山羊成纤维细胞，Cloned goat fibroblast cells，CFC）间的差异进行分析，结果显示GFC和CFC组细胞的生长曲线均呈典型“S”型，但CFC组细胞的增殖与凋亡显著高于GFC组；CFC组细胞中DNMT1（P < 0.01）、DNMT3b（P < 0.01）、TET1（P < 0.05）、TET2（P < 0.05）、H19（P < 0.05）和IGF2（P < 0.01）基因表达水平均显著低于GFC组，而DNMT3a、TET3和IGF2R在两组间无显著性差异；CFC组细胞中IGF2两个差异甲基化区域的甲基化水平均显著低于GFC组（74.1% vs. 57.8%；P < 0.01；76.8% vs. 40.0%；P < 0.01），但IGF2-H19印记基因控制区域甲基化水平显著高于GFC组（68.8% vs. 84.0%；P < 0.01）。随后用1 μM GSK-LSD1处理CFC 48 h，发现于对照组（CTL）相比，处理组（GSK-LSD1）克隆山羊成纤维细胞DNMT1（P < 0.05）、DNMT3a（P < 0.001）、DNMT3b（P < 0.05）的表达水平均显著上升，TET家族三个基因的表达水平没有显著变化；H19（P < 0.05）的表达水平显著降低，但IGF2（P < 0.001）和IGF2R（P < 0.05）的表达水平显著上升。本试验结果说明，LSD1、DNA甲基化和SCNT基因的相互作用导致印记基因座IGF2-H19的异常，可能是造成再克隆效率降低的原因之一。

The implementation of somatic cell nuclear transfer (SCNT) could not be independent without healthy matured oocytes and proper donor cells. In mammals, there are four important stages during oocyte maturation which are germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase Ⅰ (MI) and metaphase II (MII), polar body I (PBI) is visible under a microscope in mature oocyte arrested at MII. During oocyte maturation, it is vital to prepare DNA, RNA, protein and other necessary materials for oocyte to attain competence for successful fertilization and subsequent embryonic development. DNA methylation in donor cells could influence the reprogramming in SCNT embryos and the efficiency of clone. Because of few studies on epigenetic modifications involved in oocyte maturation and SCNT in goat, we used goat GV, MII oocytes and donor cells to investigate the impacts of histone methylation, lncRNA and DNA methylation on goat oocytes maturation in vitro and SCNT. The research could be divided into four parts following: 1. Comprehensive analysis of lncRNA and mRNA differential expression between GV and MII oocytes. The experiment aims to investigate the lncRNA and mRNA expression patterns during goat oocyte maturation. There are 27,033 novel lncRNAs were determined through single-cell RNA-sequencing (scRNA-seq) performed on goat GV and MII oocytes. Besides, we found 4,516 differential mRNAs and 3,019 differential lncRNAs related to many bioprocesses such as RNA splicing, mitochondrial translation, oxidative phosphorylation, cytoplasmic polyadenylation and histone methylation, indicating that there are many factors participating in goat oocyte maturation. Our results could contribute to further study of mechanisms on lncRNA and mRNA engaged in goat oocyte maturation. Moreover, the data in this experiment have provided a valuable resource used for establishing lncRNA-mRNA network in goat oocyte maturation. 2. Functional investigations of LSD1 during goat oocyte maturation. To figure out the mechanism of Lysine-specific histone demethylase 1A (LSD1) engaged in goat oocyte maturation, hence GSK-LSD1, a specific inhibitor of LSD1, was used in this study to investigate the impacts of LSD1 on goat oocyte maturation in vitro. The results showed that, GSK-LSD1 treatment obviously reduced first polar body extrusion (PBE) of goat oocytes (Control: 58.84 ± 0.95%; 2.5 μM: 52.14 ± 0.51%, P < 0.01; 50 μM: 41.22 ± 0.42%, P < 0.001; 100 μM: 29.78 ± 1.78%, P < 0.001). With the treatment of 50 μM GSK-LSD1 for 12 h, compared with control group (CTL), the H3K4me2 methylation level in GSK-LSD1 group (GSK-LSD1) was increased significantly (P < 0.001), as well as the protein level of p-H2AX (P < 0.01). Compared with CTL, the abnormal rate of spindle assembly (CTL: 25.94 ± 1.02%; GSK-LSD1: 71.15 ± 3.32%; P < 0.01), chromosome alignment (CTL: 22.93 ± 1.11%; GSK-LSD1: 76.03 ± 3.25%; P < 0.01) and cytoskeletal organization (CTL: 15.31 ± 1.60%; GSK-LSD1: 67.50 ± 3.09%; P < 0.001) was increased significantly in GSK-LSD1. Furthermore, compared with normal MII oocytes, the ratio of BCL2/BAX (P < 0.01), the mRNA level of CNOT6L (P < 0.001) and CNOT7 (P < 0.001) was decreased significantly in the oocytes with PBI treated with 50 μM GSK-LSD1 for 24 h. These results indicate that LSD1 played a vital role in goat oocyte maturation. 3. Preliminary verification of relationship between lncRNA LUZP1 and LSD1. This experiment was designed to investigate the relation of lncRNA LUZP1 and LSD1, reveal the interaction between LUZP1 and LSD1 in goat granule cells (GGC) and goat fibroblast cells (GFC), also as a proof for single-cell RNA-sequencing (scRNA-seq) data. The analysis of the location of LUZP1 and LSD1 was made, as well as their expression patterns in goat GV and MII oocytes. In the meantime, siRNAs were transfected to GGC and GFC for the studies. The results showed that, there are 1,394 bp between LUZP1 and LSD1, both RNA levels in MII goat oocytes were significantly increased compared to GV goat oocytes (P < 0.001). Interference of LUZP1 could reduce the mRNA level of LSD1 in GGC and GFC (P < 0.001), similarly, si-LSD1 could reduce the LUZP1 expression levels too in GGC and GFC (P < 0.001). All the results above indicated that LncRNA LUZP1 could interact with LSD1. 4. Influences of LSD1 on DNA methylation in cloned goat fibroblast cells. The aim of this experiment was to study the connection among LSD1, DNA methylation and SCNT, reveal the reason of poor efficiency in re-clone and provide a valuable reference to the development of SCNT. We analyzed the differences between two kinds of donor cell in SCNT (Goat fibroblast cells, GFC; Cloned goat fibroblast cells, CFC), and the results showed that both groups cell growth curves were typical “S”-shaped, but growth and apoptosis rate in CFC group was increased significantly. The expression level of DNMT1 (P < 0.01), DNMT3b (P < 0.01), TET1 (P < 0.05), TET2 (P < 0.05), H19 (P < 0.05) and IGF2 (P < 0.01) were decreased significantly in CFC group compared with GFC group. However, no statistical differences were found on the expression level of DNMT3a, TET3 and IGF2R between two groups. Compared with GFC group, the methylation level of IGF2 two differentially methylated region (74.1% vs. 57.8%; P < 0.01 and 76.8% vs. 40.0%; P < 0.01) were decreased significantly, while IGF2-H19 imprinting control region was increased significantly (68.8% vs. 84.0%; P < 0.01) in CFC group. After treated with 1 μM GSK-LSD1 for 48 h in CFC, we found that compared with control group (CTL), the expression level of DNMT1 (P < 0.05), DNMT3a (P < 0.001)、DNMT3b (P < 0.05) was increased significantly while the expression level of TETs was not changed statistically. And the expression level of H19 (P < 0.05) was decreased significantly while IGF2 (P < 0.001) and IGF2R (P < 0.05) was increased significantly in GSK-LSD1 compared with CTL. All the results above revealed that the interaction within LSD1, SCNT and DNA methylation resulted in the abnormal methylation of IGF2-H19 locus, and it could be responsible for the poor efficiency in serial re-clone.