敲除G0S2基因对绵羊卵巢颗粒细胞增殖、类固醇激素及相关基因表达的影响

doi:10.13560/j.cnki.biotech.bull.1985.2022-1398

摘要/Abstract

摘要：

本文旨在探究G0S2基因对绵羊卵巢颗粒细胞增殖，雌激素（E₂）和孕酮（P₄）分泌，以及类固醇分泌相关基因和细胞凋亡基因表达的影响。采集小尾寒羊新鲜卵巢，用割吸法收集中小卵泡的卵泡液，离心分离颗粒细胞，分为试验组和对照组，试验组采用CRISPR-Cas9基因编辑技术敲除G0S2基因，获得重组表达载体PX458-sgRNA-G0S2转染至颗粒细胞；对照组转染PX458质粒至颗粒细胞。检测颗粒细胞活力和凋亡情况，以及E₂和P₄水平及相关基因的表达量。结果表明，试验组G0S2 mRNA的表达量及蛋白水平极显著低于对照组（P<0.01），分别降低了66%和70%，G0S2敲除成功。试验组颗粒细胞的增殖活力显著高于对照组（P<0.05），细胞凋亡率极显著下降56%（P<0.01）。试验组E₂水平显著高于对照组（P<0.05），P₄水平显著低于对照组（P<0.05）。试验组颗粒细胞中StAR、CYP11、3β-HSD的表达量极显著低于对照组（P<0.01），CYP19的表达量极显著高于对照组（P<0.01）。与对照组相比，试验组颗粒细胞中Caspase3和Bax的表达极显著下调（P<0.01），Bcl-2的表达极显著上调（P<0.01）。上述结果表明，敲除G0S2基因能促进在绵羊卵巢颗粒细胞的增殖，抑制其凋亡，通过下调StAR、CYP11、3β-HSD，上调CYP19的表达影响E₂和P₄的分泌。

关键词: G0S2, 绵羊, 颗粒细胞, 增殖, 凋亡

Abstract:

The objective of this research is to explore the effects of the G0S2 gene on the cell proliferation of ovarian granulosa cells, the secretion of estrogen（E₂）and progesterone（P₄）, as well as the expressions of steroid secretion-related genes and apoptosis genes. Fresh ovaries of small-tailed sheep were collected,and the follicular fluid gathered from small and medium-sized follicles by the suction method was centrifuged to separate the granulosa cells into experimental and control groups. The G0S2 gene in the experimental group was knocked out by CRISPR-Cas9 gene editing technology and the obtained recombinant expression vector PX458-sgRNA-G0S2 was transfected to the granulosa cells. The PX458 plasmid in the control group was transfected to the granulosa cells. The viability and apoptosis of the granulosa cells were detected, as well as the E₂ and P₄ concentrations and associated gene expression. The results showed that the relative abundance and the protein level of G0S2 mRNA in the experimental group were lower（P<0.01）than those in the control group, reduced by 66% and 70% respectively, proving that the G0S2 was successfully knocked out. The proliferation activity of the granulosa cells in the experimental group was higher（P<0.05）than the control group, and the apoptosis rate reduced（P<0.01）by 56%. Compared with the control group, the E₂ concentration was higher（P<0.05）and the P₄ concentration was lower（P<0.05）in the experimental group. The expressions of genes StAR, CYP11 and 3β-HSD in the experimental group was lower（P<0.01）than the control group, and the expressions of CYP19 was significantly higher（P<0.01）than the control group. Compared with the control group, the expression of Caspase3 and Bax in the experimental group of granulosa cells was significantly downregulated（P<0.01）, and the expressions of Bcl-2 was significantly upregulated（P<0.01）. Our results demonstrate that knockout of the G0S2 gene can promote the proliferation of ovarian granulosa cells, inhibit their apoptosis, and affect the secretion of E₂and P₄ by downregulating the expression of genes StAR, CYP11, 3β-HSD and upregulating CYP19 in sheep.

Key words: G0S2, sheep, granulosa cells, proliferation, apoptosis

马钰静, 段春辉, 贺名扬, 张英杰, 杨若晨, 王泳, 刘月琴. 敲除G0S2基因对绵羊卵巢颗粒细胞增殖、类固醇激素及相关基因表达的影响[J]. 生物技术通报, 2023, 39(6): 325-334.

MA Yu-jing, DUAN Chun-hui, HE Ming-yang, ZHANG Ying-jie, YANG Ruo-chen, WANG Yong, LIU Yue-qin. Effects of Knockout of G0S2 Gene in Ovarian Granulosa Cell Proliferation, Steroids Hormones and Related Gene Expression[J]. Biotechnology Bulletin, 2023, 39(6): 325-334.

图/表 10

表1 G0S2 sgRNA引物序列

Table 1 G0S2 sgRNA primer sequences

引物名称Primer name	序列Sequence（5'-3'）
O-sp-G0S2-E1-1F	caccGTGCGAATGTACCTGCTGGG
O-sp-G0S2-E1-1R	aaacCCCAGCAGGTACATTCGCAC

图1 PX458-sgRNA-G0S2 重组质粒测序比对

Fig. 1 PX458-sgRNA-G0S2 recombinant plasmid sequencing alignment

表2 RT-qCR引物序列

Table 2 RT-qCR primers’ sequences

引物名称Primer name	序列Sequence（5'-3'）	退火温度Annealing temperature/℃	片段大小Fragment length/bp
GAPDH	F: GGTCGGAGTGAACGGATTTG	60	222
GAPDH	R: CTTGACTGTGCCGTGGAACTT	60	222
G0S2	F: GCGAAGCTGGTGCGAATGTAC	60	154
G0S2	R: CCTCCAGTGGCTTGCCTTGATC	60	154
Bcl-2	F: GATGACCGAGTACCTGAACCG	60	120
Bcl-2	R: GACAGCCAGGAGAAATCAAACA	60	120
Caspase3	F: GCTACAAGGTCCGTTATGCC	60	128
Caspase3	R: GATGCTGCCGTATTCGTTCTC	60	128
Bax	F: TGCTCACTGCCTCACTCACC	60	179
Bax	R: CCCAAGACCACTCCTCCCTA	60	179
StAR	F: ATTCAGGAGGCAAAGAGCAGC	60	270
StAR	R: TCGGGTAAGGAAAATGGGTCA	60	270
3β-HSD	F: CAGTCTATGTTGGCAATGTGGC	60	283
3β-HSD	R: CGGTTGAAGCAGGGGTGGTAT	60	283
CYP11	F: GTTTCGCTTTGCCTTTGAGTC	60	120
CYP11	R: ACAGTTCTGGAGGGAGGTTGA	60	120
CYP19	F: GCTTTTGGAAGTGCTGAACCC	60	379
CYP19	R: CATGCCGATGAACTGCAACC	60	379

图2 绵羊卵巢颗粒细胞FSHR免疫荧光染色（400×） A：FSHR染色绵羊卵巢颗粒细胞；B：DAPI染色绵羊卵巢颗粒细胞细胞核；C：叠加染色效果

Fig. 2 FSHR immunofluorescence staining of ovarian granulosa cells in sheep（400×） A: FSHR stained ovarian granulosa cells of sheep. B: DAPI stained ovarian granulosa cell nuclei of sheep. C: Overlay dyeing effect

图3 质粒载体转染绵羊卵巢颗粒细胞的效果（400×） A：转染空白PX458质粒的卵巢颗粒细胞绿色荧光表达；B：转染PX458-sgRNA-G0S2重组质粒的卵巢颗粒细胞绿色荧光表达；C：明场下未经过转染的空白卵巢颗粒细胞

Fig. 3 Effects of plasmid vector transfection of the ovarian granulosa cells in sheep（400×） A: Green fluorescence expression of ovarian granulosa cells transfected with blank PX458 plasmid. B: Green fluorescence expression of ovarian granulosa cells transfected with PX458-sgRNA-G0S2 recombinant plasmid. C: Untransfected blank ovarian granulosa cells in brightfield

图4 G0S2基因敲除对卵巢颗粒细胞中的G0S2 mRNA和蛋白表达的影响 A：G0S2敲除后mRNA表达结果统计；B：G0S2敲除后蛋白表达结果统计；C：G0S2敲除后蛋白表达灰度图；* P<0.05，** P<0.01，下同

Fig. 4 Effects of G0S2 gene knockout on the G0S2 mRNA and protein expression in ovarian granulosa cells A: Statistics of mRNA expression results after G0S2 knockout. B: Statistics of protein expression results after G0S2 knockout. C: Grayscale plot of protein expression after G0S2 knockout; * P<0.05, **P<0.01. The same below

图5 G0S2基因敲除对卵巢颗粒细胞增殖活力和细胞凋亡的影响 A：G0S2-Knockout组流式细胞凋亡检测图；B：Control组流式细胞凋亡检测图；C：流式细胞凋亡结果统计；D：细胞增殖活力结果统计

Fig. 5 Effects of G0S2 gene knockout in ovarian granulosa cell proliferation viability and apoptosis A: Flow cytometry apoptosis assay of G0S2-Knockout group. B: Control group flow cytometry apoptosis assay map. C: Flow cytometry apoptosis result statistics. D: Cell proliferation viability result statistics

图6 G0S2基因敲除对卵巢颗粒细胞E2和P4分泌的影响

Fig. 6 Effects of G0S2 gene knockout on the E2 and P4 secretions in ovarian granulosa cells

图7 G0S2基因敲除对卵巢颗粒细胞StAR、CYP11、3β-HSD、CYP19 mRNA表达的影响

Fig. 7 Effects of G0S2 knockout on the StAR, CYP11, 3β-HSD, CYP19 mRNA expressions in ovarian granulosa cells

图8 G0S2基因敲除对卵巢颗粒细胞Caspase3、Bax、Bcl-2 mRNA表达的影响

Fig. 8 Effects of G0S2 knockout on the Caspase3, Bax and Bcl-2 mRNA expressions in ovarian granulosa cells

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