Effects of Knockout of G0S2 Gene in Ovarian Granulosa Cell Proliferation, Steroids Hormones and Related Gene Expression

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

Abstract

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

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.

Figures/Tables 10

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

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

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

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

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

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

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

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

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

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

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