FST对猪卵巢颗粒细胞增殖凋亡及激素分泌的影响

doi:10.11843/j.issn.0366-6964.2025.03.024

Abstract

Abstract:

The purpose of this study was to clarify the effect of FST gene on proliferation, apoptosis, and hormone secretion of porcine ovarian granulosa cells, and provide scientific reference for investigating the role of FST gene in the development of pig follicles. In this study, immortalized porcine ovarian granulosa cells were used as experimental materials, the granulosa cells were divided into 4 groups with 3 replications per group that included overexpression negative control group, overexpression group, inhibition negative control group and inhibition group. The overexpression plasmid and siRNA of FST gene were designed and constructed according to the mRNA sequence, and transfected into porcine ovarian granulosa cells to verify the overexpression and inhibitory effects. The CCK-8 and flow cytometry were used to analyze granulosa cell proliferation and apoptosis after transfection for 24, 48 and 72 h, ELISA was used to detect estradiol and progesterone content in granulosa cell. Furthermore, RT-PCR and Western blot were used to detect mRNA and protein expression level of cell proliferation, apoptosis, and hormone synthesis related genes. The results showed that, the mRNA and protein expression level of FST gene were significantly changed by overexpression plasmid and siRNA (P < 0.01); and FST gene could inhibit cell proliferation and induce cell apoptosis after transfecting overexpression plasmid and siRNA into porcine ovarian granulosa cells, and also significantly downregulated the mRNA (P < 0.05) and protein (P < 0.01) expression level of cell proliferation and apoptosis related genes FSHR, GDF9, BCL2 and CDKN1B. After interfering with FST expression, the estradiol and progesterone content in porcine ovarian granulosa cells were extremely significantly increased (P < 0.01), and the mRNA and protein expression level of hormone synthesis related genes STAR, CYP11A1, CYP19A1, 3β-HSD, and 17β-HSD were also extremely significantly increased (P < 0.01). In summary, in porcine ovarian granulosa cells, the decrease of FST gene expression level could accelerate cell proliferation, estradiol and progesterone secretion, and this function could be due to the upregulation of cell proliferation and apoptosis related genes and hormone synthesis rate limiting enzymes expression.

Key words: FST, pig, granulosa cells, proliferation and apoptosis, estradiol, progesterone

CLC Number:

S828.3

LIU Chenlong, JI Huayuan, LU Dan, WAN Mingchun, HU Yao, ZHOU Quanyong. Effect of FST on Proliferation, Apoptosis and Hormone Secretion of Porcine Ovarian Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1242-1251.

Figures/Tables 10

Table 1

Table 2

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

References 41

1	GUPTA C , CHAPEKAR T , CHHABRA Y , et al. Differential response to sustained stimulation by hCG & LH on goat ovarian granulosa cells[J]. Indian J Med Res, 2012, 135 (3): 331- 340.
2	MATSUDA F , INOUE N , MANABE N , et al. Follicular growth and atresia in mammalian ovaries: regulation by survival and death of granulosa cells[J]. J Reprod Dev, 2012, 58 (1): 44- 50. doi: 10.1262/jrd.2011-012
3	WANG L , CHEN Y R , WU S , et al. PIM2-mediated phosphorylation contributes to granulosa cell survival via resisting apoptosis during folliculogenesis[J]. Clin Transl Med, 2021, 11 (3): e359. doi: 10.1002/ctm2.359
4	ARCHILIA E C , BELLO C A P , BATALHA I M , et al. Effects of follicle-stimulating hormone, insulin-like growth factor 1, fibroblast growth factor 2, and fibroblast growth factor 9 on sirtuins expression and histone deacetylase activity in bovine granulosa cells[J]. Theriogenology, 2023, 210, 1- 8. doi: 10.1016/j.theriogenology.2023.07.011
5	宋浩然, 冯肖艺, 张培培, 等. 奶牛卵泡颗粒细胞在卵泡发育中的作用机制[J]. 畜牧兽医学报, 2024, 55 (6): 2313- 2324. doi: 10.11843/j.issn.0366-6964.2024.06.004
	SONG H R , FENG X Y , ZHANG P P , et al. The mechanism of follicular granulosa cells in follicular development in dairy cows[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55 (6): 2313- 2324. doi: 10.11843/j.issn.0366-6964.2024.06.004
6	KIMURA F , BONOMI L M , SCHNEYER A L . Follistatin regulates germ cell nest breakdown and primordial follicle formation[J]. Endocrinology, 2011, 152 (2): 697- 706. doi: 10.1210/en.2010-0950
7	WOODRUFF T K , LYON R J , HANSEN S E , et al. Inhibin and activin locally regulate rat ovarian folliculogenesis[J]. Endocrinology, 1990, 127 (6): 3196- 3205. doi: 10.1210/endo-127-6-3196
8	PATEL O V , BETTEGOWDA A , IRELAND J J , et al. Functional genomics studies of oocyte competence: evidence that reduced transcript abundance for follistatin is associated with poor developmental competence of bovine oocytes[J]. Reproduction, 2007, 133 (1): 95- 106. doi: 10.1530/rep.1.01123
9	JORGEZ C J , KLYSIK M , JAMIN S P , et al. Granulosa cell-specific inactivation of follistatin causes female fertility defects[J]. Mol Endocrinol, 2004, 18 (4): 953- 967. doi: 10.1210/me.2003-0301
10	LI D R , QIN G S , WEI Y M , et al. Immunisation against inhibin enhances follicular development, oocyte maturation and superovulatory response in water buffaloes[J]. Reprod Fertil Dev, 2011, 23 (6): 788- 797. doi: 10.1071/RD10279
11	WANG H X , CHEN W L , SHEN P L , et al. Follistatin (FST) is expressed in buffalo (Bubalus bubalis) ovarian follicles and promotes oocyte maturation and early embryonic development[J]. Reprod Domest Anim, 2023, 58 (12): 1718- 1731. doi: 10.1111/rda.14490
12	SILVA C C , KNIGHT P G . Modulatory actions of activin-A and follistatin on the developmental competence of in vitro-matured bovine oocytes[J]. Biol Reprod, 1998, 58 (2): 558- 565.
13	LI M D , DEPAOLO L V , FORD J J . Expression of follistatin and inhibin/activin subunit genes in porcine follicles[J]. Biol Reprod, 1997, 57 (1): 112- 118. doi: 10.1095/biolreprod57.1.112
14	GUO Z , ISLAM M S , LIU D , et al. Differential effects of follistatin on porcine oocyte competence and cumulus cell gene expression in vitro[J]. Reprod Domest Anim, 2018, 53 (1): 3- 10. doi: 10.1111/rda.13035
15	ZHOU Q Y , WAN M C , WEI Q P , et al. Expression, regulation, and functional characterization of FST gene in porcine granulosa cells[J]. Anim Biotechnol, 2016, 27 (4): 295- 302. doi: 10.1080/10495398.2016.1184675
16	周泉勇, 刘敬, 刘晨龙, 等. DIRAS3诱导猪子宫内膜上皮细胞自噬及对容受性相关基因表达的影响[J]. 江西农业大学学报, 2023, 45 (1): 169- 178.
	ZHOU Q Y , LIU J , LIU C L , et al. DIRAS3 induces autophagy in porcine endometrial epithelial cells and its effect on the expression of receptivity related genes[J]. Acta Agriculturae Universitatis Jiangxiensis, 2023, 45 (1): 169- 178.
17	张焱, 张华. 哺乳动物卵巢卵泡发育调控机制研究进展[J]. 生理学报, 2020, 72 (1): 63- 74.
	ZHANG Y , ZHANG H . Research advances in regulating mechanisms of mammalian ovarian folliculogenesis[J]. Acta Physiologica Sinica, 2020, 72 (1): 63- 74.
18	ZHANG X Y , YU T , GUO X Y , et al. Ufmylation regulates granulosa cell apoptosis via ER stress but not oxidative stress during goat follicular atresia[J]. Theriogenology, 2021, 169, 47- 55. doi: 10.1016/j.theriogenology.2021.04.009
19	GAO Y Y , ZOU Y G , WU G J , et al. Oxidative stress and mitochondrial dysfunction of granulosa cells in polycystic ovarian syndrome[J]. Front Med (Lausanne), 2023, 10, 1193749.
20	凌英会, 朱露, 吴昊, 等. 山羊FST慢病毒载体构建及其对卵巢卵泡颗粒细胞增殖的影响[J]. 畜牧兽医学报, 2019, 50 (9): 1888- 1896. doi: 10.11843/j.issn.0366-6964.2019.09.017
	LING Y H , ZHU L , WU H , et al. Construction of goat FST lentivirus vectors and its effect on the proliferation of ovarian follicular granulosa cells[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50 (9): 1888- 1896. doi: 10.11843/j.issn.0366-6964.2019.09.017
21	WANG Z P , NIU W B , WANG Y J , et al. Follistatin288 regulates germ cell cyst breakdown and primordial follicle assembly in the mouse ovary[J]. PLoS One, 2015, 10 (6): e0129643. doi: 10.1371/journal.pone.0129643
22	CAI H , XIANG Y B , QU S M , et al. Association of genetic polymorphisms in cell-cycle control genes and susceptibility to endometrial cancer among Chinese women[J]. Am J Epidemiol, 2011, 173 (11): 1263- 1271.
23	WEI J , ZENG Y , YANG Q , et al. GAS5 suppresses cell proliferation, migration, and invasion via the miR-455-5p/CDKN1B axis in cutaneous squamous cell carcinoma[J]. Crit Rev Eukaryot Gene Expr, 2022, 32 (5): 63- 76.
24	KIM H S , NOH Y K , MIN K W , et al. Low CDKN1B expression associated with reduced CD8+T lymphocytes predicts poor outcome in breast cancer in a machine learning analysis[J]. J Pers Med, 2023, 14 (1): 30.
25	王雪峰, 谭峰, 陈燕英, 等. 携带bcl-2基因慢病毒感染人原代卵巢颗粒细胞的凋亡[J]. 中国组织工程研究, 2013, 17 (28): 5209- 5215.
	WANG X F , TAN F , CHEN Y Y , et al. Apoptosis of human primary ovarian granulose cells infected with lentivirus carrying bcl-2 gene[J]. Chinese Journal of Tissue Engineering Research, 2013, 17 (28): 5209- 5215.
26	EMORI C , SUGIURA K . Role of oocyte-derived paracrine factors in follicular development[J]. Anim Sci J, 2014, 85 (6): 627- 633.
27	HILKER R E , PAN B , ZHAN X S , et al. MicroRNA-21 enhances estradiol production by inhibiting WT1 expression in granulosa cells[J]. J Mol Endocrinol, 2021, 68 (1): 11- 22.
28	BABAYEV E , XU M , SHEA L D , et al. Follicle isolation methods reveal plasticity of granulosa cell steroidogenic capacity during mouse in vitro follicle growth[J]. Mol Hum Reprod, 2022, 28 (10): gaac033.
29	SHAO T , KE H N , LIU R , et al. Autophagy regulates differentiation of ovarian granulosa cells through degradation of WT1[J]. Autophagy, 2022, 18 (8): 1864- 1878.
30	GLISTER C , TANNETTA D S , GROOME N P , et al. Interactions between follicle-stimulating hormone and growth factors in modulating secretion of steroids and inhibin-related peptides by nonluteinized bovine granulosa cells[J]. Biol Reprod, 2001, 65 (4): 1020- 1028.
31	JOHNSON A L , WOODS D C . Dynamics of avian ovarian follicle development: cellular mechanisms of granulosa cell differentiation[J]. Gen Comp Endocrinol, 2009, 163 (1-2): 12- 17.
32	MOSA A , NEUNZIG J , GERBER A , et al. 2β- and 16β-hydroxylase activity of CYP11A1 and direct stimulatory effect of estrogens on pregnenolone formation[J]. J Steroid Biochem Mol Biol, 2015, 150, 1- 10.
33	TUGAEVA K V , SLUCHANKO N N . Steroidogenic acute regulatory protein: Structure, functioning, and regulation[J]. Biochemistry (Mosc), 2019, 84 (Suppl 1): S233- S253.
34	SGRÒ P , ANTINOZZI C , WASSON C W , et al. Sexual dimorphism in sex hormone metabolism in human skeletal muscle cells in response to different testosterone exposure[J]. Biology (Basel), 2024, 13 (10): 796.
35	DING Z Q , DUAN H W , GE W B , et al. Regulation of progesterone during follicular development by FSH and LH in sheep[J]. Anim Reprod, 2022, 19 (2): e20220027.
36	AN L P , MAEDA T , SAKAUE T , et al. Purification, molecular cloning and functional characterization of swine phosphatidylethanolamine-binding protein 4 from seminal plasma[J]. Biochem Biophys Res Commun, 2012, 423 (4): 690- 696.
37	GHOSH D , EGBUTA C , KANYO J E , et al. Phosphorylation of human placental aromatase CYP19A1[J]. Biochem J, 2019, 476 (21): 3313- 3331.
38	TANG X R , MA L Z , GUO S , et al. High doses of FSH induce autophagy in bovine ovarian granulosa cells via the AKT/mTOR pathway[J]. Reprod Domest Anim, 2021, 56 (2): 324- 332.
39	CHEN H Y , LIU C , JIANG H , et al. Regulatory role of miRNA-375 in expression of BMP15/GDF9 receptors and its effect on proliferation and apoptosis of bovine cumulus cells[J]. Cell Physiol Biochem, 2017, 41 (2): 439- 450.
40	TANAKA K , HAYASHI Y , TAKEHARA A , et al. Abnormal early folliculogenesis due to impeded pyruvate metabolism in mouse oocytes[J]. Biol Reprod, 2021, 105 (1): 64- 75.
41	杨贞, 江少如, 陈小燕, 等. 经后增殖方对控制性超促排卵大鼠卵巢GDF9分泌及颗粒细胞凋亡的影响[J]. 实用医学杂志, 2024, 40 (7): 918- 923.
	YANG Z , JIANG S R , CHEN X Y , et al. Effect of Jinghou Zengzhi Granules on ovarian GDF9 secretion and granulosa cells apoptosis in controlled ovarian hyperstimulation rats[J]. The Journal of Practical Medicine, 2024, 40 (7): 918- 923.

名称 Name	正义链(5'→3') Sense	反义链(5'→3') Antisense
S2-199	GGACUGAGGAGGACGUAAATT	UUUACGUCCUCCUCAGUCCTT
S3-440	CCUCAAGGCCAGAUGUAAATT	UUUACAUCUGGCCUUGAGGTT
S4-714	GGAAAGUGUAUCAAAGCAATT	UUGCUUUGAUACACUUUCCTT
S1-NC	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT

引物 Primer	引物序列(5'→3') Sequence	片段大小/bp Products size
FST	F: GCCGCTGCCAGGTCCTGTAT R: CCACGTTCTCGCACGTTTCTTT	177
FSHR	F: TGCCACCACTGGGAACAT R: CACATAAGGAACCGAGGGAC	81
GDF9	F: AGAGGGCTGTCTGAGGGTGTA R: CAGTAGCGAGGGTTGTATTTGTG	230
BCL2	F: GCGACTTTGCCGAGATGTC R: CCGAACTCAAAGAAGGCCAC	133
CDKN1B	F: TCAGGCCAACTCAGAGGACA R: CAGGTCGCTTCCTTATCCCA	104
STAR	F: TGGCTGGAAGTCCCTCAAAG R: GCAAAGTCCACCTGGGTCTG	115
CYP11A1	F: GCTCGGCAACTTGGAATCTGT R: GGCGGGATGTTGTATCGTTCT	99
CYP19A1	F: CTGGGTTGCAGTTCATTGGC R: TTGTCCAGGTGCTTGGTGATG	163
3β-HSD	F: TGAGCTTCCTGCTGAGTCCA R: AGACGGTGAACACGCTGTTG	87
17β-HSD	F: TGCTTTGCTTCATTTAGCCCTAC R: CTCATTCTTCCAGTTAGCCTTGC	111
GAPDH	F: ATTCCACCCACGGCAAGTT R: TTTGATGTTGGCGGGATCT	110

[1]	HUANG Yani, TANG Xi, LI Jingquan, WEI Jiacheng, WU Zhenfang, LI Xinyun, XIAO Shijun, ZHANG Zhiyan. Large-scale Population Analysis of Potential Causal Genes for Daily Weight Gain and Age at 100 kg in Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1100-1109.
[2]	WU Jiahao, WU Ziyi, DOU Tengfei, BAI Liyao, ZHANG Yongqian, DONG Lianhe, LI Pengfei, LI Xinjian, HAN Xuelei, LI Xiuling. Genome-wide Association Study of Copy Number Variation in Growth-Related Traits of Yunong-Black Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1110-1119.
[3]	YANG Yuting, CHEN Guoliang, CHANG Qiaoning, BAO Wu, LIU Jingchao, JI Mengting, RONG Xiaoyin, GUO Xiaohong, YANG Yang, LI Bugao. miR-375-3p Targets Fam229a to Regulate Porcine Precursor Adipocyte Differentiation [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1120-1133.
[4]	JIA Wanli, WANG Jiying, LI Jingxuan, WANG Yanping, GENG Liying, ZHANG Chuansheng, ZHAO Xueyan. Identification of Key Genes Affecting Drip Loss in Laiwu Pigs Based on Transcriptome Sequencing Technology [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1134-1146.
[5]	ZHOU Wentao, WANG Chenyu, ZHOU Hui, LIU Hongbiao, FENG Shuhuan, FAN Gaosheng, LI Tiejun, HE Liuqin. Effects of Tannic Acid on Muscle Morphology, Flavor Amino Acids, and Expression of Muscle Fiber-related Genes in Immunostressed Weaned Piglets [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1290-1301.
[6]	PAN Junyi, WU Qingyao, TAN Bi'e, GUO Qiuping, HUANG Ruilin, CHEN Jiashun. Research Progress on Precise Nutrition Supply Technology and Intelligent Farming Equipment for Growing-Finishing Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 501-512.
[7]	HE Yu, WANG Xiangyu, DI Ran, CHU Mingxing, LIANG Chen. BMP4/SMAD4 Downregulates GJA1 Gene Expression to Affect the Gap Junctional Intercellular Communication Activity in Sheep Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 679-688.
[8]	BAI Guosong, TENG Chunran, WANG Junhong, ZHONG Ruqing, MA Teng, CHEN Liang, ZHANG Hongfu. Effects of Enzymatic Corn Gluten Meal on Growth Performance and Intestinal Microorganisms of Weaned Piglets [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 953-968.
[9]	ZHOU Xianshan, HUANG Shihui, NIU Xi, RAN Xueqin, WANG Jiafu. Differential Expression Study of Structural Variation in the Ubiquitin Ligase 2 Gene of Xiang Pigs with Wrinkled Skin [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 136-146.
[10]	WU Pingxian, WANG Junge, DIAO Shuqi, CHAI Jie, ZHA Lin, GUO Zongyi, CHEN Hongyue, LONG Xi. Analysis of Genetic Architecture Characteristics and Selection Signature by Imputed Whole Genome Sequencing Data in Rongchang Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 147-158.
[11]	YANG Wenpan, LIU Xiangjie, LUO Dongxiang, CHEN Menghui, XIE Ying, FANG Yuexin, LIN Tingyan, LI Aimin, LI Wenjing, DENG Zheng, DING Nengshui. Research on Genomic Selection of Reproductive Traits in Landrace Pigs Based on Chip Data [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 213-221.
[12]	YANG Baigao, XU Jiehuan, ZHANG Liang, LONG Xi, DAI Jianjun, ZHAO Xueming, PAN Hongmei. Exploring the Effect of Vitrification on Genome Methylation Level of Porcine Parthenogenetic Activation Blastocysts by scWGBS [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 222-231.
[13]	WANG Lei, BAI Shaocheng, WANG Sen, BAO Zhiyuan, CAI Jiawei, LIU Yan, ZHAO Bohao, WU Xinsheng, CHEN Yang. Effect of SRD5A2 on the Expression of Genes Related to Proliferation, Apoptosis and Steroid Hormone Synthesis in Rabbit Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 259-268.
[14]	FAN Dingkun, ZHANG Tao, JIAO Shuai, LU Wei, FU Yuze, YANG Hong, TU Yan, SHI Lingyuan, ZHANG Naifeng. Evaluation of Relative Bioavailability of Tricalcium Phosphate Produced by High-temperature Sintering Method in Weaned Piglets Based on Slope Ratio Method [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 269-280.
[15]	WANG Xingyue, YANG Zhiyuan, LIN Jian, ZHANG Zixuan, ZHOU Yuting, CHENG Huimin, LIU Yuehuan, HU Ge. Development and Application of a Real-time Fluorescence Quantitative PCR Detection Method for Pigeon Circovirus [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 335-342.

Effect of FST on Proliferation, Apoptosis and Hormone Secretion of Porcine Ovarian Granulosa Cells

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 10

References 41

Related Articles 15

Recommended Articles

Metrics

Comments