Effect of γ-aminobutyric Acid on Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells

doi:10.13560/j.cnki.biotech.bull.1985.2023-0784

Abstract

Abstract:

【Objective】 This study was conducted to investigate the effects of γ-aminobutyric acid（GABA）on the apoptosis and the secretion of steroid hormones in ovine ovarian granulosa cells（GCs）. 【Method】 The GCs were treated with 0, 10^-8, 10^-7, 10^-6 and 10^-5 mol/L GABA for 24 h, respectively. Then, the cell proliferation and apoptosis were detected by CCK-8 and flow cytometry. And the appropriate culture concentration of GABA was screened for subsequent experiments. ELISA was used to detect the secretion levels of estrogen（E₂）and progesterone（P₄）in the culture supernatant after GCs were treated with the appropriate concentration. The qRT-PCR and Western blot were to detect the mRNA and protein expressions of GCs proliferation-promoting genes（PCNA, CCNB1, and CCND1）, anti-apoptosis genes（Bcl-2）, apoptosis-promoting genes（Bax, Caspase-3）and steroid hormone synthesis-related genes（StAR, 3β-HSD, CYP11A1, CYP19A1）.【Result】 The 10^-6 mol/L GABA treatment for 24 h significantly promoted GCs proliferation（P < 0.05）and significantly inhibited its apoptosis（P < 0.05）. The mRNA and protein expressions of PCNA, CCNB1, CCND1 and Bcl-2 in the experimental group were higher than those in the control group（P < 0.05）. Compared with the control group, the mRNA and protein expressions of Bax and Caspase-3 in the experimental group decreased significantly（P < 0.05）, as well as Bax/Bcl-2 decreased significantly（P < 0.05）. Treatment of ovine ovarian GCs with 10^-6 mol/L GABA for 24 h significantly promoted the synthesis of P₄（P < 0.05）and significantly inhibited the synthesis of E₂（P < 0.05）. Compared with the control group, the mRNA and protein expressions of StAR, 3β-HSD and CYP11A1 in the experimental group increased significantly（P < 0.05）and the mRNA and protein expressions of CYP19A1 decreased significantly（P < 0.05）. 【Conclusion】 The results demonstrate that GABA treatment could promote GCs proliferation as well as inhibit GCs apoptosis by regulating the expression of CCNB1, CCND1, PCNA, Caspase-3, Bax and Bcl-2, and promote the synthesis of P₄ as well as inhibit the synthesis of E₂ by regulating the expressions of StAR, 3β-HSD, CYP11A1 and CYP19A1.

Key words: γ-aminobutyric acid, ovine granulosa cells, apoptosis, steroid hormone

SHAN Xin-yu, LI Tai-chun, YANG Ruo-chen, DUAN Xiang-ru, KANG Jia, ZHANG Ying-jie, LIU Yue-qin. Effect of γ-aminobutyric Acid on Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells[J]. Biotechnology Bulletin, 2024, 40(3): 312-321.

Figures/Tables 10

Table 1 Primer sequence

基因Gene	引物序列Primer sequence（5'-3'）	退火温度Annealing temperature/℃	产物长度Size/bp
GAPDH	F: GGTCGGAGTGAACGGATTTG	60	222
GAPDH	R: CTTGACTGTGCCGTGGAACTT	60	222
CCND1	F：GGCGTTCGTAATCGTGTTTTG	60	185
CCND1	R：CAGGTAGAAGGAGTGAGGAATGTGT	60	185
PCNA	F：GCCGAGGAGAACAAGCAGA	60	123
PCNA	R：GAGGGTGGGTTGGAAATGAA	60	123
CCNB1	F：GGCAGGAGGACTATGACATT	60	222
CCNB1	R：TCAGGCACCCACTTCTCTTTC	60	222
Bax	F：CGAGACCTGAAGCCAGAGAC	60	183
Bax	R：GCAAAGATACAGCCAACACC	60	183
Caspase-3	F：CCCATCCTCACCATCATCAC	60	275
Caspase-3	R：GCACAAACACGCACCTCAA	60	275
Bcl-2	F：ACTTGGACCTGTCGCTGTCC	60	120
Bcl-2	R：CCTGCGTTTGGAGTGGTAGA	60	120
CYP11A1	F：AACATGGAGCTGCAGAGGAT	60	120
CYP11A1	R：CCAATGTCCAGCCCATGATG	60	120
StAR	F：TCTTTGAGTTCGGAGGGGTC	60	270
StAR	R：GGAGAAATCAAACAGGGGCC	60	270
3β-HSD	F：TGGACCCGTCGATCTGAAAA	60	283
3β-HSD	R：GCGTACAAGAAGTCTGCCTC	60	283
CYP19A1	F：AATGCTGCCCCAGGTGTT	60	379
CYP19A1	R：CGGCGTCTCTGCGATTTT	60	379

Table 2 Primary antibody information

一抗名称Name of primary antibody	品牌及货号Brand and coale	稀释度Proportion of dilution	分子量Molecular weight/kD
Caspase-3	Abcam ab13847	1∶1 000	34
StAR	Abcam ab133657	1∶1 000	32
3β-HSD	Abcam ab55268	1∶1 000	42
Bax	Abcam ab32503	1∶1 000	21
Bcl-2	Abcam ab182858	1∶1 500	26
CCNB1	Abcam ab181593	1∶1 500	55
CCND1	Abcam ab16663	1∶200	33
CYP11A1	Abcam ab272494	1∶1 000	60
CYP19A1	Abcam ab18995	1∶800	55
PCNA	Abcam ab29	1∶800	30
β-actin（C4）（内参）	Santa Cruz SC-47778	1∶1 500	43

Fig. 1 Identification of ovine ovarian granulosa cells（400×） A: FSHR staining of ovine ovarian granulosa cells. B: DAPI staining of ovine ovarian granulosa cell nucleus. C: Superimposed dyeing effect

Fig. 2 Effects of different concentrations of GABA on proliferation in ovine granulosa cells The different letters indicate the significant difference（P<0.05）, the same below

Fig. 3 Results of apoptosis in ovine granulosa cells treated with GABA A、B、C、D、E is the effects of 0, 10-8, 10-7, 10-6, 10-5 mol/L GABA on the apoptosis of ovine granulosa cells. Q1 was dead cells caused by mechanical injury; Q2 was late apoptosis; Q3 was early apoptosis; Q4 was living cells

Table 3 Effect of different concentrations of GABA on apoptosis rate in ovine granulosa cells

项目Item	正常细胞率Normal cell rate/%	早期凋亡率Early apoptosis rate/%	晚期凋亡率Late apoptosis rate/%	总凋亡率Total apoptosis rate/%
Control	81.97±0.61^a	1.58±0.22^ab	13.43±0.15^a	15.02±0.26^a
10^-8	84.97±0.49^b	1.42±0.27^a	11.43±0.38^b	12.85±0.56^b
10^-7	87.63±0.24^c	1.63±0.21^ab	9.16±0.52^c	10.78±0.31^c
10^-6	89.27±0.03^d	1.21±0.09^a	7.76±0.24^d	8.97±0.16^d
10^-5	86.80±0.58^c	2.12±0.16^b	9.70±0.17^c	11.82±0.04^e

Fig. 4 Effects of GABA on the expressions of proliferation-related genes mRNA and proteins in ovine granulosa cells A: Statistics of mRNA expression results of proliferation-related genes after GABA treatment. B: Western blot results. C: Statistics of protein expression results of proliferation-related genes after GABA treatment

Fig. 5 Effects of GABA on the expressions of apoptosis-related genes mRNA and proteins in ovine granulosa cells A: Statistics of mRNA expression results of apoptosis-related genes after GABA treatment. B: Effect of GABA on ratio of mRNA expression of Bax/Bcl-2. C: Western blot results. D: Statistics of protein expression results of apoptosis-related genes after GABA treatment

Table 4 Effects of 10-6 mol/L GABA on steroid hormone secretion of GCs in ovine granulosa cells

项目 Item	对照组 Control group	试验组 Test group
雌二醇（E₂）/（pg·mL^-1）	77.25±1.79^a	61.30±0.74^b
孕酮（P₄）/（pmol·L^-1）	344.45±7.03^a	400.66±14.78^b

Fig. 6 Effects of GABA on the expressions of steroid hormone synthesis-related genes and proteins in ovine granulosa cells A: Statistics of mRNA expression results of steroid hormone synthesis-related genes after GABA treatment. B: Western blot results. C: Statistics of protein expression results of steroid hormone synthesis-related genes after GABA treatment

References 51

[1]	Kim DH, Dasagrandhi C, Park SK, et al. Optimization of gamma-aminobutyric acid production using sea tangle extract by lactic acid bacterial fermentation[J]. LWT, 2018, 90: 636-642. doi: 10.1016/j.lwt.2018.01.011 URL
[2]	Zhou HL, Chen HY, Bao DP, et al. Recent advances of γ-aminobutyric acid: physiological and immunity function, enrichment, and metabolic pathway[J]. Front Nutr, 2022, 9: 1076223. doi: 10.3389/fnut.2022.1076223 URL
[3]	Chiu CQ, Barberis A, Higley MJ. Preserving the balance: diverse forms of long-term GABAergic synaptic plasticity[J]. Nat Rev Neurosci, 2019, 20(5): 272-281. doi: 10.1038/s41583-019-0141-5 pmid: 30837689
[4]	Kleppner SR, Tobin AJ. GABA signalling: therapeutic targets for epilepsy, Parkinson's disease and Huntington's disease[J]. Expert Opin Ther Targets, 2001, 5(2): 219-239.
[5]	Oketch-Rabah HA, Madden EF, Roe AL, et al. United states pharmacopeia(USP)safety review of Gamma-aminobutyric acid(GABA)[J]. Nutrients, 2021, 13(8): 2742. doi: 10.3390/nu13082742 URL
[6]	包华琼, 王新庄. γ-氨基丁酸(GABA)的生殖生理作用[J]. 动物医学进展, 2002, 23(3): 39-41.
	Bao HQ, Wang XZ. Effect of gamma-amino-butyric acid on reproduction and physiology[J]. Prog Vet Med, 2002, 23(3): 39-41.
[7]	许吉怡, 王志鹏, 邹宇川, 等. 食源性GABA对人体各系统的保护作用和机制的研究进展[J]. 世界科学技术-中医药现代化, 2022, 24(10): 3835-3843.
	Xu JY, Wang ZP, Zou YC, et al. Research progress on the protective effect and mechanism of oral-intake GABA on human systems[J]. Mod Tradit Chin Med Mater Med World Sci Technol, 2022, 24(10): 3835-3843.
[8]	de Bie TH, Balvers MGJ, de Vos RCH, et al. The influence of a tomato food matrix on the bioavailability and plasma kinetics of oral gamma-aminobutyric acid(GABA)and its precursor glutamate in healthy men[J]. Food Funct, 2022, 13(16): 8399-8410. doi: 10.1039/D2FO01358D URL
[9]	Hepsomali P, Groeger JA, Nishihira J, et al. Effects of oral gamma-aminobutyric acid(GABA)administration on stress and sleep in humans: a systematic review[J]. Front Neurosci, 2020, 14: 923. doi: 10.3389/fnins.2020.00923 URL
[10]	Zhong G, Shao D, Wang Q, et al. Effects of dietary supplemented of γ-amino butyric acid on growth performance, blood biochemical indices and intestinal morphology of yellow-feathered broilers exposed to a high temperature environment[J]. Ital J Anim Sci, 2020, 19(1): 431-438. doi: 10.1080/1828051X.2020.1747953 URL
[11]	Guo K, Cao HB, Zhu YJ, et al. Improving effects of dietary rumen protected γ-aminobutyric acid additive on apparent nutrient digestibility, growth performance and health status in heat-stressed beef cattle[J]. Anim Sci J, 2018, 89(9): 1280-1286. doi: 10.1111/asj.13053 pmid: 29923358
[12]	Chen J, Guo K, Song XZ, et al. The anti-heat stress effects of Chinese herbal medicine prescriptions and rumen-protected γ-aminobutyric acid on growth performance, apparent nutrient digestibility, and health status in beef cattle[J]. Anim Sci J, 2020, 91(1): e13361. doi: 10.1111/asj.v91.1 URL
[13]	Yuan XK, Zhang XY, Wu YJ, et al. Maternal amino acid mixtures supplementation during late gestation and lactation improved growth performance of piglets through improving colostrum composition and antioxidant capacity[J]. Antioxidants, 2022, 11(11): 2144. doi: 10.3390/antiox11112144 URL
[14]	Turathum B, Gao EM, Chian RC. The function of cumulus cells in oocyte growth and maturation and in subsequent ovulation and fertilization[J]. Cells, 2021, 10(9): 2292. doi: 10.3390/cells10092292 URL
[15]	Fontana J, Martínková S, Petr J, et al. Metabolic cooperation in the ovarian follicle[J]. Physiol Res, 2020, 69(1): 33-48. doi: 10.33549/physiolres.934233 pmid: 31854191
[16]	Dompe C, Kulus M, Stefańska K, et al. Human granulosa cells-stemness properties, molecular cross-talk and follicular angiogenesis[J]. Cells, 2021, 10(6): 1396. doi: 10.3390/cells10061396 URL
[17]	Krawczyk K, Marynowicz W, Pich K, et al. Persistent organic pollutants affect steroidogenic and apoptotic activities in granulosa cells and reactive oxygen species concentrations in oocytes in the mouse[J]. Reprod Fertil Dev, 2023, 35(3): 294-305. doi: 10.1071/RD21326 URL
[18]	Worku T, Rehman ZU, Talpur HS, et al. microRNAs: new insight in modulating follicular atresia: a review[J]. Int J Mol Sci, 2017, 18(2): 333. doi: 10.3390/ijms18020333 URL
[19]	Ullah A, Jahan S, Razak S, et al. Protective effects of GABA against metabolic and reproductive disturbances in letrozole induced polycystic ovarian syndrome in rats[J]. J Ovarian Res, 2017, 10(1): 62. doi: 10.1186/s13048-017-0359-7 pmid: 28915843
[20]	姜杰, 倪江, 李庆雷, 等. γ-氨基丁酸对离体大鼠黄体化颗粒细胞孕酮分泌的影响[J]. 基础医学与临床, 2000, 20(6): 547-548.
	Jiang J, Ni J, Li QL, et al. Effect of GABA on progesterone secretion in rat luteinising granulosa cells in vitro[J]. Basic Med Sci Clin, 2000, 20(6): 547-548.
[21]	杨美琼. γ-氨基丁酸对人卵巢颗粒细胞雌二醇和孕酮分泌及凋亡的影响[D]. 广州: 南方医科大学, 2009.
	Yang MQ. Effects of GABA on E₂ and P secretions and apoptosis by human ovarian granulosa cells in vitro[D]. Guangzhou: Southern Medical University, 2009.
[22]	姜杰, 倪江, 朱辉, 等. γ-GABA对大鼠离体培养颗粒细胞生成雌二醇的影响及作用机制[J]. 中国应用生理学杂志, 2000, 16(3): 272-274.
	Jiang J, Ni J, Zhu H, et al. Effect of γ-gaba on estradiol production and its mechanism by rat granulosa cells cultured in vitro[J]. Chin J Appl Physiol, 2000, 16(3): 272-274.
[23]	Xu DJ, Jiang XH, Wang YK, et al. Liver receptor homolog-1 regulates apoptosis of bovine ovarian granulosa cells by progestogen receptor signaling pathway[J]. Animals, 2022, 12(9): 1213. doi: 10.3390/ani12091213 URL
[24]	Zhu ZH, Shi ZG, Xie CL, et al. A novel mechanism of Gamma-aminobutyric acid(GABA)protecting human umbilical vein endothelial cells(HUVECs)against H₂O₂-induced oxidative injury[J]. Comp Biochem Physiol C Toxicol Pharmacol, 2019, 217: 68-75. doi: 10.1016/j.cbpc.2018.11.018 URL
[25]	温静, 余哲琪, 田佳迎, 等. γ-氨基丁酸对热应激雏鸡胰腺组织结构、抗氧化能力、消化酶活性及细胞凋亡的影响[J]. 动物营养学报, 2021, 33(5): 2927-2938. doi: 10.3969/j.issn.1006-267x.2021.05.050
	Wen J, Yu ZQ, Tian JY, et al. Effects of γ-aminobutyric acid on pancreatic tissue structure, antioxidant capacity, digestive enzyme activities and cell apoptosis of heat-stressed chicks[J]. Chin J Anim Nutr, 2021, 33(5): 2927-2938.
[26]	孙士平. GABA对LPS诱导MAC-T细胞增殖和凋亡的影响[D]. 郑州: 河南农业大学, 2018.
	Sun SP. Effect of GABA on the proliferation and apoptosis induced by LPS in MAC-T cell line[D]. Zhengzhou: Henan Agricultural University, 2018.
[27]	赵海. GABA信号在小鼠胎盘形成期的表达及功能研究[D]. 重庆: 重庆医科大学, 2014.
	Zhao H. The expressions of GABA signal and its potential role in the mouse placenta formation in mice[D]. Chongqing: Chongqing Medical University, 2014.
[28]	徐静文, 刘勇, 张德普, 等. γ-氨基丁酸能系统对肺上皮细胞增殖的影响[J]. 山东大学学报: 医学版, 2011, 49(5): 34-37.
	Xu JW, Liu Y, Zhang DP, et al. Effect of the GABAergic system on lung epithelial cell proliferation[J]. J Shandong Univ Heath Sci, 2011, 49(5): 34-37.
[29]	Peng A, Xu XY, Wang CL, et al. EZH2 promotes DNA replication by stabilizing interaction of POLδ and PCNA via methylation-mediated PCNA trimerization[J]. Epigenetics Chromatin, 2018, 11(1): 44. doi: 10.1186/s13072-018-0213-1 pmid: 30071900
[30]	Hives M, Jurecekova J, Holeckova KH, et al. The driving power of the cell cycle: cyclin-dependent kinases, cyclins and their inhibitors[J]. Bratisl Lek Listy, 2023, 124(4): 261-266. doi: 10.4149/BLL_2023_039 pmid: 36598318
[31]	Basu A. The interplay between apoptosis and cellular senescence: Bcl-2 family proteins as targets for cancer therapy[J]. Pharmacol Ther, 2022, 230: 107943. doi: 10.1016/j.pharmthera.2021.107943 URL
[32]	Czabotar PE, Lessene G, Strasser A, et al. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy[J]. Nat Rev Mol Cell Biol, 2014, 15(1): 49-63. doi: 10.1038/nrm3722
[33]	Kale J, Osterlund EJ, Andrews DW. BCL-2 family proteins: changing partners in the dance towards death[J]. Cell Death Differ, 2018, 25(1): 65-80. doi: 10.1038/cdd.2017.186 pmid: 29149100
[34]	Marquez RT, Xu L. Bcl-2: Beclin 1 complex: multiple, mechanisms regulating autophagy/apoptosis toggle switch[J]. Am J Cancer Res, 2012, 2(2): 214-221.
[35]	Yuan Z, Dewson G, Czabotar PE, et al. VDAC2 and the BCL-2 family of proteins[J]. Biochem Soc Trans, 2021, 49(6): 2787-2795. doi: 10.1042/BST20210753 URL
[36]	McComb S, Chan PK, Guinot A, et al. Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or-7[J]. Sci Adv, 2019, 5(7): eaau9433. doi: 10.1126/sciadv.aau9433 URL
[37]	Li M, Gao P, Zhang JP. Crosstalk between autophagy and apoptosis: potential and emerging therapeutic targets for cardiac diseases[J]. Int J Mol Sci, 2016, 17(3): 332. doi: 10.3390/ijms17030332 pmid: 26950124
[38]	Siddiqui WA, Ahad A, Ahsan H. The mystery of BCL2 family: Bcl-2 proteins and apoptosis: an update[J]. Arch Toxicol, 2015, 89(3): 289-317. doi: 10.1007/s00204-014-1448-7 pmid: 25618543
[39]	Brooks C, Cho SG, Wang CY, et al. Fragmented mitochondria are sensitized to Bax insertion and activation during apoptosis[J]. Am J Physiol Cell Physiol, 2011, 300(3): C447-C455. doi: 10.1152/ajpcell.00402.2010 URL
[40]	Shi SJ, Zhou XG, Li JJ, et al. miR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine granulosa cells[J]. J Anim Sci Biotechnol, 2020, 11: 94. doi: 10.1186/s40104-020-00500-y
[41]	Xiao CY, Wang J, Zhang CP. Synthesis, regulatory factors, and signaling pathways of estrogen in the ovary[J]. Reprod Sci, 2023, 30(2): 350-360. doi: 10.1007/s43032-022-00932-z
[42]	Lee EB, Chakravarthi VP, Wolfe MW, et al. ERβ regulation of gonadotropin responses during folliculogenesis[J]. Int J Mol Sci, 2021, 22(19): 10348. doi: 10.3390/ijms221910348 URL
[43]	Kolatorova L, Vitku J, Suchopar J, et al. Progesterone: a steroid with wide range of effects in physiology as well as human medicine[J]. Int J Mol Sci, 2022, 23(14): 7989. doi: 10.3390/ijms23147989 URL
[44]	van der Linden M, Buckingham K, Farquhar C, et al. Luteal phase support for assisted reproduction cycles[J]. Cochrane Database Syst Rev, 2015, 2015(7): CD009154.
[45]	Song TL, Chen JY, Yang SZ, et al. Resveratrol stimulates StAR expression and progesterone production by GPER-mediated downregulation of Snail expression in human granulosa cells[J]. J Food Drug Anal, 2023, 31(2): 315-325. doi: 10.38212/2224-6614.3460 pmid: 37335164
[46]	Fang LL, Li YR, Wang SJ, et al. Melatonin induces progesterone production in human granulosa-lutein cells through upregulation of StAR expression[J]. Aging, 2019, 11(20): 9013-9024. doi: 10.18632/aging.v11i20 URL
[47]	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. doi: 10.1016/j.jsbmb.2015.02.014 URL
[48]	Johnson AL, Woods DC. Dynamics of avian ovarian follicle development: cellular mechanisms of granulosa cell differentiation[J]. Gen Comp Endocrinol, 2009, 163(1-2): 12-17. doi: 10.1016/j.ygcen.2008.11.012 URL
[49]	Ding ZQ, Duan HW, Ge WB, et al. Regulation of progesterone during follicular development by FSH and LH in sheep[J]. Anim Reprod, 2022, 19(2): e20220027. doi: 10.1590/1984-3143-ar2022-0027 URL
[50]	An LP, 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. doi: 10.1016/j.bbrc.2012.06.016 URL
[51]	Ghosh D, Egbuta C, Kanyo JE, et al. Phosphorylation of human placental aromatase CYP19A1[J]. Biochem J, 2019, 476(21): 3313-3331. doi: 10.1042/BCJ20190633 pmid: 31652308