L-半胱氨酸对绵羊卵巢颗粒细胞增殖、凋亡和类固醇激素分泌的影响

doi:10.11843/j.issn.0366-6964.2024.01.018

Abstract

Abstract: This study was conducted to investigate the effects of L-cysteine (L-Cys) on proliferation, apoptosis and the secretion of steroid hormones (progesterone and estrogen, P₄ and E₂) in ovine ovarian granulosa cells (GCs). Thirty sexually mature ewes aged between 1-1.5 years with the similar body weight and the same feeding condition were selected, and the fresh ovaries were collected. Then, GCs were collected from the dominant follicles (2-8 mm), and the GCs were randomly divided into 5 groups with 6 replicates per group. GCs were treated with 0, 100, 300, 500 and 700 μmol· L^-1 L-Cys for 48 h. The cell proliferation, apoptosis assay and the secretion of steroid hormones were measured using the CCK-8 kit, Annexin V-FITC/PI flow cytometry and ELISA, respectively. Real-time quantitative PCR and Western blot were used to analyze the mRNA and protein expression of the cell proliferation related genes (PCNA, CCND2, CCNB1), apoptosis-related genes (BAX, Caspase-3, Bcl-2) and steroid-secretion-related genes (STAR, 3β-HSD, CYP11A1, CYP19A1). The results showed that 100, 300 and 500 μmol·L^-1 L-Cys significantly promoted the cell viability (P<0.05) and significantly inhibited the apoptotic rate (P<0.05) of GCs. The addition of 300 μmol·L^-1 L-Cys significantly inhibited the secretion of P₄ (P<0.05). The mRNA expression of proliferation-related genes (PCNA, CCND2, CCNB1) were upregulated in the 300 μmol·L^-1 and 500 μmol·L^-1 L-Cys groups, while the protein expression of proliferation-related proteins (PCNA, CCND2, CCNB1) were increased in the 300 μmol·L^-1 L-Cys group (P<0.05). The mRNA and protein expressions of apoptosis-related genes (BAX, Caspase-3) were significantly downregulated in 100 μmol·L^-1 and 300 μmol·L^-1 L-Cys groups (P<0.05). The mRNA and protein expressions of the steroid-related genes (STAR, 3β-HSD) were significantly decreased in 300 μmol·L^-1 L-Cys group (P<0.05), but the mRNA and protein expressions of the steroid-related genes (CYP11A1, CYP19A1) were significantly increased in 100 μmol·L^-1 and 300 μmol·L^-1 L-Cys groups (P<0.05). In summary, L-Cys promoted cell proliferation and inhibited the apoptosis of ovine ovarian GCs by upregulating the mRNA and protein expressions of PCNA, CCNB1, CCND2, Bcl-2 and downregulating the mRNA and protein expressions of BAX, Caspase-3; L-Cys inhibited secretion of P₄ by downregulating the mRNA and protein expressions of STAR, 3β-HSD and upregulating the mRNA and protein expressions of CYP11A1.

Key words: sheep, ovarian granulosa cells, L-cysteine, proliferation, apoptosis, steroidogenesis

CLC Number:

S826.3

DUAN Xiangru, KANG Jia, YANG Ruochen, SHAN Xinyu, LI Taichun, ZHAO Wen, ZHANG Yingjie, LIU Yueqin. Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 179-191.

References 34

[1]	SUN S, LIU S M, LUO J, et al.Repeated pregnant mare serum gonadotropin-mediated oestrous synchronization alters gene expression in the ovaries and reduces reproductive performance in dairy goats[J].Reprod Domest Anim, 2019, 54(6):873-881.
[2]	WANG Y, DUAN C H, GUO Y X, et al.Effects of glucose on glycolysis and steroidogenesis as well as related gene expression in ovine granulosa cells in vitro[J].Small Ruminant Res, 2022, 215:106766.
[3]	AN R, WANG X F, YANG L, et al.Polystyrene microplastics cause granulosa cells apoptosis and fibrosis in ovary through oxidative stress in rats[J].Toxicology, 2021, 449:152665.
[4]	AN X P, MA H D, LIU Y H, et al.Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1[J].J Anim Sci Biotechnol, 2020, 11:102.
[5]	WALSH S W, MATTHEWS D, BROWNE J A, et al.Acute dietary restriction in heifers alters expression of genes regulating exposure and response to gonadotrophins and IGF in dominant follicles[J].Anim Reprod Sci, 2012, 133(1-2):43-51.
[6]	COMIN A, PERIC T, MONTILLO M, et al.Luteal activity and effect of dietary energy restriction on follicular development in lactating cows[J].Reprod Domest Anim, 2017, 52(4):632-639.
[7]	HAN N R, KIM H Y, YANG W M, et al.Glutamic acid ameliorates estrogen deficiency-induced menopausal-like symptoms in ovariectomized mice[J].Nutr Res, 2015, 35(9):774-783.
[8]	HAN N R, KIM N R, KIM H M, et al.Cysteine prevents menopausal syndromes in ovariectomized mouse[J].Reprod Sci, 2016, 23(5):670-679.
[9]	YIN J, REN W K, YANG G, et al.L-Cysteine metabolism and its nutritional implications[J].Mol Nutr Food Res, 2016, 60(1):134-146.
[10]	CLEMENTE PLAZA N, REIG GARCÍA-GALBIS M, MARTÍNEZ-ESPINOSA R M.Effects of the usage of L-cysteine (L-Cys) on human health[J].Molecules, 2018, 23(3):575.
[11]	MOHAMMADI H, FARZINPOUR A, VAZIRY A.Reproductive performance of breeder quails fed diets supplemented with L-cysteine-coated iron oxide nanoparticles[J].Reprod Domest Anim, 2017, 52(2):298-304.
[12]	李书元.褪黑素和N-乙酰半胱氨酸对绵羊卵泡细胞增殖的影响[D].兰州:西北民族大学, 2022.LI S Y.Effects of Melatonin and N-acetyl cysteine on the proliferation of sheep follicle cells[D].Lanzhou:Northwest Minzu University, 2022.(in Chinese)
[13]	HAO E Y, WANG D H, CHANG L Y, et al.Melatonin regulates chicken granulosa cell proliferation and apoptosis by activating the mTOR signaling pathway via its receptors[J].Poult Sci, 2020, 99(11):6147-6162.
[14]	MONTE A P O, BEZERRA MÉ S, MENEZES V G, et al.Involvement of phosphorylated Akt and FOXO3a in the effects of growth and differentiation Factor-9 (GDF-9) on inhibition of follicular apoptosis and induction of granulosa cell proliferation after in vitro culture of sheep ovarian tissue[J].Reprod Sci, 2021, 28(8):2174-2185.
[15]	ELGEBALY M M, HAZAA A B M, AMER H A, et al.L-Cysteine improves bovine oocyte developmental competence in vitro via activation of oocyte-derived growth factors BMP-15 and GDF-9[J].Reprod Domest Anim, 2022, 57(7):734-742.
[16]	CHOI J Y, KANG J T, PARK S J, et al.Effect of 7, 8-dihydroxyflavone as an antioxidant on in vitro maturation of oocytes and development of parthenogenetic embryos in pigs[J].J Reprod Dev, 2013, 59(5):450-456.
[17]	JI Y, WU Z L, DAI Z L, et al.Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells[J].Amino Acids, 2016, 48(1):149-156.
[18]	李伊健, 尉斌艳, 何雨彤, 等.PCNA、AR和INHA基因在藏羊和小尾寒羊睾丸中的表达差异[J].中国草食动物科学, 2022, 42(4):27-30, 77.LI Y J, WEI B Y, HE Y T, et al.Differential expression of PCNA, AR and INHA genes in the testis of Tibetan Sheep and Small-tailed Han Sheep[J].China Herbivore Science, 2022, 42(4):27-30, 77.(in Chinese)
[19]	朱文文, 肖葛琼.CDK1和CCNB1表达水平在肝细胞癌中的临床意义[J].健康研究, 2023, 43(1):70-73, 79.ZHU W W, XIAO G Q.The clinical sighificance of CDK1 and CCNB1 expression levels in hepatocellular carcinoma[J].Health Research, 2023, 43(1):70-73, 79.(in Chinese)
[20]	MJELLE R, HEGRE S A, AAS P A, et al.Cell cycle regulation of human DNA repair and chromatin remodeling genes[J].DNA Repair (Amst), 2015, 30:53-67.
[21]	WANG Z, LIU D X, WANG F W, et al.L-Cysteine promotes the proliferation and differentiation of neural stem cells via the CBS/H2S pathway[J].Neuroscience, 2013, 237:106-117.
[22]	KASHYAP D, GARG V K, GOEL N.Intrinsic and extrinsic pathways of apoptosis:role in cancer development and prognosis[J].Adv Protein Chem Struct Biol, 2021, 125:73-120.
[23]	REED J C.Double identity for proteins of the Bcl-2 family[J].Nature, 1997, 387(6635):773-776.
[24]	HO Y S, LEE H M, MOU T C, et al.Suppression of nitric oxide-induced apoptosis by N-acetyl-l-cysteine through modulation of glutathione, bcl-2, and bax protein levels[J].Mol Carcinog, 1997, 19(2):101-113.
[25]	SHI J X, LIU C Y, CHEN M Y, et al.The interference effects of bisphenol A on the synthesis of steroid hormones in human ovarian granulosa cells[J].Environ Toxicol, 2021, 36(4):665-674.
[26]	OGUNLADE B, GBOTOLORUN S C, OGUNLADE A A.D-ribose-L-cysteine modulates lead acetate-induced hematobiochemical alterations, hormonal imbalance, and ovarian toxicity in adult female Wistar rats[J].Drug Chem Toxicol, 2022, 45(4):1606-1613.
[27]	SREERANGARAJA URS D B, WU W H, KOMRSKOVA K, et al.Mitochondrial function in modulating human granulosa cell steroidogenesis and female fertility[J].Int J Mol Sci, 2020, 21(10):3592.
[28]	PESCADOR N, SOUMANO K, STOCCO D M, et al.Steroidogenic acute regulatory protein in bovine corpora lutea[J].Biol Reprod, 1996, 55(2):485-491.
[29]	CHENG J C, HAN X Y, MENG Q X, et al.HB-EGF upregulates StAR expression and stimulates progesterone production through ERK1/2 signaling in human granulosa-lutein cells[J].Cell Commun Signal, 2022, 20(1):166.
[30]	ZHANG C W, ZHANG X L, XIA Y J, et al.Association between polymorphisms of the CYP11A1 gene and polycystic ovary syndrome in Chinese women[J].Mol Biol Rep, 2012, 39(8):8379-8385.
[31]	EIMERL S, ORLY J.Regulation of steroidogenic genes by insulin-like growth factor-1 and follicle-stimulating hormone:differential responses of cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, and 3β-hydroxysteroid dehydrogenase/isomerase in rat granulosa cells[J].Biol Reprod, 2002, 67(3):900-910.
[32]	RYTELEWSKA E, KISIELEWSKA K, GUDELSKA M, et al.The effect of orexin a on the StAR, CYP11A1 and HSD3B1 gene expression, as well as progesterone and androstenedione secretion in the porcine uterus during early pregnancy and the oestrous cycle[J].Theriogenology, 2020, 143:179-190.
[33]	PARWEEN S, DINARDO G, BAJ F, et al.Differential effects of variations in human P450 oxidoreductase on the aromatase activity of CYP19A1 polymorphisms R264C and R264H[J].J Steroid Biochem Mol Biol, 2020, 196:105507.
[34]	WANG M M, LI Y, MOLENAAR A, et al.Vitamin E and selenium supplementation synergistically alleviate the injury induced by hydrogen peroxide in bovine granulosa cells[J].Theriogenology, 2021, 170:91-106.

Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells

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