绵羊磷脂酶C-γ1对绵羊卵母细胞体外成熟的影响

doi:10.11843/j.issn.0366-6964.2022.10.018

Abstract

Abstract: The maturation rate of oocyte can be used as the index to measure the quality and development ability of oocytes cultured in vitro. The purpose of this study was to investigate whether PLC-γ1 was involved in in vitro maturation of sheep oocytes and its effects. Sheep oocytes were cultured in mature medium containing different concentrations of U73122 (PLC inhibitor) and m-3M3FBS (PLC activator), with 150 cells per concentration. The experiment was repeated 3 times. The cell maturation rate, cleavage rate and morula rate were calculated to screen out the optimal inhibitory and promoting concentration of PLC-γ1. The mRNA levels of PLC-γ1, BAK, BAX, CASP3, CASP8, P53 and BCL6 in oocytes in treatment of 0.5 μmol·L^-1 U73122 and 0.5 μmol·L^-1 m-3M3FBS were detected by qPCR for 48 h. Western blot was used to detect the expression of PLC-γ1, BAK, BAX, CASP3, CASP8, P53 and BCL6 proteins in oocytes in treatment of 0.5 μmol·L^-1 U73122 and 0.5 μmol·L^-1 m-3M3FBS for 48 h. The experiment was repeated 3 times with 150 cells per concentration. In sheep oocytes, 0.5 μmol·L^-1 U73122 was the optimal concentration to promote maturation, and 0.5 μmol·L^-1 m-3M3FBS was the optimal concentration to inhibit maturation. The cleavage rate and blastocyst rate in 0.5 μmol·L^-1 U73122 treatment group were lower than those in control group. Cleavage rate and blastocyst rate in 0.5 μmol·L^-1 m-3M3FBS treatment group were higher than those in control group. The qPCR results showed that, compared with the control group, the mRNA expression levels of BAK, BAX, CASP3, CASP8 and P53 genes in U73122 group significantly increased, while the mRNA expression levels of PLC-γ1 and BCL6 genes significantly decreased, these were contrast in m-3M3FBS group. Western blot results showed that, compared with the control group, the protein expression levels of BAK, BAX and CASP8 significantly increased in U73122 group, while the protein expression levels of PLC-γ1 and BCL6 significantly decreased, while the protein expression levels of P53 and CASP3 did not change significantly. In the m-3M3FBS group, the protein expression levels of PLC-γ1 and BCL6 significantly increased, the protein expression levels of BAX, P53 and CASP3 significantly decreased, while the protein expression levels of BAK and CASP8 did not change significantly. In conclusion, this study suggests that PLC-γ1 plays an important role in in vitro maturation culture of sheep oocytes and regulates oocyte maturation and early embryo development.

Key words: PLC-γ1, sheep oocyte, U73122, m-3M3FBS

CLC Number:

S826.3

LIU Xinjie,WU Xiaoxue,LIU Suping,YUAN Liming,CHEN Ning,SAIWU Jiafu. Effects of Sheep Phospholipase C-γ1 on in vitro Maturation of Sheep Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3459-3469.

References 40

[1]	MIYANO T.Bringing up small oocytes to eggs in pigs and cows[J].Theriogenology, 2003, 59(1):61-72.
[2]	ALAM H, LEE J, MIYANO T.Inhibition of PDE3A sustains meiotic arrest and gap junction of bovine growing oocytes in in vitro growth culture[J].Theriogenology, 2018, 118:110-118.
[3]	WIRLEITNER B, OKHOWAT J, VIŠTEJNOVÁ L, et al.Relationship between follicular volume and oocyte competence, blastocyst development and live-birth rate:Optimal follicle size for oocyte retrieval[J].Ultrasound Obstet Gynecol, 2018, 51(1):118-125.
[4]	LEAL G R, MONTEIRO C A S, SOUZA-FABJAN J M G, et al.Role of cAMP modulator supplementations during oocyte in vitro maturation in domestic animals[J].Anim Prod Sci, 2018, 199:1-14.
[5]	FUSHII M, YAMADA R, MIYANO T.In vitro growth of bovine oocytes in oocyte-cumulus cell complexes and the effect of follicle stimulating hormone on the growth of oocytes[J].J Reprod Dev, 2021, 67(1):5-13.
[6]	MOTLÍK J, FULKA J.Factors affecting meiotic competence in pig oocytes[J].Theriogenology, 1986, 25(1):87-96.
[7]	DUAN J X, CHEN H L, XU D J, et al.17β-estradiol improves the developmental ability, inhibits reactive oxygen species levels and apoptosis of porcine oocytes by regulating autophagy events[J].J Steroid Biochem Mol Biol, 2021, 209:105826.
[8]	TŮMOVÁ L, PETR J, ŽALMANOVÁ T, et al.Calcineurin expression and localisation during porcine oocyte growth and meiotic maturation[J].Anim Reprod Sci, 2013, 141(3-4):154-163.
[9]	MOCCIA F, LODOLA F, DRAGONI S, et al.Ca2+ signalling in endothelial progenitor cells:A novel means to improve cell-based therapy and impair tumour vascularisation[J].Curr Vasc Pharmacol, 2014, 12(1):87-105.
[10]	DAI L Z, ZHUANG L H, ZHANG B C, et al.DAG/PKCδ and IP3/Ca2+/CaMK IIβ operate in parallel to each other in PLCγ1-driven cell proliferation and migration of human gastric adenocarcinoma cells, through Akt/mTOR/S6 pathway[J].Int J Mol Sci, 2015, 16(12):28510-28522.
[11]	柳成荫.下丘脑PLCγ/Ca2+/GnRH通路在膳食诱导肥胖引起的小鼠雄激素下降中的作用研究[D].沈阳:中国医科大学, 2019.LIU C Y.Study the effect of hypothalamic PLCγ/Ca2+/GnRH pathway on mouse testosterone decline in diet induced obesity[D].Shenyang:China Medical University, 2019.(in Chinese)
[12]	WELLS A, GRANDIS J R.Phospholipase C-γ1 in tumor progression[J].Clin Exp Metastasis, 2003, 20(4):285-290.
[13]	JANG H J, SUH P G, LEE Y J, et al.PLCγ1:potential arbitrator of cancer progression[J].Adv Biol Regul, 2018, 67:179-189.
[14]	MING G L, SONG H J, BERNINGER B, et al.Phospholipase C-γ and phosphoinositide 3-kinase mediate cytoplasmic signaling in nerve growth cone guidance[J].Neuron, 1999, 23(1):139-148.
[15]	DITTMAR T, HUSEMANN A, SCHEWE Y, et al.Induction of cancer cell migration by epidermal growth factor is initiated by specific phosphorylation of tyrosine 1248 of c-erbB-2 receptor via EGFR[J].FASEB J, 2002, 16(13):1823-1825.
[16]	SMITH M R, COURT D W, KIM H K, et al.Overexpression of phosphoinositide-specific phospholipase Cgamma in NIH 3T3 cells promotes transformation and tumorigenicity[J].Carcinogenesis, 1998, 19(1):177-185.
[17]	袁利明, 胡广东, 刘欣杰, 等.绵羊磷脂酶C-γ1基因克隆及生物信息学分析[J].中国畜牧兽医, 2021, 48(6):1883-1893.YUAN L M, HU G D, LIU X J, et al.Cloning and bioinformatics analysis of PLC-γ1 gene in sheep[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(6):1883-1893.(in Chinese)
[18]	TIWARI M, PRASAD S, TRIPATHI A, et al.Apoptosis in mammalian oocytes:A review[J].Apoptosis, 2015, 20(8):1019-1025.
[19]	SLOT K A, VOORENDT M, DE BOER-BROUWER M, et al.Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro-and active caspase-3 in the rat ovary[J].J Endocrinol, 2006, 188(2):179-192.
[20]	XUE J, YANG G, DING H, et al.Role of NSC319726 in ovarian cancer based on the bioinformatics analyses[J].Onco Targets Ther, 2015, 8:3757-3765.
[21]	SERHATLIOGLU I, BILGIN B, KACAR E, et al.Agomelatine modulates calcium signaling through protein kinase C and phospholipase C-mediated mechanisms in rat sensory neurons[J].J Cell Physiol, 2019, 234(7):10741-10746.
[22]	陆会宁, 齐燕姣.影响绵羊卵母细胞体外成熟因素研究进展[J].黑龙江农业科学, 2013(9):147-150.LU H N, QI Y J.Research progresses on the factors influencing maturation in vitro of sheep oocyte[J].Heilongjiang Agricultural Sciences, 2013(9):147-150.(in Chinese)
[23]	BOYER J L, WALDO G L, HARDEN T K, et al.βγ-subunit activation of G-protein-regulated phospholipase C[J].J Biol Chem, 1992, 267(35):25451-25456.
[24]	KUWAHARA E, YAMAMOTO J, YANO Y, et al.Lysophosphatidic acid stimulates hyaluronan production by mouse cumulus-oocyte complexes[J].Reprod Med Biol, 2014, 13(2):95-102.
[25]	RISKE K A, DÖBEREINER H G.Diacylglycerol-rich domain formation in giant stearoyl-oleoyl phosphatidylcholine vesicles driven by phospholipase C activity[J].Biophys J, 2003, 85(4):2351-2362.
[26]	KASHIR J, NOMIKOS M, LAI F A, et al.Sperm-induced Ca2+ release during egg activation in mammals[J].Biochem Biophys Res Commun, 2014, 450(3):1204-1211.
[27]	JI Q S, WINNIER G E, NISWENDER K D, et al.Essential role of the tyrosine kinase substrate phospholipase C-γ 1 in mammalian growth and development[J].Proc Natl Acad Sci U S A, 1997, 94(7):2999-3003.
[28]	LAWSON N D, MUGFORD J W, DIAMOND B A, et al.Phospholipase C gamma-1 is required downstream of vascular endothelial growth factor during arterial development[J].Genes Dev, 2003, 17(11):1346-1351.
[29]	MA A C H, LIANG R, LEUNG A Y H.The role of phospholipase C gamma 1 in primitive hematopoiesis during zebrafish development[J].Exp Hematol, 2007, 35(3):368-373.
[30]	CUI X S, LI X Y, KIM N H.Cdc42 is implicated in polarity during meiotic resumption and blastocyst formation in the mouse[J].Mol Reprod Dev, 2007, 74(6):785-794.
[31]	CHEN H L, YANG Y F, WANG Y L, et al.Phospholipase C inhibits apoptosis of porcine primary granulosa cells cultured in vitro[J].J Ovarian Res, 2019, 12(1):90.
[32]	CHEN H L, YANG Y F, WANG Y L, et al.The effects of phospholipase C on oestradiol and progesterone secretion in porcine granulosa cells cultured in vitro[J].Reprod Domest Anim, 2019, 54(9):1236-1243.
[33]	CHEN H L, CHENG J Y, YANG Y F, et al.Phospholipase C inhibits apoptosis of porcine oocytes cultured in vitro[J].J Cell Biochem, 2020, 121(7):3547-3559.
[34]	PAPANDILE A, TYAS D, O'MALLEY D M, et al.Analysis of caspase-3, caspase-8 and caspase-9 enzymatic activities in mouse oocytes and zygotes[J].Zygote, 2004, 12(1):57-64.
[35]	QUINLAN L R, FAHERTY S, KANE M T.Phospholipase C and protein kinase C involvement in mouse embryonic stem-cell proliferation and apoptosis[J].Reproduction, 2003, 126(1):121-131.
[36]	FERREIRA R M, CHIARATTI M R, MACABELLI C H, et al.The infertility of repeat-breeder cows during summer is associated with decreased mitochondrial DNA and increased expression of mitochondrial and apoptotic genes in oocytes[J].Biol Reprod, 2016, 94(3):66.
[37]	JAIN A, JAIN T, KUMAR P, et al.Follicle-stimulating hormone-induced rescue of cumulus cell apoptosis and enhanced development ability of buffalo oocytes[J].Domest Anim Endocrinol, 2016, 55:74-82.
[38]	KIM K J, CHUN J L, LEE K B, et al.Effect of acteoside on the re-localization and abnormal morphology of mitochondria in porcine oocytes during in vitro maturation[J].J Assist Reprod Genet, 2016, 33(7):939-948.
[39]	ARIAS-ÁLVAREZ M, GARCÍA-GARCÍA R M, LÓPEZ-TELLO J, et al.In vivo and in vitro maturation of rabbit oocytes differently affects the gene expression profile, mitochondrial distribution, apoptosis and early embryo development[J].Reprod Fertil Dev, 2017, 29(9):1667-1679.
[40]	CHOI I C, ZHU J, CAMPBELL K H S.The combined treatment of calcium ionophore with strontium improves the quality of ovine SCNT embryo development[J].Zygote, 2013, 21(2):139-150.

Effects of Sheep Phospholipase C-γ1 on in vitro Maturation of Sheep Oocytes

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