PLCγ1基因对绵羊早期胚胎体外发育的影响

doi:10.11843/j.issn.0366-6964.2022.08.016

摘要/Abstract

摘要： 旨在探究磷脂酶Cγ1（phospholipase Cgamma 1，PLCγ1）基因对绵羊早期胚胎体外发育的影响，为揭示PLCγ1基因在早期胚胎发育过程中的作用机制奠定基础。本研究选取包裹3层以上颗粒细胞的绵羊卵母细胞为试验材料，体外培养成熟后将其分为两组，利用显微注射技术将PLCγ1基因导入MⅡ期（减数第二次分裂中期）卵母细胞（n=127）中为试验组，以显微注射空载体pcDNA3.1-EGFP作为对照组（n=120），经48 h后统计其卵裂率；并利用Ca²⁺探针Rhod 2-AM监测MⅡ卵母细胞和显微注射后16、48、72、96、120 h时各时期卵母细胞以及早期胚胎内Ca²⁺波动。结果显示，PLCγ1基因显微注射后卵母细胞被激活，卵裂率为（（19.67±0.2）%，P<0.01），桑椹胚发育率为（（9.00±0.17）%，P<0.05）；PLCγ1基因注射后，卵母细胞和早期胚胎不同发育时期有Ca²⁺浓度变化，可观察发现Ca²⁺主要分布在胞质中且注射48 h时可达到峰值（P<0.01）。结果表明，PLCγ1基因可以引起绵羊卵母细胞发育过程中发生Ca²⁺振荡，启动其卵裂，并促使早期胚胎的继续发育。

关键词: PLCγ1, Ca²⁺, 卵母细胞, 桑椹胚, 绵羊

Abstract: The purpose of this study was to investigate the effect of PLCγ1 on the development of sheep early embryo in vitro, and to lay a foundation for revealing the action mechanism of PLCγ1 in the development of sheep early embryo. In this study, sheep oocytes with more than 3 layers of granulosa cells were selected as experimental materials, and they were divided into two groups after mature in vitro culture. PLCγ1 was introduced into MⅡ oocytes (n=127) by microinjection as experimental group. Microinjection of empty vector pcDNA3.1-EGFP was used as the control group (n=120), and after 48 h the cleavage rate were measured. The Ca²⁺ probe Rhod 2-AM was used to monitor the Ca²⁺ fluctuation of MⅡ oocytes and early embryos at 16, 48, 72, 96 and 120 h after microinjection. The results showed that the cleavage rate was ((19.67±0.2)%, P<0.01)) and morula development rate was ((9.00±0.17)%,P<0.05)) after microinjection of PLCγ1. After injection of PLCγ1, Ca²⁺ concentration in oocytes and early embryos varied at different developmental stages.It was observed that Ca²⁺ was mainly distributed in the cytoplasm and reached the peak at 48 h after injection (P<0.01). The results showed that PLCγ1 gene could induce Ca²⁺ oscillation during ovine oocyte development, initiate cleavage, and promote the development of early embryo.

Key words: PLCγ1, Ca²⁺, oocyte, morula, sheep

中图分类号:

S826.3

吴晓雪, 袁利明, 刘欣杰, 刘素平, 陈宁, 赛务加甫. PLCγ1基因对绵羊早期胚胎体外发育的影响[J]. 畜牧兽医学报, 2022, 53(8): 2578-2585.

WU Xiaoxue, YUAN Liming, LIU Xinjie, LIU Suping, CHEN Ning, SAIWU Jiafu. Effect of PLCγ1 Gene on the Development of Sheep Early Embryo in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2578-2585.

参考文献 35

[1]	周荣, 杨秀萍.卵母细胞减数分裂的调控机制研究进展[J].国际生殖健康/计划生育杂志, 2008, 27(2):80-84.ZHOU R, YANG X P.Recent advances in understanding the regulation of oocyte meiosis[J].Journal of International Reproductive Health/Family Planning, 2008, 27(2):80-84.(in Chinese)
[2]	ZHANG M J, SU Y Q, SUGIURA K, et al.Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes[J].Science, 2010, 330(6002):366-369.
[3]	SUN L, HODEIFY R, HAUN S, et al.Ca2+homeostasis regulates Xenopus oocyte maturation[J].Biol Reprod, 2008, 78(4):726-735.
[4]	刘红林, 汪河海, 范必勤, 等.显微注射Ca2+激活小鼠MⅡ期卵母细胞[J].畜牧兽医学报, 1997, 28(3):206-210.LIU H L, WANG H H, FAN B Q, et al.Activation of mouse oocytes (M Ⅱ) by intracellular microinjection of calcium[J]. Acta Veterinaria et Zootechnica Sinica, 1997, 28(3):206-210.(in Chinese)
[5]	JANG H J, SUH P G, LEE Y J, et al.PLCγ1:potential arbitrator of cancer progression[J].Adv Biol Regul, 2017, 67:179-189.
[6]	WOO S J, JO H I, LEE H H, et al.Molecular characterization and expression analysis of olive flounder (Paralichthys olivaceus) phospholipase C gamma 1 and gamma 2[J].Fish Shellfish Immunol, 2017, 63:353-366.
[7]	JANG H J, SUH P G, LEE Y J, et al.PLCγ1:potential arbitrator of cancer progression[J].AdvBiol Regul, 2018, 67:179-189.
[8]	MANDAL S, BANDYOPADHYAY S, TYAGI K, et al.Recent advances in understanding the molecular role of phosphoinositide-specific phospholipase C gamma 1 as an emerging onco-driver and novel therapeutic target in human carcinogenesis[J].Biochim Biophys Acta Rev Cancer, 2021, 1876(2):188619.
[9]	MALCUIT C, KNOTT J G, HE C L, et al.Fertilization and inositol 1, 4, 5-trisphosphate (IP3)-induced calcium release in type-1 inositol 1, 4, 5-trisphosphate receptor down-regulated bovine eggs[J].Biol Reprod, 2005, 73(1):2-13.
[10]	郝肖琼, 贾方毅, 付旭阳.PLCγ1在小鼠卵母细胞减数分裂恢复中的作用[J].中国比较医学杂志, 2019, 29(12):16-22, 51.HAO X Q, JIA F Y, FU X Y.The role of PLCγ1 in mouse oocyte meiotic resumption[J].Chinese Journal of Comparative Medicine, 2019, 29(12):16-22, 51.(in Chinese)
[11]	RUNFT L L, CARROLL D J, GILLETT J, et al.Identification of a starfish egg PLC-γ that regulates Ca2+ release at fertilization[J].Dev Biol, 2004, 269(1):220-236.
[12]	FINLEY J.Oocyte activation and latent HIV-1 reactivation:AMPK as a common mechanism of action linking the beginnings of life and the potential eradication of HIV-1[J].Med Hypotheses, 2016, 93:34-47.
[13]	FERRER-BUITRAGO M, BONTE D, DESUTTER P, et al.Single Ca2+transients vs oscillatory Ca2+signaling for assisted oocyte activation:limitations and benefits[J].Reproduction, 2018, 155(2):R105-R119.
[14]	KRAWCZYK P, MATUSZYK J.The mechanism of phospholipase Cγ1 activation[J].Postepy Hig Med Dosw(Online), 2011, 65:470-477.
[15]	FEITOSA W B, LOPES E, VISINTIN J A, et al.Endoplasmic reticulum distribution during bovine oocyte activation is regulated by protein kinase C via actin filaments[J].J Cell Physiol, 2020, 235(7-8):5823-5834.
[16]	KAUFMAN M H.Parthenogenetic activation of oocytes[J/OL].Cold Spring Harb Protoc, 2018, 2018(1), doi:10.1101/pdb.prot094409.
[17]	SINGH K P, MOHAPATRA S K, KAUSHIK R, et al.Parthenogenetic activation of buffalo (Bubalusbubalis) oocytes:comparison of different activation reagents and different media on their developmental competence and quantitative expression of developmentally regulated genes[J].Zygote, 2021, 29(1):49-58.
[18]	LAN G C, HAN D, WU Y G, et al.Effects of duration, concentration, and timing of ionomycin and 6-dimethylaminopurine (6-DMAP) treatment on activation of goat oocytes[J].Mol Reprod Dev, 2005, 71(3):380-388.
[19]	MEHLMANN L M, CARPENTER G, RHEE S G, et al.SH2 Domain-mediated activation of phospholipase Cγis not required to initiate Ca2+release at fertilization of mouse eggs[J].Dev Biol, 1998, 203(1):221-232.
[20]	袁瑞莹.不同处理方式对小鼠卵母细胞孤雌激活钙离子振荡的影响[D].广州:南方医科大学, 2018.YUAN R Y.Effects of different treatments on the[Ca2+] i oscillations inparthenogenetic activation of mouse oocyte[D].Guangzhou:Southern Medical University, 2018.(in Chinese)
[21]	YESTE M, JONES C, AMDANI S N, et al.Oocyte activation and fertilisation:crucial contributors from the sperm and oocyte[J].Results Probl Cell Differ, 2017, 59:213-239.
[22]	SILVESTRE F, BONI R, FISSORE R A, et al.Ca2+signaling during maturation of cumulus-oocyte complex in mammals[J].Mol Reprod Dev, 2011, 78(10-11):744-756.
[23]	TAROZZI N, NADALINI M, COTICCHIO G, et al.The paternal toolbox for embryo development and health[J].Mol Hum Reprod, 2021, 27(7):gaab042.
[24]	FINKELSTEIN M, ETKOVITZ N, BREITBART H.Ca2+signaling in mammalian spermatozoa[J].Mol Cell Endocrinol, 2020, 516:110953.
[25]	MARTIN J H, BROMFIELD E G, AITKEN R J, et al.Biochemical alterations in the oocyte in support of early embryonic development[J].Cell Mol Life Sci, 2017, 74(3):469-485.
[26]	SETTE C, BEVILACQUA A, GEREMIA R.Involvement ofphospholipase Cγ1 in mouse egg activation induced by a truncated form of the C-kit tyrosine kinase present in spermatozoa[J].J Cell Biol, 1998, 142(4):1063-1074.
[27]	PAN B, LI J L.The art of oocyte meiotic arrest regulation[J].Reprod Biol Endocrinol, 2019, 17(1):8.
[28]	ANIFANDIS G, MICHOPOULOS A, DAPONTE A, et al.Artificial oocyte activation:physiological, pathophysiological and ethical aspects[J].Syst Biol Reprod Med, 2019, 65(1):3-11.
[29]	PARRINGTON J, ARNOULT C, FISSORE R A.Theeggstraordinary story of how life begins[J].Mol Reprod Dev, 2019, 86(1):4-19.
[30]	YANG F L, HUANG L, TSO A, et al.Inositol 1, 4, 5-trisphosphate receptors are essential for fetal-maternal connection and embryo viability[J].PLoS Genet, 2020, 16(4):e1008739.
[31]	LIANG S H, ZHAO J, WANG Q Y, et al.Carbon monoxide enhances calcium transients and glucose-stimulated insulin secretion from pancreatic β-cells by activating phospholipase C signal pathway in diabetic mice[J]. Biochem Biophys Res Commun, 2021, 582:1-7.
[32]	SETTE C, PARONETTO M P, BARCHI M, et al.Tr-kit -induced resumption of the cell cycle in mouse eggs requires activation of a Src-like kinase[J].EMBO J, 2002, 21(20):5386-5395.
[33]	杨霖, 罗深秋.PLC-γ1对PDGF介导的细胞内钙流的影响[J].中国医学物理学杂志, 2001, 18(2):93-94, 100.YANG L, LUO S Q.The effect of PLC-γ1on mobilization of intracellular Ca2+ induced by PDGF[J].Chinese Journal of Medical Physics, 2001, 18(2):93-94, 100.(in Chinese)
[34]	YIN X Q, ECKBERG W R.Characterization of phospholipases C β and γ and their possible roles in Chaetopterus egg activation[J].Mol Reprod Dev, 2010, 76(5):460-470.
[35]	WU H, SMYTH J, LUZZI V, et al.Spermfactor induces intracellular free calcium oscillations by stimulating the phosphoinositide pathway[J].Biol Reprod, 2001, 64(5):1338-1349.