猪胚胎早期卵裂时间与其发育潜能关系的初步研究

doi:10.11843/j.issn.0366-6964.2020.04.010

摘要/Abstract

摘要： 旨在探讨初次卵裂时间对猪孤雌胚胎发育潜能及其基因相对表达水平的影响。本试验从健康母猪卵巢上抽取卵母细胞进行体外成熟培养，将猪孤雌激活胚胎分为早期卵裂组（16~22 h）与晚期卵裂组（26~32 h）统计比较卵裂率和囊胚率，并对囊胚的多能性相关基因Oct4、Sox2、Klf4等和凋亡相关基因Bcl-xl、Bax、Caspase-3的相对表达水平进行分析检测。结果表明，猪孤雌激活胚胎在16~22 h发生卵裂的为50%~60%，而26 h之后发生卵裂的不到20%，在18 h前完成第一次卵裂的胚胎囊胚发育率为79%，42 h后发生初次卵裂的胚胎无法发育至囊胚期。猪孤雌激活胚胎早期卵裂组的囊胚发育率显著高于晚期卵裂组（P<0.05）。早期卵裂组囊胚的Oct4、Nanog、Sox2、Klf4基因的表达量显著高于晚期卵裂组（P<0.05），Oct4、Sox2、Klf4基因的相对表达量极显著高于晚期卵裂组（P<0.01），Bax和Caspase-3基因的相对表达水平极显著低于晚期卵裂组（P<0.01），而Bcl-xl作为保护因子其表达量相对于晚期卵裂胚胎（26~32 h）显著上调（P<0.05）。结果显示，初次卵裂时间较早的猪孤雌激活胚胎发育潜能显著高于较晚卵裂胚胎，其囊胚多能性相关基因表达上调，凋亡相关基因表达下调，卵裂时间可作为鉴定猪孤雌激活胚胎发育潜力的重要参数。

关键词: 猪, 初次卵裂, 发育潜能, 多能基因, 凋亡基因

Abstract: This study aimed to explore the effect of first cleavage time on the development potential and relative gene expression levels in parthenogenetic embryos of the pig. The oocytes were extracted from the ovary of a healthy sow for maturation in vitro, the pig parthenogenetic embryos were divided into two groups:early cleavage group(16-22 h) and late cleavage group (26-32 h), their cleavage rate and blastocyst rate were statistically compared. The expression levels of pluripotency-related genes Oct4, Sox2, Klf4 and other apoptosis-related genes Bcl-xl, Bax, Caspase-3 were detected and analyzed. The results showed that 50%-60% cleavage in parthenogenetically activated embryos of the pig occured at 16-22 h and less than 20% after 26 h. The embryos blastocyst development rate which completed the first cleavage before 18 h was 79%. Whereas, the embryos which completed the first cleavage after 42 h were not able to develop to the blastocyst stage. The blastocysts development rate in the early cleavage group of parthenogenetically activated pig embryos was significantly higher compared to the late cleavage group (P<0.05), the expression level of Oct4, Nanog, Sox2, and Klf4 genes in the early cleavage group were significantly higher than those in the late cleavage group (P<0.05). The expression level of Oct4, Sox2, Klf4 genes in the early cleavage group were extremely significantly higher than those in the late cleavage group (P<0.01); While the relative expression level of Bax and Caspase-3 genes in the early cleavage group were extremely significantly lower than those in the late cleavage group (P<0.01); However, the expression level of Bcl-xl as a protective factor was significantly up-regulated compared to the late cleavage embryos (26-32 h, P<0.05). The results indicate that the parthenogenetic activation potential of pigs with earlier first cleavage time is significantly higher than that of the late cleavage embryos. The expression of blastocyst pluripotency-related genes is up-regulated and the expression of apoptosis-related genes is down-regulated. The cleavage time as important parameters can be used to identify developmental potential of pig parthenogenetic activated embryos.

Key words: pig, the first cleavage, developmental potential, pluripotent gene, apoptosis gene

中图分类号:

S828.2

李田田, 熊鑫, 段林伟, 李志鹏, 伏彭辉, 徐祎雪, 石德顺, 刘庆友, 崔奎青. 猪胚胎早期卵裂时间与其发育潜能关系的初步研究[J]. 畜牧兽医学报, 2020, 51(4): 743-753.

LI Tiantian, XIONG Xin, DUAN Linwei, LI Zhipeng, FU Penghui, XU Yixue, SHI Deshun, LIU Qingyou, CUI Kuiqing. Primary Study on the Effect of Early Cleavage Time on the Developmental Potential of Pig Embryos[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(4): 743-753.

参考文献 41

[1]	YU Y,DING C H,WANG E Y,et al.Piezo-assisted nuclear transfer affects cloning efficiency and may cause apoptosis[J].Reproduction, 2007, 133(5):947-954.
[2]	WHARF E,DIMITRAKOPOULOS A,KHALAF Y,et al.Early embryo development is an indicator of implantation potential[J].Reprod Biomed Online,2004,8(2):212-218.
[3]	贾楠,韦多,郝好英,等.早卵裂对IVF助孕妊娠结局预测价值的探讨[J].医药论坛杂志,2017,38(8):1-3.JIA N,WEI D,HAO H Y,et al.Value of embryo early cleavage on clinical pregnancy of IVF[J].Journal of Medical Forum,2017,38(8):1-3.(in Chinese)
[4]	SANTOS-RIBEIRO S,TOURNAYE H,P POLYZOS N P.Trends in ectopic pregnancy rates following assisted reproductive technologies in the UK:a 12-year nationwide analysis including 160000 pregnancies[J].Hum Reprod,2016,31(2):393-402.
[5]	YOU J,KIM J,LIM J,et al.Anthocyanin stimulates in vitro development of cloned pig embryos by increasing the intracellular glutathione level and inhibiting reactive oxygen species[J].Theriogenology,2010,74(5):777-785.
[6]	李超强,蒋善芳,吴亚敏,等.胚胎早期卵裂观察在胚胎发育潜能评估中的应用分析[J].中国优生与遗传杂志,2015,23(11):114-116.LI C Q,JIANG S F,WU Y M,et al.Application analysis on embryo development potential of early cleavage observation[J].Chinese Journal of Birth Health & Heredity,2015,23(11):114-116.(in Chinese)
[7]	李萍,陈媛媛.早卵裂与胚胎质量及临床妊娠的关系[J].中国优生与遗传杂志,2016,24(2):111-113.LI P,CHEN Y Y.The correlation between early cleavage and clinical pregnancy[J].Chinese Journal of Birth Health & Heredity,2016, 24(2):111-113.(in Chinese)
[8]	MIZOBE Y,TOKUNAGA M,OYA N,et al.Synchrony of the first division as an index of the blastocyst formation rate during embryonic development[J].Reprod Med Biol,2018,17(1):64-70.
[9]	SHOUKIR Y,CAMPANA A,FARLEY T,et al.Early cleavage of in-vitro fertilized human embryos to the 2-cell stage:a novel indicator of embryo quality and viability[J].Hum Reprod,1997,12(7):1531-1536.
[10]	BASILE N,VIME P,FLORENSA M,et al.The use of morphokinetics as a predictor of implantation:a multicentric study to define and validate an algorithm for embryo selection[J].Hum Reprod,2015,30(2):276-283.
[11]	MARTÍNEZ M,SANTALÓ J,RODRÍGUEZ A,et al.High reliability of morphokinetic annotations among embryologists[J].Hum Reprod Open,2018,2018,2018(3):hoy009.
[12]	OCHOTA M,NIZANSKI W.Time of early cleavage affects the developmental potential of feline preimplantation embryos in vitro[J]. Theriogenology,2017,89:26-31.
[13]	ISOM S C,LI R F,WHITWORTH K M,et al.Timing of first embryonic cleavage is a positive indicator of the in vitro developmental potential of porcine embryos derived from in vitro fertilization,somatic cell nuclear transfer and parthenogenesis[J].Mol Reprod Dev,2012, 79(3):197-207.
[14]	LONERGAN P,A GUTIÉRREZ-ADÁN,PINTADO B,et al.Relationship between time of first cleavage and the expression of IGF-I growth factor,its receptor,and two housekeeping genes in bovine two-cell embryos and blastocysts produced in vitro[J].Mol Reprod Dev,2000, 57(2):146-152.
[15]	VAN SOOM A,YSEBAERT M T,DE KRUIF A.Relationship between timing of development,morula morphology,and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos[J].Mol Reprod Dev,1997,47(1):47-56.
[16]	BREVINI T A L,LONERGAN P,CILLO F,et al.Evolution of mRNA polyadenylation between oocyte maturation and first embryonic cleavage in cattle and its relation with developmental competence[J].Mol Reprod Dev,2002,63(4):510-517.
[17]	VANDAELE L,MATEUSEN B,MAES D,et al.Is apoptosis in bovine in vitro produced embryos related to early developmental kinetics and in vivo bull fertility?[J].Theriogenology,2006,65(9):1691-1703.
[18]	CHOUDHARY G S,AL-HARBI S,ALMASAN A.Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis[M]//MOR G,ALVERO A B.Apoptosis and Cancer:Methods and Protocols.New York:Humana Press,2015:13-21.
[19]	POIRIER M,SMITH O E,THERRIEN J,et al.Resiliency of equid H19 imprint to somatic cell reprogramming by oocyte nuclear transfer and genetically induced pluripotency[J].Biol Reprod,2019,doi:10.1093/biolre/ioz168.
[20]	VANDAELE L,MATEUSEN B,MAES D,et al.Is apoptosis in bovine in vitro produced embryos related to early developmental kinetics and in vivo bull fertility?[J].Theriogenology,2006,65(9):1691-1703.
[21]	PERS-KAMCZYC E,PAWLAK P,RUBES J,et al.Early cleaved bovine embryos show reduced incidence of chromosomal aberrations and higher developmental potential on day 4.5 post-insemination[J].Reprod Domest Anim,2012,47(6):899-906.
[22]	PARK M J,KIM E Y,KANG M J,et al.Investigation of the developmental potential and developmental kinetics of bovine parthenogenetic and somatic cell nuclear transfer embryos using a time-lapse monitoring system[J].Cell Reprogram,2017,19(4):245-254.
[23]	赵天,李谷月,潘阳阳,等.Oct 4在牦牛卵母细胞及早期胚胎发育过程中的表达[J].畜牧兽医学报,2017,48(3):577-584.ZHAO T,LI G Y,PAN Y Y.et al.The expression of octamer-binding transcription factor 4 gene (Oct 4) in yak (Bos grunneins) oocytes and preimplantation embryos produced by in vitro fertilization[J].Acta Veterinaria et Zootechnica Sinica,2017,48(3):577-584.(in Chinese)
[24]	FRUM T,HALBISEN M A,WANG C Y,et al.Oct4 cell-autonomously promotes primitive endoderm development in the mouse blastocyst[J]. Dev Cell,2013,25(6):610-622.
[25]	郭诗萌.猪早期胚胎发育中体外培养体系优化及OCT4、NANOG功能研究[D].哈尔滨:东北农业大学,2019.GUO S M.Optimization of in vitro culture system and function of OCT4 and NANOG on early embryonic development in pig[D]. Harbin:Northeast Agricultural University,2019.(in Chinese)
[26]	张艳艳,彭辉,肖天放.哺乳动物早期胚胎滋养外胚层发育的研究进展[J].畜牧兽医学报,2018,49(10):2080-2085.ZHANG Y Y,PENG H,XIAO T F.Research progress of trophectoderm development of early embryo in mammals[J].Acta Veterinaria et Zootechnica Sinica, 2018,49(10):2080-2085.(in Chinese)
[27]	CHOUDHAR G S,AL-HARBI S,ALMASAN A.Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis[M]//MOR G,ALVERO A B.Apoptosis and Cancer:Methods and Protocols.New York:Humana Press,2015:1-9.
[28]	WANG Y F,SONG Q X,HUANG X,et al.Long noncoding RNA GAS5 promotes apoptosis in primary nucleus pulposus cells derived from the human intervertebral disc via Bcl-2 downregulation and caspase-3 upregulation[J].Mol Med Rep,2019,19(3):2164-2172.
[29]	DANG-NGUYEN T Q,KIKUCHI K,SOMFAI T,et al.Evaluation of developmental competence of in vitro-produced porcine embryos based on the timing,pattern and evenness of the first cleavage and onset of the second cleavage[J].J Reprod Dev,2010,56(6):593-600.
[30]	OROZCO-LUCERO E,DUFORT I,ROBERT C,et al.Rapidly cleaving bovine two-cell embryos have better developmental potential and a distinctive mRNA pattern[J].Mol Reprod Dev,2014,81(1):31-41.
[31]	毛献宝,薛林涛,谭卫红,等.早期卵裂:移植胚胎选择的一个重要评估指标[J].中华生殖与避孕杂志,2018,38(11):875-879.MAO X B,XUE L T,TAN W H,et al.Early cleavage:an important evaluating indicator for selecting embryos to transfer[J].Chinese Journal of Reproduction and Contraception,2018,38(11):875-879.(in Chinese)
[32]	FENWICK J,PLATTEAU P,MURDOCH A P,et al.Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro[J].Hum Reprod,2002,17(2):407-412.
[33]	SALUMETS A,HYDÉN-GRANSKOG C,MÄKINEN S,et al.Early cleavage predicts the viability of human embryos in elective single embryo transfer procedures[J].Hum Reprod,2003,18(4):821-825.
[34]	LONERGAN P,KHATIR H,PIUMI F,et al.Effect of time interval from insemination to first cleavage on the developmental characteristics,sex ratio and pregnancy rate after transfer of bovine embryos[J].J Reprod Fertil,1999,117(1):159-167.
[35]	KAITH S,SAINI M,RAJA A K,et al.Early cleavage of handmade cloned buffalo (Bubalus bubalis) embryos is an indicator of their developmental competence and quality[J].Reprod Domest Anim,2015,50(2):214-220.
[36]	FU J,WANG X J,WANG Y W,et al.The influence of early cleavage on embryo developmental potential and IVF/ICSI outcome[J].J Assist Reprod Genet,2009,26(8):437-441.
[37]	LEE M J,LEE R K K,LIN M H,et al.Cleavage speed and implantation potential of early-cleavage embryos in IVF or ICSI cycles[J].J Assist Reprod Genet,2012,29(8):745-750.
[38]	VANDAELE L,MATEUSEN B,MAES D G D,et al.Temporal detection of caspase-3 and -7 in bovine in vitro produced embryos of different developmental capacity[J].Reproduction,2007,133(4):709-718.
[39]	FAIR T,MURPHY M,RIZOS D,et al.Analysis of differential maternal mRNA expression in developmentally competent and incompetent bovine two-cell embryos[J].Mol Reprod Dev,2004,67(2):136-144.
[40]	VIGNEAULT C,MCGRAW S,MASSICOTTE L,et al.Transcription factor expression patterns in bovine in vitro-derived embryos prior to maternal-zygotic transition[J].Biol Reprod,2004,70(6):1701-1709.
[41]	DODE M A N,DUFORT I,MASSICOTTE L,et al.Quantitative expression of candidate genes for developmental competence in bovine two-cell embryos[J].Mol Reprod Dev,2006,73(3):288-297.