玻璃化冷冻对牦牛未成熟卵母细胞发育能力及COC转录组的影响

doi:10.11843/j.issn.0366-6964.2020.02.010

摘要/Abstract

摘要： 旨在探讨玻璃化冷冻-解冻对牦牛未成熟卵母细胞发育能力及卵丘-卵母细胞复合体（COCs）转录组的影响，为完善牦牛COCs冷冻保存技术提供理论依据。本研究将未经成熟培养的牦牛COCs进行玻璃化冷冻-解冻后分为2组，A组：COCs体外成熟（IVM）后用普通牛精子进行体外受精（IVF），获得的受精卵在G-1胚胎培养液中培养72 h后转入G-2培养液培养96 h；B组：IVF后，受精卵在G-1培养液培养120 h后转入G-2培养液培养48 h；以未进行冷冻处理的新鲜COCs作为对照组（C组）：IVF后，受精卵在G-1培养液培养72 h后转入G-2培养液培养96 h。对牦牛新鲜COCs（n=3）和玻璃化冷冻-解冻的COCs（n=3）进行扩增、建库和转录组测序（RNA-seq）分析。结果发现，B组的卵裂率、囊胚率显著高于A组（P<0.05），但A组和B组的卵裂率、囊胚率均显著低于C组（P<0.05）。以|log₂（fold change）|≥ 2，Q<0.05为阈值，牦牛冻融COCs相对于新鲜COCs共筛选出851个差异表达基因（DEGs），其中上调846个，下调5个。GO分析表明，DEGs主要富集于生物过程、细胞组分和分子功能3大类；KEGG注释结果表明，DEGs富集到258条通路，其中16条通路显著富集（P<0.05）。研究表明，IVF后在G-1培养液中培养120 h可以提高牦牛玻璃化冷冻卵母细胞的后续发育能力；玻璃化冷冻影响牦牛COCs转录组，从而降低卵母细胞的发育潜力。该发现为完善牦牛COCs玻璃化冷冻技术提供了一定的理论基础。

关键词: 牦牛, 卵母细胞, 体外受精, 玻璃化冷冻, 转录组测序

Abstract: The objective of this study was to investigate the effect of vitrification/thawing on developmental competence of immature oocytes and cumulus-oocyte-complexes (COCs) transcriptome of yaks (Bos grunniens), in order to provide theoretical foundation to improve vitrification techniques of yak COCs. Vitrified/thawed yak immature COCs were divided into two groups. Group A:COCs were in vitro matured (IVM) and in vitro fertilized (IVF) with cattle sperms, then in vitro cultured (IVC) in G-1 for 72 h followed by IVC in G-2 for 96 h; Group B:after IVF, zygotes were IVC in G-1 for 120 h followed by IVC in G-2 for 48 h. Fresh immature yak COCs were used as the control (Group C):after IVF, zygotes were IVC in G-1 for 72 h followed by IVC in G-2 for 96 h. Yak fresh immature COCs (n=3) and vitrified/thawed immature COCs (n=3) were used for amplification, library preparation and RNA-seq analysis. The results showed that cleavage rate and blastocyst rate in Group B were significantly higher than those in Group A (P<0.05), but cleavage rate and blastocyst rate in both Group A and B were significantly lower than those in Group C (P<0.05). When|log₂(fold change)|≥ 2 and Q-value <0.05 were set as thresholds for identifying deferentially expressed genes (DEGs), a total of 851 DEGs were detected, of which, 846 were up-regulated and 5 were down-regulated in virtrified/thawed COCs compared to fresh COCs. GO analysis showed DEGs were classified into 3 categories:biological processes, cellular components and molecular functions. KEGG annotation showed that there were 258 pathways, of which, 16 were significantly enriched (P<0.05). In conclusion, the results showed that IVC in G-1 for 120 h after IVF could increase subsequent developmental competence of vitrified yak oocytes. Vitrification affected transcriptome of yak COCs, which reduced developmental potential of yak vitrified oocytes. The result provided a theoretical basis for improving vitrification techniques of yak COCs.

Key words: yak, oocyte, in vitro fertilization, vitrification, RNA-seq

中图分类号:

S823.85

杨远潇, 字向东. 玻璃化冷冻对牦牛未成熟卵母细胞发育能力及COC转录组的影响[J]. 畜牧兽医学报, 2020, 51(2): 288-298.

YANG Yuanxiao, ZI Xiangdong. Effect of Vitrification on Developmental Competence of Immature Oocytes and COC Transcriptome of Yaks[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(2): 288-298.

参考文献 48

[1]	SANCHEZ-PARTIDA L G,KELLY R D W,SUMER H,et al.The generation of live offspring from vitrified oocytes[J].PLoS One,2011,6(6):e21597.
[2]	RALL W F,FAHY G M.Ice-free cryopreservation of mouse embryos at -196℃ by vitrification[J].Nature,1985,313(6003):573-575.
[3]	MONZO C,HAOUZI D,ROMAN K,et al.Slow freezing and vitrification differentially modify the gene expression profile of human metaphase II oocytes[J].Hum Reprod,2012,27(7):2160-2168.
[4]	MOUSSA M,SHU J,ZHANG X H,et al.Cryopreservation of mammalian oocytes and embryos:current problems and future perspectives[J].Sci China Life Sci,2014,57(9):903-914.
[5]	TUCKER M J,WRIGHT G,MORTON P C,et al.Birth after cryopreservation of immature oocytes with subsequent in vitro maturation[J].Fertil Steril,1998,70(3):578-579.
[6]	AONO N,ABE Y,HARA K,et al.Production of live offspring from mouse germinal vesicle-stage oocytes vitrified by a modified stepwise method,SWEID[J].Fertil Steril,2005,84(S2):1078-1082.
[7]	THARASANIT T,COLENBRANDER B,STOUT T A E.Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes[J].Mol Reprod Dev,2006,73(5):627-637.
[8]	VIEIRA A D,FORELL F,FELTRIN C,et al.Calves born after direct transfer of vitrified bovine in vitro-produced blastocysts derived from vitrified immature oocytes[J].Reprod Domest Anim,2008,43(3):314-318.
[9]	KOHAYA N,FUJIWARA K,ITO J,et al.Generation of live offspring from vitrified mouse oocytes of C57BL/6J strain[J].PLoS One,2013,8(3):e58063.
[10]	MOAWAD A R,ZHU J,CHOI I,et al.Effect of cytochalasin B pretreatment on developmental potential of ovine oocytes vitrified at the germinal vesicle stage[J].Cryo Letters,2013,34(6):634-644.
[11]	NIU H R,ZI X D,XIAO X,et al.Developmental competence of frozen-thawed yak (Bos grunniens) oocytes followed by in vitro maturation and fertilization[J].Cryobiology,2014,68(1):152-154.
[12]	NOHALEZ A,MARTINEZ C A,GIL M A,et al.Effects of two combinations of cryoprotectants on the in vitro developmental capacity of vitrified immature porcine oocytes[J].Theriogenology,2015,84(4):545-552.
[13]	EL-SHALOFY A S,MOAWAD A R,DARWISH G M,et al.Effect of different vitrification solutions and cryodevices on viability and subsequent development of buffalo oocytes vitrified at the Germinal Vesicle (GV) stage[J].Cryobiology,2017,74:86-92.
[14]	MOAWAD A R,TAN S L,TAKETO T.Beneficial effects of glutathione supplementation during vitrification of mouse oocytes at the germinal vesicle stage on their preimplantation development following maturation and fertilization in vitro[J].Cryobiology, 2017,76:98-103.
[15]	FATHI M,MOAWAD A R,BADR M R.Production of blastocysts following in vitro maturation and fertilization of dromedary camel oocytes vitrified at the germinal vesicle stage[J].PLoS One,2018,13(3):e0194602.
[16]	NIU H R,ZI X D,XIAO X,et al.Cloning of cDNAs for H1F0,TOP1,CLTA and CDK1 and the effects of cryopreservation on the expression of their mRNA transcripts in yak (Bos grunniens) oocytes[J].Cryobiology,2014,69(1):55-60.
[17]	ARCARONS N,MORATÓ R,VENDRELL M,et al.Cholesterol added prior to vitrification on the cryotolerance of immature and in vitro matured bovine oocytes[J].PLoS One,2017,12(9):e0184714.
[18]	CHEN P,PAN Y,CUI Y,et al.Insulin-like growth factor I enhances the developmental competence of yak embryos by modulating aquaporin 3[J].Reprod Domest Anim,2017,52(5):825-835.
[19]	DINNYÉS A,DAI Y P,JIANG S,et al.High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization,and somatic cell nuclear transfer[J].Biol Reprod,2000,63(2):513-518.
[20]	WANG N,LI C Y,ZHU H B,et al.Effect of vitrification on the mRNA transcriptome of bovine oocytes[J].Reprod Domest Anim, 2017, 52(4):531-541.
[21]	MARIONI J C,MASON C E,MANE S M,et al.RNA-seq:an assessment of technical reproducibility and comparison with gene expression arrays[J].Genome Res,2008,18(9):1509-1517
[22]	RAMSK LD D,LUO S J,WANG Y C,et al.Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells[J].Nat Biotechnol,2012,30(8):777-782.
[23]	PICELLI S,BJ RKLUND Å K,FARIDANI O R,et al.Smart-seq2 for sensitive full-length transcriptome profiling in single cells[J].Nat Methods,2013,10(11):1096-1098.
[24]	兰道亮,熊显荣,林宝山,等.基于微量RNA高通量测序技术的牦牛MII期卵母细胞转录组研究[J].畜牧兽医学报,2014,45(5):722-732.LN D L,XIONG X R,LIN B S,et al.Transcriptome analysis of yak metaphase II (MII) oocytes by a micro-transcriptome sequencing method[J].Acta Veterinaria et Zootechnica Sinica,2014,45(5):722-732.(in Chinese)
[25]	WIENER G,HAN J L,LONG R J.The yak[M].Bangkok:The Regional Office for Asia and the Pacific of the Food and Agriculture Organization of the United Nations,2003.
[26]	ZI X D.Reproduction in female yaks (Bos grunniens) and opportunities for improvement[J].Theriogenology, 2003, 59(5-6):1303-1312.
[27]	YU S J,JU X H,WANG L B,et al.Successful embryo transfer in Tianzhu white yak using standard protocol[J].Sci China Ser C Life Sci,2007, 50(5):655-659.
[28]	ZI X D,YIN R H,CHEN S W,et al.Developmental competence of embryos derived from reciprocal in vitro fertilization between yak (Bos grunniens) and cattle (Bos taurus)[J].J Reprod Dev,2009,55(5):480-483.
[29]	孙永刚,徐惊涛,才让东智,等.体外受精生产犏牛胚胎与移植试验研究[J].畜牧兽医学报,2013,44(5):719-726.SN Y G,XU J T,CAIRANG D Z,et al.The study on cattle×yak in vitro fertilization and embryo transfer[J].Acta Veterinaria et Zootechnica Sinica,2013,44(5):719-726.(in Chinese)
[30]	VAJTA G,HOLM P,KUWAYAMA M,et al.Open Pulled Straw (OPS) vitrification:a new way to reduce cryoinjuries of bovine ova and embryos[J].Mol Reprod Dev,1998,51(1):53-58.
[31]	ZI X D,LIU S,XIA W,et al.Transcriptional profiles of crossbred embryos derived from yak oocytes in vitro fertilized with cattle sperm[J].Sci Rep,2018,8:11571.
[32]	KIM D,LANGMEAD B,SALZBERG S L.HISAT:a fast spliced aligner with low memory requirements[J].Nat Methods,2015, 12(4):357-360.
[33]	QIU Q,ZHANG G J,MA T,et al.The yak genome and adaptation to life at high altitude[J].Nat Genet,2012,44(8):946-949.
[34]	LI B,DEWEY C N.RSEM:accurate transcript quantification from RNA-Seq data with or without a reference genome[J].BMC Bioinformatics,2011,12:323.
[35]	LOVE M I,HUBER W,ANDERS S.Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2[J]. Genome Biol,2014,15(12):550.
[36]	BENJAMINI Y,HOCHBERG Y.Controlling the false discovery rate:a practical and powerful approach to multiple testing[J].J Roy Statist Soc Ser B,1995,57(1):289-300.
[37]	ZANDER-FOX D,CASHMAN K S,LANE M,et al.The presence of 1 mM glycine in vitrification solutions protects oocyte mitochondrial homeostasis and improves blastocyst development[J].J Assist Reprod Genet,2013,30(1):107-116.
[38]	GAO L,JIA G X,LI A,et al.RNA-Seq transcriptome profiling of mouse oocytes after in vitro maturation and/or vitrification[J].Sci Rep,2017,7(1):13245.
[39]	ČABART P,ÚJVÁRI A,PAL M,et al.Transcription factor TFIIF is not required for initiation by RNA polymerase II,but it is essential to stabilize transcription factor TFIIB in early elongation complexes[J].Proc Natl Acad Sci U S A,2011,108(38):15786-15791.
[40]	郭雪娜,诸葛斌,诸葛健.甘油代谢中甘油激酶的研究进展[J].微生物学报,2002,42(4):510-513.GO X N,ZHUGE B,ZHUGE J.Research progress on the glycerol kinase[J].Acta Microbiologica Sinica,2002, 42(4):510-513.(in Chinese)
[41]	LORENZ S,BHATTACHARYYA M,FEILER C,et al.Crystal structure of a Ube2S-ubiquitin conjugate[J].PLoS One,2016,11(2):e0147550.
[42]	NORDZIEKE S,ZOBEL T,FRILNZEL B,et al.A fungal sarcolemmal membrane-associated protein (SLMAP) homolog plays a fundamental role in development and localizes to the nuclear envelope,endoplasmic reticulum,and mitochondria[J].Eukaryot Cell,2015,14(4):345-358.
[43]	郑杰,蒲思颖,杨远潇,等.基于高通量测序的犏牛囊胚玻璃化冷冻损伤机制研究[J].畜牧兽医学报,2017,48(10):1871-1881.ZENG J,PU S Y,YANG Y X,et al.Exploring mechanism for vitrification damage of the cross-bred blastocysts of the yak via high-throughput sequencing[J].Acta Veterinaria et Zootechnica Sinica,2017,48(10):1871-1881.(in Chinese)
[44]	蒲思颖,郑杰,杨远潇,等.牦牛新鲜囊胚与玻璃化冻融囊胚转录组的比较分析[J].畜牧兽医学报,2018,49(4):709-717.P S Y,ZHENG J,YANG Y X,et al.Comparative transcriptome analysis between fresh and vitrified-thawed blastocysts of the yak (Bos grunniens)[J].Acta Veterinaria et Zootechnica Sinica,2018,49(4):709-717.(in Chinese)
[45]	LEDDA S,LEONI G,BOGLIOLO L,et al.Oocyte cryopreservation and ovarian tissue banking[J].Theriogenology, 2001,55(6):1359-1371.
[46]	MARTINO A,POLLARD J W,LEIBO S P.Effect of chilling bovine oocytes on their developmental competence[J].Mol Reprod Dev,1996,45(4):503-512.
[47]	ABOELENAIN M,KAWAHARA M,BALBOULA A Z,et al.Status of autophagy,lysosome activity and apoptosis during corpus luteum regression in cattle[J].J Reprod Dev,2015,61(3):229-236.
[48]	NAGY R A,VAN MONTFOORT A P A,DIKKERS A,et al.Presence of bile acids in human follicular fluid and their relation with embryo development in modified natural cycle IVF[J].Hum Reprod,2015,30(5):1102-1109.