玻璃化冷冻牛GV卵母细胞全基因组甲基化模式初探

旨在研究玻璃化冷冻对牛GV期卵母细胞全基因组甲基化的影响。本研究收集新鲜、玻璃化冷冻的牛GV卵母细胞,采用单细胞全基因组甲基化测序(ScWGBS)技术对新鲜、玻璃化法冷冻牛GV卵母细胞的全基因组甲基化水平进行检测,旨在揭示两者DNA甲基化模式的差异。结果表明,玻璃化冷冻不会对牛GV卵母细胞的全基因甲基化水平造成显著影响。基于基因本体(GO)和信号通路(KEGG)对140个差异甲基化区域(DMRs)进行分析,发现DMRs主要参与细胞发育、细胞骨架组织等功能,主要富集在PI3K-Akt信号通路、GnRH信号通路等,并筛选出与卵母细胞成熟(TSC2)、细胞骨架(NUDC)、细胞活力(MAFK)等相关的基因。上述结果,可为提高GV卵母细胞玻璃化冷冻效率奠定信息基础和研究方向。     

新鲜、玻璃化冷冻牛卵母细胞体外受精囊胚全基因组甲基化模式初探

旨在初步分析新鲜及玻璃化冷冻牛卵母细胞体外受精囊胚全基因组甲基化模式。本研究采用单细胞全基因组甲基化测序技术(scWGMS)检测新鲜、玻璃化冷冻牛卵母细胞体外受精囊胚全基因组甲基化水平和差异甲基化区域(DMR),探讨两者之间DNA甲基化水平上的差异。结果表明,新鲜卵母细胞体外受精囊胚的整体甲基化水平显著高于玻璃化冷冻卵母细胞体外受精囊胚的整体甲基化水平(P<0.05)。采用基因本体分析(GO)和相关信号通路(KEGG)对143个DMRs分析,发现生物学过程主要显著富集在新陈代谢、生长发育、细胞定位、细胞刺激反应等,通路主要富集在生长发育、核酸结合及组蛋白乙酰化上,并筛选出几个与之相关的候选基因(FARP2、PI4KA、FAM3D、NCOR2、ZNF827等)。本研究初步发现,玻璃化冷冻牛卵母细胞体外受精囊胚的全基因组甲基化水平显著降低,且DMR区域主要集中在ATP结合、生长发育及组蛋白乙酰化,为提高玻璃化冷冻卵母细胞体外受精囊胚质量提供信息参考。     

玻璃化冷冻牛GV卵母细胞全基因组甲基化模式初探

旨在研究玻璃化冷冻对牛GV期卵母细胞全基因组甲基化的影响。本研究收集新鲜、玻璃化冷冻的牛GV卵母细胞，采用单细胞全基因组甲基化测序（ScWGBS）技术对新鲜、玻璃化法冷冻牛GV卵母细胞的全基因组甲基化水平进行检测，旨在揭示两者DNA甲基化模式的差异。结果表明，玻璃化冷冻不会对牛GV卵母细胞的全基因甲基化水平造成显著影响。基于基因本体（GO）和信号通路（KEGG）对140个差异甲基化区域（DMRs）进行分析，发现DMRs主要参与细胞发育、细胞骨架组织等功能，主要富集在PI3K-Akt信号通路、GnRH信号通路等，并筛选出与卵母细胞成熟（TSC2）、细胞骨架（NUDC）、细胞活力（MAFK）等相关的基因。上述结果，可为提高GV卵母细胞玻璃化冷冻效率奠定信息基础和研究方向。     

钌红对玻璃化冷冻牛卵母细胞线粒体Ca~(2+)的调控研究

玻璃化冷冻会严重损伤哺乳动物卵母细胞的线粒体功能,进而极大地限制了其解冻后的发育能力。为此,本试验设置3个钌红(RR)处理组,即牛卵母细胞用含0.5、1、2μmol/L RR的玻璃化冷冻液进行冷冻,解冻后放入含0.5、1、2μmol/L RR的体外成熟液中继续培养0.5h,同时,新鲜卵母细胞一部分不进行冷冻,一部分用不含RR的冷冻液进行玻璃化冷冻,分别作为新鲜对照组和玻璃化冷冻对照组,然后共检测5组牛卵母细胞线粒体Ca~(2+)水平、ATP含量及孤雌激活后胚胎的发育能力,进而研究RR对玻璃化冷冻牛卵母细胞线粒体Ca~(2+)水平的调控作用。结果显示:(1)玻璃化冷冻显著提高了牛卵母细胞中线粒体Ca~(2+)水平(P0.05),而2μmol/L RR处理组线粒体Ca~(2+)水平显著低于冷冻对照组(P0.05),但与新鲜组相比无显著差异(P0.05);(2)玻璃化冷冻显著降低了牛卵母细胞中ATP含量(P0.05),2μmol/L RR处理组卵母细胞中ATP含量显著高于冷冻对照组及0.5、1μmol/L RR处理组(P0.05);(3)玻璃化冷冻对照组卵裂率、囊胚率显著低于新鲜对照组(P0.05),1μmol/L处理组卵裂率、囊胚率与新鲜对照组相比无显著差异(P0.05)。综上所述,RR处理能显著抑制解冻后牛卵母细胞线粒体Ca~(2+)流入,保护线粒体功能,提高其发育能力。本试验结果为正向调控玻璃化冷冻卵母细胞线粒体Ca~(2+)水平,进而提高其发育能力,促进玻璃化冷冻卵母细胞的广泛应用提供了参考依据。     

玻璃化冻存对驴卵母细胞超微结构的影响

试验旨在探究玻璃化冷冻对驴卵母细胞发育的影响,寻求驴卵母细胞冷冻的最佳条件。通过对不同发育时期的驴卵母细胞进行玻璃化冷冻,冷冻复苏后分别进行成熟培养和孤雌激活,并对GV期未冷冻组(对照组)、GV期冷冻组、IVM-MⅡ冷冻组卵母细胞微丝和线粒体超微结构进行免疫荧光标记,统计冷冻复苏后卵母细胞形态正常率、成熟率、孤雌激活卵裂率、超微结构正常率。结果表明,GV期冷冻组卵母细胞的形态正常率与GV期未冷冻组(对照组)间无显著差异(P0.05),成熟率和卵裂率均显著低于对照组(P0.05);IVM-MⅡ冷冻组的卵裂率显著低于对照组(P0.05),且卵裂后细胞发育受到阻滞。冷冻组微丝在皮质区分布明显减少的卵母细胞数目增多,冷冻组卵母细胞的线粒体数量明显低于对照组,由此可以说明冷冻对卵母细胞超微结构有损伤,从而导致复苏后成熟率下降,影响卵母细胞的受精和体外发育,且GV期冷冻组较IVM-MⅡ冷冻组在微丝与线粒体结构上有较小损伤,发育状态较好。     

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

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(P0.05),但A组和B组的卵裂率、囊胚率均显著低于C组(P0.05)。以|log_2(fold change)|≥2,Q0.05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(P0.05)。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。     

采用dCas9-SunTag-DNMT3A技术调控玻璃化冷冻牛卵母细胞IVF囊胚中IGF2R基因甲基化水平

旨在探究dCas9-SunTag-DNMT3A编辑系统对玻璃化冷冻牛卵母细胞IVF囊胚中IGF2R基因甲基化水平及胚胎发育能力的影响,为冷冻卵母细胞/胚胎特定位点DNA甲基化的精确调控奠定基础。本研究将经过体外成熟的牛卵母细胞进行玻璃化冷冻,随后进行体外受精,将受精所得到的原核胚进行dCas9-SunTag-DNMT3A编辑系统的注射,统计并计算卵母细胞的发育情况;通过亚硫酸盐测序的方式检测IGF2R基因启动子的甲基化水平,并利用荧光定量PCR检测IGF2R及相关基因的表达水平。与冷冻组相比,注射不同浓度的dCas9-SunTag-DNMT3A编辑系统后,只有40 ng·μL^-1组显著地提高了玻璃化冷冻卵母细胞IVF后的发育能力(P＜0.05),20和60 ng·μL^-1组间差异不显著(P＞0.05),但40 ng·μL^-1组发育效果仍然显著低于新鲜对照组(P＜0.05);对检测冷冻组、新鲜组、40 ng·μL^-1组IGF2R基因启动子甲基化水平分析发现,40 ng·μL^-1组水平与新鲜组相似,显著高于冷冻组(P＜0.05);荧光定量试验结果显示,40 ng·μL^-1组IGF2R基因mRNA表达水平相较于冷冻组显著降低(P＜0.05),与新鲜组相似。注射40 ng·μL^-1的dCas9-SunTag-DNMT3A甲基化编辑系统能够通过有效升高IGF2R基因启动子甲基化水平(P＜0.05)及显著降低其mRNA表达水平(P＜0.05),来正向调节玻璃化冷冻卵母细胞IVF胚胎的发育情况,提高胚胎发育能力,使得其卵裂率和囊胚率都得到显著提高(P＜0.05),同时促进胚胎发育相关基因的表达。     

小鼠卵母细胞和胚胎不同冷冻方法的比较研究

探讨程序化冷冻与玻璃化冷冻对小鼠GV期卵母细胞及二细胞期胚胎的复苏率及其发育潜能的影响。通过小鼠的卵母细胞与早期胚胎的不同冷冻方法的比较,为后续阿旺绵羊的胚胎冷冻保存提供参考。采用程序化冷冻与玻璃化冷冻技术,分别冷冻小鼠GV期卵母细胞及二细胞期胚胎,复苏后培养,比较不同冷冻处理后的复苏率、成熟率与囊胚率。小鼠GV期卵母细胞程序化冷冻复苏率(48.00%±5.29%)显著低于玻璃化冷冻复苏率(65.00%±5.00%),有统计学差异(P=0.0147<0.05);而程序化冷冻后复苏卵母细胞的发育成熟率略高于玻璃化冷冻组,但无统计学意义。小鼠二细胞期胚胎程序化冷冻组复苏率(76.00%±2.00%)显著高于玻璃化冷冻组复苏率(70.00%±2.00%),有统计学差异(P=0.0213<0.05);冷冻后复苏胚胎发育的囊胚率程序化冷冻略低于玻璃化冷冻及对照组,但无统计学意义。     

钌红对玻璃化冷冻牛卵母细胞线粒体Ca2+的调控研究

玻璃化冷冻会严重损伤哺乳动物卵母细胞的线粒体功能,进而极大地限制了其解冻后的发育能力。为此,本试验设置3个钌红（RR）处理组,即牛卵母细胞用含0.5、1、2 μmol／L RR的玻璃化冷冻液进行冷冻,解冻后放入含0.5、1、2 μmol／L RR的体外成熟液中继续培养0.5 h,同时,新鲜卵母细胞一部分不进行冷冻,一部分用不含RR的冷冻液进行玻璃化冷冻,分别作为新鲜对照组和玻璃化冷冻对照组,然后共检测5组牛卵母细胞线粒体Ca²⁺水平、ATP含量及孤雌激活后胚胎的发育能力,进而研究RR对玻璃化冷冻牛卵母细胞线粒体Ca²⁺水平的调控作用。结果显示：①玻璃化冷冻显著提高了牛卵母细胞中线粒体Ca²⁺水平（P＜0.05）,而2 μmol／L RR处理组线粒体Ca²⁺水平显著低于冷冻对照组（P＜0.05）,但与新鲜组相比无显著差异（P＞0.05）;②玻璃化冷冻显著降低了牛卵母细胞中ATP含量（P＜0.05）,2 μmol／L RR处理组卵母细胞中ATP含量显著高于冷冻对照组及0.5、1 μmol/L RR处理组（P＜0.05）;③玻璃化冷冻对照组卵裂率、囊胚率显著低于新鲜对照组（P＜0.05）,1 μmol／L处理组卵裂率、囊胚率与新鲜对照组相比无显著差异（P＞0.05）。综上所述,RR处理能显著抑制解冻后牛卵母细胞线粒体Ca²⁺流入,保护线粒体功能,提高其发育能力。本试验结果为正向调控玻璃化冷冻卵母细胞线粒体Ca²⁺水平,进而提高其发育能力,促进玻璃化冷冻卵母细胞的广泛应用提供了参考依据。     

玻璃化冻存对驴卵母细胞超微结构的影响

试验旨在探究玻璃化冷冻对驴卵母细胞发育的影响，寻求驴卵母细胞冷冻的最佳条件。通过对不同发育时期的驴卵母细胞进行玻璃化冷冻，冷冻复苏后分别进行成熟培养和孤雌激活，并对GV期未冷冻组（对照组）、GV期冷冻组、IVM-M Ⅱ冷冻组卵母细胞微丝和线粒体超微结构进行免疫荧光标记，统计冷冻复苏后卵母细胞形态正常率、成熟率、孤雌激活卵裂率、超微结构正常率。结果表明，GV期冷冻组卵母细胞的形态正常率与GV期未冷冻组（对照组）间无显著差异（P>0.05），成熟率和卵裂率均显著低于对照组（P<0.05）；IVM-M Ⅱ冷冻组的卵裂率显著低于对照组（P<0.05），且卵裂后细胞发育受到阻滞。冷冻组微丝在皮质区分布明显减少的卵母细胞数目增多，冷冻组卵母细胞的线粒体数量明显低于对照组，由此可以说明冷冻对卵母细胞超微结构有损伤，从而导致复苏后成熟率下降，影响卵母细胞的受精和体外发育，且GV期冷冻组较IVM-M Ⅱ冷冻组在微丝与线粒体结构上有较小损伤，发育状态较好。     

Cryopreservation of immature buffalo oocytes: Effects of cytochalasin B pretreatment on the efficiency of cryotop and solid surface vitrification methods

The aim of the present study was to compare the efficiency of the solid surface (SSV), cryotop (CT) vitrification methods and cytochalasin B (CB) pretreatment for cryopreservation of immature buffalo oocytes. Cumulus‐oocyte complexes (COCs) were placed for 1 min in TCM199 containing 10% dimethylsulfoxide (DMSO), 10% ethylene glycol (EG), and 20% fetal bovine serum, and then transferred for 30 s to base medium containing 20% DMSO, 20% EG and 0.5 mol/L sucrose. CB pretreated ((+)CB) or non‐pretreated ((?)CB) COCs were vitrified either by SSV or CT. Surviving vitrified COCs were selected for in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of viable oocytes after vitrification in CT groups (82%) was significantly lower (P < 0.05) than that in a fresh control group (100%), but significantly higher (P < 0.05) than those in SSV groups (71–72%). Among vitrified groups, the highest maturation rate was obtained in the CT (?)CB group (32%). After IVF, the cleavage and blastocyst formation rates were similar among vitrified groups but significantly lower than those of the control group. In conclusion, a higher survival rate of oocytes after vitrification and IVM was obtained in the CT group compared with that in the SSV group, indicating the superiority of the CT method. Pretreatment with CB did not increase the viability, maturation or embryo development of vitrified oocytes.     

Factors affecting cryopreservation of porcine oocytes

Recent improvements in cryopreservation of mammalian eggs enable the long-term preservation of female germ cells in several mammalian species. Nevertheless, cryopreservation of porcine oocytes is still considered as a challenge. Although the use of vitrification techniques result in reasonable survival rates, developmental competence of vitrified oocytes has been compromised. Alterations of zona characteristics, cytoskeleton, mitochondrial functions and antioxidant-defense ability caused by vitrification are among the most frequently observed malformations which may be responsible for the low developmental competence of cryopreserved porcine oocytes. Furthermore, in vitro maturation, fertilization and embryo culture technologies, which are indispensable for generating embryos from cryopreserved oocytes, generate high rates of abnormal fertilization (polyspermy) and additional stress in resultant embryos further compromising their developmental competence. As a result, embryo development of porcine cryopreserved oocytes is still at low level and to date no piglet has been produced from such oocytes. The aim of the present review is to summarize knowledge on viability and developmental competence of vitrified porcine oocytes and to give ideas for future perspectives for the improvement of porcine oocyte cryopreservation technology.     

Comparative Effects of Porcine Oocytes Vitrified at the GV and MⅡ Stages

This study was designed to compare the efficiency of porcine oocytes vitrified at the GV and MⅡ stages. The vitrified GV oocytes were matured in vitro and then evaluated their cortical granule distribution with immunofluorescence, CD9 protein level with Western blotting and zona pellucida dissolution time with pronse digestion method, as compared to vitrified MⅡ oocytes.The results showed that the survival percentages of oocytes vitrified at the GV stage were significantly lower than those vitrified at the MⅡ stage after 2 h of warming (P<0.05);However, there was no difference in maturation rate between vitrified and fresh oocytes (P>0.05). The oocytes vitrified at MⅡ stage resulted in significantly decreased normal cortical granule distribution and CD9 protein level (P<0.05), but no such results were found in vitrified GV oocytes after maturation (P>0.05). In addition, vitrification did not affect the dissolution time of zona pellucida in GV and MⅡ oocytes (P>0.05). The data demonstrated that despite of lower survival rate compared with MⅡ oocytes, vitrified porcine GV oocytes after maturation showed normal polar body extrusion, cortical granule distribution and CD9 protein level.     

Comparison of Ethylene Glycol and Propylene Glycol for the Vitrification of Immature Porcine Oocytes

Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P<0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P<0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC; treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P<0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development.     

Calves Born after Direct Transfer of Vitrified Bovine In Vitro-produced Blastocysts Derived from Vitrified Immature Oocytes

Vitrification has been the method of choice for the cryopreservation of bovine oocytes, as rapid cooling decreases chilling sensitivity. The aim of this study was to determine the in vitro and in vivo survival and the viability of immature oocytes vitrified using super‐cooled liquid nitrogen. Immature oocytes were randomly allocated to three groups: (i) non‐vitrified control group, (ii) vitrified in normal (?196°C) liquid nitrogen (LN₂) and (iii) vitrified in super‐cooled LN₂ (≤?200°C). Open‐pulled glass micropipettes were used as vitrification containers. Immature oocytes were in vitro‐matured, fertilized and cultured to the blastocyst stage. In vitro viability was assessed by cleavage and blastocyst rates on days 2 and 7 of culture respectively. Vitrified blastocysts derived from the immature vitrified oocytes were directly transferred to synchronous recipients. The in vitro embryo development of vitrified immature oocytes was not influenced by the LN₂ state. After direct transfer (one embryo per recipient) of 16 embryos obtained from immature vitrified oocytes (eight from each vitrified group), two healthy calves were born in each group. These results indicated that vitrification of immature bovine oocytes using glass micropipettes under normal or super‐cooled LN₂, resulted in viable blastocysts and live calves following in vitro embryo production.     

猪卵母细胞在GV期与MⅡ期的冷冻保存效果比较研究

为比较猪卵母细胞在GV期与MⅡ期的冷冻保存效果,试验在这两个成熟阶段对其进行玻璃化冷冻,GV期卵母细胞解冻后培养至成熟,MⅡ期卵母细胞解冻后恢复2 h,然后采用免疫荧光标记、Western blotting和链霉蛋白酶溶解方法分别检测它们的皮质颗粒分布、CD9蛋白表达水平和透明带消化时间上的差异。结果表明,GV期卵母细胞在解冻后2 h的存活率显著低于MⅡ期卵母细胞（P<0.05）,但极体排出率与对照卵母细胞无明显差异（P>0.05）;在冷冻MⅡ期卵母细胞中,皮质颗粒的皮质区分布比例和CD9的蛋白表达水平显著下降（P<0.05）,但冷冻GV期卵母细胞经体外成熟后则无明显变化（P>0.05）;冷冻GV期与MⅡ期卵母细胞均不会影响透明带的消化时间（P>0.05）。由此可见,猪卵母细胞在GV期的冷冻存活率虽然较MⅡ期低,但其体外成熟后极体排出率、皮质颗粒分布和CD9蛋白表达水平均未受到冷冻的影响。     

Effect of L‐carnitine on maturation,cryo‐tolerance and embryo developmental competence of bovine oocytes

In this study, the effects of the addition of L‐carnitine in in vitro maturation (IVM) medium for bovine oocytes on their nuclear maturation and cryopreservation were investigated; they were matured in IVM medium supplemented with 0.0, 0.3, 0.6 and 1.2 mg/mL of L‐carnitine (control, 0.3, 0.6 and 1.2 groups, respectively) and some of them were vitrified by Cryotop. Moreover, the effects of L‐carnitine during in vitro fertilization (IVF) and in vitro culture (IVC) on the developmental potential and quality of IVF embryos were also examined. A significantly higher maturation rate of oocytes was obtained for 0.3 and 0.6 mg/mL groups compared with the control (P < 0.05). The blastocyst formation rate in the 0.6 group was significantly improved, whereas the rate in the 1.2 group was significantly decreased when compared with the control group (P < 0.05). No significant difference was found in embryo development between the control and the L‐carnitine group after oocyte vitrification. Supplementation of IVF and IVC media with L‐carnitine had no effect on development to the blastocyst stage of IVM oocytes treated with 0.6 mg/mL L‐carnitine. In conclusion, the supplementation of L‐carnitine during IVM of bovine oocytes improved their nuclear maturation and subsequent embryo development after IVF, but when they were vitrified the improving effects were neutralized.