Ezrin对小鼠卵母细胞成熟及早期胚胎发育的影响

doi:10.7668/hbnxb.20194791

摘要/Abstract

摘要：

旨在探究Ezrin对小鼠卵母细胞成熟、受精及早期胚胎发育的影响,揭示其可能作用机制。分别向小鼠GV期卵母细胞和原核期胚胎注射Ezrin的siRNA(si-Ez)或阴性对照siRNA(si-NC),观察对小鼠卵母细胞成熟、受精和早期胚胎发育的影响,并分别通过扫描电镜和免疫荧光染色的方法观察GV期注射si-Ez对小鼠成熟卵母细胞表面微绒毛生长、纺锤体定位和皮质颗粒迁移的影响。结果显示,GV期敲低Ezrin可显著降低小鼠卵母细胞成熟率,极显著降低受精后早期胚胎的囊胚率,虽可降低成熟卵母细胞的受精率,但差异不显著;在原核期敲低Ezrin,可极显著降低小鼠囊胚细胞数,桑椹胚存活率和囊胚率虽有下降趋势,但与对照组无显著差异;扫描电镜观察发现,敲低小鼠GV期卵母细胞Ezrin后显著降低了MⅡ期卵母细胞表面微绒毛的长度和密度;免疫荧光结果显示,敲低小鼠GV期卵母细胞Ezrin影响了纺锤体和皮质颗粒的迁移。结果表明,Ezrin通过影响微绒毛形成、纺锤体定位和皮质颗粒的正常迁移阻滞了小鼠卵母细胞成熟,进而影响了受精和早期胚胎发育。

关键词: 小鼠, Ezrin, 卵子成熟, 胚胎发育

Abstract:

The aim of this study was to investigate the effect of Ezrin on the maturation of mouse oocytes,fertilization,and early embryo development,as well as to reveal its mechanisms.Mouse germinal vesicle(GV)stage oocytes and pronuclear-stage embryos were injected with siRNA targeting Ezrin(si-Ez)or a negative control siRNA(si-NC),then the rates of oocyte maturation,fertilization,and early embryo development were calculated,the length and density of microvilli on the surface of the MⅡ stage oocytes were detected with scanning electron microscopy,the spindle localization and cortical granule migration were observed using immunofluorescence staining.The results showed that knocking down Ezrin during the GV stage significantly reduced the maturation rate of mouse oocytes and extremely significantly lowered the developmental rate of early-stage embryos after fertilization.While it decreased the fertilization rate of mature oocytes,the difference was not significant.Knocking down Ezrin during the pronuclear stage extremely significantly reduced the total cell number of mouse blastocysts.Although there was a downward trend in the rates of morula and blastocyst survival,there was no significant difference compared to the control group.Scanning electron microscopy revealed that knocking down Ezrin in GV stage mouse oocytes significantly decreased the length and density of microvilli on the surface of MⅡ stage oocytes.Immunofluorescence results showed that knocking down Ezrin in GV stage mouse oocytes affected the migration of the spindle and cortical granules.These results suggest that Ezrin,through its influence on microvilli formation,spindle localization,and cortical granule migration,blocks the maturation of mouse oocytes,consequently affecting fertilization and early embryo development.

Key words: Mouse, Ezrin, Oocyte maturation, Embryo development

谢辉辉, 童彬彬, 李巧丹, 潘富强, 张博超, 廖洪艳, 贾雨珂, 崔剑坤, 李运生, 刘亚. Ezrin对小鼠卵母细胞成熟及早期胚胎发育的影响[J]. 华北农学报, 2024, 39(4): 231-238. doi: 10.7668/hbnxb.20194791.

XIE Huihui, TONG Binbin, LI Qiaodan, PAN Fuqiang, ZHANG Bochao, LIAO Hongyan, JIA Yuke, CUI Jiankun, LI Yunsheng, LIU Ya. Effect of Ezrin on Oocyte Maturation and Early Embryonic Development in Mice[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 231-238. doi: 10.7668/hbnxb.20194791.

http://www.hbnxb.net/CN/Y2024/V39/I4/231

图/表 18

表1 siRNA序列

Tab.1 Sequence of siRNA

siRNA编号 Code of siRNA	序列(5'—3') Sequence(5'—3')
siEz-1816	F: GGCAGGGACAAGUAUAAGATT
	R: UCUUAUACUUGUCCCUGCCTT
siEz-800	F: CCCUUGGACUUAACAUUUATT
	R: UAAAUGUUAAGUCCAAGGGTT
siEz-276	F: CCAGUAUGUAGACAAUAAATT
	R: UUUAUUGUCUACAUACUGGTT
siNC	F: UUCUCCGAACGUGUCACGUTT
	F: ACGUGACACGUUCGGAGAATT

图1 RT-qPCR验证Ezrin siRNA敲低效率不同字母表示差异显著(P< 0.05)。

Fig.1 Validation of siRNA efficiency by RT-qPCR Different lowercase letters indicated significant difference among groups(P<0.05).

图2 免疫印迹验证siRNA敲低效率

Fig.2 Validation of siRNA efficiency by Western Blot

图3 注射siRNA后不同时间小鼠GV期卵母细胞内Ezrin的免疫荧光图

Fig.3 Immunofluorescence images of Ezrin in mouse GV-stage oocytes at different time points after injection of siEz-1816

图4 注射siRNA 后不同时间小鼠卵母细胞内Ezrin免疫荧光相对强度不同大写字母表示差异极显著(P<0.01)。图6,8,10,12同.

Fig.4 Relative immunofluorescence of Ezrin in mouse oocytes at different time points after siRNA Different capital letters indicates extremely significant difference(P<0.01).The same as Fig.6,8,10,12.

表2 米力农抑制不同时间对GV期卵母细胞核成熟率的影响

Tab.2 Effect of the duration of milrinone treatment on the maturation rate of mouse GV-stage oocytes

分组 Groups	重复次数 Repeats	培养GV期卵母细胞数 No. of GV oocytes	卵母细胞成熟率/% Oocyte maturation rate
0 h	3	72	67.20±6.71a
6 h	3	82	65.68±4.01a
12 h	3	78	65.74±4.32a
18 h	3	70	65.41±1.36a
24 h	3	71	34.33±7.88b
30 h	3	76	25.35±5.58b
36 h	3	71	25.26±3.61b

表3 敲低Ezrin对小鼠GV期卵母细胞体外成熟的影响

Tab.3 Effect of Ezrin knockdown on in vitro maturation of GV stage mouse oocytes

组别 Groups	重复次数 Repeats	培养GV期卵母细胞数 No.of mouse oocytes culture	卵母细胞成熟率/% Oocyte maturation rate
CK	3	104	69.62±5.82a
siNC	3	102	67.26±4.81a
siEz-1816	3	108	35.09±1.78b

表4 GV期敲低Ezrin对小鼠卵母细胞体外受精及早期胚胎发育的影响

Tab.4 Effects of knocking down Ezrin at GV stage on the in vitro fertilization and developmental potential of mouse oocytes

分组 Groups	重复次数 Repeats	卵母细胞数 Oocytes	受精率/% Fertility rate	卵裂率/% Cleavage rate	囊胚率/% Blastocyst rate
siNC	8	222	62.59±3.12A	95.97±2.88A	85.54±2.95A
siEz-1816	8	118	51.34±4.94A	90.00±6.81A	48.86±8.62B

表5 原核期敲低Ezrin对小鼠早期胚胎体外发育的影响

Tab.5 Effects of knockdown Ezrin in mouse pronuclear-stage embryos on the developmental potential

分组 Groups	重复次数 No.of Repeats	注射后存活数 No. of zygotes survived	2-细胞存活率/% 2-Cell survival rate	4-细胞存活率/% 4-Cell survival rate	8-细胞存活率/% 8-Cell survival rate	桑椹胚存活率/% Morula survival rate	囊胚存活率/% Blastocyst survival
siNC	4	59	98.61±1.39A	94.44±3.21A	91.67±5.31A	90.28±6.56A	88.89±7.86A
siEz-1816	4	68	95.83±4.17A	91.67±8.33A	89.04±7.56A	75.58±3.68A	74.27±2.77A

表6 GV期敲低Ezrin对小鼠卵母细胞表面微绒毛直径和长度的影响

Tab.6 Effect of knockdown Ezrin at GV-stage on the diameter and length of mouse oocyte microvilli

组别 Groups	测量数 Number of measurement	直径/nm Diameter	长度/μm Length
siNC	100	56.76±0.73a	—
siEz-1816	100	57.82±0.79a	—
siNC	52	—	2.95±0.01a
siEz-1816	50	—	1.09±0.15b

图5 原核期注射siNC或siEz-1816的囊胚免疫荧光图 A1、A2、A3.原核期注射了siNC的囊胚;B1、B2、B3.原核期注射了siEz-1816的囊胚。

Fig.5 Immunofluorescence images of blastocysts which injected with siNC or siEz-1816 at pronuclear-stage A1,B1,C1.Blastocyst injected with siNC at pronuclear-stage;A2,B2,C2.Blastocyst injected with siEz-1816 at pronucleus stage.

图6 原核期注射siNC或siEz-1816的囊胚总细胞计数(n=21)

Fig.6 Total cell number of blastocysts which injected with siNC or siEz-1816 at pronuclear stage(n=21)

图7 不同来源MⅡ期卵母细胞中纺锤体定位 A.体内MⅡ期卵母细胞;B.体外成熟MⅡ期卵母细胞;C.注射siEz-1816的MⅡ期卵母细胞。

Fig.7 Spindle localization in MⅡ stage oocytes A.In vivo MⅡ stage oocyte;B.In vitro matured MⅡ stage oocytes;C.MⅡstage oocytes injected with siEz-1816.

图8 纺锤体位置异常率

Fig.8 Rate of abnormal spindle position

图9 原核期注射siNC或siEz-1816的小鼠成熟卵母细胞表面扫描电镜图像 A1、A2、A3.注射siNC的小鼠成熟卵母细胞扫描电镜;B1、B2、B3.注射siEz-1816的小鼠卵母细胞扫描电镜。

Fig.9 Scanning electron microscopy images of the surface of mouse mature oocytes which injected with siNC or siEz-1816 at pronuclear stage A1,A2,A3.Scanning electron microscopy images of mouse mature oocytes which injected with siNC at pronuclear stage;B1,B2,B3.Scanning electron microscopy images of mouse mature oocytes which injected with siEz at pronuclear stage.

图10 敲低Ezrin对小鼠卵母细胞表面密度的影响

Fig.10 Effect of knockdown Ezrin at GV-stage on the density of mouse oocyte microvilli

图11 敲低Ezrin对小鼠体外成熟卵母细胞中CGs分布的影响

Fig.11 Effect of Ezrin knockdown at GV satge on the CGs distribution in mouse oocytes

图12 试验组与对照组小鼠卵母细胞内CGs分布模式比例

Fig.12 The proportion that distribution patterns of CGs in mouse oocyte between control and experiment group

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