Effect of Ezrin on Oocyte Maturation and Early Embryonic Development in Mice

doi:10.7668/hbnxb.20194791

Abstract

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

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.

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Figures/Tables 18

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

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

Fig.2 Validation of siRNA efficiency by Western Blot

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

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.

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

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

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

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

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

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.

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

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.

Fig.8 Rate of abnormal spindle position

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.

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

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

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

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