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目的观察溶酶体4次穿膜蛋白β(LAPTM4β)在小鼠卵母细胞及早期胚卵中的表达,分析新基因LAPTM4β在植入前胚卵发育中的作用,为研究植入前胚卵发育的基因调控提供实验依据。方法从50只小鼠的子宫或输卵管中收集小鼠卵母细胞及从原核期到胚泡的早期胚卵共50枚。采用激光扫描共焦显微镜技术与免疫组织化学ABC法,观察小鼠卵母细胞及早期胚卵中LAPTM4β的表达;表达结果用Maticam2206图像分析系统进行半定量分析。结果激光扫描共焦显微镜下显示,小鼠卵母细胞和原核期胚卵细胞质的绿色荧光呈不均匀分布,近细胞膜处荧光信号强;2细胞期、4细胞期、8细胞期和桑椹胚卵裂球胞质中绿色荧光均呈局灶性分布,且靠近细胞膜处胞质中绿色荧光均较强,2细胞期中所见极体内也有绿色荧光信号;小鼠胚泡滋养层细胞及内细胞团中均可见中等强度的绿色荧光。免疫组织化学显示,在小鼠卵母细胞中可见LAPTM4β阳性反应颗粒呈浅棕色;原核期、2细胞期、4细胞期、8细胞期胚及桑椹胚,可见深棕色颗粒,且在卵裂球胞质中呈不均匀分布;胚泡的内细胞团及滋养层细胞中均可见棕色阳性反应颗粒。图像分析结果显示,卵母细胞与原核期胚相比,8细胞期与桑椹胚相比,桑椹胚与胚泡相比,LAPTM4β的表达量有显著差异(P<0.05)。结论LAPTM4β在正常小鼠卵母细胞及植入前各期胚卵中均有表达,且具有规律性;不同阶段早期胚卵中LAPTM4β的表达量均高于卵母细胞,推测LAPTM4β与小鼠早期胚卵的细胞分裂、增殖、分化及胚胎发育相关。
Objective To investigate the expression of LAPTM4β in mouse oocytes and early embryo eggs and to analyze the role of LAPTM4β in preimplantation embryo development. Development of gene regulation to provide experimental evidence. METHODS: Mouse oocytes were collected from the uterus or fallopian tubes of 50 mice and 50 early embryo eggs from prokaryotic stage to blastocyst stage. The laser scanning confocal microscopy and immunohistochemical ABC method were used to observe the expression of LAPTM4β in mouse oocytes and early embryo eggs. The expression results were analyzed semi-quantitatively using Maticam2206 image analysis system. Results Laser scanning confocal microscopy showed that the green fluorescence of mouse oocyte and prokaryotic embryo ova was unevenly distributed, and the fluorescence signal was strong near the cell membrane. In the 2-cell, 4-cell, 8-cell and morula embryos Green fluorescence in the cytoplasm of fissure was focal distribution, and close to the cell membrane in the cytoplasm of the green fluorescence were strong, 2-cell phase also seen polar body also has a green fluorescent signal; mouse blastocyst trophoblast cells and the inner cell mass Medium-intensity green fluorescence can be seen. Immunohistochemistry showed that LAPTM4β positive reaction particles were light brown in mouse oocytes; dark brown granules were observed in prokaryotic, 2-cell, 4-cell, 8-cell and morulae, Cytoplasm was unevenly distributed; blastocyst inner cell mass and trophoblast cells are brown positive reaction particles. The results of image analysis showed that compared with the morula, the expression of LAPTM4β in morula and the blastocyst in the 8-cell stage was significantly different (P <0.05). Conclusions LAPTM4β is expressed in normal mouse oocytes and preimplantation embryos and has regularity. The expression of LAPTM4β in early stage embryos is higher than that in oocytes at different stages, suggesting that LAPTM4β and early stage of mouse embryos Embryo egg cell division, proliferation, differentiation and embryonic development.