Factors and optimization strategies of low normal fertilization in spindle transfer of mouse oocytes
10.3760/cma.j.cn101441-20240914-00343
- VernacularTitle:小鼠卵母细胞纺锤体置换正常受精率低的原因及其优化策略
- Author:
Linlin YANG
1
;
Xiaoyu LIAO
;
Wenjuan WANG
;
Hui MIAO
;
Na LI
;
Huiling BI
;
Congxiu MIAO
Author Information
1. 长治医学院附属和平医院生殖遗传科 山西省卫健委生殖工程重点实验室，长治 046000
- Publication Type:Journal Article
- Keywords:
Spindle transfer;
Normal fertilization rate;
Mouse oocytes;
Spindle karyoplast
- From:
Chinese Journal of Reproduction and Contraception
2025;45(5):482-488
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the factors that tend to lead to a low normal fertilization rate in spindle transfer of mouse oocytes and its strategies.Methods:Oocytes in metaphase Ⅱ (MⅡ) of C57BL/6 and ICR strains mouse were obtained for observations of mitochondrial replacement operations. Spindle karyoplasts of three different sizes (large, medium, small) were isolated by micromanipulation, and their morphology was observed under bright field and polarized light at 0 min, 10 min, 20 min and 30 min after suction. For small spindle karyoplasts, 2% paraformaldehyde fixation was performed after 10 min, 20 min and 30 min respectively, and the morphology of spindle and chromosomes was observed after immunofluorescence staining. Intracytoplasmic sperm injection (ICSI) was performed on oocytes, followed by small spindle karyoplasts isolation, and then fusion was conducted after 30 min (traditional method), 10 min (modified method 1) and 0 min (modified method 2) respectively. ICSI-oocytes without spindle transfer were used as control group, and 14-16 h later, the fertilization of reconstructed oocytes was observed and statistically analyzed.Results:1) When spindle karyoplast was just retrieved (0 min), the karyoplast membrane was smooth and complete, and the spindle showed a normal spindle shape. After 10 min, 20 min and 30 min of suction, the karyoplast membrane was twisted or elongated with an irregular morphology, and the spindle was also twisted, stretched or scattered with a weaker brightness. And the smaller spindle karyoplast, the more serious the deformation was. 2) When the spindle was just taken out (0 min), immunofluorescence staining showed that the spindle was in a standard spindle shape, and the chromosomes were regularly and neatly arranged on the equatorial plate. With the prolongation of placement time, spindles were elongated and tapered or dumbbell-shaped, and chromosomes were scattered and irregularly arranged. 3) For the traditional method of spindle transfer, the normal fertilization rate of C57BL/6 mouse reconstructed oocytes was 63.16% (24/38), which was statistically different from that of control group ( P=0.002). The normal fertilization rate [68.00% (34/50)] in the modified method 1 group was significantly lower than that in control group [90.00% (36/40), P=0.019], but there was no statistical difference with the traditional method ( P=0.260). However, the normal fertilization rate [82.50% (33/40)] in the modified method 2 group was similar to control group ( P=0.422), and was significantly higher than that of the traditional method group ( P=0.010). Conclusion:The longer the spindle karyoplast of mouse oocytes is stayed after retrieval, the more serious the morphological changes, the more unstable the spindles and chromosomes, and the lower normal fertilization rate. Immediate fusion of spindle karyoplast after isolation is the best time for mitochondrial replacement micromanipulation.
