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重复控制性卵巢刺激对小鼠早期胚胎各阶段有氧糖酵解相关酶表达的影响

申路路^1,2, 史蓓蓓^1,2, 熊文艳^1,2, 史睿敢^1,2, 宋亚静^1,2, 杜惠兰^1,2,3,*

¹河北中医药大学中西医结合研究所，石家庄 050091；²河北省中西医结合肝肾病证研究重点实验室，石家庄 050091；³河北省中西医结合生殖疾病协同创新中心，石家庄 050091

摘要

本研究旨在探讨重复控制性卵巢刺激(controlled ovarian stimulation, COS)降低早期胚胎发育是否与有氧糖酵解相关酶表达水平相关。4~5 周龄ICR小鼠采用腹腔注射孕马血清促性腺激素(pregnant mare serum gonadotropin, PMSG)，48 h 后再注射人绒毛膜促性腺激素(human chorionic gonadotrophin, HCG)，并以此重复注射(0~4 次)以制备重复COS模型小鼠，这些小鼠妊娠后取2~8 细胞和囊胚，用qRT-PCR 法和免疫荧光法检测各阶段有氧糖酵解相关酶己糖激酶2 (hexokinase 2, HK2)、磷酸果糖激酶(phosphofructokinase, PFK)、丙酮酸激酶M2 (pyruvate kinase M2, PKM2)、乳酸脱氢酶A (lactate dehydrogenase A,LDHA)及葡萄糖转运蛋白1 (glucose transporter type 1, GLUT1)、单羧酸转运蛋白4 (monocarboxylate transporter 4, MCT4)mRNA和蛋白表达情况以及优质胚胎率。结果显示，相较对照组，1 次刺激各阶段优质胚胎率差异无统计学意义(P > 0.05)，随着促排刺激次数增加，胚胎形态较差，优质胚胎率降低(P < 0.05)。在2~8 细胞阶段，与对照组相比，1 次刺激对HK2、PFK、PKM2、LDHA、GLUT1、MCT4 mRNA 和蛋白表达无明显影响。2 次刺激使2 细胞阶段的PFK、PKM2、GLUT1mRNA表达水平降低，PFK蛋白表达水平降低，也使得4 细胞阶段的LDHA、MCT4 mRNA表达水平降低和PKM2、MCT4蛋白表达水平降低以及8 细胞阶段PFK、LDHA、MCT4 mRNA和蛋白表达水平降低。3 次和4 次刺激使得2、4 和8 细胞阶段的HK2、PFK、PKM2、LDHA、GLUT1、MCT4 mRNA 和蛋白表达水平均降低。在囊胚阶段，1 次刺激对HK2、PFK、PKM2、LDHA、GLUT1、MCT4 mRNA和蛋白表达无明显影响，2 次刺激使LDHA蛋白表达水平降低，3 次和4 次刺激使得HK2、PFK、PKM2、LDHA、GLUT1、MCT4 mRNA和蛋白表达水平均降低。以上结果表明，重复COS会降低早期胚胎各阶段有氧糖酵解相关酶表达水平。我们的研究结果提示重复COS降低早期胚胎发育可能与抑制有氧糖酵解相关。

关键词： 控制性卵巢刺激; 有氧糖酵解; 早期胚胎

Effects of repeated controlled ovarian stimulation on the expression of aerobic glycolysis-related enzymes at various stages of early embryo development in mice

SHEN Lu-Lu^1,2, SHI Bei-Bei^1,2, XIONG Wen-Yan^1,2, SHI Rui-Gan^1,2, SONG Ya-Jing^1,2, DU Hui-Lan^1,2,3,*

¹Institute of Integrative Medicine, Hebei University of Chinese Medicine, Shijiazhuang 050091, China；²Hebei Key Laboratory of Integrative Medicine on Liver-Kidney Patterns, Shijiazhuang 050091, China；³Hebei Collaborative Innovation Center of Integrated Traditional and Western Medicine on Reproductive Disease, Shijiazhuang 050091, China

Abstract

This study aims to investigate whether the reduction in early embryonic development due to repeated controlled ovarian stimulation (COS) is associated with the expression levels of enzymes related to aerobic glycolysis. A repeated COS mouse model was established by injecting pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotrophin (HCG) 48 h later in ICR mice. Repeated COS was performed by repeating this injection protocol. After pregnancy in these mice, 2- to 8-cell embryos and blastocysts were collected. The mRNA and protein expression levels of aerobic glycolysis-related enzymes, including hexokinase 2 (HK2), phosphofructokinase (PFK), pyruvate kinase M2 (PKM2), lactate dehydrogenase A (LDHA), as well as glucose transporter type 1 (GLUT1) and monocarboxylate transporter 4 (MCT4), were detected at each stage using qRT-PCR and immunofluorescence methods, along with the high-quality embryo rate. The results showed that, compared with the control group, there was no statistically significant difference in the high-quality embryo rate at each stage after a single stimulation (P > 0.05). As the number of stimulation cycles increased, embryo morphology deteriorated, and the high-quality embryo rate decreased. At the 2- to 8-cell stage, compared with the control group, a single stimulation had no significant effect on the mRNA and protein expression levels of HK2, PFK, PKM2, LDHA, GLUT1, and MCT4. However, two stimulations reduced the mRNA expression levels of PFK, PKM2, and GLUT1 at the 2-cell stage, the protein expression level of PFK, and also decreased the mRNA expression levels of LDHA and MCT4 and the protein expression levels of PKM2 and MCT4 at the 4-cell stage, as well as the mRNA and protein expression levels of PFK, LDHA, and MCT4 at the 8-cell stage. Three and four stimulations resulted in decreased mRNA and protein expression levels of HK2, PFK, PKM2, LDHA, GLUT1, and MCT4 at the 2-, 4-, and 8-cell stages (P < 0.05). At the blastocyst stage, a single stimulation had no significant effect on the mRNA and protein expression levels of HK2, PFK, PKM2, LDHA, GLUT1, and MCT4. Two stimulations reduced the protein expression level of LDHA, while three and four stimulations led to decreased mRNA and protein expression levels of HK2, PFK, PKM2, LDHA, GLUT1, and MCT4. These findings indicate that repeated COS reduces the expression levels of aerobic glycolysis-related enzymes at various stages of early embryonic development. Our results suggest that the reduction in early embryonic development due to repeated COS may be associated with the inhibition of aerobic glycolysis.

Key words: controlled ovarian stimulation; aerobic glycolysis; early embryo

收稿日期：　　录用日期：

通讯作者：杜惠兰　　E-mail:

DOI: 10.13294/j.aps.2026.0026

引用本文：

申路路, 史蓓蓓, 熊文艳, 史睿敢, 宋亚静, 杜惠兰. 重复控制性卵巢刺激对小鼠早期胚胎各阶段有氧糖酵解相关酶表达的影响[J]. 生理学报 2026; 78 (3): 592-604.

SHEN Lu-Lu, SHI Bei-Bei, XIONG Wen-Yan, SHI Rui-Gan, SONG Ya-Jing, DU Hui-Lan. Effects of repeated controlled ovarian stimulation on the expression of aerobic glycolysis-related enzymes at various stages of early embryo development in mice. Acta Physiol Sin 2026; 78 (3): 592-604 (in Chinese with English abstract).