补肾法通过三羧酸循环酶改善重复控制性卵巢刺激模型小鼠2－细胞胚胎发育潜能

刘孟瑞; 郝延芝; 左茜茜; 陈静; 于志芳; 段梅; 梁潇; 宋翠淼; 杜惠兰

doi:10.3969/j.issn.1006-2157.2023.02.011

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补肾法通过三羧酸循环酶改善重复控制性卵巢刺激模型小鼠2－细胞胚胎发育潜能

实验研究 | 更新时间：2023-03-07

- 补肾法通过三羧酸循环酶改善重复控制性卵巢刺激模型小鼠2－细胞胚胎发育潜能
- Kidney tonifying therapy improves the development potential of two-cell embryos in repeated controlled ovarian stimulation model mice through tricarboxylic acid cyclase
- 北京中医药大学学报 2023年46卷第2期页码：207-214
- 作者机构：
  
  河北中医学院中西医结合学院　石家庄　050200
- 作者简介：
  
  刘孟瑞，女，硕士
  #杜惠兰，女，博士，教授，主任医师，博士生导师，主要研究方向：中西医结合防治生殖调控障碍，E-mail：duhuilan@163.com
- 基金信息：
  
  国家自然科学基金面上项目(82074486)
- DOI：10.3969/j.issn.1006-2157.2023.02.011
  中图分类号： R285.5
- 纸质出版日期：2023-02-28，
  
  网络出版日期：2022-12-28，
  
  收稿日期：2022-03-18，
- 稿件说明：
移动端阅览
刘孟瑞, 郝延芝, 左茜茜, 等. 补肾法通过三羧酸循环酶改善重复控制性卵巢刺激模型小鼠2－细胞胚胎发育潜能[J]. 北京中医药大学学报, 2023,46(2):207-214.

LIU Mengrui, HAO Yanzhi, ZUO Qianqian, et al. Kidney tonifying therapy improves the development potential of two-cell embryos in repeated controlled ovarian stimulation model mice through tricarboxylic acid cyclase[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023,46(2):207-214.
刘孟瑞, 郝延芝, 左茜茜, 等. 补肾法通过三羧酸循环酶改善重复控制性卵巢刺激模型小鼠2－细胞胚胎发育潜能[J]. 北京中医药大学学报, 2023,46(2):207-214. DOI： 10.3969/j.issn.1006-2157.2023.02.011.

LIU Mengrui, HAO Yanzhi, ZUO Qianqian, et al. Kidney tonifying therapy improves the development potential of two-cell embryos in repeated controlled ovarian stimulation model mice through tricarboxylic acid cyclase[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023,46(2):207-214. DOI： 10.3969/j.issn.1006-2157.2023.02.011.

摘要

目的

通过观察胚胎合子基因组激活期(ZGA)三羧酸循环(TCA)酶和表观遗传修饰的关系，研究补肾调经方改善重复控制性卵巢刺激(COS)模型小鼠的2－细胞胚胎发育潜能。

方法

将动情周期正常的雌性小鼠按照随机数字表法分为正常组、模型组、补肾低剂量组和补肾高剂量组，每组25只。除正常组外，其余各组小鼠建立COS模型。模型组和补肾低、高剂量组每日上午9时分别灌胃蒸馏水、补肾调经方低剂量(25.6 g/kg)和高剂量(51.2 g/kg)，连续10 d。于第11天16时腹腔注射孕马血清促性腺激素(PMSG)10 IU/只，第13天16时腹腔注射人绒毛膜促性腺激素(hCG)10 IU/只；正常组注射生理盐水0.1 mL。灌胃及腹腔注射连续3个周期，每个周期间隔4 d。各组小鼠第3次腹腔注射后立即与同品系雄性小鼠合笼。次日清晨见栓或阴道涂片见精子的雌鼠记为妊娠。妊娠小鼠于末次腹腔注射hCG后44～46 h处死，在倒置显微镜下取出2－细胞胚胎观察形态并计数。免疫荧光法检测2－细胞胚胎丙酮酸脱氢酶(PDH)、柠檬酸合成酶(CS)、异柠檬酸脱氢酶(IDH3A)、性别决定区Y框蛋白2(SOX2)、组蛋白3第9位赖氨酸乙酰化修饰(H3K9ac)的蛋白表达；RT-PCR法检测2－细胞胚胎PDH、CS、IDH3A、SOX2的mRNA表达。

结果

与正常组比较，模型组2－细胞胚胎数量增加(

0.05)，形态明显破碎，发育较差；PDH、CS、IDH3A、SOX2的mRNA和蛋白表达均降低(

0.05)，H3K9ac蛋白表达升高(

0.05)；与模型组比较，补肾低、高剂量组2－细胞胚胎数差异无统计学意义(

0.05)，形态发育完整，碎片较少；PDH、CS、IDH3A、SOX2的mRNA和蛋白表达均升高(

0.05)

H3K9ac蛋白表达降低(

0.05)。

结论

补肾调经方可在ZGA影响重复COS小鼠2－细胞胚胎的表观遗传修饰，改善胚胎发育潜能，推测其作用机制可能与影响TCA酶有关。

Abstract

Objective

The potential of

Bushen Tiaojing

Recipe to improve the 2-cell embryonic development of mice with repeated controlled ovarian stimulation (COS) was studied by observing the relationship between tricarboxylic acid cycle (TCA) enzymes and epigenetic modifications during the embryonic zygotic genome activation period (ZGA).

Methods

Female mice with a normal estrous cycle were divided into the normal group

the model group

the kidney tonifying low dose group

and the kidney tonifying high dose group (

=25 mice per group). Except for the normal group

COS models were established in other groups. Mice in the model group and the treatment groups were treated with repeated controlled ovarian stimulation. Mice in these three groups were given distilled water

low dose

Bushen Tiaojing

Recipe (25.6 g/kg)

or high dose

Bushen Tiaojing

Recipe (51.2 g/kg) by gavage at 9: 00 am every day for 10 consecutive days. On the 11th day

10 IU of pregnant horse serum gonadotropin (PMSG) was intraperitoneally injected at 16: 00 pm

on the 13th day

10 IU of human chorionic gonadotropin (hCG) was intraperitoneally injected at 16: 00 pm. Mice in the normal group were injected with 0.1 mL of normal saline. There were 3 consecutive cydes with an internal of 4 days between every two cycles. After the third intraperitoneal injection

the mice were immediately caged with male mice of the same strain. In the morning of the next day

the female mice with vaginal plug or sperm on the vaginal smear were considered as pregnant. The pregnant mice were killed 44-46 h after the last intracperitoneal injection of hCG. The two-cell embryos were taken out under an inverted microscope for morphological observations and counted. The protein expression levels of pyruvate dehydrogenase (PDH)

citrate synthase (CS)

isocitrate dehydrogenase (IDH3A)

and sex determination region Y box protein 2 (SOX2) and the levels of acetylated histone 3 lysine 9 (H3K9ac) in two-cell embryos were detected by immunofluorescence. The mRNA expression levels of

PDH

IDH

and

SOX

2 in two-cell embryos were detected by RT-PCR.

Results

Compared with the normal group

the number of two-cell embryos was increased in the model group (

0.05)

the morphology was obviously disrupted

and the development was poor. The mRNA and protein expression levels of PDH

IDH3A

and SOX2 were decreased (

0.05)

while the protein expression level of H3K9ac was increased (

0.05). Compared with the model group

there was no significant difference in the number of two-cell embryos obtained in the kidney tonifying low and high dose groups (

0.05)

with complete morphological development and fewer fragments. The mRNA and protein expression levels of PDH

IDH3A

and SOX2 were increased (

0.05)

while the protein expression level of H3K9ac was decreased (

0.05).

Conclusion

Bushen Tiaojing

Recipe can (i) affect the epigenetic modifications of two-cell embryos of repeated COS mice in ZGA and (ii) improve the embryonic developmental potential. It is speculated that its mechanism of action may be related to the influence of TCA enzymes.

关键词

补肾调经方2－细胞胚胎三羧酸循环酶表观遗传修饰胚胎发育潜能小鼠

Keywords

Bushen Tiaojing Recipetwo-cell embryostricarboxylic acid cycle enzymesepigenetic modificationembryonic developmental potentialmice

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