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| Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells |
ZHANG Xue-min1*, HUANG Xiang-hua2*, WANG Jing1, XING Ying1, LIU Fang1, XIANG Jin-zhu1, WANG Han-ning1, YUE Yong-li1, LI Xue-ling1
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1 State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot 010020, P.R.China
2 Department of Urology, Inner Mongolia Autonomous Region People's Hospital, Hohhot 010017, P.R.China
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摘要
溶血磷脂酸( Lysophosphatidic acid, LPA)是一种小分子甘油磷脂，在多种动物细胞中具有生长因子和激素样活性。LPA通过结合G蛋白偶联受体,激活下游信号通路，产生促卵母细胞成熟，提高胚胎发育率，促进细胞增殖等生物学效应。绵羊是我国重要的家畜之一，其体外受精效率较其他物种偏低,且目前仍未建立真正的绵羊胚胎干细胞。本研究通过在绵羊体外受精过程及囊胚接种细胞培养过程中添加LPA，以探究LPA对绵羊体外受精以及囊胚接种细胞培养的影响。首先，我们对绵羊的体外受精体系进行了筛选，选取了两种成熟液与两种SOF液进行组合，通过比较不同体系间的成熟率、卵裂率和囊胚率，最终找到最优的体外受精的体系，即IVM Ⅱ and SOF Ⅱ。然后，在绵羊体外受精过程中添加不同浓度的LPA，探讨LPA浓度对绵羊体外受精的影响。结果显示，当LPA浓度为0.1 μmol L-1 - 10 μmol L-1之间时，随着LPA浓度的增加，绵羊卵母细胞体外成熟率、囊胚率逐渐上升(P < 0.05)，卵裂率无显著变化(P > 0.05),囊胚形态正常；当LPA浓度达到15 μmol L-1时，成熟率、卵裂率、囊胚率均出现显著下降 (P < 0.05)，且囊胚形态发生异常，胚胎内细胞团聚集不正常，囊胚内部出现分区，不能发育成为优质的囊胚。另外，随着LPA浓度在0.1 μmol L-1- 10 μmol L-1范围内逐渐增大，LPAR2、LPAR4、TE相关基因CDX-2和多能性相关基因OCT-4在绵羊早期体外受精胚胎中的mRNA表达量也逐步增加。而15 μmol L-1 LPA处理组中，早期胚胎LPAR2、LPAR4、CDX-2和OCT-4的表达量均显著低于LPA - 10 μmol L-1处理组(P<0.05)。最后，我们将绵羊体外受精囊胚接种在不同培养体系,尝试建立胚胎干细胞。 结果表明，LPA使接种后的囊胚细胞向TSC样细胞生长。随着LPA浓度从0 μmol L-1增加到10 μmol L-1, OCT-4和CDX-2的蛋白免疫荧光强度和mRNA丰度增强(P < 0.05)，而15 μmol L-1 LPA则显著降低OCT-4和CDX-2在囊胚接种细胞中的表达(P < 0.05)。同时，10 μmol L-1的LPA处理后，LPAR2和LPAR4蛋白表达显著增加。综合上述实验结果，LPA可促进绵羊体外受精早期胚胎发育,并促进囊胚接种细胞向TSC方向生长，为大动物体外受精及胚胎干细胞建系提供参考。
Abstract
Lysophosphatidic acid (LPA) is a small molecule glycerophospholipid, which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development. In this study, sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment. At first, the maturation medium containing estrus female sheep serum and synthetic oviduct fluid (SOF) were optimized for sheep IVF, and then the effects of LPA were investigated. From 0.1 to 10 μmol L–1, LPA had no significant effect on the cleavage rate (P>0.05), but the maturation rate and blastocyst rate increased dependently with LPA concentration (P<0.05), and the blastocyst morphology was normal. When the LPA concentration was 15 μmol L–1, the maturation rate, cleavage rate and blastocyst rate decreased significantly (P<0.05), and the blastocyst exhibited abnormal morphology and could not develop into high-quality blastocyst. Besides, the exogenous LPA increases the expression of LPAR2, LPAR4, TE-related gene CDX-2
and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10 μmol
L–1. The expression of LPAR2, LPAR4, CDX-2 and OCT-4 from the LPA-0.1 μmol L–1 to LPA-10 μmol L–1 groups in early embryos were extremely significant (P<0.05), while the expression of these genes significantly decreased in 15 μmol L–1 LPA-treated embryos compared with LPA-10 μmol L–1 group (P<0.05). The inner cell mass in 15 μmol L–1 LPA-treated embryos was also disturbed, and the blastocysts formation was abnormal. Secondly, the sheep IVF blastocysts were applied to establish embryonic stem cells. The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells. They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10 μmol L–1, while 15 μmol L–1 LPA decreased OCT-4 and CDX-2 expression in the derived cells. The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1 μmol L–1 group and LPA-10 μmol L–1 group extremely significantly increased (P<0.05), but there was significant decrease in LPA-15 μmol L–1 group compared with LPA-10 μmol L–1 group (P<0.05). Meanwhile, the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10 μmol L–1 concentration. This study references the IVF embryo production and embryonic stem cell research of domestic animals.
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Received: 07 December 2021
Accepted: 21 March 2022
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| Fund: This work was financially supported by the Science and Technology Major Project of the Inner Mongolia Autonomous Region of China (2020ZD0007), the Major Program of the Inner Mongolia Natural Science Foundation, China (2020ZD10), the National Natural Science Foundation of China (32160172), the Natural Science Foundation of Inner Mongolia Autonomous Region (2020BS03003 and 2020BS03022), the National Transgenic Project of China (2016ZX0801000-002 and 2016ZX08010005-001), and the Science and Technology Major Project of the Inner Mongolia Autonomous Region of China (zdzx2018065). |
| About author: ZHANG Xue-min, E-mail: 573248433@qq.com; HUANG Xiang-hua, E-mail: nmgyyhuangxianghua@126.com; #Correspondence LI Xue-ling, Tel: +86-471-3679807, E-mail: lixueling@imu.edu.cn
* These authors contributed equally to this study. |
Cite this article:
ZHANG Xue-min, HUANG Xiang-hua, WANG Jing, XING Ying, LIU Fang, XIANG Jin-zhu, WANG Han-ning, YUE Yong-li, LI Xue-ling.
2023.
Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells. Journal of Integrative Agriculture, 22(4): 1142-1158.
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