OIVC medium enhances developmental competence in porcine preimplantation embryos

doi:10.1016/j.jia.2025.03.005

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Yongjiang Yang^1,2,3,⁴^*, Ying Zhang^1,^2,⁴^*, Qiang Liu^1,^6*, Shuangjie Tian^1,²^,3,4, Wanyun Feng^1,²^,3,4, Fangwen Deng^1,²^,3,4, Guosong Qin^1,²^,4, Yanfang Wang⁵^#, Jianguo Zhao^1,2,^3,⁴^#

¹Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

²Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China.

³University of Chinese Academy of Sciences, Beijing 100049, China.

⁴Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China.

⁵Laboratory of Animal (Poultry) Genetics Breeding and Reproduction, Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China.

⁶Shaanxi University of Chinese Medicine, Xianyang 712046, Shaanxi Province, China.

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摘要

【目的】为了解决猪体外胚胎发育能力的不足，特别是内细胞团（ICM）细胞数量及囊胚总细胞数显著低于同时期体内胚胎的问题，本研究致力于开发高效稳定的猪胚胎体外培养体系，为提高体外胚胎生物技术应用的效率提供高效策略。【方法】通过优化胚胎激活第5天的培养基成分，包括Advanced DMEM/F12和FBS相结合，以及在此培养体系的基础上基于JAK/STAT3、Activin/TGF-β及FGF/ERK通路筛选出的Activin A、IGF1、IL6等生长因子的继续探究，系统性优化传统的PZM-3培养体系。通过免疫荧光染色等技术评估胚胎质量，同时利用转录组测序探讨优化培养体系的潜在分子机制。【结果】在PZM-3培养基中添加50% Advanced DMEM/F12和5% FBS（即Day 5培养体系）能够使猪孤雌激活（PA）囊胚总细胞数提高2.5倍。研究还突出FBS不能被Knockout血清替代物（KSR）替代，发现高浓度的KSR会导致囊胚率下降。在Day 5培养基中进一步加入Activin A（即OIVC培养体系），不仅能够使PA囊胚总细胞数提高4.5倍，还能提高ICM细胞数4.5倍。此外，OIVC培养体系对体细胞核移植（SCNT）和体外受精（IVF）胚胎同样有效，显著提升囊胚发育质量。转录组测序分析显示，相较于不添加Activin A的Day 5培养体系，OIVC处理的胚胎中，大多数差异表达基因下调，且主要富集的信号通路包括Activin A/TGF-β。在这些下调基因中，PAX6可能是一个潜在的关键基因，影响ICM细胞的数量，进而影响胚胎发育能力。【结论】本研究建立了一种新型的猪胚胎体外培养体系，显著提高了体外胚胎发育的质量，为早期胚胎发育机制的研究、以及生物医学研究和农业动物生产提供了高效的优质胚胎方案。

Abstract

The developmental capacity of in vitro embryos is critical for the success of embryonic biotechnology. However, in vitro embryos often exhibit suboptimal quality, with fewer inner cell mass (ICM) cells and reduced total blastocyst cell counts compared to in vivo embryos. To address this, we optimized the conventional PZM-3 culture medium by supplementing 50% Advanced DMEM/F12 and 5% FBS on the fifth day after embryo activation (Day 5 medium) and resulted in a 2.5-fold increase in the total cell numbers of parthenogenetic activation (PA) derived blastocysts. Further enhancement was achieved by incorporating Activin A in Day 5 medium, creating the OIVC (Optimized In Vitro Culture) medium, which significantly increased both the total cell numbers and the ICM cell counts by 4.5-fold in the blastocyst stage. The OIVC medium also improved the quality of pig somatic cloned and in vitro fertilized (IVF) embryos. RNA sequencing analysis revealed that in the OIVC-treated embryos, most of the differentially expressed genes were downregulated compared to the control group, with the main enriched signaling pathways including Activin A/TGF-β. Notably, among these downregulated genes, PAX6 may be as a potential key gene influencing the number of ICM cells. This study presents a novel culture system that markedly enhances pig in vitro embryo quality, providing an efficient strategy for generating cloned pigs based on somatic cell nuclear transfer (SCNT) technology.

Keywords: Blastocyst ICM PA IVF SCNT Activin A PAX6

Online: 17 March 2025

Fund:

This work was supported by the Biological Breeding-National Science and Technology Major Project (2023ZD04074, 2023ZD0404604), the National Natural Science Fund for Distinguished Young Scholars (31925036 and 32025034), the Program of National Natural Science Foundation of China (32230100, 32330099, 32470643 and 32201257), the National Key Research and Development Program of China (2020YFA0509503, 2022YFF0710700, 2021YFA0805902, 2022XAGG0121, 2022YFF1002803), the Young Elite Scientists Sponsorship Program by the China Association for Science and Technology (2019QNRC001).

About author: *: These authors contributed equally #Correspondence should be addressed to Yanfang Wang, Email: wangyanfang@caas.cn; Jianguo Zhao, Email: zhaojg@ioz.ac.cn

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Yongjiang Yang, Ying Zhang, Qiang Liu, Shuangjie Tian, Wanyun Feng, Fangwen Deng, Guosong Qin, Yanfang Wang, Jianguo Zhao. 2025. OIVC medium enhances developmental competence in porcine preimplantation embryos. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.03.005

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