Almiñana C, Dubuisson F, Bauersachs S, Royer E, Mermillod P, Blesbois E, Guignot F
UMR PRC, INRAE 0085, CNRS 7247, Université de Tours, IFCE, F, -37380, Nouzilly, France.
Functional Genomics Group, Institute of Veterinary Anatomy, VetSuisse Faculty Zurich, University of Zurich, Zürich, Switzerland.
J Anim Sci Biotechnol. 2022 Mar 15;13(1):46. doi: 10.1186/s40104-021-00672-1.
Currently, there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes. To date, vitrification (VIT) is the most efficient method for pig embryo cryopreservation. Despite a high number of embryos survives in vitro after vitrification/warming procedures, the in vivo embryo survival rates after embryo transfer are variable among laboratories. So far, most studies have focused on cryoprotective agents and devices, while the VIT effects on porcine embryonic gene expression remained unclear. The few studies performed were based on vitrified/warmed embryos that were cultured in vitro (IVC) to allow them to re-expand. Thus, the specific alterations of VIT, IVC, and the cumulative effect of both remained unknown. To unveil the VIT-specific embryonic alterations, gene expression in VIT versus (vs.) IVC embryos was analyzed. Additionally, changes derived from both VIT and IVC vs. control embryos (CO) were analyzed to confirm the VIT embryonic alterations. Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA-sequencing: (1) VIT embryos (vitrified/warmed and cultured in vitro), (2) IVC embryos and (3) CO embryos.
RNA-sequencing revealed three clearly different mRNA profiles for VIT, IVC and CO embryos. Comparative analysis of mRNA profiles between VIT and IVC identified 321, differentially expressed genes (DEG) (FDR < 0.006). In VIT vs. CO and IVC vs. CO, 1901 and 1519 DEG were found, respectively, with an overlap of 1045 genes. VIT-specific functional alterations were associated to response to osmotic stress, response to hormones, and developmental growth. While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects.
Our findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs. IVC embryos and second by an integrative transcriptome analysis including in vivo control embryos. The identified VIT alterations might reflect the transcriptional signature of the embryo cryodamage but also the embryo healing process overcoming the VIT impacts. Selected validated genes were pointed as potential biomarkers that may help to improve vitrification.
目前，在养猪业以及出于遗传多样性保存和研究目的方面，对高效的猪胚胎冷冻保存程序有很高的需求。迄今为止，玻璃化冷冻（VIT）是猪胚胎冷冻保存最有效的方法。尽管在玻璃化冷冻/复温程序后有大量胚胎在体外存活，但胚胎移植后的体内胚胎存活率在不同实验室之间存在差异。到目前为止，大多数研究都集中在冷冻保护剂和设备上，而玻璃化冷冻对猪胚胎基因表达的影响仍不清楚。少数已进行的研究是基于在体外培养（IVC）以使它们重新扩张的玻璃化冷冻/复温胚胎。因此，玻璃化冷冻、体外培养的特定改变以及两者的累积效应仍然未知。为了揭示玻璃化冷冻特有的胚胎改变，分析了玻璃化冷冻胚胎与体外培养胚胎的基因表达。此外，分析了玻璃化冷冻和体外培养两者与对照胚胎（CO）相比产生的变化，以确认玻璃化冷冻的胚胎改变。通过RNA测序分析了三组囊胚期的体内胚胎：（1）玻璃化冷冻胚胎（玻璃化冷冻/复温并在体外培养），（2）体外培养胚胎和（3）对照胚胎。
RNA测序揭示了玻璃化冷冻、体外培养和对照胚胎三种明显不同的mRNA谱。玻璃化冷冻和体外培养之间mRNA谱的比较分析确定了321个差异表达基因（DEG）（FDR < 0.006）。在玻璃化冷冻与对照以及体外培养与对照中，分别发现了1901个和1519个差异表达基因，其中有1045个基因重叠。玻璃化冷冻特有的功能改变与对渗透压应激的反应、对激素的反应以及发育生长有关。而对缺氧和线粒体自噬的反应改变与玻璃化冷冻和体外培养的综合效应有关。
我们的研究结果通过首先比较玻璃化冷冻胚胎与体外培养胚胎的差异，其次通过包括体内对照胚胎的综合转录组分析，揭示了玻璃化冷冻程序特有的胚胎基因表达改变的新见解。所确定的玻璃化冷冻改变可能反映了胚胎冷冻损伤的转录特征，但也反映了胚胎克服玻璃化冷冻影响的愈合过程。选定的经过验证的基因被指出是可能有助于改善玻璃化冷冻的潜在生物标志物。
