Oviduct epithelial spheroids during in vitro culture of bovine embryos mitigate oxidative stress, improve blastocyst quality and change the embryonic transcriptome

Background. In vitro embryo production is increasingly used for genetic improvement in cattle but bypasses the oviduct environment and exposes the embryos to oxidative stress with deleterious effects on further development. Here we aimed to examine the effect of oviduct epithelial spheroids (OES) on embryo development and quality in terms of morphology and gene expression during two co-culture times (4 days: up to embryonic genome activation at 8-16 cell stage vs. 7 days: up to blastocyst stage) and under two oxygen tension conditions (5% vs. 20%).Methods. Bovine presumptive zygotes produced by in vitro fertilization (day 0) using in-vitro matured oocytes were cultured in droplets of synthetic oviductal fluid (SOF) medium with or without (controls) OES for 4 or 7 days under 5% or 20% oxygen (4 treated and 2 control groups). Cleavage rates were evaluated on day 2 and blastocyst rates on days 7-8. Expanded blastocysts on days 7-8 were evaluated for total cell numbers and gene expression analysis by RNA-sequencing.Results. Under 20% oxygen, blastocyst rates and total cell numbers were significantly higher in the presence of OES for 4 and 7 days compared to controls (P<0.05), with no difference according to the co-culture time. Under 5% oxygen, the presence of OES did not affect blastocyst rates but increased the number of cells per blastocyst after 7 days of co-culture (P<0.05). Both oxygen level and OES co-culture had a significant impact on the embryonic transcriptome. The highest number of differentially expressed genes (DEGs) was identified after 7 days of co-culture under 20% oxygen. DEGs were involved in a wide range of functions, including lipid metabolism, membrane organization, response to stress, early embryo development, and transport of small molecules.Conclusion. OES had beneficial effects on embryo development and quality under both 5% and 20% oxygen. Four days of co-culture were enough to obtain these effects, but highest impact was observed after 7 days. This study showed the impact of OES on embryo development and revealed potential molecular targets of OES-embryo dialog involved in response to stress and early embryonic development.Keywords: Oviduct, Spheroids, Oxidative stress, In vitro culture, Embryo, RNA-seq, Transcriptome, Cattle.

研究背景。体外胚胎生产技术在牛遗传改良中的应用日益广泛，但该技术绕过了输卵管生理环境，使胚胎暴露于氧化应激之中，对胚胎后续发育产生不利影响。本研究旨在探究两种共培养时长（4天：培养至8-16细胞期的胚胎基因组激活阶段；7天：培养至囊胚期）以及两种氧张力条件（5%与20%氧气）下，输卵管上皮球体（oviduct epithelial spheroids, OES）对胚胎发育及形态学、基因表达层面品质的影响。

研究方法。本研究以体外成熟卵母细胞经体外受精获得的牛推定合子（第0天）为实验材料，在5%或20%氧气条件下，于添加或不添加OES的合成输卵管液（synthetic oviductal fluid, SOF）微滴培养基中分别培养4天或7天，共设置4个处理组与2个对照组。分别于第2天评估卵裂率，第7-8天评估囊胚率。对第7-8天的扩张囊胚进行总细胞计数，并通过RNA测序（RNA-sequencing）开展基因表达分析。

研究结果。在20%氧气条件下，与对照组相比，添加OES共培养4天或7天的实验组囊胚率与囊胚总细胞数均显著升高（P<0.05），且共培养时长对此无显著影响。在5%氧气条件下，OES共培养对囊胚率无显著影响，但共培养7天后可显著提升单个囊胚的细胞数（P<0.05）。氧张力与OES共培养均对胚胎转录组具有显著影响。在20%氧气条件下共培养7天时，鉴定到的差异表达基因（differentially expressed genes, DEGs）数量最多。这些DEGs参与了脂代谢、膜结构组织、应激响应、早期胚胎发育以及小分子物质转运等多种生物学过程。

研究结论。在5%与20%氧气条件下，OES均对胚胎发育及品质具有积极作用。共培养4天即可达到该效果，但共培养7天时的促进效果最为显著。本研究明确了OES对胚胎发育的调控作用，并揭示了OES与胚胎互作中参与应激响应及早期胚胎发育的潜在分子靶点。

关键词：输卵管、类球体、氧化应激、体外培养、胚胎、RNA测序、转录组、牛