Effects of embryo-derived exosomes on the development of bovine cloned embryos

The developmental competence of in vitro cultured (IVC) embryos is markedly lower than that of their in vivo counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT) embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced Oct-4 expression and ratio of ICM/TE), as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (in vitro development), but also following growth to term (in vivo development). Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation.体外培养（in vitro cultured, IVC）胚胎的发育能力显著低于体内发育的对应胚胎，这表明亟需优化体外培养体系。目前牛胚胎的体外培养通常在初始培养3天后更换培养基，但在当前实验条件下，该流程相较于连续不更换培养基的培养方式是否更具优势仍未明确。本研究以牛体细胞核移植（somatic cell nuclear transfer, SCNT）胚胎为模型，结果显示，相较于常规更换培养基处理组，不更换培养基的培养体系可显著提升囊胚形成率与囊胚质量：包括总细胞数增加、应激与凋亡水平降低、Oct-4表达水平升高，以及内细胞团/滋养层细胞（inner cell mass/ trophectoderm, ICM/TE）比例提升，同时可改善胚胎后续足月发育能力。随后，我们针对体细胞核移植胚胎分泌的外泌体的存在与功能展开研究，以揭示培养基更换导致胚胎发育受损的原因。通过差速离心法成功分离出外泌体，并借助电子显微镜与外泌体膜标志物CD9免疫染色对其进行了鉴定。将提取的外泌体添加至刚更换的新鲜培养基中，不仅可显著挽救囊胚形成率与囊胚质量（体外发育层面），还可改善胚胎后续的足月发育能力（体内发育层面）。值得注意的是，内细胞团/滋养层细胞比例与产犊率均提升至与不更换培养基组相当的水平。综上，本研究首次证实：1. 牛体细胞核移植胚胎可在化学成分明确的体外培养基中分泌外泌体；2. 分泌的外泌体对体细胞核移植胚胎的囊胚形成、囊胚质量及后续足月发育均不可或缺；3. 培养基更换过程会导致外泌体被移除，进而损害体细胞核移植胚胎的发育能力，而采用不更换培养基的培养流程或通过补充外泌体均可显著规避或挽救这一损伤。