Abstract
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.
Highlights
Embryos produced in vitro are generally inferior in quality and have lower developmental competence compared with their in vivo counterparts [1, 2]
Effect of exosome supplementation on bovine somatic cell nuclear transfer (SCNT) embryo in vitro development was evaluated based on blastocyst formation rate
Our results showed that whereas no obvious difference could be detected when 1 μL of exosome extract was added into freshly renewed culture medium (22.3% vs. 23.9%, P>0.05), addition of 5 μL and 10 μL both significantly improved blastocyst rate of bovine SCNT embryos (33.5% and 31.5%, respectively), with 5 μL group higher than 10 μL group but not significant (Table 2)
Summary
Embryos produced in vitro are generally inferior in quality and have lower developmental competence compared with their in vivo counterparts [1, 2]. Exosomes and bovine cloned embryos implantation, altered inner cell mass/trophectoderm (ICM/TE) ratios, higher apoptosis index, and lower total cell numbers. These embryos present blastomeres with irregular sizes, increased sensitivity to cryopreservation, and abnormal gene expression and epigenetic modification [3,4,5,6]. Suboptimal culture conditions are main factors affecting the developmental competence of embryos produced in vitro [7]. Various embryo culture conditions, such as culture medium, embryo density, supplementation of growth factors, and O2 concentration, have been extensively investigated and optimized [8,9,10], quality of embryos produced in vitro is still markedly lower, suggesting the involvement of other unknown factors that may be crucial for further optimization and improvement
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