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

• Published in: PloS one Vol. 12; no. 3; p. e0174535
• Main Authors: Qu, Pengxiang, Qing, Suzhu, Liu, Ruiqi, Qin, Hongyu, Wang, Weiwei, Qiao, Fang, Ge, Hui, Liu, Jun, Zhang, Yong, Cui, Wei, Wang, Yongsheng
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 28.03.2017; Public Library of Science (PLoS)
• Subjects: 1-Phosphatidylinositol 3-kinase; 17β-Estradiol; Actin; Activation; Adhesives; Agriculture; Agronomy; AKT protein; Ammonia; Amplification; Analysis; Animal breeding; Animal sciences; Animals; Apoptosis; Apoptosis - genetics; BAK protein; Bax protein; Bicarbonate; Bicarbonates; Biocompatibility; Biology; Biology and Life Sciences; Biotechnology; Blastocyst - cytology; Blastocyst - metabolism; Blastocysts; Blastomeres; Breeding; Cancer; Carbonates; Cattle; Cell Count; Cerebrospinal fluid; Chemicals; Cloning; Cloning, Organism - methods; Collection; Cytoplasm; Electron microscopy; Embryo; Embryo Culture Techniques; Embryo, Mammalian - embryology; Embryo, Mammalian - metabolism; Embryonic Development; Embryonic growth stage; Embryos; Epigenetics; Exosomes - metabolism; Exosomes - ultrastructure; Female; Fertility; Gene Expression; Genetic aspects; Genomes; Growth factors; Hatching; Insulin; Laboratories; Mammals; Medicine; Metabolites; MicroRNAs; Microscopy, Electron; Microscopy, Fluorescence; Neural stem cells; Nuclear Transfer Techniques; Nuclei; Nutrients; Octamer Transcription Factor-3 - genetics; Offspring; Optimization; Pain; Phosphates; Position (location); Pregnancy; Proto-Oncogene Proteins c-bcl-2 - genetics; Reproduction; Research and Analysis Methods; Reverse Transcriptase Polymerase Chain Reaction; Stress; Stresses; Studies; Surgical implants; Transcription factors; Vesicles; Veterinarians; Veterinary colleges; Veterinary medicine; Japan; China; United States > US; Massachusetts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0174535

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.