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

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.

Extracellular vesicles and melatonin benefit embryonic develop by regulating reactive oxygen species and 5‐methylcytosine

“…The effective dosage of melatonin in vitro was higher than its concentration in vivo; thus, we speculated that other important factors, such as EVs in oviduct fluid, are positively associated with melatonin function. In our previous study, we found that embryos can secrete EVs, which are essential for blastocyst formation and paracrine communication . In this study, we found that EVs exist in the ZP—consistent with a previous study—and that EVs derived from oviduct fluid are distributed on the surface of embryos, suggesting a physical interaction between EVs and embryos.…”

“…In our previous study, we found that embryos can secrete EVs, which are essential for blastocyst formation and paracrine communication. 24,43 In this study, we found that EVs exist in the ZP-consistent with a previous study 44 -and that EVs derived from oviduct fluid are distributed on the surface of embryos, suggesting a physical interaction between EVs and embryos. Notably, oviduct fluid-derived EVs decreased ROS, 5-mC, DNMT1, and DNMT3a levels and increased the blastocyst rate of embryos in the in vitro experiments.…”

Embryotrophic effects of extracellular vesicles derived from outgrowth embryos in pre‐ and peri‐implantation embryonic development in mice

“…A communication between embryos and maternal uterine milieu through secreting signals is required for proper mammalian development (O'Neill, ). Recent studies observed that EVs are secreted from various somatic cells including human embryonic stem cell (Mardpour et al, ), induced pluripotent stem cell (Bobis‐Wozowicz et al, ), ovarian follicle (da Silveira et al, ), and even embryos (Qu et al, ). Our previous study also demonstrated that membrane budding microvesicles from outgrowth embryos improved developmental competence in vitro and the implantation potential of cocultured embryos in utero as well (Kim et al, ).…”

“…Also, we did not provide data on how many EVs are adding or any quantitative measurement. According to previous studies, they calculated the concentration of EVs in the embryo culture medium by the equivalent of single embryos (Qu et al, ; Saadeldin, Kim, choi, & Lee, ). Saadeldin et al, () extracted 30 μl of exosomes/microvesicles (EXs/MVs) from 125 cloned porcine embryos.…”

Extracellular vesicles and their roles in stem cell biology

“…While investigating how these effects were mediated, the conditioned medium obtained from the SCNT embryos was shown to contain exosome sized vesicles that were positive for CD9, a classic EV marker. Interestingly, when these exosomes were added to embryos that were having their growth medium regularly replaced, a noticeable improvement in the health and development of the embryos was observed …”