Improvement of Cloned Embryos Development by Co-Culturing with Parthenotes: A Possible Role of Exosomes/Microvesicles for Embryos Paracrine Communication

Abstract

It is well known that embryos cultured in a group can create a microenvironment through secretion of autocrine and paracrine factors that can support and improve the embryos' development when compared to the embryos cultured individually. In this study, we used a co-culture system for paracrine communication between different kinds of embryos. The results showed that co-culture of porcine parthenogenetic (PA) embryos significantly improved the in vitro development of cloned (nuclear transfer, NT) embryos. To reveal the possible mechanism of communication between the two groups, we isolated exosomes/microvesicles (EXs/MVs) from the PA embryos conditioned medium (PA-CM) through differential centrifugation and identified them through transmission electron microscope and immunoflourescence against exosomal/membrane marker CD9. Furthermore, these EXs/MVs were found to contain mRNA of pluripotency genes (Oct4, Sox2, Klf4, c-Myc, and Nanog), and the PKH67-labeled EXs/MVs could be internalized by the NT embryos. The current study demonstrates that cloned embryos' developmental competence can be improved through co-culturing with PA embryos and revealed, for the first time, that in vitro-produced embryos can secrete EXs/MVs as a possible communication tool within their microenvironment. Moreover, it provides a new paradigm for embryo-to-embryo communication in vitro.

Challenges and perspectives to enhance cattle production via in vitro techniques: focus on epigenetics and cell-secreted vesicles

“…Also, transcripts for OCT4, KLF4 and NANOG were identified in extracellular vesicles isolated from the cell culture media of PA embryos; additionally, levels of these transcripts were increased in NT embryos following the co-culture with PA embryos. Furthermore, it was demonstrated that these extracellular vesicles could be up take by NT embryos independent of the zona pellucida presence, demonstrating that these vesicles are capable of crossing this structure (SAADELDIN et al, 2014). Thus, these results demonstrate that extracellular vesicles can mediate cell communication and improve embryo development in vitro during the preimplantation period.…”

“…Cellsecreted vesicles containing bioactive molecules such as miRNA, mRNA and proteins have been shown to mediate cell communication among different cell types (BURNS et al, 2014;. Recently, an experiment demonstrated the transfer of cell-secreted vesicles from parthenogenetic embryos to cloned embryos, providing a new paradigm for embryo-to-embryo communication in vitro (SAADELDIN et al, 2014). Herein, this review presents new perspectives on the role of cell-secreted vesicles during female gamete development as well as during pre-implantation period.…”

“…A mechanism to improve is performing embryo coculture systems between nuclear transfer (NT) embryos and somatic cells, offering the paracrine effects of the in vivo embryo development (SAADELDIN et al, 2014). To test the hypothesis that extracellular vesicles play an important role and can improve porcine SCNT embryo production, a co-culture system using parthenogenetic (PA) embryos in the bottom of the well with NT emrbyos on top separated by a filter was developed.…”

Cloning and its Applications

“…However, the use of this technique is limited owing to its low efficiency and several trials showed the improvement of cloning efficiencies [9]. Therefore, the current issue of "Cloning & Transgenesis" will focus on cloning and semi-cloning regarding the trials to improve their efficiency and to reveal their potential applications in biology and medicine.…”

The expression of growth factor signaling genes in co-culture IVM

“…Members of the TGFβ superfamily, including TGFβ1, TGFβ3, bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are expressed in the oocyte from very early stages and are responsible for oocyte maturation (da Silveira et al 2014). Further, few studies have focused on the effects of exposure to a combination of growth factors on oocyte IVM (Arat et al 2016), but the co-culture system also chosen to support the IVM condition (Saadeldin et al 2014;Appeltant et al 2016).…”