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.

A myriad of locally produced factors into the microenvironment of the reproductive tract is regulated, not one-way but rather, through embryonic–maternal cross-talk. In this minireview, we focused on the exosomes, which are cell-derived vesicles of 30–100 nm in diameter, as a communicating language facilitating this dialog. These nanovesicles are secreted from preimplantation embryos, oviduct epithelium, and endometrium as well as from the placenta, and contain proteins, messenger RNA (mRNA), microRNA, and DNA cargoes, and have pleiotropic effects on both embryonic and maternal environments. A better understanding of the molecular mechanisms mediating this cross-talk will lead to the development of new regulating agents, with novel diagnostic, biological, and therapeutic potential for either supporting or hindering the normal reproductive functions.

Kisspeptin (Kp) is best known as a multifunctional peptide with roles in reproduction, the cardiovascular system and cancer. In the present study the expression of kisspeptin hierarchy elements (KISS1, GNRH1 and LHB) and their receptors (KISS1R, GNRHR and LHCGR, respectively) in porcine ovary and in cumulus–oocyte complexes (COCs) were investigated, as were its effects on the in vitro maturation (IVM) of oocytes and their subsequent ability to sustain preimplantation embryo competence after parthenogenetic electrical activation. Kp system elements were expressed and affected IVM of oocytes when maturation medium was supplemented with 10–6 M Kp. Oocyte maturation, maternal gene expression (MOS, GDF9 and BMP15), blastocyst formation rate, blastocyst hatching and blastocyst total cell count were all significantly increased when oocytes were matured in medium containing Kp compared with the control group (without Kp). A Kp antagonist (p234) at 4 × 10–6 M interfered with this hierarchy but did not influence the threshold effect of gonadotrophins on oocyte maturation. FSH was critical and permissive to Kp action on COCs by increasing the relative expression of KISS1R. In contrast, Kp significantly increased apoptosis, the expression of pro-apoptotic gene, BAK1, and suppressed trophoblast outgrowths from hatched blastocysts cultured on feeder cells. The present study provides the first functional evidence of the Kp hierarchy in porcine COCs and its role in enhancing oocyte maturation and subsequent developmental competence in an autocrine–paracrine manner. However, Kp supplementation may have a harmful impact on cultured hatched blastocysts reflecting systemic or local regulation during the critical early period of embryonic development.