Parthenogenetic reconstruction is one major strategy to create patient-specific stem cells. The aim of this study was to find the best artificial activation protocol for parthenogenetic activation of mouse and human oocytes comparing different methods. In a first set of experiments, in-vivo matured mouse oocytes and human failed-fertilized, in-vitro and in-vivo matured oocytes were artificially activated by a chemical (ionomycin) or electrical stimulus. In a second set of experiments, a combination of activating agents (electrical pulses followed by ionomycin or SrCl(2)) was applied in an aim to improve developmental competence. All embryos were evaluated daily until day 6 after activation. Mouse blastocysts were differentially stained to evaluate blastocyst quality. For mouse oocytes and human failed-fertilized oocytes, blastocyst development was significantly higher after electrical activation (P<0.05). For human in-vitro and in-vivo matured oocytes, blastocyst formation was only obtained after electrical activation of in-vitro matured oocytes. After combining activating agents, no differences in development could be observed. In conclusion, this study revealed that for both mouse and human oocytes development to the blastocyst stage was significantly better after electrical activation compared with chemical activation. Combining activating agents had no further positive effect on developmental potential.

In embryonic stem cell culture, small molecules can be used to alter key signaling pathways to promote self-renewal and inhibit differentiation. In mice, small-molecule inhibition of both the FGF/MEK/Erk and the GSK3b pathways during preimplantation development suppresses hypoblast formation, and this results in more pluripotent cells of the inner cell mass (ICM). In this study, we evaluated the effects of different small-molecule inhibitors of the FGF/ MEK/Erk and GSK3b pathway on embryo preimplantation development, early lineage segregation, and subsequent embryonic stem cell derivation in the humans. We did not observe any effect on blastocyst formation, but smallmolecule inhibition did affect the number of OCT3/4-and NANOG-positive cells in the human ICM. We found that combined inhibition of the FGF/MEK/Erk and GSK3b pathways by PD0325901 and CHIR99021, respectively, resulted in ICMs containing significantly more OCT3/4-positive cells. Inhibition of FGF/MEK/Erk alone as well as in combination with inhibition of GSK3b significantly increased the number of NANOG-positive cells in blastocysts possessing good-quality ICMs. Secondly, we verified the influence of this increased pluripotency after 2i culture on the efficiency of stem cell derivation. Similar human embryonic stem cell (hESC) derivation rates were observed after 2i compared to control conditions, resulting in 2 control hESC lines and 1 hESC line from an embryo cultured in 2i conditions. In conclusion, we demonstrated that FGF/MEK/Erk and GSK3b signaling increases the number of OCT3/4-and NANOG-positive cells in the human ICM, but does not improve stem cell derivation.

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Parthenogenetic activation of oocytes is a helpful tool to obtain blastocysts, of which the inner cell mass may be used for derivation of embryonic stem cells. In order to improve activation and embryonic development after parthenogenesis, we tried to use sperm injection and subsequent removal of the sperm head to mimic the natural Ca2+ increases by release of the oocyte activating factor. Visualization of the sperm could be accomplished by Hoechst staining and ultraviolet (UV) light irradiation. To exclude negative effects of this treatment, we examined toxicity on activated mouse oocytes. After activation, oocytes were incubated in Hoechst 33342 or 33258 stain and exposed to UV irradiation. The effects on embryonic development were evaluated. Our results showed that both types of Hoechst combined with UV irradiation have toxic effects on parthenogenetically activated mouse oocytes. Although activation and cleavage rate were not affected, blastocyst formation was significantly reduced. Secondly, we used MitoTracker staining for removal of the sperm. Sperm heads were stained before injection and removed again after 1 h. However, staining was not visible anymore in all oocytes after intracytoplasmic sperm injection. In case the sperm could be removed, most oocytes died after 1 day. As MitoTracker was also not successful, alternative methods for sperm identification should be investigated.

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We have demonstrated that FGF signalling is crucial for hypoblast generation and small molecules can be efficiently used to inhibit this process both in zygotes and parthenogenetic embryos.