147 AGGREGATION OF IN VIVO FERTILIZED AND PARTHENOGENETICALLY ACTIVATED MOUSE EMBRYOS

Abstract

Embryonic chimerism – mixing of cells originated from at least two different fertilizations – has been used as a tool for stem cell pluripotency diagnosis, transgenic rodent production, and organogenesis studies. Additionally, parthenotes are used for studies related to gene imprinting and the ability of their cells to compose the placenta and/or an adult animal. The aim of this work was to validate the production and characterize developmental kinetic of parthenogenetic embryos, obtained from C57BL/6 EGFP mice (EGFP), to determine their potential to produce chimeras, and to localize parthenogenetic cells on the produced blastocyst. Embryos were harvested from superovulated females. For the aggregation, pre-compaction IVF (Swiss Webster/SW strain) or parthenogenetic activated (PGA; SW and EGFP) embryos were used. For PGA, strontium chloride hydrate (5 mM for 6 h) was used. Two experiments were outlined in order to: i) evaluate the development and kinetic from IVF (IVF and in vitro developed; 2-cell stage embryos, n = 53) and from PGA (parthenogenetically activated and in vitro developed; n = 409 oocytes) techniques, both from SW; ii) evaluate the aggregation between pairs of control embryos (C; n = 20, 4-cell in vivo produced embryos from SW) or pairs of 4-cell in vivo produced SW embryos and 4- to 8-cell PGA EGFP embryos (parthenogenetic embryo, PG; n = 40). After manipulation (removal of the zona pellucida and approximation of pairs, for C and PG groups), all the groups were kept in vitro culture (37°C, 5% CO₂, and saturated humidity) for 48 to 60 h (C and PG), or up to blastocyst stage (IVF and PGA). Chimerism rate and parthenogenetic cells fluorescence (pre- and post-culture) were evaluated under an inverted microscope with epifluorescence source, and digital images were captured (Eclipse Ti and NIS-Elements, Nikon, Japan, respectively), by merging visible and UV light images. The rate of PGA (EGFP oocytes) was 54.5% (66/121), assessed 6 h post-activation by the presence of at least one pronucleus. The assessment was performed with 20× magnification and Hoffman modulation contrast in an inverted microscope. The rate of blastocyst production between IVF and PGA was significantly different (71.4 and 12.9%, respectively; P < 0.001, Fisher’s exact test). When developmental kinetic was evaluated there a difference in the average time to the majority of embryos reach blastocyst stage for IVF (48 h) and PGA (120 h) was observed. The chimerism rate was assessed by the presence of a single and cohesive cell mass (C) or by the incorporation of EGFP cells in the SW embryo (PG). There was difference (P = 0.006; Fisher’s exact test) between C (55.0%; 11/20) and PG (17.5%; 7/40) chimerism rates, assessed from 48 to 60 h of culture. In PG group, the incorporation of EGFP cells in the obtained chimeras was mainly detected in the trophectoderm although one chimeric blastocyst contained EGFP cells only in the inner cell mass. It was concluded that parthenogenetic embryos have a slower developmental kinetic (until 120 h of culture) than embryos derived from IVF.

Acknowledgment to FAPESP for fellowship and funding.