Characterization of a diverse secretome generated by the mouse preimplantation embryo in vitro

Abstract

This study investigates the suitability of surface-enhanced laser desorption and ionization time-of-flight (SELDI-TOF) and electrospray ionization (ESI) mass spectrometry for analysis of the proteins released by the mouse preimplantation embryo in vitro. SELDI-TOF analysis with CM10 or IMAC30 (but not Q10) protein chips detected a protein peak at m/z approximately 8570 released by both C57BL6 and hybrid embryos. No other peaks unique to the presence of the embryo were identified with this method. ESI mass spectrometry of tryptic digests of embryo-conditioned media identified a total of 20 proteins released during development from the zygote to blastocyst stage. Four proteins were expressed in at least 7 out of 8 cultures tested, one of these (lactate dehydrogenase B) was in all cultures. A further five proteins were in at least half of the cultures and 11 more proteins were in at least one culture. The expression of two of these proteins is essential for preimplantation embryo development (NLR family, pyrin domain containing 5 and peptidyl arginine deiminase, type VI). A further four proteins detected have roles in redox regulation of cells, and three others are capable of inducing post-translational modifications of proteins. This study shows the feasibility of ESI mass spectrometry for identifying the proteins secreted by the preimplantation embryo in vitro. This analysis identifies a range of targets that now require detailed functional analysis to assess whether their release by the embryo is an important property of early embryo development.

Figure 1

SELDI-TOF analysis of conditioned culture media from (A) F1 blastocysts and (B) media alone on the CM10 protein chip. Chip profile shows m/z 3000-70,000 in both trace and gel views. Protein peaks were detected in the media at m/z values coinciding with the molecular weight of BSA proteins (66, 33, 22 and 13.7kDa). A protein peak of ~8570 (*) was detected in the conditioned culture media (a'), but not in the media only samples (b') which contained no developing embryos.

Figure 2

SELDI-TOF analysis of conditioned culture media taken 24, 48, 72 and 96 h after culture of F1 zygotes using the CM10 chip. The relative changes in the amplitude of the 8570 m/z peak for media from F1 embryos. Results are the mean + SEM.

Figure 3

SELDI-TOF analysis of conditioned culture media from F1 (A), C57 (B) animals and media alone (C) on the IMAC30 protein chip. Chip profile shows m/z 3000-70,000. Similar to the CM10 chip, peaks were detected in the media at molecular weights coinciding with BSA proteins. A) Gel and trace view of F1 conditioned media. B) Gel view of C57 conditioned media. C) Gel view of media that did not contain developing embryos. A protein peak of ~8570 (*) was detected in the conditioned culture media from both F1 (a') and C57 (b') media, but not in the media only samples (c')