340. ANALYSIS OF PROTEINS SECRETED BY THE PREIMPLANTATION MOUSE EMBRYO
R. Gentles A and C. O’Neill ASydney Centre for Developmental and Regenerative Medicine, University of Sydney, Sydney, NSW, Australia.
Reproduction, Fertility and Development 22(9) 140-140 https://doi.org/10.1071/SRB10Abs340
Published: 6 September 2010
Abstract
Many embryos generated by assisted reproductive technologies do not have the capacity for full development. A non-destructive test that allows the most viable embryos to be identified would allow development of a range of strategies to improve these technologies. Analysis of the proteinaceous secretome of the preimplantation mouse embryo by electrospray mass spectrometry of tryptic digests of embryo-conditioned culture media identified 20 peptides.1 Lactate dehydrogenase beta (LDH) and protein disulfide isomerase (PDI) were consistently among the most abundant peptides identified. PDI has been found to be secreted and surface expressed in a number of other cell types suggesting a biologically significant role. LDH-beta is a marker of plasma membrane integrity and is widely used in medicine for assessment of cardiac health. The aim of this study was to assess the expression of these proteins in the early embryo to gain some understanding of the potential significance of their presence within the embryonic secretome. Zygotes (B6CBF2) were collected 20–22 h post hCG. They were cultured in groups of 10 embryos for 96 h in 10 mL of modHTF 30 µg BSA/mL media. Embryonic protein was extracted for western blot analysis or embryos subjected were to immunolocalization studies for PDI and LDH. Western blot analysis revealed presence of proteins of expected molecular mass. Staining for both proteins occurred at high levels at each stage of development throughout pre-implantation development. Staining was predominantly cytoplasmic and excluded from nuclei. Cultured blastocysts had less LDH than fresh but the level for PDI was similar for both. Staining of non-permeabilized embryos revealed extracellular staining of PDI. The results show high levels of cellular expression of two proteins reported to be released by the embryo in vitro.
(1) Beardsley A, Li Y, O’Neill C (2010). Characterization of a diverse secretome generated by the mouse preimplantation embryo in vitro. Reproductive Biology and Endocrinology 8, 71.
