Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, α chain), and CD11a (LFA-1, α chain) on mouse oocytes, and pre-and peri-implantation stage embryos was examined by quantitative indirect immunofluorescence microscopy. ICAM-1 was most strongly expressed at the oocyte stage, gradually declining almost to undetectable levels by the expanded blastocyst stage. NCAM, also expressed maximally on the oocyte, declined to undetectable levels beyond the morula stage. On the other hand, CD44 declined from highest expression at the oocyte stage to show a second maximum at the compacted 8-cell/morula. This molecule exhibited high expression around contact areas between trophectoderm and zona pellucida during blastocyst hatching. CD49d was highly expressed in the oocyte, remained significantly expressed throughout and after blastocyst hatching was expressed on the polar trophectoderm. Like CD44, CD49d declined to undetectable levels at the blastocyst outgrowth stage. Expression of both VCAM-1 and CD11a was undetectable throughout. The diametrical temporal expression pattern of ICAM-1 and NCAM compared to CD44 and CD49d suggest that dynamic changes in expression of adhesion molecules may be important for interaction of the embryo with the maternal cellular environment as well as for continuing development and survival of the early embryo.
This study investigated the suitability of surface-enhanced laser desorption and ionisation time-of-flight (SELDI-TOF) and electro-spray ionisation (ESI) mass spectrometry for the analysis of the proteins released by the mouse preimplantation embryo in vitro. SELDI-TOF analysis with CM10 or IMAC30 (but not Q10) chips detected a protein peak at m/z 8570 released by both C57BL6 and hybrid embryos. No other peaks unique to 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 blastocysts 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 putative proteins were detected in at least one culture. The pattern of protein secretion was not obviously different for C57BL6 or hyrid embryos. The expression of two of these proteins is essential for preimplantation embryo development (NLR family, pyrin domain containing 5 protein 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 modification of proteins. This study shows the feasibility of ESI mass spectrometry and the limitations of SELD-TOF mass spectrometry for identifying the proteins scereted 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 the early embryo, or an artefact of in vitro culture.
Epigenetic modification of the genome is one potential mechanism for the persistence of altered gene expression. Recent evidence indicates that the growth environment of the preimplantation embryo may have long-term consequences in the gene expression of progeny (Mahsoudi et al. 2007; Watkins et al. 2008). This study assessed whether culture of zygotes induced persistent epigenetic changes in gene expression. The agouti viable yellow (Avy) allele is epigenetically sensitive, and is a widely used model for studying epigenetic phenomena. It has active (hypomethyalted) and inactive (hypermethylated) epialleles. Progeny with an active epiallele have yellow fur; those with an inactive epiallele have an agouti coat. Some progeny are mosaics for the two epigenetic states and have a mottled coat color. Males (Avy/a C57BL/6) were mated to wildtype females (A/A FVB). Embryo treatments were: (1) zygotes collected and cultured for 96 h in Sydney IVF Media suite and transferred to day 3 pseudopregnant Swiss females; (2) blastocysts collected and transferred to recipients without being subjected to culture; (3) naturally mated females left to give birth without interventioin. The number, sex, genotype, and epi-phenotype of the resulting progeny were recorded. The culture of zygotes caused a significant shift in the expression of the epialleles compared with embryos that were transferred without culture (P = 0.014), or those born following natural matings (P < 0.001). Offspring that resulted from culture are more likely to have active epialleles, resulting in a higher proportion of progeny with yellow fur. The results of this study show that culture of mouse zygotes changed the expected expression of the Avy allele. This study provides evidence for the epigenetic basis for the long-term ‘memory’ of preimplantation embryo’s growth environment, and for robust and readily accessible methodology for addressing the underlying molecular bases for this phenomenon. (1) Mahsoudi B, Li A, O’Neill C. Assessment of the Long-Term and Transgenerational Consequences of Perturbing Preimplantation Embryo Development in Mice. Biol Reprod 2007; 77: 889–896. (2) Watkins AJ, Ursell E, Panton R, Papenbrock T, Hollis L, Cunningham C, Wilkins A, Perry VH, Sheth B, Kwong WY, Eckert JJ, Wild AE, Hanson MA, Osmond C, Fleming TP. Adaptive responses by mouse early emb.
Platelet activating factor (paf) is an autocrine survival factor for preimplantation embryo. Binding of paf to its receptor activates PI3kinase, causing an IP3-dependent release of Ca2+ from intracellular stores as well as activation of Ca2+ influx via a dihydropyridine-sensitive Ca2+ channel. These actions result in the generation of a defined intracellular calcium ([Ca2+]i) transient in the 2-cell embryo[1]. By using combined whole-cell patch-clamp and real-time [Ca2+]i analyses, we have shown that paf also induces a concomitant hyperpolarisation of the membrane potential in 2-cell embryos, accompanied by an increased net outward ion current. Both the membrane hyperpolarisation and outward current were dependent upon the occurrence of the paf-induced [Ca2+]i transient[2]. The aim of this study was to investigate the characteristics of the paf-induced outward current in 2-cell embryos and to assess whether it has a role in normal mouse preimplantation development. We show that: (1) removal of extracellular anions or treatment with niflumic acid (NFA, 100 μM, a Ca2+-activated Cl- channel blocker) prevented activation of the outward current by paf but had no effect on the paf-induced [Ca2+]i transient; and (2) The culture of embryos with NFA (100 μM) from the 1-cell to late 2-cell stage significantly reduced their development to the blastocyst stage (P < 0.001), but treatment with NFA from the late 2-cell stage had no effect on development. The results show that paf induces an increase in [Ca2+]i which in turn activates a Ca2+-activated Cl- channel. The activity of this NFA-sensitive channel during the zygote to 2-cell stage is required for normal embryo development. (1) C. O’Neill (2008) The potential roles of embryotrophic ligands in preimplantation embryo development. Hum Reprod Update 14:275–288. (2) Y. Li, M.L. Day & C. O’Neill (2007) Autocrine activation of ions currents in the two-cell mouse embryo. Exp Cell Res. 313:2785–2794.
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