240 DEVELOPMENT, DIFFERENTIATION, AND Trk EXPRESSION IN PARTHENOGENETIC BOVINE BLASTOCYSTS

Abstract

Parthenogenetic embryos allow study of the roles of paternal and maternal genomes in early mammalian development. Nevertheless, pregnancies established with parthenotes arrest around 48 days. Genomic imprinting alterations, fewer cells, and apoptotic index are higher in parthenotes than in IVF embryos and are likely to contribute to the failure to reach full-term development. Neurotrophins are a family of anti-apoptotic cytokines that mediate survival, growth, and differentiation by binding to two types of cell surface receptors, tyrosine kinase (Trk) and the low affinity p75 neurotrophin receptor (p75). Trk and p75 receptors have been localized in early bovine in vitro-produced embryos. At present there are no available data on expression of Trk and numbers of cells in the inner cell mass (ICM) and the trophectoderm (TE) of parthenogenetic embryos. The aim of this study was to evaluate the quality of bovine parthenotes in terms of cell allocation and blastocyst development, and to analyze TrkA, TrkB, and TrKC expression in the ICM and TE. Starting from in vitro-matured slaughterhouse oocytes, embryos were produced by conventional IVF, while parthenotes resulted from ionomycin activation followed by 6-dimethylaminopurine. Zygotes were cultured in SOF + 6 gL^{– 1} BSA. In vitro development was assessed for IVF embryos on (and referred to on) Days 3, 6, 7, and 8 after fertilization, and 24 h before these time points for parthenotes. Data were analyzed by the GLM procedure of SAS SAS Institute, Inc., Cary, NC, USA). Parthenotes cleaved at rates similar to IVF embryos (80.8 ± 3.9 v. 85.8 ± 3.9, respectively), but percentages of 5–8 and 8–16 cell stages were lower in parthenotes (40.4 ± 4.3 v. 67.9 ± 4.3, P < 0.005, and 9.7 ± 3.5 v. 25.3 ± 3.5, P < 0.01, respectively). However, parthenogenetic blastocyst rates were higher than those in IVF embryos (Day 6: 33.6 ± 2.6 v. 11.0 ± 2.6, P < 0.005; Day 7: 49.2 ± 4.1 v. 30.0 ± 4.1, P < 0.02). Double staining showed fewer TE cells in parthenotes (78.7 ± 8.5) than in IVF embryos (111.0 ± 8.6, P < 0.02). This reduction accounted for a reduced number of total cells in parthenotes (105.3 ± 9.9) v. controls (144.0 ± 9.8, P < 0.01), while numbers of cells in the ICM were comparable (27.9 ± 3.5 v. 31.1 ± 3.5, in parthenotes and controls, respectively). As in the case of IVF embryos, immunocytochemical analysis showed positive staining for Trk receptors in parthenotes. Although parthenotes showed blastocyst development rates higher than in IVF embryos, the reduced amount of TE cells in parthenotes could negatively affect implantation. Interestingly, parthenotes do not contain abnormally reduced cell numbers in their ICM, and they express Trks. Therefore, specific stimulation of these receptors with appropriate cytokines could improve blastocyst development and embryonic stem cell derivation.

This work was supported by the Spanish Ministry of Science and Education (AGL2005-04479). Dr. Muñoz was supported by FICYT.