164 HEAT STRESS ENHANCES THE DEVELOPMENT OF PORCINE PARTHENOGENETIC EMBRYOS, BUT NOT IN VITRO-FERTILIZED EMBRYOS

Abstract

Recent research in our laboratory has demonstrated that a borderline supra-physiological heat stress (42°C) of moderate duration (9 h) can have beneficial effects on the development of porcine parthenogenetic embryos depending on the timing of heat stress application relative to oocyte activation. A 9-h heat stress (HS) applied to presumptive parthenotes immediately after oocyte activation by electrical stimulus (0-9 h post-activation [hpa]; 09HS) significantly accelerated the timing of the first cleavage event: only 26% of the non-heat stressed (NHS) controls had cleaved by 24 hpa, but more than 60% of the 09HS embryos had cleaved after 24 h (P < 0.0001; ANOVA as determined using the PROC GLM commands in SAS; SAS Institute, Inc., Cary, NC, USA). This discrepancy had all but disappeared by 48 hpa (P > 0.1). After 7 days in culture, however, 38.0% of the 09HS embryos developed to blastocyst whereas 18.5% of the NHS embryos developed to that stage (P < 0.0001). Pharmaceutical and biomolecule inhibitors were used to block several candidate biochemical pathways and processes that were thought to be involved in bringing about the phenotype observed in the 09HS embryos: alpha amanitin (an RNA polymerase inhibitor), roscovitine (a cdc2/MPF inhibitor), cordycepin (an inhibitor of polyadenylation), SP600125 (a stress-activated protein kinase inhibitor), LY294002 (a phosphoinositide 3-kinase inhibitor), and MG132 (a 26S proteasome inhibitor). NHS and 09HS embryos were incubated with and without inhibitors. At least three replicates of each experiment were performed. Although some interesting data have been generated, none of these pathways individually appears to be responsible for the observed beneficial effect of the heat stress on early embryo development in parthenotes (P > 0.01). Efforts to replicate in in vitro-fertilized (IVF) embryos the beneficial effects of the heat stress observed in parthenogenetic embryos have been largely unsuccessful, even if the timing of stressor application was altered. IVF embryos were heat stressed at 42°C for 9 h at different time points after insemination: 4-13 h post-insemination (hpi; 413HS), 13-22 hpi (1322HS), or 22-31 hpi (2231HS). The 24- and 48-h cleavage rates, and Day 7 blastocyst rates were assessed. These observations were repeated at least five times. Although the heat stress was able to alter the 24-h cleavage rates (P < 0.0001), 48-hpi cleavage rates and Day 7 blastocyst rates were not significantly different between groups (P > 0.1). We have observed similar results in nuclear transfer (NT) embryos (Lai et al. 2005), leading us to postulate that the nuclear remodeling that is necessary in IVF and NT embryos prevents these embryos from responding positively to the HS in a manner similar to that in the parthenogenetic embryos. Experiments to test this hypothesis as well as further genomic and proteomic analyses of this phenomenon are ongoing in our laboratory.

This work was funded in part by Food for the 21st Century.