237 EFFECTS OF IN VITRO V. IN VIVO CULTURE ON EXPRESSION OF EMBRYO DERIVED GENE TRANSCRIPTS INVOLVED IN APOPTOSIS IN SINGLE BOVINE EMBRYOS

Abstract

Earlier studies reported that the level of apoptosis in in vitro-produced bovine blastocysts is higher then in in vivo developed blastocysts (Gjorret et al., 2003: Biol. Reprod, in press). The molecular basis for this difference has not yet been studied. The regulation and execution of apoptosis is dependent on a cascade of events in which many proteins are involved. The aim of the present study was to analyze expression of BAX, a pro-apoptotic, and BCL-XL, a anti-apoptotic member of the BCL-2 family, and heat shock protein (HSP 70.1) transcripts in blastocysts cultured in vitro or in vivo. Furthermore, to verify if these transcripts detected in the blastocysts stages were newly expressed from the embryonic genome, a RNA polymerase II specific inhibitor, α-amanitin, was added to the culture medium from the zygote stage onwards, to block transcription. For the in vitro group, embryos were obtained from abattoir oocytes after IVM/IVF and IVC in SOF medium. For the in vivo group, embryos were collected from normally cyclic cows, superovulated with 3000 IU eCG (Intergonan;; Intervet, Tönisvorst, Germany) at day 7 po by non-surgical uterine flushing. The developmental stage of the embryos was determined with stereomicroscopy, and early blastocysts (eb), blastocysts (b), and expanded blastocysts (xb) were collected and frozen at −80°C. For transcription inhibition experiment, embryos were cultured in vitro as for the in vitro group until the 8- to 16-cell stage at 100 h after the start of fertilization, one group with 10 mM α-amanitin added to the culture medium (α-amanitin group) and the other group without (control group). A highly sensitive semi-quantitative RT-PCR assay (Wrenzycki et al., 1999) Mol. Reprod. Dev. 53, 8–18) was used to determine the relative levels of gene transcripts in single blastocysts and pooled 8- to 16-cell embryos. The relative abundance was calculated on a per cell basis per embryo. Assays were repeated on average eight times. Data on the relative expression of transcripts in blastocysts were analyzed by Anova followed by multiple pairwise comparisons using the Tukey test. No significant differences in relative abundance between in vitro and in vivo cultured embryos, for any of the developmental stages, were found for the three apoptosis related genes. The molecular basis for the difference in level of apoptosis between in vitro and in vivo cultured blastocysts is not related to the level of expression of BAX, BCL-XL and HSP transcripts but other genes involved in the apoptotic cascade may be responsible for the reported differences. The expression of BCL-XL and HSP 70.1 transcripts in the blastocysts was from embryonic origin as no expression of these transcripts was detected in the 8- to 16-cell stage embryos treated with α-amanitin. The expression of BAX gene transcripts was not affected by α-amanitin. Probably the maternally derived transcripts are very stable and not yet degraded at the 8- to 16-cell stage.

Table 1 
Relative abundance (±SEM) of BAX, BCL-XL, and HSP 70.1 transcripts in bovine single in vitro or in vivo-produced eb, b, and xb and in vitro produced pooled 8- to 16-cell stage embryos treated with or without α-amanitin