Poly(ADP-ribosyl)ation is post-translational modification of cellular proteins related to cell survival, cell death, cellular proliferation and epigenetic events. It has recently been shown to be important for pre-implantation development of mouse embryos. However, its function during early embryonic development of pig is not clear. This study investigated the importance of poly(ADP-ribosyl)ation during in vitro development of pig embryos produced by in vitro fertilization(IVF) or parthenogenetic activation (PA). Results showed that, chemical inhibition of PARP by 3-aminobenzamide (3-AB) did not influence the in vitro development of pig embryos up to morula stage (20±3.1 vs. 28.1±1.2%; p>0.05) but significanlty reduced the rate of blastocyst formation (5.2±2.1 vs. 20±3.1%; p<0.05) when compared to non-treated controls. Furthermore, culture of morula stage embryos in the pressence of 3-AB for 24h significantly reduced the rate of blastocyst formation (19.6± 4.6 vs. 41.4±5.3%; p<0.05) and expansion (4.7±3.0 vs. 28.1±6.1; p<0.05). The proportion of large-sized blastocyst (>200 μm) having higher blastocoel volume (15.3×106 μm3) was significantly reduced (p<0.05) in treatment group (32.2±7.8%) compared to non-treated control group (65.7±9.0%). TUNEL assay revealed that poly(ADP-ribosyl)ation-inhibited blastocyst had significantly increased indices of apoptosis than those of non-treated controls (10.88±0.02 vs. 2.71±0.01; p<0.05). These data suggest that Poly(ADP-ribosyl)ation may be important for blastocyst formation in pig embryo.

Autophagy, the process of bulk degradation and recycling of long-lived proteins, macromolecular aggregates, and damaged intracellular organelles, has recently been shown to be important for pre-implantation development and cavitation in mouse embryos. This study investigated the occurrence of autophagy and its importance in determining the in vitro development of pig embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). Western blot analysis for autophagy marker, microtubule associated protein light chain 3 (MAP-LC3), revealed the temporal pattern of LC3-conversion with intense changes during 10 20 h post-insemination and at morula-blastocyst transition in pig embryos. Specific inhibition of autophagy in 2 4 cell stage pig embryos, by treatment with 3-methyladenine (3MA), did not affect their embryonic development up to morula stage (p>0.05) but completely blocked their progression to the blastocyst stage (0.0±0.0 vs. 28.5±1.7% p<0.05). On the other hand, autophagy-inhibition in morula stage embryos significantly inhibited the formation of blastocoel (14.9±3.6 vs. 37.5±7.2%) and reduced the proportion of expanded blastocysts (5.6±2.6 vs. 29.6± 4.6% p<0.05). TUNEL assay revealed that autophagy-inhibited embryos had significantly increased indices of apoptosis (10.2±0.4 vs. 2.3±0.2) and DNA fragmentation (0.8± 0.1 vs. 0.3±0.1) than those of controls (p<0.05). Interestingly, while anti-oxidants reduced (p<0.05) the apoptosis and improved the blastocyst formation rate in pig embryos, it had no influence (p>0.05) on the expression of MAP-LC3. These data therefore, suggest that autophagy may have essential role during blastocyst formation in pig embryos.