64 SIN3 transcription regulator family member A regulates porcine early embryonic development by modulating CCNB1 expression

SIN3 transcription regulator family member A (SIN3A) is the central scaffold protein of the SIN3/HDAC (histone deacetylase) transcriptional repressor complex. We previously found that SIN3A participates in the mouse pre-implantation development by finetuning HDAC1 expression. However, it remains unresolved whether this functional significance of SIN3A is conserved in other mammals. The objective of this work was thus to characterise the expression profiles and the functional role of SIN3A in pre-implantation development using non-rodent animal models. RNA sequencing results show that a large amount of SIN3A mRNA is present in oocytes and early embryos before embryonic genome activation and a low amount thereafter, suggesting a maternal origin of SIN3A in all species examined. Interestingly, immunofluorescence data show that SIN3A protein level peaks at the 4-cell stage in pigs compared with the morula stage in cattle, suggesting a differential role of SIN3A among species. To explore the function of SIN3A in early embryonic development, we used a short interfering (si)RNA-mediated knockdown approach in porcine parthenogenetic activated (PA) embryos. Immunocytochemical analysis showed that SIN3A levels were diminished ∼80% compared with nonspecific siRNA (NC) injected control (n=3). To monitor the developmental potential of embryo depleted of SIN3A, we injected SIN3A-siRNA into MII stage oocytes, followed by parthenogenetic activation, and percent cleavage and blastocyst formation were recorded. We found that SIN3A knockdown (KD) did not affect the cleavage rate (NC vs. KD, 83.63±3.63% vs. 80.08±4.66%, n=5), but significantly reduced blastocyst rate compared with the NC group (NC vs. KD, 36.64±4.28% vs. 6.33±3.12%, n=5). Specifically, SIN3A depletion in early embryos causes developmental arrest at 2-cell stage in pigs but does not affect early embryonic development in bovines. In contrast with mouse data, SIN3A depletion results in only a slight decrease and even no difference in HDAC1 expression in porcine and bovine early embryos, respectively. In addition, HDAC1 knockdown does not cause 2-cell block but leads to a reduced blastocyst rate, suggesting that the effect of SIN3A depletion on porcine early embryos is independent of HDAC1. RNA-Seq analysis was used to compare the global transcript content between NC and KD 2-cell embryos. A total of 23 genes (14 upregulated and 9 downregulated) had undergone significant changes. Interestingly, cyclin B1 (CCNB1) ranked second among downregulated genes. To test whether knockdown of CCNB1 would display a similar phenotype in porcine early embryos, we injected CCNB1-siRNA into pronuclear stage. CCNB1 KD resulted in a similar phenotype as SIN3A depletion. Injection of exogenous CCNB1 mRNA into SIN3A-depleted embryos could partly rescue embryonic development. In conclusion, our results indicate SIN3A plays an essential role in porcine early embryonic development, probably involving the regulation of CCNB1 expression. This work was funded by National Natural Science Foundation of China, the Anhui Provincial Natural Science Foundation and China Postdoctoral Science Foundation.