71 Identifying PRDM family members potentially involved in epigenetic reprogramming after fertilization in porcine embryos

Dynamic changes in epigenetic marks occur before zygotic genome activation (ZGA) and are critical for establishing pluripotency. Upon fertilization, a rapid decrease in the DNA methylome is observed in multiple species. Recent studies point out that DNA demethylation is orchestrated by the 10–11 translocation (TET1/2/3) enzyme family. Transcripts of TET3 are abundant in oocytes and zygotes, while TET1 is highly expressed in the relatively late-stage embryos of mice, pigs, cows, and sheep. The PRDI-BF1 and RIZ homology domain containing (PRDM) family are transcription factors known to possess epigenetic reprogramming capacity. For example, TET1 co-functions with PRDM to promote lineage differentiation in blastocysts. The structure of the PRDM family is highly conserved, suggesting that multiple PRDM family members may be involved in epigenetic reprogramming during early development. We hypothesize that maternal transcripts of PRDM family members are expressed in oocytes and transcribed to promote TET3 activity. In this study, we characterized the transcript patterns of 11 PRDM family members during early embryo development in porcine oocytes. Porcine ovaries were harvested in a local abattoir. Cumulus–oocyte complexes were isolated and incubated in maturation medium for 42–44 h. After maturation, cumulus cells were removed, and MII oocytes were collected, fertilized in vitro, and then cultured for up to 7 days. Throughout in vitro embryo production, oocytes and embryos were randomly selected and collected, and cumulus cells and zona pellucida were removed and then flash frozen in 10 μL of PBS. Porcine oocytes/embryos were collected at germinal vesicle (GV), MII, 2-cell, 4-cell, 8-cell, and blastocyst stages of development. RNA was extracted from each stage, and real-time quantitative polymerase chain reaction was used to quantify PRDM family member expression. Exogenous green fluorescent protein transcripts were used to normalize target gene threshold cycle (Ct) values, and relative ratios were calculated using the 2^−ΔΔ Ct method with the GV stage used as the control. Five biological replications were performed, and data were analyzed using ANOVA. As expected, PRDM14 was expressed in 8-cell embryos and blastocysts but could not be detected in other developmental stages (P < 0.01). The transcript level of PRDM family members in general dynamically changed throughout embryonic development. The expression of PRDM1, PRDM2, PRDM4, PRDM5, and PRDM6 was detected in GV and MII oocytes, and their transcript abundance remained low during embryo development (P < 0.02). The expression patterns of PRDM1, PRDM2, PRDM5, and PRDM6 indicate that these genes are maternal transcripts in swine that are not reactivated post-ZGA. Future studies will elucidate their function during oocyte-mediated reprogramming and potential function as epigenetic modulators.

This work was supported in part by USDA-NIFA 2022-67015-36299.