PADI6 is a maternal effect gene (MEG) that is expressed throughout oocyte maturation and appears to be critical for early embryo development and embryonic genome activation (EGA). It is described as the fifth member of the oocyte-specific subcortical maternal complex (SCMC), which is involved in transcription, epigenetic regulation and cytoskeletal organisation. PADI6 is also essential for the maintenance of critical structures in the oocyte called cytoplasmic lattices that are believed to function as storage sites for maternal transcripts and proteins. It is hypothesised that PADI6 is involved in translational regulation in the oocyte in preparation for EGA. Experiments were conducted on bovine oocytes as a physiologically relevant model for functional investigations of the role of PADI6 and its interaction with the SCMC during human oocyte maturation and embryo development.
Initial studies characterising the expression of PADI family genes in different bovine somatic tissues showed that PADI6 expression was restricted to the oocyte and early embryo. PADI6 expression patterns were mapped across bovine oocyte meiotic maturation following the in vitro maturation (IVM) of oocytes and during preimplantation embryogenesis in embryos derived following in vitro fertilisation and embryo culture to the blastocyst stage. Quantitative PCR analysis of Smart-seq2 cDNA libraries from individual oocytes and embryos indicated that PADI6 was highly expressed in the germinal vesicle (GV) and metaphase II (MII) oocytes and embryos prior to EGA (8-16 cell stage in bovine) and thereafter was significantly reduced by the blastocyst stages. This expression pattern was in marked contrast to the tissue distribution of other members of the PADI family PADI1-4 which showed no expression in oocytes and early embryo development in the bovine model. The expression patterns of the other members of the SCMC – namely KHDC3L, NLRP5, OOEP and TLE6 were also mapped across oocyte maturation and preimplantation embryo development.
Functional analysis of the role of PADI6 during bovine oocyte maturation was conducted using a validated system for the microinjection of double-stranded short-interfering RNAs (dsiRNAs) into cumulus-enclosed GV oocytes (n=17) relative to duplex buffer injected (n=22) and scrambled siRNA (n=19) controls. Gene knockdown (KD) was assessed following 24 hr of IVM and was found to reduce the expression of PADI6 by an average of 74±3.6% (p<0.05). KD of PADI6 did not affect oocyte meiotic progression to metaphase II or cumulus expansion after IVM. However, targeted real-time PCR highlighted 7 transcripts (DNMT3A, DPPA3, PLAGL1, PRDX1, TRIM28, ZP1 and ZFP57) associated with oocyte and embryo developmental competence and epigenetic regulation that were significantly dysregulated in PADI6KD oocytes. Full transcriptome analysis by RNA sequencing was conducted on 6 individual PADI6KD MII oocytes and 12 controls. Bioinformatic analysis identified 452 differentially expressed genes (DEGs): 165 genes were downregulated and 287 genes were upregulated following PADI6 KD. Network analysis revealed that 61 DEGs were directly or indirectly linked to PADI6. A diverse number of transcripts were involved in RNA processing and translation. Genes from 7 downstream networks and 10 downstream networks were associated with infertility and organisation of the cytoskeleton, respectively. Finally, KD of PADI6 appeared to impact oocyte metabolism as the regulation of 5 amino acids was altered in PADI6KD oocytes compared to controls.
Overall, the results of this thesis suggest a role for PADI6 in translational control of maternal transcripts which may impact on oocyte developmental competence in the bovine.
