(348) The Role of Connexin 37 in Early Antral Bovine Folliculogenesis

Bidirectional communication between oocytes and their supportive somatic cells is critical for ovarian follicle development. One method of this communication is via gap junctions, protein channels that directly link the cytoplasm of adjacent cells for diffusion of small molecules. Among those, connexin 37 (CX37) connects oocytes to adjacent cumulus cells. To better understand the role of CX37 in supporting follicle development, we sought to block CX37 function in bovine cumulus-oocyte complexes (COCs) via introducing a 13 amino acid mimetic peptide that blocks function of the protein’s hemichannels. COCs were collected from early antral follicles ( < 2 mm) to examine CX37 function prior to transcriptional silencing and nuclear maturation. COCs were cultured in mimetic peptide (MIM), a scrambled version of the peptide (SCR), or vehicle control (VEH) at 38.5°C. Heat stress (HS, 41°C), known to impair gap junction communication in COCs, was used as a positive control. After 16 hours of culture, oocytes were denuded of cumulus cells (CCs), and the two cell types were processed separately. 

Oocytes were examined for chromatin state via Hoechst DNA labeling and global mRNA transcription via the Click-iT fluorescence kit. Overall, there was an effect of treatment on chromatin remodeling (P < 0.05), with HS oocytes having the lowest proportion of oocytes at GV0, VEH having the highest, and MIM and SCR oocytes having intermediate proportions. As expected, global mRNA transcription was higher in GV0 oocytes than in GVc oocytes, and HS and MIM oocytes had numerically higher values than SCR or VEH oocytes. However, mRNA transcription in oocytes was unaffected by treatment (P = 0.5). 

We performed bulk RNA-sequencing on oocytes and CCs from all treatments and found 16,503 genes expressed in oocytes and 14,305 in CCs. Of these, 12,725 genes shared between the two cell types. Within the CC, there were 78 differentially expressed genes (DEGs) between MIM and VEH, 93 DEGs between HS and VEH, and 144 genes DEGs between MIM and HS. Oocytes had very few ( < 15) DEGs between any treatments. Gene ontology analysis was performed using DAVID. In mimetic-treated CCs compared with vehicle-treated CCs, the most enriched upregulated pathway was the spliceosome (U1, U2, and U4 spliceosomal RNA) and the most enriched downregulated pathway was positive regulation of transcription by RNA polymerase II (WNT5A, PROX1, HOXA4). In heat stressed CCs compared with vehicle-treated CCs, the most enriched upregulated pathway was stress response (DNAJB1, HSP90AA1, HSPH1, AHSA2, HSPB1, HSPA1A) and the most downregulated was cell cycle (CCNB3, ASPM, TPX2, CCNB1, CDCA3, MKI67). When comparing heat stressed CCs to mimetic-treated CCs, the most enriched upregulated pathway was stress response (HSP90AA1, HSPH1, CHORDC1, AHSA2, HSPA2, SGK1, HSPA1A) and the most enriched downregulated pathway was the spliceosome (RBM3, CIRBP). 

Overall, results show that blocking CX37 in early antral COCs via mimetic peptide had no effect on chromatin remodeling or global mRNA transcription in oocytes. Transcriptomic profiling indicates changes in spliceosome gene expression in CCs exposed to MIM. Heat stress, which blocks both CX37 and connexin CX43 among other effects, affects CCs via different pathways than blockage of CX37 alone, predominantly via heat stress response. These results suggest that inhibition of gap junctional communication between the oocyte and CCs disrupts follicular function and does so in a distinct manner from global gap junction inhibition via heat stress.