(148) Dysregulation of Canonical WNT Signaling is Observed in the Cumulus Cell Transcriptome in Both Human and Murine Models with Reproductive Aging

Abstract Authors: Shawn M. Briley¹, Mary E. Haywood¹, Blair R. McCallie¹, William B. Schoolcraft¹, Mandy G. Katz-Jaffe¹

1. CCRM Fertility, Lone Tree, CO, United States

Abstract Text:

Granulosa cells are a critical component of the ovarian follicle and are required for the optimal development of a competent egg. In antral follicles, two populations of granulosa cells are present: mural granulosa cells and cumulus cells (CCs), the latter being released from the follicle during ovulation with the egg. CC expansion is critically associated with ovulation and has been found to be impaired in aged mice. The aim of this study was to investigate the impact of mammalian ovarian aging on the cumulus cell transcriptome in both human and murine models.

“Young” infertility patients (< 35 years; n=16; mean age=29.0 years) and advanced maternal age “AMA” women ( >40 years; n=18; mean age=41.6 years) undergoing infertility treatment donated their discarded CCs at the time of oocyte retrieval, with IRB approval and patient consent. RNA from CC samples (Young n=7, mean age=26.9 years; AMA n=9, mean age=42.1 years) was isolated (PicoPure RNA Isolation kit) followed by library build using the NEBNext Low Input RNA Library Prep Kit and RNA sequencing (NextSeq, Illumina). Young (3mo) and aged (15mo) mice were stimulated with 5IU PMSG prior to harvesting ovaries and collection of germinal vesicle (GV) oocytes with surrounding cumulus cells. GV oocytes were cultured in maturation medium for 24 hours, and CC RNA from GVs that reach metaphase II with normal spindle morphology and chromosome alignment were utilized for RNA isolation and sequencing as described above.  Reads were processed with gsnap and featureCounts. Differential expression analysis was performed using DESeq2 (padj< 0.05). Statistical analysis included Student’s t-test or Welch’s t-test using GraphPad Prism (p< 0.05).

Unsupervised hierarchical clustering separated “Young” and “AMA” CC samples, with RNA-sequencing analysis identifying 336 differentially expressed genes (DEGs) in human CCs and 1,149 DEGs in mouse CCs (padj< 0.05). Of specific interest in human CCs, was WNT5A, known to be essential for normal folliculogenesis and ovulation. RNA-sequencing data revealed significantly decreased transcription of WNT5A and altered transcription of several of its regulators, including FZD5, a WNT5A receptor, and KLF4, an activating transcription factor (padj< 0.05). Additionally, GREM1 and GREM2, two inhibitors of TGFβ (also an activator of WNT5A transcription), showed increased expression with CC aging (p< 0.05). KEGG pathway analysis of young and aged mouse DEGs identified WNT signaling as a top affected pathway, suggesting WNT signaling is altered in aged mouse CCs. Fzd7, a WNT receptor, was found to have increased expression in aged mice CCs, as were Dvl1, a signal transducer in the canonical WNT pathway, and Lgr6, an R-spondin receptor that potentiates canonical WNT signaling. Additionally, c-Jun, a downstream target of the canonical WNT signaling pathway also showed increased expression in aged mice CCs.

In mouse models, suppression of canonical WNT signaling by Wnt5a is required for proper folliculogenesis and ovulation. CCs from aged mice were found to have increased expression of genes involved in canonical WNT signaling. Likewise, human CCs from AMA women were found to have decreased expression of WNT5A and altered expression of critical genes and regulators known to inhibit canonical WNT signaling. In summary, decreased egg quality observed with ovarian aging may be partially explained by increased canonical WNT signaling and decreased non-canonical WNT signaling.