(293) Pre-granulosa cell specific knockout of Vegfa increase steroidogenic and fibrotic gene expression in pubertal mouse ovaries

Corrine F. Monaco¹, Manjula Magamage^1,2, Rachel R. Reith¹, Scott G. Kurz¹, Ailenn Castillo¹, Brooke Rudloff¹, Katie Hoffman¹, Andrea S. Cupp¹

¹Department of Animal Science, University of Nebraska-Lincoln; Lincoln, NE, USA.

² Department of Livestock Production, Sabaragamuwa University of Sri Lanka; Belihuloya, Sri Lanka.

Abstract Text:

Our laboratory identified that granulosa cell-specific elimination of Vascular Endothelial Growth Factor A (Vegfa) is critical for follicle development and fertility. In the current study, we eliminated Vegfa in pre-granulosa cells utilizing Vegfa^-/^-; Sry-Cre mice (knockout). We hypothesized that Vegfa elimination in pre-granulosa cells would affect gene transcripts altering ovarian function at puberty. Ovaries from mice (n = 6 control, n = 4 knockout) were harvested on postnatal day 30 (d30), (puberty). Ovaries were collected for histology and RNA sequencing. Statistical analysis of transcript counts was performed with DESeq2 package in R. Genes with an adjusted p < 0.05 and log2 fold change ≤ │2│ (knockout vs control) were considered for Ingenuity Pathway Analysis (IPA). Histology of d30 ovaries revealed that there was no difference in follicular progression between controls and knockouts (p > 0.99). RNA sequencing revealed 1100 differentially expressed genes (adjusted p value < 0.05), 66% of the which were upregulated. IPA predicted activation of 35 pathways (p < 0.05, z-score = 2) in the d30 knockouts vs. controls, including steroidogenesis, connective tissue, and remodeling of cytoskeleton. Levels of steroid acute regulatory protein (Star) (p = 4.71E-12) and hydroxysteroid B dehydrogenase B2 (Hsd17b2) (p = 2.13E-07) were elevated in d30 knockout ovaries, which indicates that steroidogenesis may be altered in pubertal ovaries in the absence of Vegfa. Additionally, molecules known to be upstream of steroidogenesis such as follicle stimulating hormone, cAMP, and FSK, were predicted by IPA to be upstream regulators. Beta-estradiol was another predicted upstream regulator. Fibroblast growth factor (Fgf) signaling (p < 0.001, z-score = 1.89) and PI3K signaling (p < 0.05, z-score = 2) were also predicted to be elevated in d30 KO ovaries based on expression of Fgfs -1, -18, and -19, which could indicate fibrosis and/or compensatory angiogenic signaling in other cell types due to the cell-specificity of the knockout. Growth of connective tissue was also predicted increased based on increased expression of Fgf family members as well as increased expression of Lgals7 (p = 1.3E-15), Eln, which encodes elastin (p = 0.002), and other regulators of cell proliferation. Our data indicate that there may be elevated steroidogenesis and a shift towards a fibrotic phenotype in d30 knockouts, before follicular arrest is even observed. These data reveal the importance of Vegfa in ovarian development and steroidogenesis, even if a visible phenotype is not yet observed.