Fig. 7
Altered gene expression in granulosa cells of Gdf9Q308X/S415T mice explored by RNA sequencing. A Differentially expressed genes (DEGs) in granulosa cells collected from Gdf9wt/wt and Gdf9Q308X/S415T mice (three samples in each group, and each samples contains granulosa cells from three mice) 48 hours after PMSG stimulation. DEGs were defined as genes with adjusted P value < 0.05 and |log2FC|>1. B,C Enrichment analysis of GO-BP (Gene Ontology- Biological Process) terms (B) and Reactome pathways (C) using GSEA method. Normalized enrichment score (NES)>0 represent terms/pathways up-regulated in Gdf9Q308X/S415T mice. NES<0 represent terms/pathways down-regulated in Gdf9Q308X/S415T mice. D FPKM (Fragments Per Kilo bases per Million reads) of Has2, P4ha2, Cldn15, and Aqp5 in granulosa cells collected from Gdf9wt/wt and Gdf9Q308X/S415T mice as a result of RNA-sequencing. *P < 0.05. E Expression of P4ha2 in granulosa cells of Gdf9wt/wt (n=3) and Gdf9Q308X/S415T (n=3) mice after 48 hours of PMSG stimulation detected by qPCR. *P < 0.05. F P4HA2 immunostaining in ovaries of Gdf9wt/wt and Gdf9Q308X/S415T mice. The integrated density of P4HA2 staining of cumulus cells (CC) and mural granulosa cells (GC) were calculated separately. *P < 0.001. G mRNA levels of P4HA2 in human primary luteinized granulosa cells treated in vitro with GDF9 protein with or without 10μM SB431542 for 24 hours. SB-431542 is an inhibitor of ALK5, which is the receptor of GDF9. *P < 0.001. H Protein levels of P4HA2 in human primary luteinized granulosa cells treated in vitro with GDF9 protein with or without 10μM SB431542 for 24 hours and 48 hours. Meanwhile, protein levels of STAR and CYP19A1 after 48 hours treatment were shown. *P < 0.05