Regulation of FOXO3 subcellular localization by Kit ligand in the neonatal mouse ovary

Abstract

Purpose Foxo3 protein is required in the oocyte nucleus for the maintenance of primordial follicles in a dormant state. PI3K/AKT-dependent phosphorylation of Foxo3 leads to its relocalization to the cytoplasm and subsequent follicular activation. However, the nature of the upstream signals controlling Foxo3 activity and subcellular localization remains unknown. We aimed to study the in vitro effects of Kit ligand (stem cell factor) on the subcellular localization of Foxo3 in primordial follicles within the postnatal mouse ovary. Methods This was an in vitro study using explants of intact neonatal mouse ovaries. The study was performed in laboratory animal facility and basic science research laboratory at a University Hospital. The animals used for this study were FVB mice. Neonatal FVB mice ovaries at postnatal day 7 (PD7) were harvested and incubated in culture medium (DMEM) at 37°C and 5 % CO 2 for 60-90 min with (n=3) or without (n=3) Kit ligand at 150 ng/mL (8 nM). Similar experimental conditions were used to establish a dose-response curve for the effects of Kit ligand and assess the effects of imatinib (small molecule inhibitor of the Kit receptor). Immunofluorescence was used to identify the subcellular location of Foxo3 in oocytes. Proportions of cytoplasmic versus nuclear Foxo3 in primordial follicles were determined. Results Kit ligand treatment increased the cytoplasmic localization of Foxo3 from 40 % in the untreated ovaries to 74 % in the treated group (p=0.007 in paired samples and p=0.03 in unpaired samples). Furthermore, this effect was reversible with imatinib (p=0.005). A dose-response curve for Kit ligand treatment showed that maximum effect was seen at 150 ng/mL. Conclusion Kit ligand treatment in vitro increases the proportion of cytoplasmic Foxo3 in primordial follicles at PD7, lending support to the idea that Kit receptor/ligand controls Foxo3 activity in the context of primordial follicle activation

SMAD3 directly regulates cell cycle genes to maintain arrest in granulosa cells of mouse primordial follicles

“…In cell culture models, changes in SMAD3-regulated gene expression have been observed as little as 30 minutes after stimulation with TGFβ ligands 30,31 . Furthermore, a 60–90 min exposure of kit ligand or the pathway inhibitor imatinib to post-natal day 7 mouse ovaries in culture was sufficient to cause a significant shift in FOXO3a translocation in oocytes 32 . We therefore evaluated effects on gene expression and signalling following a brief 2-hour exposure to TGFβ1 ligand and A83-01, a Type I receptor inhibitor, in day 4 ovaries in vitro .…”

Transcriptome Analyses Identify Potential Key microRNAs and Their Target Genes Contributing to Ovarian Reserve

“…Foxo3 and Fshr are also mainly regulated by miRNAs. Foxo3 is involved in the apoptosis of granulosa cells [ 58 ] and involved in the early development of follicles on its own [ 59 ] or with other genes [ 60 ]. Foxo3 interacts with mmu-miR-153-3p, which is known to suppress tumor growth in ovarian carcinoma [ 61 ].…”

Imatinib mesylate does not counteract ovarian tissue fibrosis in postnatal rat ovary

“…Inactivity or mutation of these transcription factors has been related to POF [42]. The effect of imatinib is based on binding to c-Abl kinase and inactivating the PI3K/ Akt/FOXO axis [28,45]. FOXOs have also shown to play a potential role in tissue fibrosis in other organs e.g.…”

“…Imatinib inhibit FOXO3 phosphorylation, i.e. its translocation to cytoplasm of primordial follicles in vitro via PI3K/Akt pathway [45,54]. However, besides phosphorylation, FOXO3 can be also regulated by acetylation, methylation, glycosylation, redox modulation, and ubiquitination, which will determine FOXO3 function [55].…”