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


CONCLUSIONS
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.
Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX, 75390-9072, USA. ezzatim@pamf.org.|Palo Alto Medical Foundation, Palo Alto, CA, USA. ezzatim@pamf.org.
M Max
MM
Ezzati
Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX, 75390-9072, USA.
Michael D
MD
Baker
Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX, 75390-9072, USA.
Hatice D
HD
Saatcioglu
Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX, 75390-9072, USA.
Gina M
GM
Aloisio
Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX, 75390-9072, USA.
Christopher G
CG
Pena
Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX, 75390-9072, USA.
Yuji
Y
Nakada
Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX, 75390-9072, USA.
Ileana
I
Cuevas
Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX, 75390-9072, USA.
Bruce R
BR
Carr
Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX, 75390-9072, USA. Diego.Castrillon@UTSouthwestern.edu.
Diego H
DH
Castrillon
D000818:Animals; D005260:Female; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D000068877:Imatinib Mesylate / Q000494:pharmacology; D066298:In Vitro Techniques; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D006080:Ovarian Follicle / Q000254:growth & development / Q000378:metabolism; D010053:Ovary / Q000378:metabolism*; D019089:Stem Cell Factor / Q000378:metabolism / Q000502:physiology*
D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; C474033:FoxO3 protein, mouse; D019089:Stem Cell Factor; D000068877:Imatinib Mesylate
Foxo3; Kit; Kit ligand; Primordial follicle; Stem cell factor