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

M. Max Ezzati, Michael D. Baker, Hatice D. Saatcioglu, Gina M. Aloisio, Christopher G. Pena, Yuji Nakada, Ileana Cuevas, Bruce R. Carr, Diego H. Castrillon

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 % CO2 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

Original languageEnglish (US)
Pages (from-to)1741-1747
Number of pages7
JournalJournal of Assisted Reproduction and Genetics
Volume32
Issue number12
DOIs
StatePublished - Dec 1 2015

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Stem Cell Factor
Ovary
Oocytes
Laboratory Animals
Phosphatidylinositol 3-Kinases
Fluorescent Antibody Technique
Culture Media
Cytoplasm
Therapeutics
Maintenance
Phosphorylation
Research

Keywords

  • Foxo3
  • Kit
  • Kit ligand
  • Primordial follicle
  • Stem cell factor

ASJC Scopus subject areas

  • Obstetrics and Gynecology
  • Reproductive Medicine
  • Developmental Biology
  • Genetics
  • Genetics(clinical)

Cite this

Regulation of FOXO3 subcellular localization by Kit ligand in the neonatal mouse ovary. / Ezzati, M. Max; Baker, Michael D.; Saatcioglu, Hatice D.; Aloisio, Gina M.; Pena, Christopher G.; Nakada, Yuji; Cuevas, Ileana; Carr, Bruce R.; Castrillon, Diego H.

In: Journal of Assisted Reproduction and Genetics, Vol. 32, No. 12, 01.12.2015, p. 1741-1747.

Research output: Contribution to journalArticle

Ezzati, M. Max ; Baker, Michael D. ; Saatcioglu, Hatice D. ; Aloisio, Gina M. ; Pena, Christopher G. ; Nakada, Yuji ; Cuevas, Ileana ; Carr, Bruce R. ; Castrillon, Diego H. / Regulation of FOXO3 subcellular localization by Kit ligand in the neonatal mouse ovary. In: Journal of Assisted Reproduction and Genetics. 2015 ; Vol. 32, No. 12. pp. 1741-1747.
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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 {\%} CO2 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",
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AU - Baker, Michael D.

AU - Saatcioglu, Hatice D.

AU - Aloisio, Gina M.

AU - Pena, Christopher G.

AU - Nakada, Yuji

AU - Cuevas, Ileana

AU - Carr, Bruce R.

AU - Castrillon, Diego H.

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