Purpose Foxo3 in female reproduction has been reported to regulate proliferation of granulose cells that form follicles. birth and lasted until PPW 3. After PPW 3, most Foxo3 expression occurred in the nuclei of Leydig cells; however, at PPW 5, Foxo3 was expressed in both the nucleus and cytoplasm. When R2C cells were treated with luteinizing hormone, Foxo3 phosphorylation levels by AKT increased. After blocking the PI3K pathway, LH-induced phosphorylated Foxo3 levels decreased, indicating that LH signaling regulates Foxo3 localization. When active FOXO3-TM adenovirus was introduced into a Leydig tumor cell line, the concentrations of testosterone and StAR protein decreased. When FOXO3 and a StAR promoter vector were co-transfected into HEK293 cells for a reporter assay, FOXO3 inhibited the StAR promoter. Conclusion FOXO3 affects testosterone synthesis by inhibiting the formation of StAR protein. LH hormone, meanwhile, influences Foxo3 localization, mediating its function. Keywords: Foxo3, Leydig cell, testosterone, StAR INTRODUCTION The Iguratimod main functions of the testes are Iguratimod testosterone production and spermatogenesis. These two functions are controlled by the hypothalamus-pituitary-gonad axis. Gonadotropin-releasing hormone (GnRH) from the hypothalamus stimulates the production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the anterior pituitary.1 FSH binds to its receptor on the surface of Sertoli cells to regulate spermatogenesis.2,3 LH binds to its receptor on the Leydig cell membrane to stimulate testosterone production. The testosterone produced by LH negatively regulates GnRH production in the hypothalamus.4,5,6 There are four types of forkhead box class O (Foxo) transcription factors: Foxo1 (FKHR, forkhead in rhabdomyosarcoma), Foxo3 (FKHRL2, Rabbit Polyclonal to OR10A4 FKHR-like1), Foxo4 (AFX, acute-lymphocytic-leukemia-1), and Foxo6. These Foxo proteins regulate stress responses, aging, insulin sensitivity, and ontogenesis,7,8 and their transcription is inhibited by phosphoinositide 3-kinase (PI3K). PI3K signaling phosphorylates AKT, which then phosphorylates Foxo3 at Ser24, Thr32, and Ser56 residues. These phosphorylated sites recruit 14-3-3 protein to guide Foxo3 from the nucleus into the cytoplasm. Finally, Foxo3 is removed by proteasomes.9 In female reproduction, Foxo1, Foxo3, and Foxo4 are expressed in the granulosa cells at various stages of follicle development.10 Foxo1 in granulosa cells inhibits cyclin D2 gene expression and increases the nuclear localization of p27kip proteins, which makes it a key regulator of G1/S transition. Foxo proteins also play an important role in regulating ovarian function by pituitary gonadotropins.10,11 In a previous study, Foxo3-null female mice exhibited age-dependent fertility issues and were completely sterile at 10 weeks or older. In Foxo3-/- ovaries at 9.5 weeks, oocytes in developing follicles appeared to have degenerated, reflecting atretic change. At 12 weeks, Foxo3-/- ovaries had no developing follicles. These indicated that Foxo3 is important in ovarian follicular development.11,12,13 Meanwhile, in males, germ line specific Foxo1 KO mice showed defective proliferative expansion and small testes, which was Iguratimod not due to cell death, but rather to renewal of spermatogonial stem cells.14 However, the function of Foxo3 in Leydig cells is not clear.15 Foxo3 is important not only in females, but also in males. Foxo3 expression and location are likely to be dynamic throughout life. In this study, Foxo3 expression and location were investigated from mouse embryonic stage to 12 weeks, and the role of Foxo3 in Leydig cells was investigated to outline the function and regulation of Leydig cells. MATERIALS AND METHODS Animals and testis preparation C57/BL6 (Jackson Labs, CA, USA) male mice were housed in a barrier facility under normal light and dark conditions and fed ad libitum. Testes were isolated at postpartum days (PPD) 1 and 5 and postpartum weeks (PPW) 3, 4, 5, and 12. Testes were removed, fixed in 10% formalin, and embedded in paraffin. All procedures were approved by the Animal Care and Use Committees at Yonsei University College of Medicine and Northwestern University. Plasmid and adenovirus construction The mStARp-Luc plasmid was constructed by inserting the mouse steroidogenic acute regulatory (StAR) promoter (2730 bps) into the multiple cloning site of pGL3 basic vector. As synthetic poly A (spa) region in PGL3 basic vector contained two FOXO binding sequences, we removed them using NotI and KpnI.16 The human FOXO3 triple mutant (FOXO3-TM) was generated by substituting Thr32, Ser253, and Ser315 with alanine residues. This FOXO3-TM cannot be phosphorylated by Akt and is constitutively activated.17,18 Recombinant adenoviral vectors carrying FOXO3 [wild type (WT) or TM] were.
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