The role of signaling pathways on proliferation and self-renewal of cultured bovine primitive germ cells
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ORIGINAL ARTICLE
The role of signaling pathways on proliferation and self-renewal of cultured bovine primitive germ cells Mahesh Sahare • Ayagi Otomo • Kana Komatsu Naojiro Minami • Masayasu Yamada • Hiroshi Imai
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Received: 4 June 2014 / Accepted: 13 July 2014 Ó Japan Society for Reproductive Medicine 2014
Abstract Purpose Gonocytes are primitive male germ cells residing in the neonatal testes and are unipotent in nature, but also have pluripotent stem cell ability in mice under appropriate culture conditions. This study was performed to elucidate the molecular mechanisms of self-renewal and survival of cultured bovine gonocytes. Methods Gonocytes were isolated from neonatal bull calves and were cultured in DMEM/F12 supplemented with 15 % knock-out serum replacement (KSR) and glial cell-derived neurotrophic factor (GDNF). Cells were analyzed six days after culturing for cell-signaling molecular markers. Results Colony formation was observed 3–4 days after being cultured. Addition of GDNF enhanced mitogenactivated protein kinase 1/2 (MAPK1/2) phosphorylation and activated the MAPK signaling pathway. Inhibition of MAPK signaling reduced cell proliferation and abolished colony formation. However, inhibition of phosphoinositide 3-kinase-AKT (PI3K-AKT) signaling, a dominant pathway for self-renewal of mouse germ cells, did not show any effects on cultured bovine gonocytes. Expression of cell cycle-related regulators cyclin D2 and cyclin-dependent kinase 2 (CDK2) was downregulated with inhibition of MAPK signaling. Conclusions These results indicate activation of MAPK plays a critical role in self-renewal and survival of bovine gonocytes via cyclin D1 and CDK2.
M. Sahare A. Otomo K. Komatsu N. Minami M. Yamada H. Imai (&) Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan e-mail: [email protected]
Keywords Cell cycle regulators Gonocytes MAPK Self-renewal Signaling pathways Testes
Introduction Gonocytes reside mostly in the center of the seminiferous tubules and remain quiescent [1]. These cells resume proliferation, migrate to the basement membrane and are transformed to spermatogonial stem cells (SSCs) after arriving at a stem cell niche. The niche refers to a specialized microenvironment that provides architectural support, stimulates secretion of growth factors and provides extrinsic signals for synchronizing self-renewal and differentiation [2]. Understanding the niche factor that regulates germ cell function in rodents has been greatly aided by transplantation assays to immunodeficient mice and the development of a long-term culture system [3]. Culture conditions that support long-term self-renewal and maintenance proliferation of germ cells have been established in various species including mice [4–6], rats [7], hamsters [8] and rabbits [9]. GDNF was shown to be a critical factor for self-renewal of cultured germ cells in these culture systems. Global gene expression profiling has identified several intrinsic downstream targ
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