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The role of signaling pathways on proliferation and self-renewal of cultured bovine primitive germ cells

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Reproductive Medicine and Biology

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 mitogen-activated 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.

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Acknowledgments

The authors thank Drs. K. Konishi and Y. Hashiyada (National Livestock Breeding Centre), Dr. Y. Hoshino (Gifu Prefectural Livestock Research Institute) and H. Yoshioka, Y. Nagase, S. Kitamura, E. Itoyama and H. Murakami of Livestock Farm (Graduate School of Agriculture, Kyoto University) for providing bovine teste samples. This study was supported by a grant from the Japan Society for the Promotion of Science (JSPS) to MS and a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to HI.

Conflict of interest

Mahesh Sahare, Ayagi Otomo, Kana Komatsu, Naojiro Minami, Masayasu Yamada, and Hiroshi Imai declare that they have no conflict of interest.

Animal studies

All institutional and national guidelines for the care and use of animals were followed.

Human rights

This article does not contain any studies with human subjects performed by any of the authors.

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Authors and Affiliations

  1. Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan

    Mahesh Sahare, Ayagi Otomo, Kana Komatsu, Naojiro Minami, Masayasu Yamada & Hiroshi Imai

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  1. Mahesh Sahare
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  2. Ayagi Otomo
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  3. Kana Komatsu
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Correspondence to Hiroshi Imai.

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Sahare, M., Otomo, A., Komatsu, K. et al. The role of signaling pathways on proliferation and self-renewal of cultured bovine primitive germ cells. Reprod Med Biol 14, 17–25 (2015). https://doi.org/10.1007/s12522-014-0189-x

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  • DOI: https://doi.org/10.1007/s12522-014-0189-x

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