Biotechnology and Bioprocess Engineering 2021; 26(4): 677-687  
Effect of Epidermal Growth Factor on the Colony-formation Ability of Porcine Spermatogonial Germ Cells
Ran Lee, Hyun-Jung Park, Won-Young Lee, Min-Gi Han, Ji Hu Park, Jeonghwan Moon, Dae An Kwon, and Hyuk Song
Ran Lee, Hyun-Jung Park, Min-Gi Han, Ji Hu Park, Jeonghwan Moon, Dae An Kwon, Hyuk Song*
Department of Stem Cell and Regenerative Biology, Konkuk University, Seoul 05029, Korea
Tel: +82-2-450-0562
E-mail: songh@konkuk.ac.kr
Hyun-Jung Park
Department of Animal biotechnology, College of Life Science and Natural Resources, Sangji University, Wonju 26339, Korea
Won-Young Lee
Department of Beef & DairyScience, Korea National College of Agricultures and Fisheries, Jeonju 54874, Korea
These authors contributed equally to this work
Received: November 26, 2020; Accepted: December 3, 2020; Published online: August 30, 2021.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Spermatogonial stem cells (SSCs) are crucial for maintaining spermatogenesis, studying germ line stem cell biology, and producing transgenic animals. Growth factors, including leukemia inhibitory factor (LIF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and glial cell line-derived neurotrophic factor (GDNF), are essential for in vitro culture of SSCs as well as their selfr-enewal and maintenance. In this study, we investigated the effects of these growth factors on porcine spermatogonial germ cell (pSGC) colony formation. We determined round alkaline phosphatase (AP)-positive pSGC colonies in the presence and absence of growth factors after 7 days of pSGC culture. EGF was found to be essential to support the formation of AP-positive pSGC colonies. The expression of epidermal growth factor receptor (EGFR) and Erb-B2 receptor tyrosine kinase 2 (ERBB2) was also altered in cultured pSGCs compared to that in feeder cells. We verified the effect of EGF signaling on pSGC colony formation using AG1478 as an EGFR inhibitor and AG879 as an ERBB2 inhibitor. pSGC colonies were observed in low dose AG1478-treated groups with EGF, whereas a high dose of AG1478 suppressed pSGC colony formation. APpositive colonies were also observed in all AG879-treated groups. Taken together, EGFR and EGF signaling play a critical role in the initiation of colony formation of pSGCs. Our study provides insights into the mechanisms of EGF-mediated colony formation by SGCs derived from porcine testes, and will aid the development of transplantation techniques for the production of transgenic offspring.
Keywords: porcine spermatogonial germ cells (pSGCs), epidermal growth factor (EGF), colony formation, epidermal growth factor receptor (EGFR)


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