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Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes

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Abstract

Purpose

To date, the methods available for isolating spermatogonial stem cells (SSCs) from porcine testicular cells have a low efficiency of cell separating. Therefore, we tried to develop a novel isolation technique with a high-yield cell separating ability to isolate SSCs from porcine testes.

Methods

We confirmed the presence of SSCs by measuring alkaline phosphatase (AP) activity and SSC-specific gene expression in neonatal porcine testis-derived testicular cells. Subsequently, the isolation of SSCs from testicular cells was performed using different techniques as follows: differential plating (DP), double DP, Petri dish plating post-DP, magnetic-activated cell sorting (MACS), and MACS post-DP. Positive AP staining was used to assess and compare the isolation efficiency of each method.

Results

Petri dish plating post-DP resulted in the highest isolation efficiency. The putative SSCs isolated using this method was then further characterized by analyzing the expression of SSC-specific genes and -related proteins, and germ cell-specific genes. OCT4, NANOG, EPCAM, THY1, and UCHL1 were expressed transcriptionally, and OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, and PLZF were expressed translationally in 86 % of the isolated SSCs. In contrast, no difference was observed in the percentage of cells expressing luteinizing hormone receptor (LHR), a Leydig cell-specific protein, or GATA4, a Sertoli cell-specific protein, between SSCs and negative control cells. In addition, transcriptional expression of VASA, a primordial germ cell-specific marker, and DAZL, a premeiotic germ cell-specific marker, wasn’t and was detected, respectively.

Conclusions

We successfully developed a novel high-yield technique to isolate SSCs from porcine testes to facilitate future porcine SSC-related research.

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Acknowledgments

This research was supported by Agricultural Biotechnology Development Program (IPET112015-4), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.

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Authors and Affiliations

  1. Department of Animal Life Science, Kangwon National University, Chuncheon, 200-701, South Korea

    Min Hee Park, Min Seong Kim, Hye Jin Park & Seung Tae Lee

  2. Division of Applied Animal Science, Kangwon National University, Chuncheon, 200-701, South Korea

    Ji Eun Park & Seung Tae Lee

  3. College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, 200-701, South Korea

    Kwon Young Lee, Jung Im Yun, Jung Hoon Choi & Eun song Lee

  4. Department of Animal Life Science and Division of Applied Animal Science, Kangwon National University, Dongsangdae 2-#105-1, Chuncheon, 200-701, South Korea

    Seung Tae Lee

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  1. Min Hee Park
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Correspondence to Seung Tae Lee.

Additional information

Capsule Petri dish plating post-DP is a high-yield technique that isolates SSCs from porcine neonatal testis.

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Park, M.H., Park, J.E., Kim, M.S. et al. Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes. J Assist Reprod Genet 31, 983–991 (2014). https://doi.org/10.1007/s10815-014-0271-7

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  • DOI: https://doi.org/10.1007/s10815-014-0271-7

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