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Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers

  • Stem Cell Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Introduction

Presently the techniques for making transgenic animals are cumbersome, required costly instruments and trained man-power. The ability of spermatogonial stem cells (SSCs) to integrate foreign genes has provided the opportunity for developing alternate methods for generation of transgenic animals. One of the big challenges in this field is development of the methods to identify and purify donor SSCs by antibody mediated cell sorting.

Purpose

The present study was aimed to identify goat subpopulations of SSCs using polyclonal antibodies against PGP9.5 and c-kit molecular markers as well as the growth characteristics of SSCs during short term culture.

Methods

One month old goats’ testicular samples were subjected for immunohistochemical and immunocytochemical evaluations. The enzymatically isolated SSCs were cultured in DMEM plus FCS supplemented with (treatment) or without (control) growth factors (GDNF, LIF, FGF, and EGF) for 2 weeks. At the end of culture the morphological characteristics of SSCs colonies and immunocytochemical staining were evaluated.

Results

The number and size of colonies in treatment groups were significantly (P < 0.01) higher than corresponding values in controls. The presence of PGP 9.5 and c-kit antigens was confirmed in immunocytochemical evaluation. In immunocytochemical evaluation, the proportion of c-kit and PGP9.5 positive cells were significantly (P < 0.001) higher in control and treatment groups, respectively.

Conclusions

The presence of PGP9.5 and c-kit antigens was confirmed in goat SSCs. Moreover, culture medium supplementation with growth factors could effectively retain the undifferentiation status of SSCs, reflected as a higher population of PGP9.5 positive cells, after short term culture.

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Acknowledgment

The authors would like to thank the Avicenna Research Institute for technical and financial supports, ACECR, Tehran, Dr. K. Kamali for assistance with the statistical analysis, and Dr. H. Soltangharaee for histological evaluation.

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Authors and Affiliations

  1. Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, P. O. Box: 19615-1177, Tehran, Iran

    Banafsheh Heidari, Maryam Rahmati-Ahmadabadi, Mohammad Mehdi Akhondi, Amir Hassan Zarnani, Mahmood Jeddi-Tehrani, Abolfazl Shirazi, Mohammad Mehdi Naderi & Bahareh Behzadi

  2. Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University, P. O. Box: 115, Shahrekord, Iran

    Abolfazl Shirazi

Authors
  1. Banafsheh Heidari
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  2. Maryam Rahmati-Ahmadabadi
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  3. Mohammad Mehdi Akhondi
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  4. Amir Hassan Zarnani
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  5. Mahmood Jeddi-Tehrani
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  6. Abolfazl Shirazi
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  7. Mohammad Mehdi Naderi
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  8. Bahareh Behzadi
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Corresponding author

Correspondence to Abolfazl Shirazi.

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Capsule

This study confirmed the different expression of PGP9.5 and c-kit markers in subpopulation of undifferentiated and differentiated goat type A spermatogonia.

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Heidari, B., Rahmati-Ahmadabadi, M., Akhondi, M.M. et al. Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers. J Assist Reprod Genet 29, 1029–1038 (2012). https://doi.org/10.1007/s10815-012-9828-5

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  • DOI: https://doi.org/10.1007/s10815-012-9828-5

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