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In vitro culture and morphological characterization of prepubertal buffalo (Bubalus bubalis) putative spermatogonial stem cell

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

Abstract

Purpose

Spermatogonial stem cells (SSCs) have the unique ability both to self-renew and to produce progeny that undergo differentiation to spermatozoa. The present study has been carried out to develop a method to purify and enrich the pure populations of spermatogonial stem cell like cells in buffalo.

Methods

The spermatogonial cells were isolated from testes of 3–7 month old buffalo calves and disaggregated by double enzymatic digestion. Mixed population of isolated cells were then plated on Datura stramonium agglutinin (DSA) lectin coated dishes for attachment of Sertoli cells. The desired cells were obtained from suspension medium after 18 h of incubation and then loaded on discontinuous density gradient using percoll (20–65 %) and different types of spermatogonia cells were obtained at interface of each layer. These cells were cultured in vitro.

Results

Spermatogonial cells isolated have spherical outline and two or three eccentrically placed nucleoli, created a colony after proliferation during first week or immediately after passage. After 7–10 days of culture, the resulted developed colonies of spermatogonial cells expressed the spermatogonial specific genes like Plzf and VASA; and other pluripotency related markers viz. alkaline phosphtase, DBA, CD9, CD90, SSEA-1, OCT-4, NANOG and REX-1.

Conclusion

Our results show that the isolated putative spermatogonial stem cells exhibit the expression of pluripotency related and spermatogonial specific genes. This study may help to establish a long term culture system for buffalo spermatogonia.

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Acknowledgments

This work was supported by NAIP Stem Cell C-067&075, Indian Council of Agriculture Research, New Delhi.

Conflict of interests

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India

    S. Kala, R. Kaushik, K. P. Singh, P. H. Kadam, M. K. Singh, R. S. Manik, S. K. Singla, P. Palta & M. S. Chauhan

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  1. S. Kala
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  2. R. Kaushik
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  3. K. P. Singh
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  4. P. H. Kadam
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Correspondence to M. S. Chauhan.

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Capsule

Present study showed the in vitro culture of enriched buffalo spermatogonial stem cell like cells and thereby characterized for some of the pluripotency markers.

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Kala, S., Kaushik, R., Singh, K.P. et al. In vitro culture and morphological characterization of prepubertal buffalo (Bubalus bubalis) putative spermatogonial stem cell. J Assist Reprod Genet 29, 1335–1342 (2012). https://doi.org/10.1007/s10815-012-9883-y

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  • DOI: https://doi.org/10.1007/s10815-012-9883-y

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