Expression of promyelocytic leukaemia zinc-finger in ovine testis and its application in evaluating the enrichment efficiency of differential plating
Uyunbilig Borjigin A B C , Rhonda Davey A C , Keryn Hutton A C and Muren Herrid A C DA CSIRO Livestock Industries, FD McMaster Laboratory, Armidale, NSW 2350, Australia.
B Key Laboratory of Ministry of Education of China for Mammalian Reproduction Biology and Biotechnology of Inner Mongolia University.
C CSIRO Food Futures National Research Flagship, Australia.
D Corresponding author. Email: muren.herrid@csiro.au
Reproduction, Fertility and Development 22(5) 733-742 https://doi.org/10.1071/RD09237
Submitted: 14 September 2009 Accepted: 5 November 2009 Published: 7 April 2010
Abstract
Identification and enrichment of spermatogonial stem cells (SSCs) are critical steps in testis germ cell transplantation. The present study shows that expression of the protein promyelocytic leukaemia zinc-finger (PLZF) does not occur in all cells, only in gonocytes in neonatal testis (Stage 1) and a subpopulation of Type A spermatogonia in peripubertal (Stage 2), prepubertal (Stage 3) and post-pubertal (Stage 4) ovine testes. Dolichos biflorus agglutinin (DBA) lectin binding does not occur at any stage of testis development. The numbers of putative undifferentiated spermatogonia, germ cells and Sertoli cells were assessed by PLZF, VASA and vimentin staining, respectively. In paraffin sections, the percentage of PLZF-positive cells per tubule in samples derived from Stage 2 testis (12.2 ± 2.8%) was twofold higher than that from Stage 1 testis (6.4 ± 0.4%), but the percentages decreased in Stage 3 and Stage 4 testes (4.6 ± 0.7% and 3.1 ± 0.6%, respectively). Single cell suspensions from Stage 1 and Stage 2 testis were generated by two-step enzymatic digestion. The spermatogonia were enriched by 2 h and 2 + 16 h (overnight) differential plating on 0.2% gelatin-coated coated flasks. For Stage 1 testes, a sixfold increase in PLZF-positive cells was observed in 2 h differential plating and an almost 10-fold increase was produced following 2 + 16 h enrichment. There was less than a twofold increase in PLZF-positive cells between the 2 h and 2 + 16 h differential plating. A similar level of enrichment efficiency was also obtained for Stage 2 testis, but the percentage of PLZF-positive cells in the final enrichment was approximately one-third of that Stage 1. The efficiency of isolation and/or enrichment of PLZF-positive cells appears to depend on the maturity of the testis and the neonatal testis is better suited for isolation of gonocytes and/or putative SSCs.
Additional keywords: immunostaining, sheep, spermatogonial stem cell.
Acknowledgements
The authors thank Brendan Hatton and Andrew Eichorn for assistance with experimental animal management and funding from the CSIRO Food Futures National Research Flagship and CSIRO Livestock Industries. The authors are also grateful to Dr Keith Ellis and Sally Stockwell for critical reading of the manuscript. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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