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BOR - Papers in Press, published online ahead of print October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.004986
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Submitted February 26, 2002
Returned for revision March 13, 2002
Accepted July 16, 2002

Testis


Proliferation and Differentiation of Bovine Type A Spermatogonia During Long-Term Culture

Fariborz Izadyar 1*, Krista den Ouden 2, Laura B. Creemers 2, George Posthuma 1, Martti Parvinen 3, Dirk G. de Rooij 2
1 University Medical Center Utrecht
2 Dept. of Endocrinology, Biology Faculty, Utrecht University
3 Institute of Biomedicine, University of Turku, Finland

* To whom correspondence should be addressed. E-mail: f.izadyar{at}vet.uu.nl.

Abstract

This study was aimed to develop a method for long-term culture of bovine type A-spermatogonia. Testes from 5-month-old calves were used and pure populations of type A-spermatogonia were isolated. Cells were cultured in MEM or KSOM and different concentrations of FCS, for 2-4 weeks at 32°C or 37°C. Culture in MEM resulted in more viable cells and more proliferation than in KSOM and better results were obtained at 37°C. After one week of culture in the absence of serum only 20% of the cells were alive. However, in the presence of 2.5% FCS about 80% of cells were alive and proliferating. Higher concentrations of FCS, only enhanced numbers of somatic cells. In long-term culture, spermatogonia continued to proliferate and eventually A-spermatogonial colonies were formed. The majority of colonies consisted mostly of groups of cells connected by intercellular bridges. Most of the cells in these colonies underwent differentiation as they were c-kit positive and ultimately cells with morphological and molecular characteristics of spermatocytes and spermatids were formed. Occasionally, large round colonies consisting of single c-kit negative A spermatogonia, presumably spermatogonial stem cells, were observed. For the first time a method was developed, allowing proliferation and differentiation of highly purified type A-spermatogonia, including spermatogonial stem cells during long-term culture.



Key words: Developmental biology • Spermatogenesis


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