Spermatogonial stem cells in the bull

In the testis a complex process, called spermatogenesis, generates millions of spermatozoa per day. At the start of this process there are spermatogonial stem cells (SSCs) that have the ability to divide either into new stem cells (self-renewal) or daughter cells committed to develop into spermatozoa (differentiation). SSCs are the only cells among the adult stem cell systems capable of transmitting genetic information to future generations. This offers possibilities for in vitro SSC manipulation for instance with the goal of transferring relevant genes across bovine herds. As bovine species have a long generation interval, a system through which genes could be delivered to cattle through SSCs would prove effective and time-saving. SSCs and the first generations of differentiating spermatogonia are morphologically indistinguishable and are called type A spermatogonia. Therefore, we started with finding out at which age type A spermatogonia could be best isolated. We first investigated the onset of spermatogenesis in Brahman bulls, a widely used breed of Asian origin, and compared our results with the available information on European breeds. The best time to obtain A spermatogonia from European and Asian breeds was found to be the age period between the start of spermatogenesis (appearance of the first spermatogonia) to the age at which the first spermatocytes are formed. During this period the purity of the spermatogonia is optimal since no other differentiating germ cells have yet appeared. We then developed a culture system to propagate these A spermatogonia. Using StemPro medium and adding a number of growth factors we could successfully culture the cells for weeks. The usefulness of adding the growth factors GDNF, EGF, LIF and FGF2 was studied. Each of these growth factors was found to exert a specific effect on the culture. EGF, LIF and FGF2 specifically affected the somatic cells in culture while GDNF had a pronounced effect on the numbers of single type A spermatogonia, presumably SSCs. Overall the best results were obtained when the culture medium contained all four growth factors. A 365-fold increase in the numbers of A spermatogonia was found after 4 weeks of culture. To proof that GDNF actually stimulated the propagation of SSCs, a spermatogonial stem cell transplantation assay was carried out in which the cultured cells were transplanted into recipient mouse testes. The transplanted bovine SSCs colonized these testes and the extent of the colonization was taken as a measure of the numbers of bovine SSCs transplanted. A 10.000–fold increase in SSC numbers was found after a culture of 4.5 weeks. During culture in a liquid medium, many germ cells are lost during medium refreshments. To prevent this cell loss, we tested a semisolid culture medium (agar). Indeed, with an agar medium four times as many bovine spermatogonial colonies were able to grow. This indicates that it will be worthwhile to study the advantages of using an agar medium in further detail. In conclusion, we have developed a culture system for the propagation of bovine SSC which will allow further experimentation/manipulation and will eventually lead to more efficient ways of genetical improvement of cattle populations