Propagation of bovine spermatogonial stem cells in vitro

Abstract

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

In vitro propagation of male germline stem cells from piglets

“…To reduce the interference of somatic cells, the subculturing MGSCs are maintained in media supplemented with growth factors. Glial cell linederived neurotrophic factor (GDNF) has far been demonstrated to play vital roles in SSC self-renewal and long-term cultures of SSCs across species [2,9,16,17,19,21,[25][26][27]32]. However, we observed that GDNF showed little positive effects on porcine MGSC colony formation (data not shown), which is in line with the previous report by Kuijk et al [20].…”

“…bFGF, as well as EGF, has far been used to stimulate in vitro proliferation of MGSCs from mice, rats, pigs and bulls [2,15,19,20,25]. In our study, despite the addition of bFGF at 1, 5 ng/ml significantly promoted the formation of large colonies, a devastating effect of bFGF at 10 ng/ml was observed.…”

In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus)

“…Among the livestock species, goats are often considered as unique for transgenesis because of their small body size, shorter gestation period, prolificacy and relatively higher protein content in their milk [29]. There are several reports of SSC culture in different species including mice [22,30], rat [31,32], cattle [11,33], buffalo [34,35] and human [13,36]. But the isolation and enrichment as well as establishment of long term culture system for SSCs have not yet been exploited substantially in goats except a very recent report [10].…”

Effects of extracellular matrices and lectin Dolichos biflorus agglutinin on cell adhesion and self-renewal of bovine gonocytes cultured in vitro

“…The higher number of colonies on the DBA-precoated plates than on the plates precoated with the ECM components (Fig. 3) suggests that proper stimulation of somatic cells, including Leydig cells, Sertoli cells and endothelial cells (which are necessary for the survival and proliferation of germ cells; Aponte et al 2008), supports the colony formation of gonocytes on the DBA-precoated plates. In fact, the colony formation of bovine gonocytes can be supported on GN-coated plates for the first week of culture (Fujihara et al 2011).…”

“…The colony formation of testicular cells in culture depends on the presence of germ cell populations (Aponte et al 2008), and these cell populations were strongly associated with the expression of NANOG and POU5F1 (Fig. 5b).…”