Improvement of Expression of α6 and β1 Integrins by the Co-culture of Adult Mouse Spermatogonial Stem Cells with SIM Mouse Embryonic Fibroblast Cells (STO) and Growth Factors

Abstract

Objective(s): Spermatogonial Stem Cells (SSCs) maintain spermatogenesis throughout the life of the male. Because of the small number of SSCs in adult, enriching and culturing them is a crucial step prior to differentiation or transplantation. Maintenance of SSCs and transplantation or induction of in vitro spermio-genesis may provide a therapeutic strategy to treat male infertility. This study investigated the enrichment and proliferation of SSCs co-cultured with STO cells in the presence or absence of growth factors.

Materials and methods: Spermatogonial populations were enriched from the testis of 4-6 week-old male mice by MACS according to the expression of a specific marker, Thy-1. Isolated SSCs were cultured in the presence or absence of growth factors (GDNF, GFRα1 and EGF) on STO or gelatin-coated dishes for a week. Subsequently, the authors evaluated the effects of growth factors and STO on SSCs colonization by alkaline phosphates (AP) activity and flow cytometery of α6 and β1 integrins.

Results: SSCs co-cultured with STO cells and growth factors developed colonization and AP activity as well as expression of α6 and β1 integrins (P≤0/05).

Conclusion: Our present SSC-STO co-culture provides conditions that may allow efficient maintenance and proliferation of SSCs for the treatment of male infertility.

Keywords: Growth factors; SSCs; STO cells; α6 and β1 integrins.

Figure 1

Flow cytometric analysis for detection of α6 and β1-integrin in SSCs. While there are no cells with expression of β1 and α6 integrins prior to MACS (A), there are considerable cells with positive β1 and α6 integrins following MACS purification( B)

Figure 2

Microscopy morphology of SSCs derived from 4-6 week-old male mice in different groups. SSCs were single on the first day of cell culture (A). Following 2-3 days of cell culture, small SSCs colonies were formed (B), on the seventh day of cell culture the size of colonies was increased significantly (C) and were positive for alkaline Phosphatase activity (D)

Figure 3

Flow cytometric analysis for the propagation of SSCs. Expression of α6 and β1-integrin in cultured SSCs on gelatin coated dishes absent growth factors (A), gelatin coated dishes with growth factors (GDNF, GFRa1, and EGF) (B), STO coated dishes without growth factors (C), STO coated dishes with growth factors (D). Comparison expression of α6 and β1-integrin in cultured SSCs in a different culture medium (E). (P≤0.05)