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

Abstract

Abstract. Surface molecules of primitive male germ cells, gonocytes, are essential components for regulating cell adhesion and maintaining self-renewal in mammalian species. In domestic animals, the stage-specific glycan epitope a-N-acetylgalactosamine (GalNAc) is recognised by the lectin Dolichos biflorus agglutinin (DBA) and is found on the surface of gonocytes and spermatogonia. Gonocytes from bovine testis formed mouse embryonic stem-like cell colonies on plates that had been coated with DBA or extracellular matrix (ECM) components, such as gelatin (GN), laminin (LN) and poly-L-lysine (PLL). The number of colonies on the DBA-coated plate was significantly higher than that on the GN-, LN-and PLL-coated plates. Pretreating gonocytes with DBA to neutralise the terminal GalNAc residues strongly suppressed colony formation. Furthermore, expression of a germ cell-specific gene and pluripotency-related transcription factors was increased considerably on the DBA-coated plates. These results suggest that the GalNAc residues on gonocytes can recognise precoated DBA on plates and the resulting GalNAc-DBA complexes support germ cell and stem cell potentials of gonocytes in vitro. These glycan complexes, through the GalNAc epitope, may provide a suitable microenvironment for the adhesion and cell proliferation of gonocytes in culture.

Factors supporting long-term culture of bovine male germ cells

“…3) was several times higher than the ratios on the other coated dishes. The gonocytes-to-somatic cells ratio was calculated by using DBA staining as a germ-cell marker as shown by Kim et al (2014) to count actual germ-cell numbers (Fig. 4).…”

“…In addition, poly-L-lysine is a key component in a defined culture medium for mouse ES cells (Harb et al 2008). In short-term culture of bovine germ cells from 3.5-month-old prepubertal testis, DBA-coated dishes were observed to be a better support for the binding and survival of cells than gelatin-, poly-L-lysine-and laminin-coated dishes (Kim et al 2014). This indicates that there are age-specific differences in the requirements of DBA-and poly-L-lysine-coated dishes for selfrenewal and proliferation of bovine neonatal and pre-pubertal germ cells.…”

“…Gonocytes were isolated by a three-step enzymatic digestion method as described previously (Kim et al 2014) with minor modifications. Briefly, the testes were encapsulated, minced and digested with collagenase Type IV (1 mg mL À1 ; Sigma-Aldrich, St. Louis, MO, USA) at 378C for 45 min with constant agitation.…”

“…DBAcoated dishes support cell adhesion and induce proliferation of bovine gonocytes (Kim et al 2014). Culture dishes were washed once with PBS and further utilised for culture experiments.…”

Recent advances of in vitro culture systems for spermatogonial stem cells in mammals

“…However, no long‐term culture system for livestock species has been developed. In the authors’ laboratory, growth factors, matrix substrates for culture dishes, and serum‐free supplements have been examined in order to develop a defined system for culturing primitive GCs (gonocytes) from the neonatal bovine testis . Poly‐L‐lysine is a suitable substrate for the selective inhibition of the growth of somatic cells and makes it possible to maintain gonocytes.…”