Biotechnological evaluation of extracellular matrix proteins expressed by cultured testicular cells

Sertoli cells and peritubular myoid cells play an important role in germ cell maturation and contribute in the process of spermatogenesis. The production of extracellular matrix components (ECM) by these cells is thought to mediate testicular development. We have selected primary culture of Sertoli cells, secondary culture of peritubular myoid cells and a transformed mouse cell line (MSC-1 cells) to evaluate production of selected ECM proteins. Previously we have reported biosynthetic cooperation between Sertoli cells and peritubular myoid cells with respect to production of ECM proteins and in particular, the production of fibronectin by peritubular myoid cells in vitro was enhanced when cultured in association with Sertoli cells. Here we evaluated primary culture of Sertoli cells, secondary culture of peritubular myoid cells, and the transformed MSC-1 cells in terms of expression of laminin and fibronectin by using various biotechniques. Laminin, but not fibronectin was detectable by immunocytochemistry in primary culture of Sertoli cells when cultured on glass coverslips. Secondary culture of peritubular myoid cells produced both laminin and fibronectin. In MSC-1 cells, both laminin and fibronectin were detected. Levels of soluble laminin and fibronectin secreted by the three types of cells were determined by an ELISA. The dot-blot analysis using specific antibodies for laminin and fibronectin was undertaken to demonstrate the expression of these ECM proteins. Primary Sertoli cells in culture secreted laminin, but did not secrete detectable amounts of fibronectin. Peritubular myoid cells and MSC-1 cells released high amounts of both laminin and fibronectin into the medium. To further confirm the difference in fibronectin expression by these cells we have conducted western blot analysis. A specific band of fibronectin at 220 kDa was observed in peritubular myoid cells and MSC-1 cells. Again, Sertoli cells in primary culture did not express a significant level of fibronectin. Furthermore, Sertoli cells demonstrated a much higher electrical resistance than the secondary culture of peritubular myoid cells or transformed MSC-1 cells when cultured on Matrigel coated Millipore-HA filters in bicameral chambers. Through biotechnological evaluation and screening we demonstrated the differences between these cultured testicular cells, which may be used to better understand the biology of the seminiferous tubule, and capture nuances of the in vivo cellular microenvironment.