Isolation and primary Sertoli cell cultures.

Sertoli cells isolated using 20-day-old-rat testes were used for primary cultures as earlier described (62). These cells were differentiated and similar to Sertoli cells isolated from adult rat testes both functionally and morphologically as earlier reported (45, 57). Freshly isolated Sertoli cells were seeded on Matrigel-coated (BD Biosciences, San Jose, CA) culture dishes (either 6-, 12-, or 24-well), coverslips (placed in 12-well dishes), and bicameral units (Millipore, Billerica, MA; placed in 24-well dishes) at a density 0.3~0.4, 0.03~0.04, and 1.0 × 10⁶ cells/cm², respectively. Residual germ cells were lysed by a hypotonic treatment using 20 mM Tris (pH 7.4 at 22°C for 2 min) as described (27, 62). Sertoli cells were cultured in serum-free DMEM/F-12 (Sigma-Aldrich, St. Louis, MO) medium supplemented with growth factors and gentamicin in a humidified atmosphere of 95% air-5% CO₂ (vol/vol) at 35°C (62). For 6- and 12-well dishes, each well contained 5 and 3 ml F12/DMEM medium for specific biochemical assays or for immunoblottings (IB). For immunofluorescence (IF), each well (with Sertoli cells cultured on a coverslip) contained 2 ml F12/DMEM. For bicameral units placed in 24-well dishes, the apical and the basal chamber contained 0.5 ml F12/DMEM. All media were supplemented with growth factors [bovine insulin (10 µg/ml), human transferrin (5 µg/ml), EGF (2.5 ng/ml), bacitracin (5 µg/ml), and gentamicin (20 µg/ml)] as described (62). These Sertoli cell cultures were used for experiments on day 3 with an established function tight junction (TJ)-permeability barrier, and ultrastructures of TJ, basal ES, gap junction, and desmosome that mimicked the Sertoli cell blood-testis barrier (BTB) in vivo were also detected as earlier described (47, 53, 82), consistent with earlier reports by others (11, 38). In fact, this in vitro system has been widely used to study Sertoli cell BTB dynamics by others (16, 24, 40, 64, 70). These Sertoli cell cultures were >98% pure with negligible contamination of germ cells, Leydig cells, and/or peritubular myoid cells using corresponding primer pairs for specific cell markers by PCR as described (44).