Study on the formation of specialized inter-Sertoli cell junctions in vitro

Abstract

An in vitro culture system using Sertoli cells was employed to assess the expression of component genes pertinent to occluding junctions (OJ) (such as zonula occludens-1, ZO-1), anchoring junctions (AJ) (such as N-cadherin and beta-catenin), and communicating gap junctions (GJ) (such as connexin 33, Cx33) when they are being formed in vitro. Freshly isolated Sertoli cells from 20-day-old rats with a purity of greater than 90% were cultured either at low- (2.5 x 10(4) cells/cm(2)) or high-cell density (0.6 x 10(6) cells/cm(2)) on Matrigel-coated dishes for 7 days in vitro to allow the establishment of specialized junctions. In low cell density Sertoli cell cultures, specialized OJ such as tight junctions did not form during the entire culture period when assessed by the transepithelial electrical resistance (TER). In high cell density cultures, there was an increase in ZO-1 expression in days 1 to 3 preceding the establishment of tight junctions by day 4. When Sertoli cells were cultured at both cell densities, there was a transient increase in Sertoli cell N-cadherin expression, which peaked by days 4-5, suggesting the time course for the establishment of AJ may overlap with the OJ. A significant increase in the expression of Sertoli cell beta-catenin was also detected by days 5-7 in the high but not low cell density cultures. The expression of Cx33 was also enhanced at days 4-5 in both high and low density cultures. These results suggest that OJ, AJ, and GJ are formed between Sertoli cells in high density cultures, whereas OJ cannot be formed in low density cultures. A full-length cDNA clone coding for rat testicular beta-catenin was also isolated. The deduced amino acid sequence of rat beta-catenin yielded a 781 amino acid polypeptide which displayed a 99.9% identity with the mouse homolog. Conditioned medium of germ cells induced a dose-dependent stimulation on Sertoli cell beta-catenin expression, suggesting germ cells may affect the N-cadherin/beta-catenin-mediated signal transduction pathway. In summary, this study illustrates several target genes can be used as molecular markers to monitor the inter-Sertoli junction formation. This system should be applicable to screen new male contraceptives in vitro targeted at the interference of junction formation by disrupting the timely expression of genes necessary for junction establishment and/or maintenance.

Intercellular adhesion molecule-1 is a regulator of blood–testis barrier function

“…Cells were seeded at a density of 0.5610 6 cells/cm 2 in 12-well plates for lysate preparation, RNA isolation and overexpression experiments, at a density of 1.2610 6 cells/cm 2 in Millicell inserts (Millipore Corp., Billerica, MA) for transepithelial electrical resistance (TER) measurements and overexpression experiments, or at a density of 0.05610 6 cells/ cm 2 on round glass coverslips (18 mm; Thomas Scientific, Swedesboro, NJ) for immunofluorescent staining. It is worth noting that functional junctions were assembled and maintained in control Sertoli cells cultured at all densities used in this study, a conclusion that is based on nearly two decades of research from this and other laboratories Chung et al, 1999;Dym and Papadopoulos, 1992;Florin et al, 2005;Hadley et al, 1987;Janecki et al, 1991;Janecki and Steinberger, 1986;Lie et al, 2011;Lui et al, 2001;Siu et al, 2003;Su et al, 2010b;Tung and Fritz, 1993). All substrata were coated with Matrigel TM basement membrane matrix as previously described (Xiao et al, 2011).…”

“…On day 2, cells were transfected with empty pCI-neo vector (MOCK, control), ICAM-1, sICAM-1 or ICAM-2 plasmids using EffecteneH transfection reagent (Qiagen). It should be noted that by this time in vitro, Sertoli cells had formed a polarized epithelium with functional TJs, basal ESs, desmosomes and GJs Lie et al, 2009;Siu et al, 2005;Wong et al, 2000), thereby mimicking the BTB in vivo (Byers et al, 1986;Chung et al, 1999;Siu et al, 2005;Steinberger and Jakubowiak, 1993;Yan et al, 2008). Thus, this excellent in vitro model has been, and continues to be, used by many investigators to study BTB dynamics.…”

Anchoring Junctions As Drug Targets: Role in Contraceptive Development

“…3). After decades of morphological and biochemical studies, we know that Sertoli cells isolated from rat testes become polarized and establish functional junctions when they are plated on a reconstituted basement membrane such as Matrigel at high cell density (ϳ0.5-1.0 ϫ 10 6 cells/cm 2 ) in a two-dimensional environment (Chung et al, 1999;Grima and Cheng, 2000;Wong et al, 2000;Chung and Cheng, 2001;Lee et al, 2003). We also know that Sertoli cells cultured under these conditions are functionally similar to those found in the testis in vivo, because they secrete transferrin, androgen binding protein (ABP), testin, and ␣ 2 -macroglobulin in a polarized fashion (Grima et al, 1992).…”

Claudin-11 expression and localisation is regulated by androgens in rat Sertoli cells in vitro

“…In the current study, we have used a well-established in vitro model of Sertoli cells in bicameral culture (Hadley et al 1987, Handelsman et al 1989, Onoda et al 1990, Janecki et al 1991a, Djakiew & Onoda 1993 to study the effects of androgens and FSH on TJs. TJ function in this culture system is readily monitored by the determination of transepithelial electrical resistance (TER; Janecki et al 1991a, 1991b, Chung et al 1999, Fanning et al 1999, Li et al 2001, Lui et al 2003a, Siu et al 2003. Testosterone and FSH, either alone or in combination, are known to increase rat Sertoli cell TER two to threefold in this model (Janecki et al 1991a, 1991b, Steinberger & Klinefelter 1993 indicating that Sertoli cell TJ function can be regulated by gonadotrophins.…”