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First published online 20 January 2004
doi: 10.1242/jcs.00900

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Regulation of blood-testis barrier dynamics: an in vivo study

Population Council, Center for Biomedical Research, 1230 York Avenue, New York, NY 10021, USA

View larger version (130K): [in a new window]   Fig. 2. Changes in cytokine levels, different TJ- and AJ-associated molecules, cytoskeleton components, proteases and protease inhibitors in rat testes during CdCl2-induced BTB damage. (A) Rats that had been treated with CdCl2 [3 mg/kg body weight by intraperitoneal injection (b.w., i.p.)] were sacrificed and testis lysates were prepared as described in Materials and Methods. Proteins (100 µg per lane) from these lysates were resolved by SDS-PAGE and subjected to immunoblot analysis using the corresponding primary antibody as indicated. (B) Immunoblot analysis to quantify the levels of 2-MG of testis lysates from rats receiving 1 mg/kg b.w. CdCl2. (C-N) Densitometry scans of immunoblots, such as those shown in A. The protein level of a corresponding target protein at time 0 was arbitrarily set at 1. Each bar is mean±s.d. of three to five determinations from three to five rats of each experiment. Two other sets of experiments yielded virtually identical results. nd, non-detectable; ns, not significantly different by ANOVA where a given sample was compared to all other samples within an experimental group; *significantly different, P<0.05; **significantly different, P<0.01.

View larger version (134K): [in a new window]   Fig. 3. Immunohistochemical localization of ZO-1 in the seminiferous epithelium of adult rat testes during CdCl2-induced BTB damage. (A) Low magnification of a cross section of a rat testis at 0 hours immunostained with an anti-ZO-1 antibody, and (B) a control section where normal rabbit serum was substituted for the primary antibody. (C-H) Micrographs at high magnifications of rat testes after CdCl2 treatment (3 mg/kg b.w.i.p.). Immunoreactive ZO-1 appears as reddish-brown precipitates localized at the basal compartment consistent with the site of the BTB (see arrowheads in A). These micrographs are typical of the results from at least 120 cross sections from three testes of different rats. Scale bar: (A,B) 200 µm; (C-H) 80 µm; (C inset, applies to insets in D-G) 20 µm.

View larger version (67K): [in a new window]   Fig. 4. Immunofluorescence microscopy to identify occludin and ZO-1 in the seminiferous epithelium of rat testes at the BTB site during CdCl2-induced BTB damage. (A-O) Immunofluorescence micrographs of cross sections of normal rat testes (A-C) and rat testis at 7 hours (D-F), 12 hours (G-I) and 24 hours (J-O) after CdCl2 treatment. Occludin appears as red fluorescence (Cy-3, white arrowheads in A), ZO-1 as green fluorescence (FITC, arrowheads in B). (C,F,I,L,O) Merged images of the corresponding immunofluorescence micrographs for occludin and ZO-1, in which co-localization appears orange (arrowheads in C). White broken lines in M-O indicate the approximate location of the basement membrane of the seminiferous epithelium. Scale bar: 20 µm.

View larger version (87K): [in a new window]   Fig. 1. Ultrastructural analysis of the integrity of the blood-testis barrier (BTB) following treatment of rats with CdCl2 (3 mg/kg b.w. via i.p.). (A,B) Electron micrographs of (A) the BTB between two adjacent Sertoli cells at the seminiferous epithelium of a control rat, as an example of a tight junction (TJ), which appears as electron dense material (arrowhead). Magnification, x67,500; (B) a rat testis 24 hours after CdCl2 treatment illustrating that the TJ were disrupted (arrowheads). Magnification, x45,000. LD, lipid droplet; SC, Sertoli cell; BM, basement membrane; C, collagen network; MC, myoid cell layer.

View larger version (120K): [in a new window]   Fig. 5. Immunohistochemical localization of protease inhibitor 2-MG in the seminiferous epithelium of rat testes at the BTB site during CdCl2-induced BTB damage. (A-D) Immunoreactive 2-MG in the seminiferous epithelium after treatment of rats with CdCl2 (3 mg/kg b.w., i.p.). Shown are different magnifications of cross sections of testes at time 0 hours, 20 hours, 24 hours and 96 hours. Immunoreactive 2-MG appears as reddish-brown precipitates. (E) Control in which normal rabbit serum was substituted for the primary antibody, illustrating that the staining for 2-MG shown in A-D is specific. The smaller images in A, B and E are higher magnifications of the representative micrographs shown on the left panel that illustrate the cellular localization of 2-MG in the seminiferous epithelium. Bars in left panel of A and E, 200 µm; bars in C, in middle panel of A and right panel of E, 80 µm; bar in right panel of A, 20 µm. Bars shown in A are applicable to B, and bar in C is applicable to D.

View larger version (45K): [in a new window]   Fig. 6. Studies by co-immunoprecipitation to investigate the association of the protease inhibitor 2-MG with TGF-ß3, cathepsin L, and TJ-associated, AJ-associated and cytoskeletal proteins during CdCl2-induced BTB damage. Immunoprecipitation was performed using either anti-2-MG or anti-TGF-ß3 antibody and lysates of testes from rats that had received CdCl2 (3 mg/kg b.w., i.p.) at 0, 24 and 96 hours earlier. For positive controls, lysates of testes from rats (150 µg protein per lane) at 20 hours after CdCl2 treatment without immunoprecipitation were used since at this time most target proteins pertinent to our studies were induced (see Fig. 2), confirming the specificity of the antibodies. Only a set of representative data is shown, but two other experiments using different rat testis lysates yielded identical results.

View larger version (40K): [in a new window]   Fig. 7. A study to assess if a blockade of the p38 MAP kinase by SB202190 can delay the CdCl2-induced target protein losses from the sites of BTB and AJs in the testis during BTB damage. Normal rat testes (negative controls) (Ctrl), and testes injected with 1% DMSO in saline (vol/vol, intratesticular administration) followed by CdCl2 treatment served as positive controls (CdCl2/vehicle). For test groups (n=3-5 rats), testes of rats were pre-treated with 8 nmol SB202190 in 200 µl saline with 1% DMSO (i.e. 5 µM assuming a volume of 1.6 ml per testis), followed by CdCl2 treatment (3 mg/kg b.w.i.p.) (CdCl2 + SB202190), and rats were sacrificed 16 hours later. Lysates were prepared from these samples for immunoblotting with antibodies against occludin, ZO-1, N-cadherin, E-cadherin, ß-catenin, nectin-3, l-afadin, cathepsin L, 2-MG, and TGF-ß3.

View larger version (150K): [in a new window]   Fig. 8. Immunohistochemical localization of occludin, ZO-1, N-cadherin and 2-MG in the seminiferous epithelium of rat testes with and without pretreatment with either SB202190 or vehicle (1% DMSO) (via intratesticular injection) prior to CdCl2 administration to induce BTB damage. (A-L) Micrographs of seminiferous epithelium of normal rats (A,D,G,J, negative controls), rats pretreated with vehicle (1% DMSO) followed by CdCl2 (B,E,H,K; positive controls), and rats pretreated with 5 µM SB202190 followed by CdCl2 (C,F,I,L; test). A-C, D-F, G-I and JL show the immunohistochemical localization of occludin, ZO-1, N-cadherin and 2-MG, respectively. It is noted that after the pre-treatment of testes with SB202190, the CdCl2-induced losses of occludin (C compared with B), ZO-1 (F compared with E) and N-cadherin (I compared with H) were blocked. However, the induction of 2-MG expression by CdCl2 was not affected (L compared with K). Scale bar in A: 80 µm, applicable to B-L.

View larger version (31K): [in a new window]   Fig. 9. A schematic drawing that illustrates how protease inhibitors, such as 2-MG, may be involved in the regulation of TJ and AJ dynamics in the seminiferous epithelium. This proposal is based on the results reported herein and other recent studies (Siu et al., 2003a; Siu et al., 2003b; Siu and Cheng, 2004; Mruk et al., 2003; Lui et al., 2003a; Lui et al., 2003b). This diagram also illustrates the current yet simplified molecular architecture of different TJ and AJ protein complexes in the testis (for reviews, see Cheng and Mruk, 2002; Lui et al., 2003b; Lui et al., 2003c). Occludin and ZO-1/-2/-3 are the major constituent proteins of TJ fibrils found in Sertoli cells at the BTB site. The cadherin/catenin and nectin/afadin complexes are the putative AJ protein complexes in the testis. They are connected to the actin cytoskeleton network, partly by -catenin. When TJs and AJs are `closed', the junction-associated multiprotein complexes of two apposed cells interact with each other, whereas the level and/or activity of cytokines and proteases apparently are kept low by 2-MG. However, when TJ and AJ disassemble (or `open'), such as when treated with CdCl2, junction functionality is perturbed. This may be mediated by cytokines, such as TGF-ß3 and TNF that bind to their receptors and lead to the rapid turnover (possibly undergoing proteolysis) of junction components (represented by broken lines) via the p38 MAP kinase signal pathway. Proteases activities may also be induced, facilitating junction dissociation. At this time, the amount of the protease inhibitor 2-MG in the seminiferous tubules increases, binding to the excessive cytokines to limit their action, or inhibiting proteases to protect the integrity of the BTB. As such, protease inhibitors may act as `rescuers' during junction remodeling in the testis, regulating the homeostasis of proteins that constitute both TJs and AJs.

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