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. 2005 Nov 15;102(46):16696-700.
doi: 10.1073/pnas.0506084102. Epub 2005 Nov 7.

Androgens regulate the permeability of the blood-testis barrier

Jing Meng  1 , Robert W Holdcraft, James E Shima, Michael D Griswold, Robert E Braun
Affiliations

Androgens regulate the permeability of the blood-testis barrier

Jing Meng et al. Proc Natl Acad Sci U S A. .

Abstract

Within the mammalian testis, specialized tight junctions between somatic Sertoli cells create basal and apical polarity within the cells, restrict movement of molecules between cells, and separate the seminiferous epithelium into basal and adluminal compartments. These tight junctions form the basis of the blood-testis barrier, a structure whose function and dynamic regulation is poorly understood. In this study, we used microarray gene expression profiling to identify genes with altered transcript levels in a mouse model for conditional androgen insensitivity. We show that testosterone, acting through its receptor expressed in Sertoli cells, regulates the expression of claudin 3, which encodes a transient component of newly formed tight junctions. Sertoli cell-specific ablation of androgen receptor results in increased permeability of the blood-testis barrier to biotin, suggesting claudin 3 regulates the movement of small molecules across the Sertoli cell tight junctions. These results suggest that androgen action in Sertoli cells regulates germ cell differentiation, in part by controlling the microenvironment of the seminiferous epithelium. Our studies also indicate that hormonal strategies for male contraception may interfere with the blood-testis barrier.

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Figures

Fig. 1.
Fig. 1.
Cldn3 expression is regulated by androgens. (A Upper) Northern blot analysis on total RNA from testes of 2-month-old wild-type and Arinvflox(ex1-neo)/Y;Tg (Amh-Cre) mice and probed with a 32P-labeled Cldn3 cDNA fragment. (Lower) 28S rRNA was used as a loading control. (B) Western blot analysis of total testis protein extracts prepared from wild-type and Ar mutant mice probed with anti-Cldn3 antibody (Upper) and an anti-actin antibody as a loading control (Lower). (C) Semiquantitative RT-PCR analysis of Cldn3 expression in immortalized Sertoli-like TM4 cells transiently transfected with a cDNA encoding the androgen receptor. RNA was harvested from cells 48 h after treatment with varying doses of dihydrotestosterone (DHT), reverse transcribed, and amplified by PCR with primers specific for Cldn3 (Upper) or Actb (Lower). Reactions were terminated after 25, 30, and 35 cycles. (D) Schematic drawing of a transverse section through the seminiferous epithelium. The epithelium is segregated into basal and adluminal compartments by the formation of TJs between neighboring Sertoli cells (15). As germ cells move from the basal to the adluminal compartment, new TJs form and old TJs are disassembled. Expression of the androgen receptor in Sertoli cell nuclei is maximal during the stages of new TJ formation (8, 16). SC, Sertoli cell; PL, preleptotene spermatocyte; PS, pachytene spermatocyte; BL, basal lamina. (E) Immunofluorescence detection of Cldn3 and Cldn11 on serial sections from wild-type and Ar mutant testes. Both proteins are localized to TJs in the basal compartment of the seminiferous epithelium, although the staining of Cldn3 (arrow in Upper Left) appears to extend beyond that of the staining of Cldn11 (arrow in Lower Left). Expression of Cldn3 is absent in the region of Sertoli cell TJs in Ar mutants (Upper Right). The basal lamina of the tubule is outlined with a dotted line. Expression of Cldn11 is retained in Ar mutant testis (Lower Right). (Scale bar: 50 μm.)
Fig. 2.
Fig. 2.
Expression of Cldn3 is restricted to newly formed TJs. (A–D) Immunofluorescence localization of Cldn3 in adult testis. Nuclei were stained with DAPI and the tubules staged based on the particular association of germ cells within a tubule (44). The arrows indicate regions of Cldn3 detection. High magnification images show the absence of Cldn3 in Stages IV and X and its presence in Stage VIII. (Scale bars: A, 50 μm; B, 20 μm for B–D). (E) Western analysis of Cldn3 in extracts prepared from seminiferous tubule fragments identified by transillumination (30). Actin was used as a loading control.
Fig. 3.
Fig. 3.
Cldn3 expression is developmentally regulated. (A) Immunofluorescence detection of Cldn3 in testis sections during puberty. Cldn3 is first detected at day 15 postpartum and is diffusely distributed. By day 20 postpartum, Cldn3 is localized to Sertoli cell TJ. The sections were counterstained with DAPI. The basal lamina of the tubules is outlined with a dashed line. (Scale bar: 50 μm.) (B) Western blot showing that Cldn3 protein is first detected at day 15 postpartum. A portion of the gel was stained with Coomassie blue as a control for equal loading of sample. (C) RT-PCR (35 cycles) showing that Cldn3 RNA is first detected at day 15 postpartum. Actb was used as a control.
Fig. 4.
Fig. 4.
Loss of androgen signaling in Sertoli cells increases the permeability of the BTB. (A) Penetration of the seminiferous tubules of a 15-day-old wild-type mouse by a biotin tracer 30 min after injection into the interstitial space. (B and C) Restriction of the biotin tracer to the interstitial space and basal compartment of 25-day-old and adult mice. (D) High magnification image of TJ and exclusion of the biotin tracer from the adluminal compartment of a sexually mature mouse. The TJs were stained with anti-occludin antibody, and the nuclei were counterstained with DAPI. (E) Penetration of biotin into the adluminal compartment of several seminiferous tubules 30 min after it was injected into the interstitial space of an adult Ar mutant. In some tubules, penetration was considerable and was detected as aggregates throughout the adluminal compartment. (F and G) Examples of seminiferous tubules from adult Ar mutants showing lesser degrees of biotin diffusion into the adluminal compartment. @, tubules with moderate biotin accumulation; #, light diffuse staining throughout the tubule; *, tubule with no biotin detected in the adluminal region. (H) High magnification image of TJ and penetration of biotin into the adluminal compartment of a seminiferous tubule from an adult Ar mutant. The basal lamina is outlined with a dashed line. (Scale bars: A, 50 μm for A–C and E–G; D, 20 μm for D and H.)
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