Gonadotropins regulate rat testicular tight junctions in vivo

Abstract

Sertoli cell tight junctions (TJs) are an essential component of the blood-testis barrier required for spermatogenesis; however, the role of gonadotropins in their maintenance is unknown. This study aimed to investigate the effect of gonadotropin suppression and short-term replacement on TJ function and TJ protein (occludin and claudin-11) expression and localization, in an adult rat model in vivo. Rats (n = 10/group) received the GnRH antagonist, acyline, for 7 wk to suppress gonadotropins. Three groups then received for 7 d: 1) human recombinant FSH, 2) human chorionic gonadotropin (hCG) and rat FSH antibody (to study testicular androgen stimulation alone), and 3) hCG alone (to study testicular androgen and pituitary FSH production). TJ proteins were assessed by real-time PCR, Western blot analysis, and immunohistochemistry, whereas TJ function was assessed with a biotin permeation tracer. Acyline treatment significantly reduced testis weights, serum androgens, LH and FSH, and adluminal germ cells (pachytene spermatocyte, round and elongating spermatids). In contrast to controls, acyline induced seminiferous tubule permeability to biotin, loss of tubule lumens, and loss of occludin, but redistribution of claudin-11, immunostaining. Short-term hormone replacement stimulated significant recoveries in adluminal germ cell numbers. In hCG +/- FSH antibody-treated rats, occludin and claudin-11 protein relocalized at the TJ, but such relocalization was minimal with FSH alone. Tubule lumens also reappeared, but most tubules remained permeable to biotin tracer, despite the presence of occludin. It is concluded that gonadotropins maintain Sertoli cell TJs in the adult rat via a mechanism that includes the localization of occludin and claudin-11 at functional TJs.

Figure 1

Stereological assessment of the number of germ cells (millions/testis) counted in the following subgroups: A, Type A/intermediate spermatogonia; B, Type B/preleptotene spermatocytes; C, Leptotene/zygotene spermatocytes; D, Pachytene spermatocytes stages I–VIII; E, Pachytene spermatocytes stages IX–XIV; and F, Round spermatids steps 1–8. Spermatogenesis was suppressed by GnRH antagonist treatment (acyline, 7 wk), then restored over a further 7-d period with acyline and either hrecFSH or hCG in combination with a polyclonal sheep antiserum raised against rat FSH (for details, see Materials and Methods). Data are mean ± sd, n = 5 rats/group. Significant differences (P < 0.05 or greater) between treatment groups are denoted by different letters, with full details given in the text. Note the logarithmic y-axes for the two lower right graphs.

Figure 2

Assessment of TJ functionality using biotin tracer. A and B, TJs in adult rat testis sections were visualized by staining for the TJ protein occludin (red, indicated by arrows) in conjunction with the nuclear counterstain 4′,6-diamidino-2-phenylindole (blue). A, The TJ functional tracer biotin (green) remained localized to the interstitial space and only permeated around cells on the basal side of TJs in the seminiferous epithelium (arrowheads). B, The animal received the vehicle control for the biotin tracer. C, Biotin tracer permeation in a prepubertal (9 d) rat testis that lacks a functional BTB. Nonspecific staining (insets) was assessed by omitting streptavidin Alexa-488 from the probing solutions (A) or by substituting the occludin antibody with a nonspecific rabbit IgG at similar concentration (B). Scale bar, 50 μm.

Figure 3

Immunofluorescence microscopy of occludin (red, indicated by arrows) and biotin functional tracer (green) in testes from rats given hormone suppression and replacement. TJ structure and function was initially visualized in a normal control testis (A) and compared with testes which had been hormonally suppressed with acyline alone for 8 wk (B) or with acyline for 8 wk plus the FSH Ab in the final eighth week (C). After hormone suppression, the effect of short-term hormone replacement with hrecFSH alone (D), hCG + FSH Ab to investigate the effect of androgen production alone (E), and hCG to stimulate both androgens and endogenous rat FSH (F) on TJ function and structure was analyzed. G–I, A much smaller proportion of TJ phenotypes observed with each of the short-term hormone replacement treatments listed above, respectively. Such phenotypes were not observed in acyline ± FSH Ab treatments alone. Insets are three times enlarged portions of detail in the same panel. Scale bar, 50 μm.

Figure 4

Effect of hormone suppression and replacement on the localization of claudin-11, JAM-A, and ZO-1 by immunofluorescence microscopy to the testicular TJ. Inset in control panels shows negative control for each TJ protein. Scale bar, 50 μm. Arrows, Basally located staining of TJ proteins; *, TJ protein staining adjacent to germ cells.

Figure 5

Effect of hormone suppression and replacement on TJ protein expression. Western blot analysis of occludin (64 kDa, 40 μg total protein per lane, reducing conditions) (A) and claudin-11 (22 kDa, 120 μg total protein per lane, nonreducing conditions) (B). Protein samples were from single animals from each treatment group. The housekeeping protein β-actin (43 kDa) was also probed in each lane on the claudin-11 gel. Lanes are as follows: 1, Control; 2, acyline; 3, acyline + FSH Ab; 4, acyline + FSH; 5, acyline + hCG + FSH Ab; 6, acyline + hCG; 7, adult rat testis positive control; and 8, adult rat testis negative control, with nonimmune IgG (occludin) or rabbit serum (claudin-11) at matching concentrations.

Figure 6

Effect of hormone suppression and replacement on TJ mRNA expression. Real-time PCR quantitation was conducted for the TJ proteins claudin-11 (A), occludin (B), and JAM-A (C), as well as the cytoplasmic plaque protein, ZO-1 (D). As a positive control for hormone suppression and replacement, the androgen-regulated testicular gene INSL-3 (note log scale; E) was also assessed. All data are presented as a ratio to the housekeeper protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To check for any changes in GAPDH mRNA across the various treatment groups, a second housekeeper, β-actin (F), was also quantitated. Data are mean ± sd, n = 5/group. Differences between letters are P < 0.001.