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Gonadotropins Regulate Rat Testicular Tight Junctions in Vivo

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Gonadotropins Regulate Rat Testicular Tight Junctions in Vivo REPRODUCTION-DEVELOPMENT Gonadotropins Regulate Rat Testicular Tight Junctions in Vivo Mark J. McCabe, Gerard A. Tarulli, Sarah J. Meachem, David M. Robertson, Peter M. Smooker, and Peter G. Stanton Prince Henry’s Institute (M.J.M., G.A.T., S.J.M., D.M.R., P.G.S.), Monash Medical Centre, Clayton, Victoria 3168, Australia; School of Applied Sciences (M.J.M., P.M.S.), Royal Melbourne Institute of Technology University, Bundoora, Victoria 3083, Australia; and Department of Biochemistry and Molecular Biology (P.G.S.), Monash University, Clayton, Victoria 3800, Australia 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 ϭ 10/group) received the GnRH antagonist, acyline, for 7 wk to suppress gonado- tropins. 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, immuno- staining. Short-term hormone replacement stimulated significant recoveries in adluminal germ cell numbers. In hCG Ϯ 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 local- ization of occludin and claudin-11 at functional TJs. (Endocrinology 151: 2911–2922, 2010) he blood-testis barrier (BTB) is a permeability barrier, Formation of the BTB at puberty in animal models (2, Twhich functions to sequester germ cells undergoing 4) and humans (5) requires the gonadotropins FSH and meiotic and postmeiotic differentiation from the vascular LH (6, 7), a requirement that is also supported by in vitro environment. Endothelial, peritubular myoid, and Sertoli models (8–12). Once formed, Sertoli cell TJs undergo a cells contribute to the barrier (1), but it is recognized that cyclic remodelling during spermatogenesis, as early mei- tight junctions (TJs) found basally between Sertoli cells are otic preleptotene spermatocytes translocate to the adlu- the major component in the adult testis (1). Sertoli cell TJs minal compartment where they complete meiosis and are required for fertility, and it is well established that their spermiogenesis (2). An expanding list of cytokines and disruption leads to germ cell atresia and cessation of sper- growth factors, including TGF-␤, TNF-␣, growth differ- matogenesis (2). Changes in BTB permeability and constit- entiation factor-9, activins, inhibins, and interleukins, uent proteins have been noted in men with infertility (3). produced by both Sertoli and germ cells (10, 13–16; for ISSN Print 0013-7227 ISSN Online 1945-7170 Abbreviations: BTB, Blood-testis barrier; FSH Ab, FSH antibody; hCG, human chorionic Printed in U.S.A. gonadotropin; hrec, human recombinant; JAM, junctional adhesion molecule; T, testos- Copyright © 2010 by The Endocrine Society terone; TJ, tight junction; ZO, zona occludens. doi: 10.1210/en.2009-1278 Received October 27, 2009. Accepted March 2, 2010. First Published Online March 31, 2010 Endocrinology, June 2010, 151(6):2911–2922 endo.endojournals.org 2911 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 January 2015. at 16:54 For personal use only. No other uses without permission. All rights reserved. 2912 McCabe et al. Regulation of Rat Testicular TJs Endocrinology, June 2010, 151(6):2911–2922 review, see Ref. 17) mediate the local cross talk required Experimental design for this translocation step. Circulating FSH and LH/testosterone (T) were suppressed by Sertoli cell TJs contain three main transmembrane pro- weekly sc injections of the GnRH antagonist, acyline (donated by Richard Blye; National Institutes of Health, Bethesda, MD) for tein groups, the claudins, particularly claudin-11 (18, 19) 7 wk into adult rats at 1.5 mg/kg in 5% mannitol/sterile MilliQ and claudin-3 (6), occludin (20, 21), and the junctional water (34), administered as single injections to the hind flank or adhesion molecule (JAM) family (22). Both androgens and shoulder (400 ␮l/site). Control rats received a sc injection of the FSH can regulate these proteins during TJ formation in vehicle alone (n ϭ 10). ϭ vivo (6, 22) and in vitro (9–11). However, the extent to After 7 wk of gonadotropin suppression, rats (n 10/group) in the eighth week received an additional dose of acyline in con- which gonadotropins contribute to the maintenance of the junction with short-term hormone replacement (daily, for 7 d) by established BTB in the normal adult remains unknown. sc injections with one of the following: 1) human recombinant Animal studies that have previously ablated gonadotropin (hrec) FSH (25 IU/kg) (Puregon; Organon, Oss, The Nether- action on the testis via hypophysectomy (23–25), GnRH lands), 2) human chorionic gonadotropin (hCG) (2.5 IU/kg) ϩ antagonism (26, 27), or GnRH immunization (28, 29) (Pregnyl; Organon) FSH antibody (FSH Ab) (sheep antirat, 2 mg/kg) (29) to study testicular production of androgens alone, have either not examined BTB function or are inconclusive and 3) hCG (2.5 IU/kg) ϩ normal sheep immunoglobulins (2 on this point. Regulation of the BTB has potential impor- mg/kg) to study the production of androgens and endogenous rat tance in understanding the mechanism(s) of action of male FSH (29). Three more groups of rats (n ϭ 10/group) continued hormonal contraception, which suppresses spermatogen- to receive 1) acyline vehicle, 2) acyline ϩ FSH Ab (daily, sheep ϩ esis via suppression of circulating gonadotropins (for re- antirat, 2 mg/kg) to suppress residual FSH, and 3) acyline normal sheep immunoglobulins (daily, 2 mg/kg) for the eighth view, see Ref. 30). week. Doses and treatment intervals were as described elsewhere Precedents exist for a regulatory role for gonadotropins (29, 35). Previous studies demonstrate that endogenous rat FSH on Sertoli cell TJs in adult animals. In the seasonal breed- levels rise under the influence of T stimulation (28, 36, 37). ing, adult Djungarian hamster, circulating gonadotropins, At the end of the experiment, rats were killed by CO2 as- and spermatogenesis are both suppressed during the non- phyxiation, and testes were excised and weighed immediately. Five of the left testes per treatment group were snap-frozen in dry breeding winter months when the BTB is nonfunctional ice for RT-PCR and Western blot analyses. The remaining five (31). We recently demonstrated that claudin-3 and clau- left testes from each group were injected with either the quali- din-11, occludin, and JAM-A were no longer present at tative TJ functional tracer biotin (10 mg/ml, EZ-Link Sulfo- organized Sertoli cell TJs in these animals and that admin- NHS-LC-Biotin; Pierce, Rockford, IL) (n ϭ 4) (6) in a volume of 10% testis weight, or the biotin vehicle, PBS (pH 7.4), and 1 istration of FSH restored BTB functionality and localiza- ϭ mM CaCl2 (n 1). Testes were then incubated at room tem- tion of TJ proteins at Sertoli cell TJs (7, 32). It remains to perature for 30 min before immersion fixation in Bouin’s fixative be seen whether a similar gonadotropin dependence of TJs for 5 h and were then randomly sampled by a systematic uniform occurs in animal models that do not undergo seasonal approach (29, 38) for stereological or immunohistochemical changes. assessment. Shortly after killing rats, blood was taken by cardiac puncture The aim of this study was to profoundly suppress cir- and stored overnight at 4 C before being spun down in a cen- culating gonadotropins and spermatogenesis in adult male trifuge. Serum was then collected by centrifugation and snap- rats with the GnRH antagonist acyline (33, 34) and then frozen before storage at Ϫ80 C for hormone analysis. selectively replace testicular androgens and/or FSH via short-term replacement regimens (29). Changes in germ Immunohistochemistry cell numbers were quantified by stereology, whereas TJ Tissues were embedded in paraffin wax, and 5-␮m sections function was assessed using a qualitative biotin perme- were mounted onto Superfrost-Plus slides (HD Scientific, Mel- ation tracer (6). Changes in TJ proteins were examined bourne, Australia). Antigen retrieval was performed by heating using immunohistochemistry, real-time RT-PCR, and the sections for 10 min in 600 ml 1 mM EDTA-NaOH (pH 8.0) Western blot analysis. (39) in an 800-W microwave. Primary antibodies [rabbit anti- occludin (no. 71-1500, 2.5 ␮g/ml, 30 min; Zymed, South San Francisco, CA), rabbit anti claudin-11 (no. 36-4500, 2 ␮g/ml, 1 h; Zymed), rabbit anti zona occludens (ZO)-1 (no. 61-7300, 1.25 ␮g/ml, 1 h; Zymed), and rabbit anti JAM-A (no. 36-1700, Materials and Methods 0.63 ␮g/ml, overnight; Zymed)] were diluted in 10% normal goat serum (Chemicon International, Temecula, CA) in PBS and Animals then applied to the sections for the times specified at 25 C.
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