Androgens regulate the permeability of the blood–testis barrier

Abstract

Spermatogenesis in mammals requires the actions of a complex assortment of peptide and steroid hormones. These hormonal messengers are critical not only for regulation of germ cell differentiation, but also for the proliferation and function of the somatic cell types required for proper development of the testis (1, 2). These cells include the interstitial steroidogenic Leydig cells, whose primary function is to produce testosterone (3), the myoid cells that surround the seminiferous tubules and secrete basal lamina components (4), and the Sertoli cells, whose direct contact with proliferating and differentiating germ cells within the seminiferous tubules makes them essential for providing both physical and nutritional support for spermatogenesis (5). Each of these cell types is essential for unimpaired male fertility and is a direct target of one or more of the reproductive hormones.

Genetic and endocrine ablation studies suggest that androgens, testosterone and its derivatives, regulate several steps in mammalian spermatogenesis (1, 6, 7). In sexually mature mice, the androgen receptor (Ar) is expressed in Sertoli cells in a stage-dependent pattern (8), and androgen withdrawal experiments support the notion that androgens exert their influence during the stages of highest androgen receptor expression (9–11). Sertoli cell-specific ablation of the gene encoding the androgen receptor indicates that androgens are required for progression of germ cells through meiosis I, again during the transition from round to elongating spermatids, and finally during the terminal stages of spermiogenesis (12–14).

Mammalian spermatogenesis is a continuous process that occurs within a tubular seminiferous epithelium formed by a radial clustering of somatic Sertoli cells. Diploid germ cells, which include spermatogonial stem cells, transit-amplifying spermatogonia, and preleptotene spermatocytes, reside in the basal compartment in direct contact with the basal lamina and the somatic Sertoli cells. Further progression of germ cell differentiation occurs after the movement of the premeiotic cells off the basal lamina and into the adluminal compartment of the epithelium. As the germ cells move into the adluminal compartment, tight junctions (TJs) between adjacent Sertoli cells form behind the germ cells creating the blood–testis barrier (BTB) (Fig. 1D) (15, 16). The BTB segregates the meiotic and postmeiotic cells into the immunologically privileged adluminal compartment. The androgen receptor is expressed at highest levels during stages when new TJs form and premeiotic cells move from the basal to the adluminal compartment of the seminiferous epithelium (8, 16, 17).

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 Ar^{invflox(ex1-neo)/Y};Tg (Amh-Cre) mice and probed with a ^P-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.)

TJs are specialized anchoring junctions composed of several integral and peripheral membrane proteins (18, 19). The membrane-spanning proteins include the occludins, claudins, and junction adhesion molecules (20–22). Occludin, present in mouse Sertoli TJs but not human or guinea pig, appears to be essential for TJ function in the testis of mice, because a targeted mutation in the occludin gene causes male sterility (23). More than 20 different claudins have been described since the initial discovery of Cldn1 and Cldn2 in the late 1990s (21). Among these proteins, claudins 1, 3, 4, 5, 7, 8, and 11 have been shown to be expressed in the testis (reviewed in ref. 24). Targeted deletion of Cldn11 has revealed that it is essential for normal Sertoli cell TJs, and the myelin sheaths of oligodendrocytes, because the mice have both reproductive and CNS defects (25). Two junction adhesion molecules, Jam1 and Jam2, are expressed in the testis (22, 26). Associated with the cytoplasmic tails of the membrane spanning proteins are several peripheral membrane proteins, including zonula occludens, cingulin, and symplekin (24). Almost nothing is known about the regulation of the Sertoli cell TJ components and their assembly and disassembly during the cycle of the seminiferous epithelium, although TGFβ3 has been shown to affect the expression of Cldn11 in cell culture (27).

We have previously investigated the function of the androgen receptor in Sertoli cells by creating mice with a hypomorphic inverted floxed allele of Ar (Ar^{invflox(ex1-neo)}) and crossing them with mice expressing Cre recombinase driven by the Sertoli cell-specific promoter of the anti-Müllerian hormone gene (Amh). In this model of incomplete and conditional androgen insensitivity, Ar levels are reduced in all cells where it is normally expressed because of an aberrant and incomplete splicing reaction and are completely absent in Sertoli cells because of loxP-mediated inversion of the first exon. The mice are azoospermic and show a block in the transition from round to elongating spermatids (14). Accompanying the arrest are increased germ cell apoptosis and sloughing of spermatids into the lumen of the seminiferous epithelium.

In hopes of discovering the mechanisms by which androgens control spermatogenesis, it is necessary first to identify transcriptional targets of the androgen receptor. In this study we used microarray gene expression profiling to investigate the transcriptional consequences after the loss of androgen signaling in Sertoli cells. We found that claudin 3 (Cldn3) is down-regulated in Ar^{invflox(ex1-neo)/Y};Tg (Amh-Cre) mice, suggesting that androgens may regulate the assembly or function of Sertoli cell TJs. We tested this hypothesis and found that the permeability of the BTB is altered in mice with conditional ablation of Ar expression in Sertoli cells.

Acknowledgments

Notes

Author contributions: J.M., R.W.H., and R.E.B. designed research; J.M., R.W.H., and J.E.S. performed research; J.M., R.W.H., J.E.S., M.D.G., and R.E.B. analyzed data; and J.M., M.D.G., and R.E.B. wrote the paper.

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: Ar, androgen receptor; BTB, blood–testis barrier; Cldn3, claudin 3; TJ, tight junctions.