Claudin-11 and occludin are protein components in tight junctions (TJs) between Sertoli cells which are important for the maintenance of the blood-testis barrier. Barrier formation occurs during puberty, with evidence suggesting hormonal regulation of both claudin-11 and occludin. This study aimed to investigate the regulation of claudin-11 and occludin mRNA expression by testosterone (T) and FSH and their immunolocalisation at rat Sertoli cell TJs in vitro, and to correlate any steroid regulation with the functional capacity of TJs. Sertoli cells formed functional TJs within 3 days as assessed by transepithelial electrical resistance (TER). Both T and dihydrotestosterone significantly (P!0.01) increased TER twofold and claudin-11 mRNA two-to threefold within 3 days. FSH partially stimulated TER and claudin-11 mRNA, but estradiol had no effect. T also promoted claudin-11 localisation into extensive inter-cellular contacts. In contrast to claudin-11, T and FSH did not change occludin mRNA expression, however, T promoted localisation of occludin at cell contacts in a similar manner to claudin-11. Addition of flutamide to T-stimulated cells caused a twofold decrease in both TER and claudin-11 mRNA expression, and resulted in the loss of both proteins from cell contacts. This effect was reversible following flutamide removal. It is concluded that androgens i) co-regulate claudin-11 mRNA expression and TER, implicating claudin-11 in TJ formation and ii) promote the localisation of claudin-11 and occludin at Sertoli cell contacts. Hence, the ability of androgens to maintain spermatogenesis in vivo is partly via their effects on TJ proteins and regulation of the blood-testis barrier.

The production of mature sperm is reliant on androgen action within the testis, and it is well established that androgens act on receptors within the somatic Sertoli cells to stimulate male germ cell development. Mice lacking Sertoli cell androgen receptors (AR) show late meiotic germ cell arrest, suggesting Sertoli cells transduce the androgenic stimulus co-ordinating this essential step in spermatogenesis. This study aimed to identify germ cell proteins responsive to changes in testicular androgen levels and thereby elucidate mechanisms by which androgens regulate meiosis. Testicular androgen levels were suppressed for 9 weeks using testosterone and estradiol-filled silastic implants, followed by a short period of either further androgen suppression (via an AR antagonist) or the restoration of intratesticular testosterone levels. Comparative proteomics were performed on protein extracts from enriched meiotic cell preparations from adult rats undergoing androgen deprivation and replacement in vivo. Loss of androgenic stimulus caused changes in proteins with known roles in meiosis (including Nasp and Hsp70–2), apoptosis (including Diablo), cell signalling (including 14-3-3 isoforms), oxidative stress, DNA repair, and RNA processing. Immunostaining for oxidised DNA adducts confirmed spermatocytes undergo oxidative stress-induced DNA damage during androgen suppression. An increase in PCNA and an associated ubiquitin-conjugating enzyme (Ubc13) suggested a role for PCNA-mediated regulation of DNA repair pathways in spermatocytes. Changes in cytoplasmic SUMO1 localisation in spermatocytes were paralleled by changes in the levels of free SUMO1 and of a subunit of its activating complex, suggesting sumoylation in spermatocytes is modified by androgen action on Sertoli cells. We conclude that Sertoli cells, in response to androgens, modulate protein translation and post-translational events in spermatocytes that impact on their metabolism, survival, and completion of meiosis.

Sertoli cell tight junctions (TJs) form at puberty as a major component of the blood-testis barrier (BTB), which is essential for spermatogenesis. This study characterized the hormonal induction of functional Sertoli cell TJ formation in vivo using the gonadotropin-deficient hypogonadal (hpg) mouse that displays prepubertal spermatogenic arrest. Androgen actions were determined in hpg mice treated for 2 or 10 days with dihydrotestosterone (DHT). Follicle-stimulating hormone (FSH) actions were studied in hpg mice expressing transgenic human FSH (hpg+tgFSH) with or without DHT treatment. TJ formation was examined by mRNA expression and immunolocalization of TJ proteins claudin-3 and claudin-11, and barrier functionality was examined by biotin tracer permeability. Immunolocalization of claudin-3 and claudin-11 was extensive at wild-type (wt) Sertoli cell TJs, which functionally excluded permeability tracer. In contrast, seminiferous tubules of hpg testes lacked claudin-3, but claudin-11 protein was present in adluminal regions of Sertoli cells. Biotin tracer permeated throughout these tubules, demonstrating dysfunctional TJs. In hpg+tgFSH testes, claudin-3 was generally absent, but claudin-11 had redistributed basally toward the TJs, where function was variable. In hpg testes, DHT treatment stimulated the redistribution of claudin-11 protein toward the basal region of Sertoli cells by Day 2, increased Cldn3 and Cldn11 mRNA expression, then induced the formation of functional TJs containing both proteins by Day 10. In hpg+tgFSH testes, TJ protein redistribution was accelerated and functional TJs formed by Day 2 of DHT treatment. We conclude that androgen stimulates initial Sertoli cell TJ formation and function in mice, whereas FSH activity is insufficient alone, but augments androgen-induced TJ function.

BackgroundDuring the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL) 11 regulates human endometrial epithelial cells (hEEC) adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach.MethodsUsing a 2D-differential in-gel electrophoresis (DIGE) electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2) and flotillin-1 (FLOT1), were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa) and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle).Results2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary hEEC and ECC-1 whilst, the lipid-raft protein FLOT1 demonstrated punctate staining in the apical surface of ECC-1 plasma membranes and was upregulated in the epithelium in the receptive phase of the menstrual cycle (p lower or equal 0.05).ConclusionsThis is the first study to use a proteomics approach to identify hEEC plasma membrane proteins that may be useful as infertility markers or pharmacological targets for fertility regulation.

The Sertoli cell tight junction (TJ) is the key component of the blood-testis barrier, where it sequesters developing germ cells undergoing spermatogenesis within the seminiferous tubules. Hormonally regulated claudin-11 is a critical transmembrane protein involved in barrier function and its murine knockout results in infertility. We aimed to assess quantitatively the significance of the contribution of claudin-11 to TJ function, in vitro, using siRNA-mediated gene silencing. We also conducted an analysis of the contribution of occludin, another intrinsic transmembrane protein of the TJ. Silencing of claudin-11 and/or occludin was conducted using siRNA in an immature rat Sertoli cell culture model. Transepithelial electrical resistance was used to assess quantitatively TJ function throughout the culture. Two days after siRNA treatment, cells were fixed for immunocytochemical localization of junction proteins or lyzed for RT-PCR assessment of mRNA expression. Silencing of claudin-11, occludin, or both resulted in significant decreases in TJ function of 55% (P < 0.01), 51% (P < 0.01), and 62% (P < 0.01), respectively. Data were concomitant with significant decreases in mRNA expression and marked reductions in the localization of targeted proteins to the Sertoli cell TJ. We provide quantitative evidence that claudin-11 contributes significantly (P < 0.01) to Sertoli cell TJ function in vitro. Interestingly, occludin, which is hormonally regulated but not implicated in infertility until late adulthood, is also a significant (P < 0.01) contributor to barrier function. Our data are consistent with in vivo studies that clearly demonstrate a role for these proteins in maintaining normal TJ barrier structure and function.

Communication between the testicular somatic (Sertoli, Leydig, peritubular myoid, macrophage) and germ cell types is essential for sperm production (spermatogenesis), but the communicating factors are poorly understood. We reasoned that identification of proteins in the testicular interstitial fluid (TIF) that bathes these cells could provide a new means to explore spermatogenic function. The aim of this study was to map the proteome of TIF from normal adult rats. Low-abundance proteins in TIF were enriched using ProteoMiner beads and identified by MALDI-MS/MS, recognizing 276 proteins. Comparison with proteomic and genomic databases showed these proteins originated from germ cells, somatic cells (Sertoli, peritubular myoid, Leydig), and blood plasma. In silico analysis revealed homologues of >80% TIF proteins in the human plasma proteome, suggesting ready exchange between these fluids. Only 36% of TIF proteins were common with seminiferous tubule fluid that transports mature spermatids to the epididymis, indicating these two fluids are quite different. This TIF proteome provides an important new resource for the study of intercellular communication in the testis.

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