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research-article |
Department of Biology, Monmouth University, West Long Branch, New Jersey 07764
ABSTRACT
Protecting developing and maturing spermatozoa and reproductive tissues from microbial damage is an emerging aspect of research in reproductive physiology. Bacterial, viral, and yeast infections of the testis and epididymis can hinder maturation and movement of spermatozoa, resulting in impaired fertility. Toll-like receptors (TLRs) are a broad family of innate immunity receptors that play critical roles in detecting and responding to invading pathogens. Objectives of this study were to determine if organs of the rat male reproductive tract express mRNAs for members of the TLR family, to characterize expression patterns for TLRs in different regions of the epididymis, and to determine if TLR adaptor and target proteins are present in the male reproductive tract. Messenger RNA for Tlr1–Tlr9 was abundantly expressed in testis, epididymis, and vas deferens, as determined by RT-PCR, while Tlr10 and Tlr11 were less abundantly expressed. Tlr mRNA expression showed no region-specific patterns in the epididymis. Immunoblot analysis revealed relatively equal levels of protein for TLRs 1, 2, 4, and 6 in testis, all regions of the epididymis and vas deferens, and lower levels of TLRs 3, 5, and 9–11. TLR7 was primarily detected in the testis. The TLR adapter proteins, myeloid differentiation primary response gene 88 and TLR adaptor molecule 1, as well as v-rel reticuloendotheliosis viral oncogene homolog and NFKBIA, were prominent in testis, epididymis, and vas deferens. The abundant expression of a majority of TLR family members together with expression of TLR adaptors and activation targets provides strong evidence that TLRs play important roles in innate immunity of the male reproductive tract.
epididymis, immunology, male reproductive tract, testis
Toll-like receptors (TLRs) are a large family of highly conserved proteins that are essential pathogen-specific recognition sensors of the innate immune system, which are also involved in induction of adaptive immune responses [1–3]. To date, 11 distinct TLRs have been identified in humans. These transmembrane proteins are expressed by both immune cells, such as B and T cells, mast cells, dendritic cells and macrophages, and nonimmune cell types, particularly epithelia of the lung, kidney, heart, liver, spleen, small intestine, and the female reproductive tract [4–6].
TLRs can recognize Gram-positive and Gram-negative bacteria, viruses, fungi, and parasites, and are activated by a variety of ligands derived from microorganisms, including lipopolysaccharides, bacterial flagellin, lipoproteins, peptidoglycans, CpG bacterial DNA, as well as single-stranded and double-stranded viral RNA [1, 3, 7–9]. Pathogen recognition by TLRs elicits highly conserved intracellular signaling responses that ultimately result in activation of v-rel reticuloendotheliosis viral oncogene homolog (RELA, previously known as nuclear factor kappa B, NF-
B), a key transcriptional regulator of inflammation. Activation of RELA in turn stimulates transcription of a variety of proinflammatory cytokines, including tumor necrosis factor (TNF) 
, interleukins (IL) 6, 1ß, and 12 [10], and antimicrobial peptides, such as defensins [11, 12] that constitute the host response to pathogens, which results in pathogen clearance under normal circumstances.
Extracellular, leucine-rich repeat regions of TLRs are largely responsible for pathogen recognition and cytoplasmic Toll/interleukin 1 receptor (TIR) domains are involved in intracellular signal transduction via two key intracellular adaptor proteins, myeloid differentiation primary response gene (MYD) 88 and TLR adaptor molecule (TICAM) 1, previously known as TIR domain-containing adaptor protein-inducing interferon ß or TRIF [7, 13, 14]. These adaptors are essential for mediating TLR signaling, leading to activation of RELA. MYD88 is a central adaptor that appears to be coupled to all TLRs except TLR3, whereas TICAM1 is primarily involved in signaling of TLR3 and MYD88-independent signaling of TLR4 [7]. An understanding of the roles TLRs play in innate immunity has rapidly increased in recent years, as has insight about the involvement of TLRs in human diseases and infectious disease susceptibility [15–19].
Antimicrobial protection of male reproductive organs is an essential aspect of reproductive physiology [20]. Spermatozoa must be protected from microbes during their formation in the testis, and during subsequent maturation, transit, and storage in the epididymis [21]. Because of the role of the epididymis in sperm maturation and storage, it is also critical that the epithelium of the male reproductive tract be protected from a variety of pathogens that can invade the tract, including pathogens that cause sexually transmitted diseases [20, 22, 23]. For example, Neisseria gonorrhoeae and Chlamydia trachomatis infections affect the epididymis, leaving males less fertile or, in severe cases, infertile [24, 25]. Chlamydia and Escherichia coli infections can cause inflammation of the testis and both acute and chronic epididymiditis, leading to blockage of epididymal tubules [24–29]. Bacterial lipopolysaccharides induce alterations in Leydig cell steroidogenesis [30]. A number of viruses also infect the male reproductive tract [23].
In recent years, research on antimicrobial protection of spermatozoa and male reproductive organs has become a rapidly emerging area of male reproductive physiology. A number of known or putative antimicrobial defense systems are present in the testis and epididymis—most notably defensins [31–41], but also several others, including eppin [42], human cationic antimicrobial protein 18 [43, 44], lipopolysaccharide-binding protein [45], and sperm antigen 11 [46, 47]. In addition to their roles in innate immunity of male reproductive organs, members of the ß-defensin family [38, 40] and the protein, eppin [48], have been shown to be sperm-associated, and may play roles in antimicrobial protection of sperm in the female reproductive tract.
Because of the emerging importance of TLRs in innate immunity, we hypothesize that TLRs perform fundamental roles in pathogen recognition in male reproductive organs. The objectives of this study were to determine if organs of the rat male reproductive tract express mRNA and protein for members of the TLR family, to characterize expression patterns for TLRs in different regions of the epididymis, and to determine if TLR adaptor and target proteins are present in the testis, epididymis, and vas deferens.
Adult male Sprague-Dawley rats (475–600 g) were purchased from Charles River Laboratories (Stoneridge, NY). Animals were housed one per cage at Monmouth University under controlled light (12L:12D) and temperature, with free access to food and water. Animal research protocols were reviewed and approved by the Monmouth University Institutional Animal Care and Use Committee, and animal studies were conducted according to the Guide for Care and Use of Laboratory Animals (copyright 1996, National Academy of Science).
Antibody vendors and the working concentrations of each antibody used for immunoblotting experiments were as follows: antibodies to TLR1 (IMG-5012; 1 µg/ml), TLR2 (IMG-545; 1:1000 dilution), TLR5 (IMG-580; 1:750 dilution), TLR6 (IMG-527; 1 µg/ml), TLR7 (IMG-581; 1 µg/ml), TLR9 (IMG-305A; 1 µg/ml), TLR11 (IMG-5034; 0.5 µg/ml), and MYD88 (IMG-178; 0.5 µg/ml) were obtained from Imgenex (San Diego, CA). Antibodies for TLR3 (sc-28999; 1:500 dilution) and TLR8 (sc-13213; 1:1000 dilution) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to TLR4 (24–9004; 1:3000 dilution) and TLR7 (14–9079, 1.5 µg/ml) were purchased from eBioscience (San Diego, CA). TLR10 antibody was from ImmunoKontact (211-MG-1TLR10, 3 µg/ml). Antibody for RELA (KAP-TF112; 0.5 µg/ml) was obtained from Stressgen Biotechnologies Corporation (San Diego, CA), and antibody for NFKBIA (9242; 1:1000 dilution) was obtained from Cell Signaling Technology (Danvers, MA). Antibody for TICAM1 (ab13810; 1:1000 dilution) was purchased from Abcam, Inc. (Cambridge, MA). Actin monoclonal antibody (A2066; 1:2000) was acquired from Sigma-Aldrich, Inc. (St. Louis, MO).
Mouse RAW 264.7 + LPS/IFN-
Abelson-transformed macrophage whole cell lysate (sc-24767), SW480 human colorectal adenocarcinoma whole cell lysate (sc-2219), and Daudi cell (Burkitt lymphoma) whole cell lysate (sc-2415) were purchased from Santa Cruz Biotechnology. Mouse heart tissue lysate (40102) and Ramos cell lysate (40175) was from Imgenex.
RNA Isolation and RT-PCR Analysis of TLR mRNA Expression
Rats were killed with carbon dioxide and tissues excised, trimmed free of fat, and then immersed in liquid nitrogen prior to storage at –80°C. Total RNA was isolated from frozen tissues using TRIReagent (Molecular Research Center Inc., Cincinnati, Ohio), according to the manufacturer's instructions. Primers were designed using Primer3 software (URL: http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) [49] and synthesized by MWG-Biotech (High Point, NC). Sequences for PCR primer pairs and expected sizes of PCR products are shown in Table 1.
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TLR PCR products were coamplified by relative RT-PCR analysis with mouse ß-actin (Stratagene, La Jolla, CA) primers as internal controls. Primer concentrations were optimized to amplify ß-actin PCR products of 514 bp in the same linear range as TLR PCR products by carrying out independent amplifications with different concentrations of actin or TLR primers and uniform amounts (1 µg) of RNA from each reproductive organ. PCR products from primer amplification titration reactions were then separated by agarose gel electrophoresis and product amounts quantitated using Quantity One software (v. 4.4; Bio-Rad Laboratories, Inc., Hercules, CA) to produce standard curves indicating linear ranges of each primer concentration. Amplifications were carried out in a Bio-Rad MyCycler thermal cycler. Total RNA (1 µg) was treated with deoxyribonuclease I (Invitrogen, Carlsbad, CA), then reverse transcribed and amplified by the SuperScriptIII one-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen) in a 50-µl reaction volume, containing 50 pmol of each forward and reverse TLR primer and 10 pmol of each ß-actin primer. RT-PCR amplification was carried out at 55°C for 30 min, followed by denaturation at 94°C for 2 minutes and 40 cycles of PCR (94°C for 15 sec, 55°C for 30 sec, 68°C for 2 min), with a final extension at 68°C for 5 min. Aliquots (8 µl) of PCR products were electrophoresed through 2% agarose gels in 1x sodium borate (SB) buffer at 300V (Faster Better Media, LLC, Baltimore, MD) and gels stained with ethidium bromide. Gel images were captured with a ChemiDoc gel documentation system (Bio-Rad). RT-PCR controls included a single primer pair positive control amplification, and no reverse transcriptase negative controls. All RT-PCR products were verified by cloning amplicons into pGEM-T Easy plasmid vectors (Promega Corp.) followed by cycle sequencing and analysis using a LI-COR 4300L DNA sequencer (LI-COR Biosciences, Lincoln, NE).
Protein Isolation and Immunoblot Analysis
Frozen tissues were homogenized on ice in three volumes of 10 mM Tris-HCl, 1.5 mM MgCl2, and 10 mM KCl containing 1 mM dithiothreitol (DTT), 1 mM of the phosphatase inhibitor, Na3VO4, and protease inhibitor cocktail (P8340; Sigma-Aldrich) using a glass dounce homogenizer. The homogenate was centrifuged for 5 min at 5000 x g at 4°C, and cytoplasmic protein extracts stored at –80°C. Protein concentrations were determined by the Bradford assay (Bio-Rad) using BSA as the standard.
Equal aliquots of proteins were separated by denaturing SDS-PAGE through 10% polyacrylamide gels (Cambrex Bio Science, Rockland, ME), according to the method of Laemmli. Commercially available whole cell lysates were included as positive controls for antibody specificity, along with rat protein extracts from lung, spleen, kidney, and small intestine. Proteins were transferred to Trans-Blot nitrocellulose (Bio-Rad) by electroblotting, and blots were stained with 0.005% Ponceau S in 1% acetic acid to confirm protein transfer. Blots were blocked in 1x Western wash (50 mM Tris, pH 7.6, 30 mM NaCl, 0.001% Tween 20) containing 5% nonfat dry milk for 30 min at room temperature with gentle agitation, then incubated with primary antibodies diluted in 1x Western wash and 5% nonfat dry milk overnight at 4°C with gentle agitation.
Blots were washed three times in 1x Western wash, then incubated with a 1:20 000 dilution of horseradish peroxidase-conjugated secondary antibodies in 1x Western wash, 5 % nonfat dry milk, for 1 h at room temperature with gentle agitation. Blots were washed in three changes of 1x Western wash, then developed by enhanced chemiluminescence using Pierce SuperSignal West Pico or Pierce SuperSignal West Femto substrate (Pierce, Rockford, IL) and exposed to x-ray film (BioMax ML; Kodak, Rochester, NY).
Blots were stripped of bound antibody in Restore Western blot stripping buffer (Pierce) for 15 min at 37°C, washed in 1x Western wash for 5 min, and then blocked prior to subsequent reprobing. Actin was detected on immunoblots as a loading control for protein quantitation using a 1:2 000 dilution of anti-actin antibody (A2066; Sigma-Aldrich). Control experiments for antibody specificity were carried out by incubating blots with preimmune sera, followed by incubation with secondary antibodies and enhanced chemiluminescence.
Quantitation of Results and Statistical Analysis
RT-PCR gel images were captured using a ChemiDoc gel documentation system and quantitation of results carried out with Quantity One software (v. 4.4; Bio-Rad). Integrated peak areas for TLR PCR products were normalized to integrated peak areas for ß-actin PCR products. RT-PCR data were analyzed by one-way ANOVA, and results were considered significantly different at P < 0.05.
Expression of Tlr mRNAs in the Male Rat Reproductive Tract
To determine if Tlr mRNAs are expressed in the rat testis, epididymis, and vas deferens, total RNA was isolated from these organs and analyzed by RT-PCR using primers shown in Table 1. Region-specific gene expression in the epididymis is important for regulating regional functions of the epididymis [50, 51]; therefore, different segments of the epididymis were analyzed for Tlr mRNA expression. RNA was also analyzed from kidney, lung, and spleen as positive control tissues. As shown in Figure 1, mRNA for a majority of Tlr family members—in particular, Tlr1 through Tlr9—were detected and abundantly expressed in the testis, epididymis, and vas deferens. Compared to Tlr1 through Tlr9, lower levels of Tlr10 were expressed in the testis and epididymis (Fig. 1B). Tlr11 mRNA was faintly detected in testis and vas deferens (Fig. 1B).
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In the epididymis, very low levels of Tlr11 expression were detected, primarily in initial segment and caput epididymidis, but several experiments showed similarly faint expression in corpus and cauda epididymidis (Fig. 1B). None of the TLRs detected showed evidence of region-specific expression in the epididymis.
When PCR data were quantitated, no statistically significant differences were observed in mRNA expression profiles for individual Tlrs in testis, epididymis, or vas deferens (P < 0.05), indicating that the relative expression of each Tlr in these male reproductive organs is similar.
Immunoblot Analysis of TLR Proteins in the Testis, Epididymis, and Vas Deferens
To determine which TLR family members in the testis and epididymis detected by RT-PCR were actively translated, and thus likely to be involved in innate immunity of these organs, cytoplasmic protein extracts were subjected to immunoblot analysis using commercially available antibodies for TLRs. These experiments showed that TLRs 1, 2, 4, and 6 were most abundantly detected when blots were developed using SuperSignal West Pico substrate (Fig. 2). TLRs 3, 5, and 7–11 were not detected using SuperSignal West Pico substrate, but when the more sensitive substrate, SuperSignal West Femto, was used for enhanced chemiluminescence, these TLRs were detected in the testis and epididymis (Fig. 2).
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TLRs 3, 5, and 9 were detected in the testis, epididymis, and vas deferens. TLR7 was primarily detected in the testis and vas deferens, but does not appear to be a prominent protein in the epididymis. Three different commercially available antibodies were used in immunoblotting experiments for TLR8. These experiments demonstrated that TLR8 does not appear to be abundant in the male reproductive tract. A faint signal for TLR8 was detected in testis (Fig. 2). In the epididymis, a very faint and diffuse signal, perhaps indicative of degradation, was consistently detected in initial segment and caput epididymidis only after prolonged overexposure of blots (Fig. 2). This result may reflect regional differences in translation of TLR8 in the epididymis. A similar pattern was observed for TLR10.
TLR11 was detected in all regions of the epididymis, as well as the vas deferens, but TLR11 at the expected size of 
90 kDa was not detected in the testis (Fig. 2), although we consistently detected a protein at 110 kDa in the testis. The significance of this band, and whether it may be a testis-specific isoform of TLR11, is unclear. Clearly TLRs 7, 8, 10, and 11 are less abundant in the testis and epididymis than other TLRs.
TLR Adaptor Proteins MYD88 and TICAM1, and the Inflammatory Regulator RELA, Are Present in the Testis, Epididymis, and Vas Deferens
Once we had evaluated TLR expression patterns in the testis and epididymis, we were interested in determining if key adaptor proteins for TLRs are also present in these tissues. MYD88 appears to be an essential adaptor for all TLRs, except TLR3. The TICAM1 protein is important for signaling via TLR3 and MYD88-independent signaling of TLR4. Immunoblotting experiments showed that MYD88 and TICAM1 are present in testis and all regions of the epididymis, as well as the vas deferens (Fig. 3).
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To determine if the transcription factor, RELA, a primary downstream target of TLR signaling, is present in rat male reproductive organs, blots were probed for the NFKBIA inhibitory subunit of RELA, the RELA p50 (lower band) and p65 subunits (upper band; Fig. 3). Both NFKBIA and RELA were present in the testis, epididymis, and vas deferens. These results demonstrate that important adaptor proteins for TLRs and RELA are prevalent, supporting our hypothesis that TLRs are essential for innate immune responses in the male reproductive tract.
In recent years, the role of the epididymis in protecting both spermatozoa and the epididymal epithelium from microbes has emerged as a new area of research in reproductive physiology [20, 21, 52]. In particular, a number of antimicrobial peptides, such as defensins, have been detected in the testis and epididymis [34, 37, 38, 40, 41, 53, 54].
Bacterial, viral, and yeast infections of the male reproductive tract are known to contribute to impaired fertility. For example, E. coli and Chlamydia infections of the epididymis are a common cause of epididymitis and epididymal tubal blockage, which can compromise movement of spermatozoa through the excurrent ductal system and contribute to decreased fertility [25, 26, 29]. Infections of the male reproductive tract and concomitant production of reactive oxygen species (ROS), particularly by macrophages, is known to cause oxidative damage of spermatozoa [55]. Similarly, ROS production following lipopolysaccharide-induced inflammation of the testis is known to inhibit steroidogenesis of testicular Leydig cells [30, 56].
While defensins and other antimicrobial systems in the male reproductive tract have received significant attention from reproductive biologists, relatively little is known about other systems involved in innate immunity of these organs. The goal of this study was to characterize mRNA and protein expression patterns for TLRs in the male reproductive tract by examining the presence of TLRs in the testis, different regions of the epididymis, and the vas deferens.
A number of studies have reported the presence of TLR family members in the female reproductive tract of mice [57] and humans [4, 58–60]. However, unlike the male reproductive tract, female reproductive organs appear to primarily express Tlr1 through Tlr6. In humans, Tlr2 and Tlr4 appear to show differential expression patterns in the fallopian tube, endometrium, cervix, and ectocervix [4]. As reviewed by Wira et al. [61], it is becoming clear that TLRs are important for innate immunity of the female reproductive tract; yet, by comparison, little is known about the significance of TLRs in the male reproductive tract.
Nishimura and Naito [62] provided evidence that Tlr1 through Tlr10 are present in the human testis, as detected by real-time PCR, but these experiments were limited to including the testis among organs examined for tissue-specific studies of TLR mRNA expression. In addition, studies by Malm et al. [45] characterized the expression of lipopolysaccharide-binding protein (LBP) in the human epididymis and found LBP localized to the epididymal epithelium and spermatozoa. This protein binds lipopolysaccharides of Gram-negative bacteria, and is known to elicit host-defense responses through signaling with CD14 and TLR4.
This study presents the first comprehensive analysis of TLR expression in organs of the rat male reproductive tract, and demonstrates that TLR family members are abundantly expressed in the testis, epididymis, and vas deferens. Messenger RNA for Tlr1 through Tlr9 were highly expressed in the testis, all regions of the epididymis, and the vas deferens. Lower levels of Tlr10 were detected. Tlr11 does not appear to be abundantly expressed in male reproductive organs, as only faint levels of Tlr11 were detected. The relative abundance of Tlr10 and Tlr11 compared to Tlr1 through Tlr9 suggest that Tlr10 and Tlr11 are perhaps not as important as the other TLRs for innate immunity responses in the testis in epididymis. Alternatively, 10 and 11 may be inducible TLRs that are stimulated by the presence of pathogens.
The importance of TLR11, in detecting uropathogenic bacteria in the male urinary tract has been reported by Zhang and colleagues [63]. They demonstrated that Tlr11 mRNA is expressed in kidney and bladder and showed no expression in the testis and several other tissues, and that Tlr11 knockout mice are susceptible to kidney infections [63]. As a result of this work, TLR11 has been described as being a urogenital TLR. The Zhang data, along with our work, indicate that, while TLR11 may be important for detecting uropathogenic microbes in the bladder or kidney, it may not play significant roles in the testis and epididymis.
When TLR proteins were examined by immunoblotting, results generally confirmed RT-PCR studies. Because TLRs 1, 2, 4, and 6 could be detected with SuperSignal, whereas TLRs 3, 5, and 7–11 could only be detected with Femto substrate, these results suggest TLRs 1, 2, 4, and 6 are more abundant than other TLRs. We cannot rule out that these detection differences are due to differences in antibody affinity, but, for TLR7 and TLR8, similar results were observed when three different antibodies were used. Compared with mRNA expression, TLR8 protein levels were low, perhaps suggesting translational regulation of TLR 8. TLR8 also showed evidence for region-specific translational differences in the epididymis, because it was primarily detected in initial segment and caput epididymidis, and not distal regions of the duct.
TLRs are broadly distributed in immune cells, but less widely distributed in mucosa, so we were surprised by the wide range of TLR family members detected in the male reproductive tract [6]. With the possible exception of spleen, we are unaware of any organs that show higher levels of relative expression of a majority of TLR family members than the epididymis and testis. This observation underscores the importance of these receptors in innate immune responses of the male reproductive tract.
The abundance of TLRs in the male reproductive tract suggests that they provide broad-spectrum detection of bacteria and viruses that may enter the tract to protect both spermatozoa and the epithelial linings of reproductive organs. MYD88 and TICAM1 proteins were also abundantly expressed in the testis, all regions of the epididymis, and the vas deferens, as was RELA, and with the inhibitory subunit NFKBIA.
In summary, the widespread and abundant expression of a majority of TLR family members together with the expression of key adaptor proteins and the inflammatory regulator, RELA, provides strong evidence that TLRs are important for innate immunity of male reproductive organs. Additional studies are underway to determine which cell types express TLRs in the testis and epididymis, and we are also investigating TLR activation and signaling patterns following microbial challenge of these organs. Understanding the roles TLRs play in innate immunity responses of male reproductive organs will provide significant insight about mechanisms that male reproductive organs use to detect and respond to invading microbes.
ACKNOWLEDGMENTS
We thank Joe Cusick for help with preliminary experiments and Mary-Katherine Dughi and Michael Savarese for help with sequencing PCR products.
FOOTNOTES
1Supported by a 2005 American Society for Microbiology Summer Undergraduate Research Fellowship awarded to T.A.J., the Monmouth University Biology Department, and a LI-COR Genomics Education Matching Fund grant from LI-COR Biosciences. Presented in part at the 31st Annual Meeting of the American Society of Andrology, Chicago, Illinois, April 2006; the 106th General Meeting of the American Society for Microbiology, Orlando, Florida, May 2006; and the Fourth International Conference on the Epididymis, Châtel Guyon, France, December 2006. 
Correspondence: 2M.A. Palladino, Department of Biology, Monmouth University, 400 Cedar Avenue, West Long Branch, NJ 07764. FAX: 732 263 5243; e-mail: mpalladi{at}monmouth.edu
Received: 18 December 2006.
First decision: 16 January 2007.
Accepted: 12 February 2007.
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