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Male Reproductive Tract |
a Biology Department, Monmouth University, West Long Beach, New Jersey 07764
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, gene regulation, male reproductive tract, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition, microbes present a threat to the health of the epididymis and to sperm. Sexually transmitted pathogens, such as Chlamydia trachomatis and Neisseria gonorrhoeae, as well as Escherichia coli can cause a range of inflammatory conditions of the genital tract, including epididymitis and urinary tract infections [4, 5]. Microbial infections of the epididymis are the most common cause of epididymitis [6]. Infections of the epididymis and other male reproductive organs can cause obstructions of the reproductive tract, thus compromising movement of spermatozoa through the excurrent ductal system and contributing to decreased fertility [6]. Surprisingly, an understanding of possible mechanisms involved in antimicrobial protection of male reproductive organs is relatively limited.
In mammals, most epithelial tissues serve as major barriers for protection against microorganisms, in part by producing antimicrobial peptides that can kill or inhibit the growth of pathogens [7–9]. A family of cationic peptides, called defensins, are widely expressed antimicrobial molecules that play an essential role in the innate immune system of virtually all life forms, from insects and plants to crustaceans, amphibians, and mammals [10]. Defensins are a large, highly conserved multigene family [11]. Defensin genes encode peptides of approximately 12–50 amino acids in length that are rich in basic amino acid residues [12]. More than 80 different defensins have been characterized, and three subfamilies have been identified in mammals: 
-defensins, ß-defensins, and 
-defensins [12]. Human tissues widely express - and ß-defensins. In rodents and humans, -defensins are primarily expressed by neutrophils and Paneth cells in the small intestine, whereas ß-defensins are more widely expressed in epithelial tissues, including the linings of the trachea, salivary glands, kidney, skin, lung, small intestine, and liver [7, 10].
Some members of the defensin family are constitutively expressed, whereas the expression of other defensins is regulated by proinflammatory molecules, including cytokines and bacterial lipopolysaccharides (LPS) [9]. Defensins display broad-spectrum antimicrobial activity against Gram-positive bacteria (e.g., Staphylococcus aureus), Gram-negative bacteria (e.g., Pseudomonas aeruginosa, Klebsiella pneumoniae, and E. coli), and yeast (Candida albicans) [9].
Recent studies have provided evidence for expression of defensins in the male reproductive tract [13, 14]. An androgen-dependent gene with sequence homology to members of the ß-defensin family, EP2 is expressed in the epididymis of humans [15], chimpanzees [15, 16], and rats [14]. Another defensin family member, Bin1b, a gene with homology to bovine ß-defensins, has been cloned from the mouse epididymis [13]. Two defensins, ß-defensin (RBD)-1 and RBD-2, are primarily expressed in rat tissues [17]. RBD-1 mRNA has been detected in the rat testis; however, RBD-1 and RBD-2 mRNA expression in the male reproductive tract has not been extensively studied [17]. The goals of the present study were to determine if mRNA for RBD-1 and RBD-2 is expressed in the adult rat male reproductive tract and to examine the effects of bacterial LPS and androgens on the expression of RBD-1 and RBD-2 mRNA in the epididymis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult male Sprague-Dawley rats (weight, 450–600 g) were purchased from Charles River Laboratories (Stoneridge, NY). Animals were housed at Monmouth University under controlled light (12L:12D) and temperature with free access to food and water. All aspects of animal handling and surgery were conducted in accordance with appropriate animal welfare criteria established by the National Institutes of Health Public Health Service Policy on Humane Care and Use of Laboratory Animals (1996).
Surgical Manipulations
For LPS studies, rats were anesthetized with halothane and surgeries carried out through a midline laparotomy. The testis and epididymis were exposed through the incision site, and 400-µg amounts of LPS from E. coli (serotype 055:B5; Sigma-Aldrich Chemical Company, St. Louis, MO) or P. aeruginosa (serotype 10; Sigma-Aldrich) dissolved in sterile, distilled, deionized water were administered unilaterally to each region of the epididymis through two intraparenchymal injections (volume, 50 µl each) using a 30-gauge syringe. Studies were also carried out with 25- to 100-µg amounts of LPS. Rats were allowed to recover and were then killed at 1, 4, or 24 h after treatment to harvest tissues for RNA isolations. Alternating-side injections (left vs. right) were used on different animals from each treatment group. Normal, untreated rats and sham-operated, vehicle-injected (sterile, distilled, deionized water) animals served as controls.
For androgen-regulation studies, animals were anesthetized with halothane and orchiectomy (orch) surgeries performed via a midline laparotomy as previously described [18]. Rats were divided into three groups: 1) bilaterally sham-orch controls simultaneously implanted s.c. with an empty 3.5-cm polydimethylsiloxane capsule (Silastic medical-grade tubing; Dow-Corning, Midland, MI); 2) bilateral orch rats implanted with an empty, 3.5-cm capsule; and 3) bilateral orch rats implanted with a 3.5-cm capsule filled with testosterone (Sigma-Aldrich). Capsules were prepared and presoaked for 48 h in PBS (pH 7.3) containing 4% (w/v) BSA according to the procedure of Berndtson et al. [19] as described previously [18]. Following orch, the epididymis was returned to the scrotum and the abdominal muscle and skin sutured closed. Animals were killed with carbon dioxide at 1, 5, and 15 days after surgery, organs trimmed free of fat, and epididymides dissected into four regions (initial segment, caput, corpus, and cauda). Tissues were immediately frozen in liquid nitrogen and stored at -70°C before RNA isolation.
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction Analysis of mRNA Expression
Total RNA was isolated from frozen tissues using TRIReagent according to the manufacturer's instructions (Molecular Research Center, Inc., Cincinnati, OH). Rat defensin primers for reverse transcription-polymerase chain reaction (RT-PCR) analysis were designed from published cDNA sequences as follows: RBD-1 gene [17] (GenBank acc. no. AF068860), 5'-GACCCTGACTTCACCGACAT-3' (forward) and 5'-CCTGCAACAGTTGGGCTTAT-3' (reverse); and RBD-2 gene (GenBank acc. no. AF068861), 5'-ATTTCTCCTGGTGCTGCTGT-3' (forward) and 5'-TCCACAAGTGCCAATCTGTC-3' (reverse). Primers amplify a 225-base pair (bp) fragment of the RBD-1 gene and a 132-bp fragment of the RBD-2 gene. For initial experiments, RBD-1 and RBD-2 primers were generously provided by Dr. Paul McCray, Jr. (University of Iowa College of Medicine, Iowa City, IA), and later purchased from MWG-Biotech, Inc. (High Point, NC).
RBD-1 and RBD-2 were coamplified by relative RT-PCR with either mouse 18S rRNA primers and competimers (Ambion, Inc., Austin, TX) or mouse ß-actin (Stratagene, La Jolla, CA) primers as internal controls, which amplified products of 489 and 514 bp, respectively. Optimal PCR cycle number for each primer set was determined, and primer and competimer concentrations were optimized (2:8 [v/v] ratio) to amplify control PCR products in the same linear range as defensin PCR products.
One microgram of total RNA was reverse transcribed and amplified by the one-step Access RT-PCR procedure (Promega Corp., Madison, WI) in a 50-µl reaction containing 50 pmol of each RBD-1 or RBD-2 primer and either 25 pmol of each 18S primer or 12.5 pmol of each ß-actin primer in the presence of avian myeloblastosis virus reverse transcriptase and Tfl DNA polymerase. The RT was carried out at 48°C for 45 min, and 40 cycles of PCR were performed with a denaturing step at 94°C for 30 sec, an annealing step at 60°C for 1 min, an elongation step at 68°C for 2 min, and a final extension at 68°C for 7 min. Equal aliquots of PCR products were electrophoresed through 2% agarose gels and stained with ethidium bromide. The RT-PCR controls routinely run included a single-primer-pair positive-control amplification, no RNA template (negative control), RNase-treated negative control, and a no-reverse-transcriptase negative control.
Defensin PCR products from initial experiments were verified first by direct sequencing (MWG-Biotech) and in subsequent experiments by Southern blot analysis using digoxigenin-labeled RBD-1 (GenBank acc. no. AF068860) and RBD-2 (GenBank acc. no. AF068861) cDNA probes [17]. Defensin cDNAs cloned into pBluescript II phagemids were provided by Dr. Paul McCray, Jr. XL1-Blue strain E. coli (Stratagene) were transformed with defensin cDNA constructs and plasmids isolated and purified by the QIAFilter Maxi protocol (Qiagen, Inc., Valencia, CA). BamHI and HindIII double-digests were used to liberate fragments of 122 and 134 bp for RBD-1 and RBD-2, respectively. Inserts were agarose gel-purified using Amicon Ultrafree DA spin tubes (Millipore Corp., Bedford, MA), random prime labeled with digoxigenin-dUTP using a DIG High Prime DNA Labeling system, and hybridized to Southern blots of RT-PCR products according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN). Probe hybridized to defensin PCR products was immunodetected with a 1:10 000 (v/v) dilution of sheep anti-digoxigenin-alkaline phosphatase Fab-fragments followed by incubation with the chemiluminescence substrate CSPD and exposure to x-ray film (Kodak BioMax ML; Eastman Kodak Co., Rochester, NY).
Serum Testosterone Assays
Blood was collected from the inferior vena cava at the time that animals were killed for tissue collection and clotted overnight at 4°C. Erythrocytes were pelleted by centrifugation at 13 000 x g for 5 min at 4°C and sera stored at -70°C. Chemiluminescence immunoassays of serum testosterone were carried out at the University of Virginia Health System General Clinical Research Center Core Laboratory (Charlottesville, VA) using a Bayer ACS 180 system (Block Scientific, Inc., Englewood, NJ). Inter- and intraassay coefficients of variations were 6.82% and 8.68%, respectively.
Quantitation of Results and Statistical Analysis
The RT-PCR gel images were digitized using a flatbed scanner and densitometric quantitation of results carried out with ONE-DScan software (Scanalytics Inc., Fairfax, VA). Integrated peak areas for RBD-1 and RBD-2 PCR products were normalized to integrated peak areas for either 18S rRNA or ß-actin PCR products to control for variations in intersample amplification and gel loading. Data were analyzed by one-way ANOVA, and results were considered to be significantly different at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RBD-1 mRNA was expressed in all male reproductive tissues examined by relative RT-PCR analysis (Fig. 1). Quantitation of RT-PCR results revealed relatively equal expression of RBD-1 mRNA in all segments of the epididymis and similar expression in ductus deferens (Fig. 2). Expression of RBD-1 mRNA in the epididymis was 
1.5-fold higher (P < 0.05) than expression in the kidney, which was used as a positive-control tissue (Figs. 1 and 2). Expression of RBD-1 mRNA in the testis was 2-fold lower than expression in the epididymis, and statistically similar levels of expression (P < 0.05) were detected in seminal vesicles, prostate, and penis (Fig. 2). Expression of RBD-2 mRNA was primarily restricted to the penis (Fig. 1B), which showed an average 200-fold higher level of expression (±59-fold [SEM]) compared to the lung, which was included as a positive-control tissue.
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LPS Do Not Induce Expression of RBD-1 and RBD-2 mRNA in the Epididymis
Effects of inflammatory challenge on defensin mRNA expression in the testis and epididymis were examined in adult rats administered unilateral subcapsular or intraparenchymal injections of LPS. Administration of 400 µg of LPS from E. coli or P. aeruginosa for 24 h had no effect on expression of RBD-1 mRNA in the testis (data not shown), initial segment, or caput epididymidis (Fig. 3). Expression of RBD-1 mRNA in the corpus and cauda epididymidis was unaffected by LPS from P. aeruginosa or E. coli (Fig. 4). No statistically significant differences in expression of RBD-1 mRNA in LPS-treated epididymides were observed compared with controls (P < 0.05) (Figs. 3B and 4B). Studies carried out for 4 h did not result in changes in RBD-1 mRNA expression in the testis or epididymis (data not shown). Similarly, preliminary studies with 25 and 100 µg of LPS had no effect on RBD-1 and RBD-2 mRNA expression in the testis and epididymis (data not shown). No significant expression of RBD-2 mRNA was detected in the testis or in any regions of the epididymis following LPS treatment (data not shown).
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Androgen Regulation of RBD-1 mRNA Expression in the Epididymis
Androgen-regulation studies were carried out to determine whether circulating androgens are sufficient to maintain expression of RBD-1 mRNA in the epididymis. Serum testosterone concentrations (mean ± SEM) in adult rats after 1, 5, and 15 days of sham orch, bilateral orch, and orch with testosterone replacement (orch + T) were as follows: sham orch, 0.99 ± 0.12 ng/ml; 1-day orch, 0.27 ± 0.11 ng/ml; 5-day orch, 0.22 ± 0.10 ng/ml; 15-day orch, 0.17 ± 0.08 ng/ml; 1-day orch + T, 2.19 ± 0.20 ng/ml; 5-day orch + T, 1.77 ± 0.16 ng/ml; and 15-day orch + T, 1.41 ± 0.09 ng/ml.
Expression of RBD-1 mRNA in the initial segment and caput was unchanged compared with sham-operated controls following 1-day orch but showed androgen-sensitive reductions in expression after 5 and 15 days of orch (Fig. 5). Quantitation revealed that RBD-1 mRNA expression in the initial segment decreased by approximately 40% and 90% after 5- and 15-day orch, respectively, whereas expression in caput epididymidis decreased by approximately 50% after 5- and 15-day orch, respectively (Fig. 5). Circulating androgens maintained RBD-1 mRNA expression in both initial segment and caput of orch + T rats (Fig. 5).
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Expression of RBD-1 mRNA expression in the corpus and cauda was not significantly affected by orch or orch + T (Fig. 6). Levels of RBD-1 mRNA expression in the kidney, examined as a control tissue, appeared unchanged by androgen treatment at all time points, demonstrating that androgen effects on RBD-1 mRNA expression are epididymis-specific (Fig. 7). RBD-2 mRNA was not detected in any segments of the epididymis following orch or orch + T (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we have demonstrated that RBD-1 mRNA is highly expressed in the adult rat male reproductive tract. RBD-1 mRNA is equally expressed in all segments of the rat epididymis at levels higher than in the kidney, which has been previously reported to be a rich source of RBD-1 [17]. Unlike the widespread expression pattern of RBD-1 mRNA in the male reproductive tract, expression of RBD-2 mRNA, though also found in the lung, appears to be restricted primarily to the penis. The high expression of RBD-2 mRNA in the penis is an interesting finding that warrants further investigation.
Other investigators have demonstrated that LPS can stimulate defensin expression. Heat-killed Pseudomonas and Pseudomonas LPS can induce expression of RBD-1 (also called tracheal antimicrobial peptide) mRNA in cultured bovine tracheal epithelial cells [21]. Similarly, mRNA for human ß-defensin (hBD)-2, which is expressed in respiratory tissues, is also induced by mucoid forms of P. aeruginosa in bronchial and tracheal cells [22]. Results from our LPS experiments suggest that RBD-1 mRNA expression in the epididymis is not influenced by bacterial endotoxins. Perhaps expression of RBD-1 in the epididymis may be sufficiently high to provide adequate antimicrobial protection without LPS induction, although intraluminal deliver of native microbes might influence RBD-1 mRNA expression in the epididymis.
Human BD-1 is constitutively expressed in epithelia, including kidney, and prostate and is highly expressed in the vaginal epithelium [23]. Unregulated expression has also been reported for the mouse homologue, mBD-1 [24], so it appears that RBD-1 in the testis and epididymis may be unaffected by bacterial endotoxins. Alternatively, in addition to androgens, other factors may affect RBD-1 mRNA expression. For instance, Fehlbaum et al. [25] demonstrated that L-isoleucine can induce ß-defensin production in cultured kidney cells. Epididymal luminal fluid contains a number of different amino acids, including isoleucine [26]. Human BD-2, which is expressed in skin, lung, intestine, and other tissues, can be induced by bacterial infection and cytokines such as tumor necrosis factor and interleukin-1ß through transcriptional activation by nuclear factor-
B [27], but we did not detect RBD-2 in the epididymis before or after LPS treatment.
A variety of different genes in the epididymis are expressed in an androgen-dependent manner [28–30]. We examined if RBD-1 mRNA expression is androgen-regulated. Androgen regulation of RBD-1 mRNA is epididymal region-specific. Expression of RBD-1 mRNA was androgen sensitive in the initial segment and caput epididymidis but not in the corpus and cauda epididymidis. The relatively slow decrease in RBD-1 mRNA expression in the initial segment and caput following androgen withdrawal after 5 and 15 days suggests that RBD-1 is androgen sensitive, but this result may be caused by indirect actions of androgens on metabolism of the epididymal epithelium rather than by direct effects of androgens on transcription of RBD-1. Genomic DNA sequences for RBD-1 are not available for promoter analysis of putative androgen-response elements.
The length of the male urogenital tract and urine flow are frequently cited as deterrents against microbial infections; however, we hypothesize that these features may be insufficient as sole mechanisms for providing antimicrobial protection of male reproductive organs and spermatozoa. Until recently, the literature contained relatively few reports of antimicrobial mechanisms in male reproductive organs. Seminalplasmin, a protein in bull semen that is effective against growth of Gram-positive and Gram-negative bacteria as well as yeast such as Saccharomyces and Candida, was one of the first antimicrobial molecules isolated from a tissue of the male reproductive tract [31]. During our studies over the last year, other investigators have provided strong evidence for antimicrobial protection as an important function of the epididymis. However, none of the antimicrobial genes studied to date are the same as the RBD-1 and RBD-2 examined in our study. In particular, a gene called EP2, originally identified as HE2 in humans, has been shown to be an epididymis-specific, androgen-regulated gene with sequence homology to ß-defensins [14, 15, 32]. Unlike RBD-1, which is equally expressed throughout the epididymis, rat EP2 is primarily expressed in caput epididymidis [14].
Similarly, Li et al. [13] identified a rat epididymis-specific defensin, called Bin1b, that is expressed in the caput and is androgen-regulated [13]. In vitro experiments with primary cultures of caput tissue demonstrated that Bin1b mRNA expression is up-regulated by E. coli and that secreted Bin1b shows an ability to suppress microbial growth [13]. Another antimicrobial peptide, sperm oocyte-binding 3 (SOB3), is localized within the acrosome and on the neck region of human spermatozoa. Originally named because of its role in secondary binding to the zona pellucida, SOB3 shares sequence similarity with two antimicrobial proteins [33]. In fact, SOB3 is 98% identical to preproprotein FALL-39 and 100% identical to human cationic antimicrobial peptide (hCAP-18), both of which are members of the cathelicidin family of antimicrobial molecules and defensin-like relatives [33]. In the human epididymis, SOB3 mRNA expression is restricted to the corpus and cauda [33]. The SOB3/hCAP-18 protein has been detected in semen at high levels and associated with spermatozoa, suggesting a role in innate immunity of the male reproductive system [34]. It has also been suggested that SOB3/hCAP-18 coating of spermatozoa may be designed to prevent microbes from adhering to sperm as they move into the female tract [35].
Lastly, a role for defensins in antimicrobial protection of the male reproductive tract was recently demonstrated by Morrison et al. [36]. Knockout mice deficient in mBD-1, the murine homologue of RBD-1, showed a significantly higher incidence of Staphylococcus infections of the bladder compared to wild-type mice and heterozygotes, suggesting a role for mBD-1 in protection against urinary tract infections [36].
In summary, the results of the present study and of the work of other investigators provide strong evidence supporting a role for the epididymis in antimicrobial protection. The findings presented in this paper support our working hypothesis that RBD-1 may be critically important for antimicrobial protection of the epididymis and other male reproductive organs. Additional studies are underway to further examine the functions of RBD-1 in the epididymis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Michael A. Palladino, Biology Department, Monmouth University, 400 Cedar Avenue, West Long Branch, NJ 07764. FAX: 732 263 5243; mpalladi{at}monmouth.edu
Received: 2 July 2002.
First decision: 24 July 2002.
Accepted: 27 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-glutamyl transpeptidase messenger ribonucleic acid transcripts in the adult rat epididymis is differentially regulated by androgens and testicular factors in a region-specific manner. Endocrinology 1994 135:1146-1156[Abstract], and IL-1ß, but not IL-6, induce human ß-defensin-2 in respiratory epithelia. Am J Respir Cell Mol Biol 2000 22:714-721B-mediated transcriptional regulation of human ß-defensin-2 gene following lipopolysaccharide stimulation. J Leukoc Biol 2002 71:154-162, new members of an epididymis-specific family of androgen-regulated proteins in the human. Endocrinology 2000 141:1245-1253This article has been cited by other articles:
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  M.A. Palladino, T.A. Johnson, R. Gupta, J.L. Chapman, and P. Ojha Members of the Toll-Like Receptor Family of Innate Immunity Pattern-Recognition Receptors Are Abundant in the Male Rat Reproductive Tract Biol Reprod, June 1, 2007; 76(6): 958 - 964. [Abstract] [Full Text] [PDF]
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 Y. Sang, M. T. Ortega, F. Blecha, O. Prakash, and T. Melgarejo Molecular Cloning and Characterization of Three {beta}-Defensins from Canine Testes Infect. Immun., May 1, 2005; 73(5): 2611 - 2620. [Abstract] [Full Text] [PDF]
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M. C. W. Avellar, L. Honda, K. G. Hamil, S. Yenugu, G. Grossman, P. Petrusz, F. S. French, and S. H. Hall Differential Expression and Antibacterial Activity of Epididymis Protein 2 Isoforms in the Male Reproductive Tract of Human and Rhesus Monkey (Macaca mulatta) Biol Reprod, November 1, 2004; 71(5): 1453 - 1460. [Abstract] [Full Text] [PDF]
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S. Yenugu, R. T. Richardson, P. Sivashanmugam, Z. Wang, M. G. O'Rand, F. S. French, and S. H. Hall Antimicrobial Activity of Human EPPIN, an Androgen-Regulated, Sperm-Bound Protein with a Whey Acidic Protein Motif Biol Reprod, November 1, 2004; 71(5): 1484 - 1490. [Abstract] [Full Text] [PDF]
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 S. Yenugu, K. G. Hamil, Y. Radhakrishnan, F. S. French, and S. H. Hall The Androgen-Regulated Epididymal Sperm-Binding Protein, Human {beta}-Defensin 118 (DEFB118) (Formerly ESC42), Is an Antimicrobial {beta}-Defensin Endocrinology, July 1, 2004; 145(7): 3165 - 3173. [Abstract] [Full Text] [PDF]
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M.A. Palladino, J.D. Powell, N. Korah, and L. Hermo Expression and Localization of Hypoxia-Inducible Factor-1 Subunits in the Adult Rat Epididymis Biol Reprod, April 1, 2004; 70(4): 1121 - 1130. [Abstract] [Full Text] [PDF]
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A. Zanich, J. C. Pascall, and R. Jones Secreted Epididymal Glycoprotein 2D6 That Binds to the Sperm's Plasma Membrane Is a Member of the {beta}-Defensin Superfamily of Pore-Forming Glycopeptides Biol Reprod, December 1, 2003; 69(6): 1831 - 1842. [Abstract] [Full Text] [PDF]
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