Antimicrobial proteins and peptides constitute an important part of the innate immune system of multicellular organisms including plants, insects and mammals 1. The wide variety identified in recent years can be categorized into different structural classes including the amphipathic alpha helical peptides such as magainins in frog skin, cecropins in insects and other animals, cathelicidins in mammals and other vertebrates and the beta sheet proteins including the 2-β-strand bactenecins in mammals and the 3-β-strand defensins found in plants and animals http://www.bbcm.units.it/~tossi/pag1.htm. Among the best studied, the defensins are low molecular weight (<20 kDa) cationic peptides containing a well conserved 6 cysteine motif that forms 3 disulfide linkages. They are classified as α-, β-, and θ-defensins depending on their disulfide bond pairing and secondary structure. The α-defensins are primarily expressed in the paneth cells and polymorphonuclear leukocytes (PMNs) whereas β-defensins are expressed primarily in the epithelial cells 2. β-defensins exhibit remarkable antibacterial, antifungal and antiviral activities against a wide variety of microorganisms 3. Their mechanisms of action are well documented and involve the permeabilization of target cell membranes and interference with the basic metabolic processes 3.

Infections of the male reproductive tract are common and pose a threat to fertility. Epididymitis can lead to epididymal tubule damage and occlusion of the ductules by peritubular fibrosis resulting in transient or permanent sterility 4. The innate immune responses in the male reproductive tract to microbial attack are poorly understood in spite of their likely importance in preventing the establishment and spread of sexually transmitted diseases. Microbial protection of the male tract represents such a crucial function for individual and species survival that it alone may have driven the evolution of more than 30 beta-defensin genes in 5 separate chromosomal regions 5. This protective role is suggested by in vivo and in vitro demonstrations of their antimicrobial action. 6789101112, However, β-defensins in the male reproductive tract 13141516 are also recognized key effector molecules in reproductive processes of sperm maturation and capacitation 171819. In addition, β-defensins exhibit chemokine properties 20. They opsonize bacteria, inhibit the production of cortisol, act as inhibitors of protein kinase C 2 and thus may serve as an important bridge between the innate and adaptive immune systems.

In the rat, several recent studies conducted to characterize and analyze the expression of novel defensins were focused on rat genes orthologous to those on human chromosome 8p 2122232425. In a comprehensive mammalian evolutionary study, Patil et al. 16 reported genitourinary tract expression of 37 rat defensin genes in four clusters including those in this study 22. Here we report a more detailed analysis specifically focused on rat Defb21, Defb24, Defb27, Defb30 and Defb36 demonstrating developmental regulation for all five genes in both epididymis and testis. We also extend the observations of Patil et al. by analyzing androgen dependent expression and antibacterial function. Despite the demonstration in several other species of representative defensin antibacterial activity 72627, direct quantitative analysis of rat β-defensin bactericidal action has not been reported. In this study, we demonstrate potent dose and time dependent antibacterial activity of DEFB24 and DEFB30.

Five novel β-defensin genes, Defb21, Defb24, Defb27, Defb30 and Defb36 were discovered in the rat genome. Defb21, Defb24, Defb27 and Defb36 are located on chromosome 3q41 and represent orthologs of the cluster on human chromosome 20q (DEFB118, DEFB119, DEFB122 and DEFB123). Orthologs of DEFB120 and DEFB121 were not found in rat. Defb30 on chromosome 15p12 is similar to DEFB121, but is orthologous to DEFB135 (Figure 1A and 1B). Genomic versus cDNA sequence comparisons reveal that each gene, like most β-defensin genes contains two exons (see Additional Data File 1A-C). The first exon encodes the predicted signal peptide and the second encodes a C-terminal peptide containing the characteristic β-defensin 6-cysteine motif. A PROSITE scan revealed consensus post-translational modification sites including N-glycosylation, casein kinase II phosphorylation and protein kinase C phosphorylation sites (Figure 2). Other general characteristic features of these β-defensins are listed in Table 2.

In order to understand the environment in which these Defb transcripts are expressed, we investigated their presence in a series of different tissues. In the male reproductive tract, Defb21 was expressed in all the three regions of the epididymis as well as in testis, but was not detected in the seminal vesicle (Figure 3). Defb24 and Defb27 were expressed in all male reproductive tissues analyzed (Figure 3) and many other tissues as well including the female reproductive tract (Figure 4). Expression of Defb30 was the most restricted, detected only in the epididymis (Figures 3 and 4). Defb36 expression was also restricted, detected in distal epididymis, it appeared highly expressed in testis (Figure 3) and it was also found in spleen (Figure 4).

The male reproductive tract is dependent on testosterone for normal development and mature function 28. To investigate whether androgen regulates expression of Defb transcripts identified in this study, age-dependent expression was analyzed in 10 to 60 day old rats. Epididymis expression of the β-defensins was variable during early development. Defb27 was not expressed fully until 40–50 days of age while Defb21 and Defb30 reached full expression between 20–30 days of age and Defb36 between 10–20 days (Figure 5). In testis, however, Defb21 expression was not observed until late puberty indicating a relationship to spermatogenesis (Figure 6). By contrast, Defb24, Defb27 and Defb36 were expressed in testis throughout this age range consistent with regulation by testosterone as well as other factors. To determine the role of testosterone in the adult epididymis, the effects of androgen ablation and replacement were investigated. Androgen ablation by castration resulted in down regulation of Defb expression (Figure 7). Testosterone supplementation maintained the expression of all. This result suggests testosterone involvement in regulating these defensin genes in the epididymis of adult rat.

Although broad spectrum antimicrobial activity of defensins and defensin-like peptides expressed in human and primate male reproductive tracts was reported previously 810, direct demonstration of rat β-defensin antibacterial activity has not been described. To determine if representatives of this group of rat defensins possess antibacterial capacity, their capacity to kill E. coli was analyzed. Both recombinant DEFB24 and DEFB30 proteins exhibited potent dose and time-dependent antibacterial activity suggesting that these rat defensins too have a role in male reproductive tract immunity (Figure 8).

Cationic antimicrobial peptides form an important component of innate immunity and are known to play a role in preventing the onset of infection in many organisms 29. Systematic studies to identify and characterize novel antimicrobial proteins and peptides are revealing that the majority of defensins are expressed predominantly in the male reproductive tract 16. Moreover, evidence is accumulating that male reproductive tract defensins not only contribute to innate immunity, but also play important roles in sperm maturation in the epididymis and capacitation 181930. What evolutionary advantage might accrue through this particular pairing of activities is not clear. It is long established and well understood that male tract functions including sperm maturation are androgen dependent 3132. Protection against pathogens during active sperm production years could be an inherent mechanism linked to the androgen dependent expression of these defensins and other defensin-like proteins reported previously 33343536.

Although expression of all the epididymis β-defensins exhibited some degree of androgen dependence, Defb27 expression appeared the least androgen dependent and yet its mRNA did not appear until 40–50 days of age in contrast to Defb36 that was expressed between 10–20 days. The substantial differences in age of onset of expression of the epididymis β-defensins suggest their promoters are regulated by transcription factors expressed in different developmental time frames. Further evidence indicates that this timing of expression of the epididymis β-defensins is dependent on maturation of the epithelium and consequent expression of relevant gene regulatory factors. Such factors act in concert with androgens (testosterone and dihydrotestosterone) and the androgen receptor, present in rat epididymis from the first week of postnatal life 37. Epididymis tissue androgen decreases from birth until 20 days but remains at a substantial level of approximately 10 ng/g tissue (~35 nM) until approximately 40 days when it begins to increase to adult levels of between 15–20 ng/g 37. The most abundant androgen in epididymis tissue during this period of postnatal development is likely dihydrotestosterone synthesized in epididymis by metabolic conversion of 5α-androstane-3α 17βdiol. During postnatal development 5α-androstane-3α 17βdiol is the major androgen produced through the actions of 5α reductase and 3α hydroxysteroid dehydrogenase and secreted by the mouse testis 38. Testosterone is synthesized by the immature rat testis but is converted rapidly to 5α-androstane-3α 17βdiol 394041. Serum testosterone levels in the rat remain low and do not begin to increase to adult levels until 35–40 days 41. Down regulation of defensin expression in the adult castrated rats in this study and the maintenance of their expression upon testosterone replacement suggests that they are regulated primarily by androgen in the adult. Androgen regulation of epididymal defensin-like gene expression was reported earlier in different species 282930313233. However, androgen-regulation of defensins outside the male tract has not been reported.

Our analyses of these five novel rat β-defensins differ on specific points from those recently reported 16. In the male reproductive tract, we detected generally broader expression than Patil et al. 16. In somatic tissues, Patil et al. reported Defb36 expression in numerous organs, whereas we found highly restricted Defb36 expression. The widespread expression of Defb24 and Defb27 that we report was not analyzed by Patil et al. but is similar to rat β-defensins RBD-1, RBD-2 and Defb4 which were also found expressed in various tissues including the male reproductive tract 1622. The highly restricted expression of Defb30 in our study is consistent with a role in protection against microorganisms specifically transmitted through the reproductive tract.

In presenting the first direct demonstration of bacterial killing by rat β-defensins we confirm the hypothesis that these proteins, related by amino acid sequence to the known antibacterial defensins of other species, indeed also possess this protective capacity. Further studies may reveal that other and perhaps all male reproductive tract defensins in rat exhibit broad spectrum antimicrobial activities and are responsible for protecting this species against pathogens in vivo. Male tract defensin-like bactericidal activities extend beyond E. coli to include Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis 11. The antibacterial mechanisms of β-defensins in the male reproductive tract involve membrane permeabilization and inhibition of macromolecular synthesis 891042. Similar mechanisms may mediate DEFB24 and DEFB30 action. In controlling bacterial proliferation, these proteins may protect against fertility loss due to tissue damage and fibrotic occlusion of the epididymal ducts. Our studies defining the genomic, mRNA and protein sequences of these rat defensins will give impetus to further analyses to broaden our understanding of the biology of defensins, their structure-function relationships and their regulation within and beyond the male reproductive tract.

SY performed the in silico analysis, PCRs, recombinant protein expression, antibacterial assays and wrote majority of the manuscript. VC and CJW conducted the androgen ablation studies. YR contributed to the genomic sequences. SHH and FSF supervised and coordinated the work and the preparation of the manuscript. All authors read, commented upon and approved the final manuscript.