Susan H. Hall

Antimicrobial Activity of Human EPPIN, an Androgen-Regulated, Sperm-Bound Protein with a Whey Acidic Protein Motif

Abstract The role of epididymal sperm-binding proteins in reproductive tract immunity is now well recognized in addition to their role in sperm maturation. Spermatozoa acquire forward motility and fertilizing ability during their passage through the epididymis, where they acquire a wide variety of proteins belonging to different classes. Previously, we demonstrated that EPPIN (epididymal protease inhibitor), an androgen-regulated, sperm-binding protein containing protease-inhibitory motifs, is expressed specifically in the testis and epididymis. In the present study, we investigated the antibacterial activity of EPPIN against Escherichia coli and the mechanism of antimicrobial action. EPPIN exhibited dose- and time-dependent antibacterial activity that was relatively insensitive to salt. However, EPPIN lost its antibacterial activity completely on reduction and alkylation of its cysteines, indicating the importance of disulfide bonds for its activity. EPPIN permeabilized the outer and inner membranes of E. coli, which is consistent with its ability to induce striking morphological alterations of E. coli membranes as shown by scanning electron microscopy. EPPIN did not cause disruption of eukaryotic membranes in the rat erythrocyte hemolytic assay. The present results indicate that EPPIN has a role in the innate immune system of human epididymis.

Protein inhibitors of activated STAT resemble scaffold attachment factors and function as interacting nuclear receptor coregulators.

Protein inhibitor of activated STAT1 (PIAS1) functions as a nuclear receptor coregulator and is expressed in several cell types of human testis. However, the mechanism of PIAS1 coregulation is unknown. We report here that PIAS1 has characteristics of a scaffold attachment protein. PIAS1 localized in nuclei in a speckled pattern and bound A-T-rich double-stranded DNA, a function of scaffold attachment proteins in chromatin regions of active transcription. DNA binding was dependent on a 35-amino acid sequence conserved among members of the PIAS family and in scaffold attachment proteins. The PIAS family also bound the androgen receptor DNA binding domain, and binding required the second zinc finger of this domain. PIAS1 contained an intrinsic activation domain but had bi-directional effects on androgen receptor transactivation; lower expression levels inhibited and higher levels increased transactivation in CV1 cells. Other PIAS family members also had dose-dependent effects on transactivation, but they were in a direction opposite to those of PIAS1. When coexpressed with PIAS1, other PIAS family members counteracted PIAS1 coregulation of androgen receptor transactivation. The interaction of PIAS1 with other members of the PIAS family suggests a transcription coregulatory mechanism involving a multicomponent PIAS nuclear scaffold.

Antibacterial properties of the sperm-binding proteins and peptides of human epididymis 2 (HE2) family; salt sensitivity, structural dependence and their interaction with outer and cytoplasmic membranes of Escherichia coli.

During passage through the epididymis, sperm interact with secreted epididymal proteins that promote maturation, including the acquisition of motility and fertilization competence. Viewed previously as distinct from sperm maturation, host defence capabilities are now recognized functions of the human epididymis 2 (HE2) family of sperm-binding proteins. We analysed the potent dose and time-dependent bactericidal activity of recombinant HE2alpha, HE2beta1 and HE2beta2 and found that the full-length proteins (10 microg/ml or approximately 1 microM) caused more than a 50% decrease in Escherichia coli colony forming units within 15 min. By contrast, human beta-defensin-1, at a similar concentration, required more than 90 min to exhibit similar antibacterial activity. The epididymis-specific lipocalin, LCN6, failed to kill bacteria. Higher concentrations (25-100 microg/ml) of HE2 proteins and a longer duration of treatment resulted in near total inhibition of bacterial growth. The C-terminal peptides of HE2alpha, HEbeta1 and HEbeta2 proteins exhibited antibacterial activity similar to their full-length counterparts, indicating that the antibacterial activity of HE2 proteins resides in these C-terminal regions. Antibacterial activities of HE2 proteins and peptides were slightly inhibited by NaCl concentrations of up to 150 mM, while human beta-defensin-1 activity was nearly eliminated. Reduction and alkylation of disulphide bonds in HE2 proteins and their C-terminal peptides abolished their antibacterial activity. Consistent with the ability to kill bacteria, full-length HE2 proteins and C-terminal peptides caused rapid dose-dependent permeabilization of outer and cytoplasmic E. coli membranes. A much longer exposure time was required for human beta-defensin-1-mediated permeabilization of membranes, suggesting a possible difference in mode of action compared with the HE2 antibacterial peptides.

Activation of Toll-Like Receptor 4 (TLR4) by In Vivo and In Vitro Exposure of Rat Epididymis to Lipopolysaccharide from Escherichia Coli

Abstract This study provides the first evidence that rat epididymis is fully capable of initiating an inflammatory response to lipopolysaccharide (LPS) from Escherichia coli through activation of Toll-like receptor 4 (TLR4). TLR4 functionality was demonstrated by in vivo LPS challenge, which induced a time- and dose-dependent activation of the transcription factor nuclear factor kappa B (NFKB) in caput and cauda epididymides. NFKB activation by LPS in caput epididymidis was abrogated when rats were pretreated with the NFKB inhibitor PDTC, confirming the specificity of this response. Within 2 h of LPS treatment (0.01 and 1 mg/kg, i.v.), NFKB activation in caput and cauda was accompanied by upregulation of Il1b, Nfkbia, and Cd14, but not Tlr4, mRNA. These effects, however, were not sustained after 24 h of LPS treatment. Lipopolysaccharide systemic effects were not restricted to epididymides, since Il1b, Nfkbia, and Cd14 mRNAs were also upregulated in other male reproductive tissues from LPS-treated rats (1 mg/kg, i.v., 2 h). Constitutive TLR4 was immunolocalized in some, but not all, epididymal epithelial cells and in interstitial cells, some of them identified as resident ED2-positive macrophages. No change in TLR4 immunostaining pattern was observed when epididymides from control and LPS-treated rats were compared (1 mg/kg, i.v., 2 h and 24 h). Significant NFKB activation was also achieved within 1 min of in vitro incubation of caput epididymidis with LPS (0.01–5 μg/ml), confirming that components for TLR4 signaling cascade activation are fully active in this tissue. This study contributes to a better understanding of the innate immune response in the epididymis and other tissues from the male reproductive tract.

Antimicrobial actions of the human epididymis 2 (HE2) protein isoforms, HE2alpha, HE2beta1 and HE2beta2

BackgroundThe HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis.MethodsE. coli treated with 50 micro g/ml of HE2alpha, HE2beta1 or HE2beta2 peptides for 30 and 60 min were visualized using transmission and scanning electron microscopy to investigate the impact of these peptides on bacterial internal and external structure. The effects of HE2alpha, HE2beta1 and HE2beta2 on E. coli macromolecular synthesis was assayed by incubating the bacteria with 2, 10 and 25 micro g/ml of the individual peptides for 0–60 min and measuring the incorporation of the radioactive precursors [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N)]leucine into DNA, RNA and protein. Statistical analyses using Students t-test were performed using Sigma Plot software. Values shown are Mean ± S.D.ResultsE. coli treated with HE2alpha, HE2beta1 and HE2beta2 peptides as visualized by transmission electron microscopy showed extensive damage characterized by membrane blebbing, thickening of the membrane, highly granulated cytoplasm and appearance of vacuoles in contrast to the smooth and continuous membrane structure of the untreated bacteria. Similarly, bacteria observed by scanning electron microscopy after treating with HE2alpha, HE2beta1 or HE2beta2 peptides exhibited membrane blebbing and wrinkling, leakage of cellular contents, especially at the dividing septa, and external accumulation of fibrous materials. In addition, HE2alpha, HE2beta1 and HE2beta2 peptides inhibited E. coli DNA, RNA and protein synthesis.ConclusionsThe morphological changes observed in E. coli treated with epididymal HE2 peptides provide further evidence for their membrane dependent mechanism of antibacterial action. HE2 C-terminal peptides can inhibit E. coli macromolecular synthesis, suggesting an additional mechanism of bacterial killing supplementary to membrane permeabilization.

Genome-wide profiling of segmental-regulated transcriptomes in human epididymis using oligo microarray

Sperm maturation during passage through the epididymis depends on regionalized gene expression which maintains the progressively changing environment within the epididymal tubule. Towards defining the genes that drive the sequential maturation of spermatozoa, we profiled regionally regulated gene expression pattern in the epididymis of a fertile young male donor using Affymetrix human genome U133 plus 2.0 microarray representing approximately the whole human genome. Over 15000 transcripts, almost one-third of the total on the array were identified in whole epididymis. Among them, 65% were detected in all three regions of the epididymis, 410 or 2.6% were present only in one region and the remaining 32.4% were distributed in two regions. Region-specific transcripts observed in caput (264), corpus (61) and cauda (81) epididymides were further classified as empirically determined reported genes or ESTs. This study revealed for the first time, the expression in human epididymis of a number of region-specific genes. The original data will be made publicly available on the Shanghai Science and Technology Database (http://www.scbit.org/human_epididymis_transcriptomes).

Identification, characterization, and evolution of a primate β-defensin gene cluster

Defensins are members of a large diverse family of cationic antimicrobial peptides that share a signature pattern consisting of six conserved cysteine residues. Defensins have a wide variety of functions and their disruption has been implicated in various human diseases. Here we report the characterization of DEFB119–DEFB123, five genes in the human β-defensin cluster locus on chromosome 20q11.1. The genomic structures of DEFB121 and DEFB122 were determined in silico. Sequences of the five macaque orthologs were obtained and expression patterns of the genes were analyzed in humans and macaque by semiquantitative reverse transcription polymerase chain reaction. Expression was restricted to the male reproductive tract. The genes in this cluster are differentially regulated by androgens. Evolutionary analyses suggest that this cluster originated by a series of duplication events and by positive selection. The evolutionary forces driving the proliferation and diversification of these defensins may be related to reproductive specialization and/or the host–parasite coevolutionary process.

Characterization of mouse Eppin and a gene cluster of similar protease inhibitors on mouse chromosome 2.

We have recently described a novel gene on human chromosome 20q 12-13.2 called Eppin (Epididymal protease inhibitor) that expresses three mRNAs encoding two isoforms of a cysteine-rich protein containing both Kunitz-type and WAP-type (four disulfide core) consensus sequences (Richardson et al., 2001). To further our studies on Eppin, we have cloned, sequenced and characterized mouse Eppin and report that it lies within a 200 Kb cluster of putative Eppin-like genes on mouse chromosome 2. Analysis of the homologies between the genes in the human and mouse Eppin clusters indicates that the first part of the cluster immediately surrounding Eppin represents a conserved linkage because the order of homologous genes is conserved. Sequencing of reverse transcription polymerase chain reaction (RT-PCR) products confirmed the expression of five of these novel Eppin-like genes in the mouse, which include the mouse homologue of HE-4. These genes are characterized by having either one or both of the Kunitz-type and WAP-type consensus sequences. Additional RT-PCR experiments revealed that expression of some of the Eppin-like genes is restricted to epididymis and testis while others are expressed in several somatic tissues. Northern blot analysis of 22 different mouse tissues identified Eppin transcripts only in the epididymis and testis. Immunostaining of Eppin with anti-recombinant mouse Eppin demonstrated Eppin predominantly on the postacrosomal region of mouse spermatozoa, in Sertoli cells, Leydig cells, and round spermatids in the testis, and in the principal cells of the cauda epididymidis epithelium. Eppin is first expressed by Sertoli cells of 12-day-old mice and subsequently in round spermatids, which is consistent with androgen regulation. Our results demonstrate that mouse chromosome 2 contains a conserved linkage of Eppin-like protease inhibitor genes that are expressed in the epididymis.

Differential Expression and Antibacterial Activity of Epididymis Protein 2 Isoforms in the Male Reproductive Tract of Human and Rhesus Monkey (Macaca mulatta)

Abstract The epididymis protein 2 (EP2) gene, the fusion of two ancestral β-defensin genes, is highly expressed in the epididymis and subject to species-specific regulation at the levels of promoter selection, transcription, and mRNA splicing. EP2 mRNA expression is also androgen dependent, and at least two of the secreted proteins bind spermatozoa. Alternative splicing produces more than 17 different EP2 mRNA variants. In this article, the expression of EP2 variants was profiled in different tissues from the human and rhesus monkey (Macaca mulatta) male reproductive tract using reverse transcriptase-polymerase chain reaction. Different EP2 mRNA variants were identified not only in human and rhesus testis and epididymis but also in the novel sites, seminal vesicle and prostate. Immunolocalization of EP2 protein in epithelial cells from rhesus and human seminal vesicle demonstrated that EP2 transcripts are translated in these tissues. In addition, two novel splicing variants, named EP2R and EP2S, were discovered. EP2C was the only splice variant expressed in all tissues tested from rhesus monkey. However, expression was not detected in human testis or seminal vesicle. For the first time, bactericidal function was demonstrated for EP2C, EP2K, and EP2L. Taken together, the results indicate that EP2 expression is more widespread in the male reproductive tract than realized previously. Whereas the activity of every EP2 variant tested thus far is antibacterial, further investigation may reveal additional physiological roles for EP2 peptides in the primate male reproductive tract.

LCN6, a novel human epididymal lipocalin

BackgroundThe lipocalin (LCN) family of structurally conserved hydrophobic ligand binding proteins is represented in all major taxonomic groups from prokaryotes to primates. The importance of lipocalins in reproduction and the similarity to known epididymal lipocalins prompted us to characterize the novel human epididymal LCN6.Methods and ResultsLCN6 cDNA was identified by database analysis in a comprehensive human library sequencing program. Macaca mulatta (rhesus monkey) cDNA was obtained from an epididymis cDNA library and is 93% homologous to the human. The gene is located on chromosome 9q34 adjacent LCN8 and LCN5. LCN6 amino acid sequence is most closely related to LCN5, but the LCN6 beta-barrel structure is best modeled on mouse major urinary protein 1, a pheromone binding protein. Northern blot analysis of RNAs isolated from 25 human tissues revealed predominant expression of a 1.0 kb mRNA in the epididymis. No other transcript was detected except for weak expression of a larger hybridizing mRNA in urinary bladder. Northern hybridization analysis of LCN6 mRNA expression in sham-operated, castrated and testosterone replaced rhesus monkeys suggests mRNA levels are little affected 6 days after castration. Immunohistochemical staining revealed that LCN6 protein is abundant in the caput epithelium and lumen. Immunofluorescent staining of human spermatozoa shows LCN6 located on the head and tail of spermatozoa with the highest concentration of LCN6 on the post-acrosomal region of the head, where it appeared aggregated into large patches.ConclusionsLCN6 is a novel lipocalin closely related to Lcn5 and Lcn8 and these three genes are likely products of gene duplication events that predate rodent-primate divergence. Predominant expression in the epididymis and location on sperm surface are consistent with a role for LCN6 in male fertility.