Celebrating the Silver Anniversary of the North American Testis Workshop.

OBJECTIVE: To provide an overview of the history of the North American Testis Workshop (NATW), of its relationship to the American Society of Andrology (ASA), and of the publications that resulted from the first 25 workshops. METHODS: The collection of volumes and journal articles that relate to the NATW was searched. DISCUSSION AND CONCLUSION: During the first twenty-five meetings of the NATW, a remarkable number of breakthroughs regarding every aspect of the testis were presented. We anticipate that with the acceleration of new genetic, epigenetic, and molecular knowledge of the functions of testicular cells, we will continue to learn about the discovery of new and clinically important aspects of testicular function during the next twenty-five NATWs.

Parting messages from current and former editors of the Journal of Andrology.

The proposal to produce this final commemorative issue for the Journal of Andrology arose during our regular discussions as current editors soon after it was announced that the Journal would complete its own life course and merge into a new publication (to be named Andrology) with the International Journal of Andrology. We considered the momentous occasion to be one that should be celebrated with an enduring tribute in recognition of the Journal's exceptional 33-year existence. Among the various contributions sought for inclusion in this issue, we envisioned an article assembling collected short essays from all living former editors drawing on notable events and highlights, if not less well-known challenges and successes arising during their editorship eras. We thought that any such production of musings, viewpoints, and most of all words of wisdom from those who have had major roles in the direction and accomplishments of the Journal would offer an illuminating read for the society's members and friends and provide all readers another venue to share in and enjoy the Journal's great history. We are enthralled to have gathered these collections, all personal compositions of the former editors-in-chief, and for their effort that has helped us complete this special endeavor we express to them our tremendous gratitude. Serving as the Journal's last editors, we are also grateful to contribute our essay at the very end as part of this joyous chronicle.

DNA demethylation-dependent AR recruitment and GATA factors drive Rhox5 homeobox gene transcription in the epididymis.

Mammalian male fertility depends on the epididymis, a highly segmented organ that promotes sperm maturation and protects sperm from oxidative damage. Remarkably little is known about how gene expression is controlled in the epididymis. A candidate to regulate genes crucial for epididymal function is reproductive homeobox gene on X chromosome (RHOX)5, a homeobox transcription factor essential for optimal sperm motility that is expressed in the caput region of the epididymis. Here, we report the identification of factors that control Rhox5 gene expression in epididymal cells in a developmentally regulated and region-specific fashion. First, we identify GATA transcription factor-binding sites in the Rhox5 proximal promoter (Pp) necessary for Rhox5 expression in epididymal cells in vitro and in vivo. Adjacent to the GATA sites are androgen-response elements, which bind to the nuclear hormone receptor androgen receptor (AR), and are responsible for the AR-dependent expression of Rhox5 in epididymal cells. We provide evidence that AR is recruited to the Pp in a region-specific and developmentally regulated manner in the epididymis that is dictated not only by differential AR availability but differential methylation of the Pp. Site-specific methylation of the Pp cytosine and guanine separated by one phosphate, most of which overlap with androgen-response elements, inhibited both AR occupancy at the Pp and Pp-dependent transcription in caput epididymal cells. Together, our data support a model in which DNA methylation, AR, and GATA factors collaborate to dictate the unique developmental and region-specific expression pattern of the RHOX5 homeobox transcription factor in the caput epididymis, which in turn controls the expression of genes critical for promoting sperm motility and function.

Organic cation/carnitine transporter, OCTN2, transcriptional activity is regulated by osmotic stress in epididymal cells.

Protection of cells from osmotic stress is crucial for their survival. Exposure to high osmolarity promotes rapid diffusion of water across cell membranes, dramatically increasing cellular ionic strength, leading to disruption of key proteins/DNA resulting in cell-cycle arrest and apoptosis. The luminal microenvironment of the epididymis is hypertonic; therefore, epididymal cells adapt to the higher osmolarity by accumulating organic osmolytes, such as L-carnitine. Osmolytes do not perturb cells when accumulated in high concentrations, nor do they affect key proteins or damage DNA. Therefore, osmolytes and their transporters are crucial for cell survival. Transporters that are responsible for the accumulation of organic osmolytes have been shown to be regulated at the transcriptional level by hypertonicity. The present study examines the gene expression of known osmoprotective/stress genes in epididymal cells exposed to changes in tonicity. We demonstrate that the osmoprotective/stress pathways present in other organs, such as the kidney, operate in the epididymis, potentially aiding in the protection of its luminal cells and spermatozoa. Further, it was also seen that OCTN2, a transporter that is thought to be responsible for the accumulation of L-carnitine in the epididymal lumen, is regulated in response to changes in tonicity.

Epididymis-specific lipocalin promoters.

Our goal is to decipher which DNA sequences are required for tissue-specific expression of epididymal genes. At least 6 epididymis-specific lipocalin genes are known. These are differently regulated and regionalized in the epididymis. Lipocalin 5 (Lcn5 or mE-RABP) and Lipocalin 8 (Lcn8 or mEP17) are homologous genes belonging to the epididymis-specific lipocalin gene cluster. Both the 5 kb promoter fragment of the Lcn5 gene and the 5.3 kb promoter fragment of the Lcn8 gene can direct transgene expression in the epididymis (Lcn5 to the distal caput and Lcn8 to the initial segment), indicating that these promoter fragments contain important cis-regulatory element(s) for epididymis-specific gene expression. To define further the fragments regulating gene expression, the Lcn5 promoter was examined in transgenic mice and immortalized epididymal cell lines. After serial deletion, the 1.8 kb promoter fragment of the Lcn5 gene was sufficient for tissue-specific and region-specific gene expression in transgenic mice. Transient transfection analysis revealed that a transcription factor forkhead box A2 (Foxa2) interacts with androgen receptor and binds to the 100 bp fragment of the Lcn5 promoter between 1.2 kb and 1.3 kb and that Foxa2 expression inhibits androgen-dependent induction of the Lcn5 promoter activity. Immunohistochemistry indicated a restricted expression of Foxa2 in the epididymis where endogenous Lcn5 gene expression is suppressed and that the Foxa2 inhibition of the Lcn5 promoter is consistent with the lack of expression of Lcn5 in the corpus and cauda. Our approach provides a basic strategy for further analysis of the epididymal lipocalin gene regulation and flexible control of epididymal function.

The role of forkhead box A2 to restrict androgen-regulated gene expression of lipocalin 5 in the mouse epididymis.

Murine epididymal retinoic acid-binding protein [or lipocalin 5 (Lcn5)] is synthesized and secreted by the principal cells of the mouse middle/distal caput epididymidis. A 5-kb promoter fragment of the Lcn5 gene can dictate androgen-dependent and epididymis region-specific gene expression in transgenic mice. Here, we reported that the 1.8-kb Lcn5 promoter confers epididymis region-specific gene expression in transgenic mice. To decipher the mechanism that directs transcription, 14 chimeric constructs that sequentially removed 100 bp of 1.8-kb Lcn5 promoter were generated and transfected into epididymal cells and nonepididymal cells. Transient transfection analysis revealed that 1.3 kb promoter fragment gave the strongest response to androgens. Between the 1.2-kb to 1.3-kb region, two androgen receptor (AR) binding sites were identified. Adjacent to AR binding sites, a Foxa2 [Fox (Forkhead box) subclass A] binding site was confirmed by gel shift assay. Similar Foxa binding sites were also found on the promoters of human and rat Lcn5, indicating the Foxa binding site is conserved among species. We previously reported that among the three members of Foxa family, Foxa1 and Foxa3 were absent in the epididymis whereas Foxa2 was detected in epididymal principal cells. Here, we report that Foxa2 displays a region-specific expression pattern along the epididymis: no staining observed in initial segment, light staining in proximal caput, gradiently heavier staining in middle and distal caput, and strongest staining in corpus and cauda, regions with little or no expression of Lcn5. In transient transfection experiments, Foxa2 expression inhibits AR induction of the Lcn5 promoter, which is consistent with the lack of expression of Lcn5 in the corpus and cauda. We conclude that Foxa2 functions as a repressor that restricts AR regulation of Lcn5 to a segment-specific pattern in the epididymis.

Epididymis-specific promoter-driven gene targeting: a transcription factor which regulates epididymis-specific gene expression.

Mammalian spermatozoa undergo several modification and finally acquire the ability to fertilize during epididymal transit. One of the distinct features of the epididymis is that it displays a highly regionalized pattern of gene expression. This tissue-, region-, and cell-specific pattern of gene expression is critical for the maintenance of a fully functional epididymis. One would hypothesize that disrupting this process provides an ideal approach to male contraception, since it would not interfere with testicular endocrine output or sperm production. To achieve this purpose, we studied a cluster of epididymis-specific lipocalin genes for understanding the specific mechanisms involved in the control of gene expression in the epididymis. We have identified six epididymis-specific lipocalin genes that are differently regulated and regionalized in the epididymis. Lipocalin 5 [Lcn5 or epididymal retinoic acid-binding protein (E-RABP)] is a member of this epididymis-specific lipocalin gene cluster, which binds hydrophobic molecules such as retinoic acid. We have previously shown that the 5kb promoter fragment of the Lcn5 gene confers both androgen-dependent regulation and epididymis-specific gene expression in transgenic mice whereas 0.6 kb promoter fragment does not. To further narrow down the important cis-regulatory elements that regulate gene expression in the epididymis, we studied the Lcn5 promoter in both transgenic mice and immortalized epididymal cells. We have found that 1.8kb promoter fragment of the Lcn5 gene was sufficient for tissue- and region-specific expression in transgenic mice, and that a transcription factor Forkhead box A2 (Foxa2) interacts with the androgen receptor and binds to the 100 bp fragment of the Lcn5 promoter between 1.2 and 1.3 kb. Our finding provides a framework for further analysis of the epididymal lipocalin gene regulation and modulated control of epididymis-specific expression.

Foxa1 and Foxa2 interact with the androgen receptor to regulate prostate and epididymal genes differentially.

Previous studies from our group have shown that Foxa1 is expressed in the prostate and interacts with the androgen receptor (AR) to regulate prostate-specific genes such as prostate-specific antigen (PSA) and probasin (PB). We report here that Foxa2 but not Foxa1 is expressed in the epididymis. Further, Foxa2 interacts with the AR to regulate the mouse epididymal retinoic acid binding protein (mE-RABP) gene, an epididymis-specific gene. Binding of Foxa2 to the mE-RABP promoter was confirmed by gel-shift and chromatin immunoprecipitation (ChIP) assays. Overexpression of Foxa2 suppresses androgen activation of the mE-RABP promoter while overexpression of Foxa2 with prostate-specific promoters activates gene expression in an androgen-independent manner. GST pull-down assays determined that both Foxa1 and Foxa2 physically interact with the DNA binding domain of the AR. The interaction between Foxa proteins and AR was further confirmed by gel-shift assays where Foxa protein was recruited to AR binding oligomers even when Foxa binding sites were not present, and AR was recruited to Foxa binding oligomers even in the absence of an AR binding site. Given that Foxa1 and Foxa2 proteins are expressed differentially in the prostate and epididymis, these data suggest that the Foxa proteins have distinct effects on AR-regulated genes in different male reproductive accessory organs.

Molecular evolution of epididymal lipocalin genes localized on mouse chromosome 2.

We previously identified two murine secretory proteins, mE-RABP(Lcn5) and mEP17(Lcn8), belonging to the lipocalin family and specifically expressed in the epididymis. The genes are contiguous and localized on mouse chromosome 2. We now show that five other related lipocalin genes, Lcn9, Lcn10, Lcn11, Lcn12, and Lcn13, that evolved by in situ tandem duplication are present on the same locus. Lcn9, Lcn10, Lcn12, and Lcn13 genes, like Lcn5 and Lcn8 genes, are specifically expressed in the mouse epididymis. However, each gene has a distinct spatial expression within the epididymis and different regulation. Analysis of the human genome sequence shows the presence of genes encoding lipocalins with genomic organization, chromosomal arrangement, and orientation similar to that of the corresponding murine genes, indicating that the epididymal cluster is evolutionary conserved. A phylogenetic analysis of the new epididymal proteins reveals their spread position in the lipocalin protein family tree. This suggests the preservation of the regulatory sequences, while protein sequences have greatly diverged, reflecting functional diversity and possibly multifunctionality. In terms of the cluster ancestry, epididymal expression possibly appeared in a PGDS-like lipocalin in amniotes, and the duplications generating the cluster occurred at least in the common ancestor of rodents and primates. The presence and conservation of a cluster of five genes encoding epididymal lipocalins, differently regulated and regionalized in the epididymis, strongly suggests that these proteins may play an important role for male fertility.

Immortalization by large T-antigen of the adult epididymal duct epithelium.

The SV40 large T-antigen has been widely used to convert various cell types to a transformed phenotype, and also to induce progressive tumours in transgenic animals. The objectives of this review are to compare and discuss three different approaches to generate epididymal epithelial cell lines using the large T-antigen. In the first approach, retroviral transfection of primary cultures was used to immortalize canine epididymal cells in vitro; the other two approaches used transgenic mice expressing the large T-antigen. In one of these in vivo approaches, a construct consisting of the coding sequence of a temperature sensitive (ts) SV40 large T-antigen was inserted in a mouse genome. When the cells are exposed to the permissive temperature of 33 degrees C, functional expression of the large T-antigen occurs and cells start to proliferate. In the second in vivo approach a tissue-specific promoter, the 5kb GPX5 promoter, was used to direct expression of the large T-antigen to the epididymal duct epithelium.

Profiling and imaging proteins in the mouse epididymis by imaging mass spectrometry.

Different aspects of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) have been used as discovery tools to obtain global and time-correlated information on the local proteomic composition of the sexually mature mouse epididymis from both qualitative and semiquantitative points of view. Tissue sections and laser captured microdissected cells and secretory products were analyzed by MALDI-MS and from the recovered protein profiles, over 400 different proteins were monitored. Over 50 of these, some of which have been identified, displayed regionalized behavior from caput to cauda within the epididymis. Combining the information obtained from high-resolution imaging mass spectrometry and laser captured microdissection experiments, numerous proteins were localized within the epididymis at the cellular level. Furthermore, from the signal intensities observed in the different protein profiles organized in space, semiquantitative information for each protein was obtained.

Gene and protein expression in the epididymis of infertile c-ros receptor tyrosine kinase-deficient mice.

Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein beta, PEA3) were unchanged. Genes normally absent from the initial segment (gamma-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive.

Disruption of androgen regulation in the prostate by the environmental contaminant hexachlorobenzene.

Hexachlorobenzene (HCB) is a persistent environmental contaminant that has the potential to interfere with steroid hormone regulation. The prostate requires precise control by androgens to regulate its growth and function. To determine if HCB impacts androgen action in the prostate, we used a number of methods. Our in vitro cell-culture-based assay used a firefly luciferase reporter gene driven by an androgen-responsive promoter. In the presence of dihydrotestosterone, low concentrations (0.5-5 nM) of HCB increased the androgen-responsive production of firefly luciferase and high concentrations of HCB (> 10 microM) suppressed this transcriptional activity. Results from a binding assay showed no evidence of affinity between HCB and the androgen receptor. We also tested HCB for in vivo effects using transgenic mice in which the transgene was a prostate-specific, androgen-responsive promoter upstream of a chloramphenicol acetyl transferase (CAT) reporter gene. In 4-week-old mice, the proportion of dilated prostate acini, a marker of sexual maturity, increased in the low HCB dose group and decreased in the high HCB dose mice. In the 8-week-old mice, there was a significant decrease in both CAT activity and prostate weight upon exposure to 20 mg/kg/day HCB. Therefore, in vitro and in vivo data suggest that HCB weakly agonizes androgen action, and consequently, low levels of HCB enhanced androgen action but high levels of HCB interfered. Environmental contaminants have been implicated in the rising incidence of prostate cancer, and insight into the mechanisms of endocrine disruption will help to clarify their role.

The 5'-flanking region of the murine epididymal protein of 17 kilodaltons gene targets transgene expression in the epididymis.

A murine epididymal retinoic-acid-binding protein (mE-RABP) is specifically expressed in the mid/distal caput epididymidis and is androgen regulated. The murine epididymal protein of 17 kDa (mEP17) gene, a novel gene homologous to mE-RABP, is located within 5 kb of the 5'-flanking region of the mE-RABP gene. In contrast, expression of the mEP17 gene is restricted to the initial segment and regulated by factor(s) contained in testicular fluid. To identify cis-DNA regulatory element(s) involved in the tissue- and region-specific expression of the mEP17 gene in transgenic mice, we have studied the expression of a transgene containing 5.3 kb of the 5'-flanking region of the mEP17 gene (5.3mEP17) linked to chloramphenicol acetyltransferase (CAT) reporter gene. Significant caput epididymidis-specific CAT activity was detected in transgenic mouse lines; and CAT gene expression is restricted to the initial segment, as is the expression of the endogenous mEP17 gene. Ontogenic expression and testicular factor dependency also mimic that of endogenous mEP17 gene. These results suggest that the 5.3mEP17 fragment contains all the information required for spatial and temporal expression in the mouse epididymis. The 5.3mEP17 fragment will be useful to express a foreign gene of interest in the epididymis in an initial segment-specific manner.

Identification, immunolocalization, regulation, and postnatal development of the lipocalin EP17 (epididymal protein of 17 kilodaltons) in the mouse and rat epididymis.

Several lipocalins are present in the mouse epididymis and are thought to play a role in sperm maturation by transporting lipophilic molecules. We have previously reported that two lipocalin genes, mERABP (mouse epididymal retinoic acid binding protein), and mEP17 (mouse epididymal protein of 17 kDa), derived from an ancestral gene, are specifically expressed in the epididymis. In the present study, a polyclonal antibody was raised against a recombinant protein to investigate the presence and the regulation of mEP17. mEP17 was detected in the supranuclear region of the principal cells of the initial segment, the clear cells of the caput epididymidis, and the lumen of the mid/distal caput but not of the distal epididymis. Initial segment and caput tissue extracts were subjected to HPLC separation. After electrophoresis of the immunoreactive mEP17-enriched fractions, the immunoreactive band was analyzed by mass spectrometry to identified mEP17 unambiguously. After two-dimensional electrophoresis, mEP17 appeared as a train of five 22-kDa spots with a range of pI (isoelectric point) from 5.8-6.7. N-glycanase digestion gave rise to a single spot of 17 kDa and pI 6, the predicted mass and pI. During ontogeny, mEP17 was detected as early as 3 wk of age and increased afterward. After bilateral orchiectomy, mEP17 disappeared 2 d after surgery and was not restored after testosterone replacement. After unilateral orchiectomy, mEP17 levels decreased only in the orchiectomized side. After cryptorchidism or busulfan treatment, mEP17 levels were either greatly diminished or not detected. This suggests that mEP17 is dependent on testicular factor(s) that may have a germ cell origin. Altogether, our data demonstrate that mEP17 spatial expression, regulation, and fate are different from that of the highly related mouse epididymal retinoic acid binding protein. This suggests that these two related proteins exhibit distinct functions in the mouse epididymis.

Immortalized epididymal cell lines from transgenic mice overexpressing temperature-sensitive simian virus 40 large T-antigen gene.

Epididymal epithelium is well known as a site of secretion of various proteins present in epididymal luminal fluid. Although there have been many reports of primary cultures of epididymal epithelial cells, their growth is limited over time. We have established immortalized epididymal epithelial cell lines from primary cultures of epididymal cells from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene in order to study the regulatory mechanisms of epididymal function, including specific factor secretion. These cell lines (PC1 from proximal caput; and DC1, DC2, and DC3 from distal caput) have been maintained for more than 1 year and show temperature-dependent growth and expression of cytokeratin, a marker of epithelial cells. These cells express the androgen receptor as well as markers of the murine epididymal epithelium, PEB-like protein (ie, phosphatidye ethanolamine binding protein), E-RABP (ie, epididymal retinoic acid-binding protein), and EP17 (ie, epididymal protein of 17 kd). The androgen-regulated 5-kilobase mE-RABP promoter DNA fragment ligated to the neomycin-resistant gene was used for stable transfection of DC1 cells. Because the mE-RABP gene is specifically expressed in the distal caput, neomycin selection provides a pure population of epithelial cells from that segment. This neomycin-resistant immortalized cell line from the distal caput was cultured for more than 6 months. Such immortalized cell lines should be valuable tools for studying the regulation of tissue-specific gene expression, and may be used to identify one or more epididymal specific transcription factors involved in the expression of epididymal specific proteins.

Epididymal lipocalin-type prostaglandin D2 synthase: identification using mass spectrometry, messenger RNA localization, and immunodetection in mouse, rat, hamster, and monkey.

This study identified prostaglandin D2 synthase (PGDS) in murine epididymal fluid using a proteomic approach combining two-dimensional (2D) gel electrophoresis and mass spectrometry (MS). The caudal epididymal fluid was collected by retroperfusion, and proteins were separated by 2D gel electrophoresis followed by matrix-assisted laser desorption ionization MS analyses after trypsin digestion. The identification was based on the protein-specific peptide map as well as on sequence information generated by nano-electrospray ionization MS/MS. By in situ hybridization, the mRNA was detected in caput, corpus, and cauda, but it was not detected in the initial segment. The PGDS protein was mostly detected in the corpus and cauda by Western blot analysis and immunohistochemistry using a specific polyclonal antibody. In caudal fluid, PGDS was distributed among several isoforms (pI range, 6.5-8.8), suggesting that this protein undergoes posttranslational modification of its primary sequence. After N-glycanase digestion, the molecular mass decreased from 20-25 to 18.5 kDa, its theoretical mass. The PGDS was also detected in the epididymis of rat, hamster, and cynomolgus monkey from the caput to the cauda. In conclusion, MS is a powerful and accurate technique that allows unambiguous identification of the murine epididymal PGDS. The protein is 1) present throughout the epididymis, except in the initial segment, with an increasing luminal concentration from distal caput to cauda; 2) a major protein in caudal fluid; 3) an N-glycosylated, highly polymorphic protein; and 4) conserved during evolution.