The Rhox5 homeobox gene regulates the region-specific expression of its paralogs in the rodent epididymis.

The mechanisms by which the region-specific expression patterns of clustered genes evolve are poorly understood. The epididymis is an ideal organ to examine this, as it is a highly segmented tissue that differs significantly in structure between closely related species. Here we examined this issue through analysis of the rapidly evolving X-linked reproductive homeobox (Rhox) gene cluster, the largest known homeobox gene cluster in metazoans. In the mouse, we found that most Rhox genes are expressed primarily in the caput region of the epididymis, a site where sperm mature and begin acquiring forward motility. This region-specific expression pattern depends, in part, on the founding member of the Rhox cluster--Rhox5--as targeted mutation of Rhox5 greatly diminishes the expression of several other family members in the caput region. In the rat, Rhox5 expression switches from the caput to the site of sperm storage: the cauda. All Rhox genes under the control of Rhox5 in the mouse epididymis display a concomitant change in their regional expression in the rat epididymis. Our results lead us to propose that widespread changes in the region-specific expression pattern of genes over evolutionary time can be the result of alterations of one or only a few master regulatory genes.

Identification of epididymis-specific transcripts in the mouse and rat by transcriptional profiling.

As part of our efforts to identify novel contraceptive targets in the epididymis we performed transcriptional profiling on each of the 10 and 19 segments of the mouse and rat epididymidis, respectively, using Affymetrix whole genome microarrays. A total of 17 096 and 16 360 probe sets representing transcripts were identified as being expressed in the segmented mouse and rat epididymal transcriptomes, respectively. Comparison of the expressed murine transcripts against a mouse transcriptional profiling database derived from 22 other mouse tissues identified 77 transcripts that were expressed uniquely in the epididymis. The expression of these genes was further evaluated by reverse transcription polymerase chain reaction (RT-PCR) analysis of RNA from 21 mouse tissues. RT-PCR analysis confirmed epididymis-specific expression of Defensin Beta 13 and identified two additional genes with expression restricted only to the epididymis and testis. Comparison of the 16 360 expressed transcripts in the rat epididymis with data of 21 other tissues from a rat transcriptional profiling database identified 110 transcripts specific for the epididymis. Sixty-two of these transcripts were further investigated by qPCR analysis. Only Defensin 22 (E3 epididymal protein) was shown to be completely specific for the epididymis. In addition, 14 transcripts showed more than 100-fold selective expression in the epididymis. The products of these genes might play important roles in epididymal and/or sperm function and further investigation and validation as contraceptive targets are warranted. The results of the studies described in this report are available at the Mammalian Reproductive Genetics (MRG) Database (http://mrg.genetics.washington.edu/).

Segment boundaries of the adult rat epididymis limit interstitial signaling by potential paracrine factors and segments lose differential gene expression after efferent duct ligation.

The epididymis is divided into caput, corpus and cauda regions, organized into intraregional segments separated by connective tissue septa (CTS). In the adult rat and mouse these segments are highly differentiated. Regulation of these segments is by endocrine, lumicrine and paracrine factors, the relative importance of which remains under investigation. Here, the ability of the CTS to limit signaling in the interstitial compartment is reviewed as is the effect of 15 days of unilateral efferent duct ligation (EDL) on ipsilateral segmental transcriptional profiles. Inter-segmental microperifusions of epidermal growth factor (EGF), vascular endothelial growth factor (VEGFA) and fibroblast growth factor 2 (FGF2) increased phosphorylation of mitogen activated protein kinase (MAPK) in segments 1 and 2 of the rat epididymis and the effects of all factors were limited by the CTS separating the segments. Microarray analysis of segmental gene expression determined the effect of 15 days of unilateral EDL on the transcriptome-wide gene expression of rat segments 1-4. Over 11,000 genes were expressed in each of the four segments and over 2000 transcripts in segment 1 responded to deprivation of testicular lumicrine factors. Segments 1 and 2 of control tissues were the most transcriptionally different and EDL had its greatest effects there. In the absence of lumicrine factors, all four segments regressed to a transcriptionally undifferentiated state, consistent with the less differentiated histology. Deprivation of lumicrine factors could stimulate an individual gene's expression in some segments yet suppress it in others. Such results reveal a higher complexity of the regulation of rat epididymal segments than that is generally appreciated.

Sonic hedgehog pathway inhibition alters epididymal function as assessed by the development of sperm motility.

The sonic hedgehog (Shh) signaling pathway plays a role in pattern orientation in the developing embryo and has been shown to be required for development of the prostate and external genitalia. Recent evidence has shown that important elements of the Shh pathway are also expressed in the adult mouse epididymis at both the gene and protein levels. The objective of the present investigation was to refine the expression pattern of Shh in the mouse epididymis and to determine if the Shh pathway is important for epididymal function vis-à-vis sperm maturation. The former was achieved by microarray analysis of Shh expression in all segments of the mouse epididymis, and the latter was determined by 14-day administration of cyclopamine, a Shh pathway inhibitor, followed by a microassay for the activation and duration of cauda epididymal sperm motility. Shh pathway inhibition was monitored by semiquantitative reverse transcriptase-polymerase chain reaction for expression of epididymal Gli1 and Gli3. The Gli family of transcription factors is commonly activated and regulated by Shh pathway activation. Cyclopamine treatment reduced Gli1 expression by 61% and initiation of cauda sperm motility by 50%. Gli3 expression was reduced by approximately 50%. Subsequent cluster analysis using the microarray data on epididymal gene expression highlighted several potential target genes for the Shh pathway, the most prominent of which is prostaglandin D2 synthase. These results indicate that an operating Shh pathway is important in the murine epididymis for the development of sperm motility and implies a role for Shh signaling in adult epididymal function.

Testicular torsion alters the presence of specific proteins in the mouse testis as well as the phosphorylation status of specific proteins.

Testicular torsion followed by torsion repair induces an ischemia-reperfusion injury to the testis that can render the testis aspermatogenic. Previous results have demonstrated this loss of spermatogenesis to be the result of germ cell apoptosis induced by oxidative stress. The present work reports protein changes occurring in the mouse testis 24 hours after repair of a testicular torsion known to induce germ cell apoptosis and severe seminiferous impairment. Total proteins were extracted from sham-operated testes and testes having had 2-hour 720 degrees torsion 24 hours previously. Testicular proteins were separated by 2-dimensional electrophoresis and the resulting gel images were analyzed with image analysis software. Of the over 1100 proteins detected on the average gel, over 700 were consistently appearing in multiple gels, and those protein spot intensities were averaged within sham and torsion groups and compared between the 2 groups. Twenty-three proteins were consistently increased after torsion repair and 48 were decreased. Six proteins, 3 of which increased and 3 of which decreased after torsion repair, were identified by mass spectrometry. The 3 proteins that increased after torsion repair, beta2-tubulin and 2 isoforms of serum albumin, as well as the 3 proteins that decreased after torsion repair, vimentin, phosphoglycerate kinase, and t-complex protein 1beta, were for the most part associated with various aspects of cell stress responses. The number of proteins phosphorylated on tyrosine residues exceeded the number of proteins phosphorylated on serine/threonine residues, but among 6 stress-related proteins specifically examined for phosphorylation in sham testes and those examined after torsion repair, increases in threonine phosphorylation of c-Jun NH2 terminal kinase and activating transcription factor 2 were the most prominent. Knowing these proteins and the pathways to which they point will aid in the search for new therapies of oxidative stress in the testis.

Activation of the nuclear factor kappa B pathway following ischemia-reperfusion of the murine testis.

Ischemia-reperfusion (IR) of the testis results in testicular oxidative stress and germ cell-specific apoptosis. Nuclear factor kappa B (NF-kappaB) is a nuclear transcription factor involved in the control of a number of cellular processes, and its activation is part of the cellular stress response to a variety of factors including cytokine stimulation, irradiation, and IR. The present study investigates NF-kappaB activation after IR of the murine testis and potential downstream target genes of that activation. Mice were subjected to a period of testicular ischemia followed by 0-4 hours of reperfusion. Activation of NF-kappaB was assessed by 1) Western blot analysis of the NF-kappaB inhibitory protein, IkappaBalpha; 2) immunohistochemistry for IkappaBalpha; and 3) TranSignal NF-kappaB target gene array (107 genes) analysis. Results demonstrate that IkappaBalpha is phosphorylated on serine 32 reaching a peak by 2 hours after IR of the testis. A decrease in total IkappaBalpha was also noted at 2 hours after IR, consistent with the rapid degradation of the phosphorylated protein. Phosphorylation and degradation of IkappaBalpha is indicative of NF-kappaB activation. Immunolocalization revealed IkappaBalpha specifically in Sertoli cells of the murine testis. Results of the TranSignal target gene array revealed that the expression of 9 genes was consistently changed 2 hours after IR of the testis, 3 of which increased in expression and 6 of which were down-regulated. Most notably, high-mobility group nucleosomal binding domain 1 increased in expression while platelet-derived growth factor B and Wilms tumor homolog decreased. These results suggest that testicular IR releases the suppression of NF-kappaB by IkappaBalpha in Sertoli cells. Activation of the NF-kappaB pathway in the testis resulted in an alteration of expression of potential NF-kappaB target genes, some increased while others decreased. The specific roles of these genes in the testicular response to IR remains to be determined.

Exploring the epididymis: a personal perspective on careers in science.

Science is a profession of inquiry. We ask ourselves what is it we see and why our observations happen the way they do. Answering those two question puts us in the company of those early explorers, who from Europe found the New World, and from Asia reached west to encounter Europe. Vasco Núñez de Balboa of Spain was such an explorer. He was the first European to see or "discover" the Pacific Ocean. One can imagine his amazement, his excitement when he first saw from a mountain top that vast ocean previously unknown to his culture. A career in science sends each of us seeking our own "Balboa Moments," those observations or results that surprise or even amaze us, those discoveries that open our eyes to new views of nature and medicine. Scientists aim to do what those early explorers did: discover what has previously been unknown, see what has previously been unseen, and reveal what has previously been hidden. Science requires the scientist to discover the facts from among many fictions and to separate the important facts from the trivial so that knowledge can be properly developed. It is only with knowledge that old dogmas can be challenged and corrected. Careers in science produce specific sets of knowledge. When pooled with other knowledge sets they eventually contribute to wisdom and it is wisdom, we hope, that will improve the human condition.

Sonic hedgehog pathway genes are expressed and transcribed in the adult mouse epididymis.

One role of the hedgehog (hh) signaling pathway during development is to assist in establishing pattern orientation in the embryo. The structure and function in the adult epididymis is highly patterned, and since the sonic hedgehog (Shh) pathway is known to be functional in the developing male tract and the expression of other pattern-influencing genes has recently been found in the adult epididymis, we have examined the adult mouse epididymis for Shh pathway molecules. Examination was at both the gene and protein level. Shh, the secreted signal molecule, patched (Ptc), its membrane receptor, and Gli-1, a downstream transcription factor, were detected at the gene level with semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and at the protein level with Western blot analysis. Immunohistochemical localization further detected Shh specifically in the epididymal epithelium. It was hypothesized that efferent duct ligation (EDL) would alter epididymal segmentation within 30 days of the ligation, especially in the proximal segments of the caput epididymis. It was further hypothesized that these alterations would be correlated with changes in the expression of genes in the Shh pathway. EDL did not alter epididymal segmentation, but Shh, Ptc, and Gli1 expression was significantly altered at specific times after the ligation. The presence of the signaling pathway in the adult epididymis is a novel finding, as is the fact that in the distal epididymis, the specific gene expressions are altered by EDL. This suggests that the genes are capable of being regulated in a manner that is influenced by testicular contribution, and it implies that those genes have a function in the epididymis subject to that regulation.

Peptide and nonpeptide reactive oxygen scavengers provide partial rescue of the testis after torsion.

Ischemia-reperfusion (IR) of the testis results in germ cell-specific apoptosis, followed by a reduction in testis weight and daily sperm production (DSP). This has been associated with an increase in the adhesion of neutrophils to testicular subtunical venules and an increase in reactive oxygen species (ROS). The present study investigated: 1) the effects of a direct, non-IR-related ROS insult to the testis and 2) the effects of catalase, superoxide dismutase (SOD), and a novel nonpeptide mimic of SOD, M40403, on neutrophil recruitment, ROS production, testis weight, and DSP following IR of the rat testis. Results revealed that the infusion of H2O2 increased testicular lipid peroxidation 1 hour after administration and increased germ cell apoptosis within 24 hours of administration. Four hours after the repair of torsion plus vehicle infusion, there was a significant increase in myeloperoxidase (MPO) activity, an indicator of neutrophil accumulation, and thiobarbituric acid reactive substances (TBARS), a measure of ROS production, compared to equivalent data in sham-treated testes. Animals sacrificed 30 days after the torsion plus vehicle infusion revealed a significant decrease in testis weight and DSP compared to the same parameters in sham-operated animals. The treatment of animals with catalase plus SOD or M40403 showed a significant decrease in MPO activity and TBARS 4 hours after IR of the testis. Animals treated with SOD, SOD plus catalase, and M40403 provided a partial rescue of DSP 30 days after IR of the testis. These results demonstrate that oxidative stress can directly cause germ cell apoptosis, even outside the IR model, and confirm the importance of oxidative stress in testicular IR injury. Also, following testicular IR, there is a recruitment of neutrophils and an increase in ROS production in the testis. The administration of ROS scavengers significantly reduced the IR-induced responses. Interestingly, the administration of all ROS scavengers also blocked neutrophil recruitment to the testis. The mechanism by which ROS modulates neutrophil adhesion to venules is presently under investigation, as are additional therapeutic regimens to block oxidative stress.

Looking to the future of epididymal research: why this, why now?

Epididymal biology is an area of science at risk. Never a large field to begin with (the number of papers produced by laboratories studying the epididymis is roughly only 20% of the number produced by laboratories studying the testis), it tends to shrink even further in times of funding crisis. This matters because the numbers of laboratories, investigators, and trainees in any area of science affects the number of new people coming into the field, the new ideas that new people can bring, and the number of interested scientists on important grant review panels. How can epididymal biologists face the current challenges? First, great ideas are the key. They prompt compelling hypotheses that can be challenged with interesting experiments. Second, it must be recognized that the past is past. The fact that studies on sperm maturation, epididymal histology, or tubule physiology are significant parts of the past does not mean that they are no longer interesting, but it does mean that truly original questions in those areas will likely be difficult to find. In the real world of competitive science, national granting agencies require applications that clearly answer the questions, why is this of interest and why is it important now? Productive areas of future research may include lumicrine regulation of the epithelium, immunobiology of the epididymis, and cell-cell communication between epididymal epithelial cells and cells in the peritubular/interstitial space.

Cytokine responses to E. coli-induced epididymitis in the rat: blockade by vasectomy.

OBJECTIVES: To determine cytokine responses in rat epididymal tissues after retrograde Escherichia coli inoculation of the cauda epididymidis via the intact and obstructed vas deferens. METHODS: Adult male Sprague-Dawley rats were divided into 3 groups: bilateral sham vasectomy followed by unilateral sham retrograde inoculation in the vas deferens (group A), bilateral sham vasectomy followed by unilateral retrograde inoculation of E. coli (group B), and bilateral vasectomy followed by left-sided inoculation of E. coli (group C). Three days later, the cauda epididymides and proximal vasa were subjected to histologic examination and assay for 9 cytokines. RESULTS: Groups A and C showed no histologic evidence of epididymal inflammation. Group B had leukocyte infiltrates in the inoculated tissue. Cytokine levels in the injected cauda epididymides were low in groups A and C; however, interleukin (IL)-1α, IL-1ß, and IL-4 were significantly increased in group B. The 6 other cytokines showed no significant change after E. coli infection though tumor necrosis factor-α, and IL-6 did show strong trends for increase. Contralateral epididymides never showed an inflammatory response. CONCLUSIONS: Experimental epididymitis induced by retrograde movement of bacteria in the vas deferens results in different responses by different cytokines. The cytokine responses and the histologically evident inflammation are prevented by vasectomy.

Hypoxia-inducible factor-1alpha is constitutively expressed in murine Leydig cells and regulates 3beta-hydroxysteroid dehydrogenase type 1 promoter activity.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that plays an essential role in oxygen homeostasis. HIF-1alpha is constitutively made in cells; however, it is ubiquitinated and degraded under normoxic conditions. Hypoxia prevents the ubiquitination of HIF-1alpha, resulting in stabilization of the protein and activation of target genes. Because of its vascular arrangement and the high metabolic demand of spermatogenesis, the testis has been described previously as functioning on the brink of hypoxia; thus, we have hypothesized that HIF-1alpha is constitutively expressed and stabilized in the testis, where it could play a role in testicular homeostasis. Western blot analysis using nuclear proteins from liver, kidney, and testis revealed the presence of HIF-1alpha only in the testis. Immunohistochemistry confirmed this result and revealed that HIF-1alpha was specifically located in interstitial Leydig cells. Electromobility shift assays employing nuclear extracts from the TM3 Leydig cell line revealed that these cells express HIF-1alpha that is capable of binding DNA under normoxic conditions. Furthermore, we found that protein levels can be increased further when the TM3 cells are cultured under hypoxic conditions. Finally, transient transfections of TM3 Leydig cells revealed that the promoter of the mouse 3beta-hydroxysteroid dehydrogenase type 1 (Hsd3b1) gene, which encodes a key enzyme in testosterone production, is a potential target of HIF-1alpha. In conclusion, HIF-1alpha is constitutively present in the Leydig cells of the murine testis, where it potentially regulates Hsd3b1 transcription, and thus male reproductive function.

De Graaf's thread: the human epididymis.

The epididymis consists of a single, highly coiled and convoluted tubule that Antoine De Graaf, the famous 17th-century anatomist, likened to a thread thickening to a string. The uncoiled tubule is several meters long and sperm in transit through it become functionally mature under the under the influence of the tubule lumen's microenvironment. The regulation of that microenvironment and the manner by which it influences sperm maturation have been the topic of investigation for many years, though the study of the human epididymis directly is fraught with problems related to sample availability and condition. Nevertheless, investigations using a variety of mammalian tissue sources, human included, have resulted in significant advances in our understanding of both the biology and pathology of the organ. The epididymal functions of transporting, concentrating, maturing, and storing sperm are important to male fertility and their absence or significant impairment can be a factor in male infertility.

Oxidative stress: a common factor in testicular dysfunction.

Oxidative stress results from the production of oxygen radicals in excess of the antioxidant capacity of the stressed tissue. Many conditions or events associated with male infertility are inducers of oxidative stress. X-irradiation, for example, or exposure to environmental toxicants and the physical conditions of varicocele and cryptorchidism have been demonstrated to increase testicular oxidative stress, which leads to an increase in germ cell apoptosis and subsequent hypospermatogenesis. Such stress conditions can cause changes in the dynamics of testicular microvascular blood flow, endocrine signaling, and germ cell apoptosis. Testicular oxidative stress appears to be a common feature in much of what underlies male infertility, which suggests that there may be benefits to developing better antioxidant therapies for relevant cases of hypospermatogenesis.

Caspase-9-dependent pathway to murine germ cell apoptosis: mediation by oxidative stress, BAX, and caspase 2.

Ischemia-reperfusion (IR) of the testis results in germ-cell-specific apoptosis (GCA) and a reduction in daily sperm production. This has been correlated with and is dependent upon neutrophil recruitment to the testis. In a rat model of testicular IR, this has also been correlated with an increase in reactive oxygen species (ROS). We have investigated ROS in the mouse testis after IR and determined whether the observed GCA is mediated via a mitochondrial caspase-9-dependent pathway involving the upstream mediators caspase 2 and BAX. Mice were subjected to a 2-h period of testicular ischemia followed by reperfusion. An accumulation of 8-isoprostane, a marker of oxidative stress, occurred 4 h after reperfusion. Activation of a mitochondrial dependent pathway to GCA after testicular IR was determined based on the observations that both BAX and caspase 2 translocated to the mitochondria, and that an increase occurred in cytoplasmic cytochrome c. Moreover, microinfusion of a specific caspase 9 inhibitor significantly reduced active caspase 3 after testicular IR and the number of apoptotic germ cells. These results suggest that oxidative stress products accumulate in the testis following IR and demonstrate that the observed GCA is stimulated through a mitochondrial caspase-9-dependent pathway. The identification of the germ-cell apoptotic pathway induced after testicular IR, including the key players in the pathway subsequent to ROS (BAX, caspase 9, and caspase 2), aids our understanding of IR injury in the testis and provides a wider background for the development of therapeutic interventions to rescue testis function.

Differential gene expression among the proximal segments of the rat epididymis is lost after efferent duct ligation.

The epididymis has traditionally been divided into the caput, corpus, and cauda regions, which are further organized into intraregional segments. In the rat and mouse, these segments have high degrees of transcriptional differentiation, and what has traditionally been called the initial segment of the rat epididymis actually consists of three transcriptionally different intraregional segments. These segments are regulated by endocrine, lumicrine, and paracrine factors, whose relative importance remains a topic of investigation. In the present study, 15-day unilateral efferent duct ligation (EDL) was used to deprive ipsilateral rat epididymides of lumicrine regulation. Segments 1-4 of EDL epididymides and contralateral, sham-operated tissues were collected individually. Microarray analysis of gene expression was used to determine the effect of lumicrine factor deprivation on the transcriptome-wide gene expression of each segment studied. More than 11 000 genes were detected as being expressed in each of the four segments examined. More than 2000 genes responded significantly to EDL in segment 1, although this number of genes declined in each succeeding segment. Segments 1 and 2 of control tissues were the most different transcriptionally and the most affected by EDL. In the absence of lumicrine factors, the four segments regressed to a transcriptionally undifferentiated state, which was consistent with the less-differentiated histology seen after EDL. Interestingly, for an individual gene, lumicrine factor deprivation could stimulate expression in some segments and suppress expression in other segments. These results reveal a higher complexity to the regulation of rat epididymal segments than heretofore appreciated.

The rat epididymal transcriptome: comparison of segmental gene expression in the rat and mouse epididymides.

Regional differences along the epididymis are essential for the establishment of the luminal environment required for sperm maturation. In the current study, 19 morphologically distinct segments of the rat epididymis were identified by microdissection. Total RNA was isolated from each segment and subjected to microarray analysis. Segmental analysis of epididymal gene expression identified more than 16,000 expressed qualifiers, whereas profiling of RNA from whole rat epididymis identified approximately 12,000 expressed qualifiers. Screening a panel of normal rat tissues identified both epididymal-selective and epididymal-specific transcripts. In addition, more than 3500 qualifiers were shown to be present and differentially upregulated or downregulated by more than fourfold between any two segments. The present study complements our previous segment-dependent analysis of gene expression in the mouse epididymis and allows for comparative analyses between datasets. A total of 492 genes was shown to be present on both the MOE430 (mouse) and RAE230_2 (rat) microarrays, expressed in the epididymis of both species, and differentially expressed by more than fourfold in between segments in each species. Moreover, in-depth quantitative RT-PCR analysis of 36 members of the beta defensin gene family showed highly conserved patterns of expression along the lengths of the mouse and rat epididymides. These analyses elucidate global gene expression patterns along the length of the rat epididymis and provide a novel evaluation of conserved and nonconserved gene expression patterns in the epididymides of the two species. Furthermore, these data provide a powerful resource for the research community for future studies of biological factors that mediate sperm maturation and storage.