Epididymal genomics and the search for a male contraceptive.

This report represents the joint efforts of three laboratories, one with a primary interest in understanding regulatory processes in the epididymal epithelium (TTT) and two with a primary interest in identifying and characterizing new contraceptive targets (DSJ and SAJ). We have developed a highly refined mouse epididymal transcriptome and have used it as a starting point for determining genes in the human epididymis, which may serve as targets for male contraceptives. Our database represents gene expression information for approximately 39,000 transcripts, of which over 17,000 are significantly expressed in at least one segment of the mouse epididymis. Over 2000 of these transcripts are up- or down-regulated by at least four-fold between at least two segments. In addition, human databases have been queried to determine expression of orthologs in the human epididymis and the specificity of their expression in the epididymis. Genes highly regulated in the human epididymis and showing high tissue specificity are potential targets for male contraceptives.

Association of segmentation of the epididymal interstitium with segmented tubule function in rats and mice.

The epithelium of the epididymal tubule has different biological functions in different regions of the tubule. Each region is further organized into lobules or intra-regional segments surrounded by connective tissue septa (CTS). Epididymal segmentation has received little direct attention, yet there is considerable evidence that expression of mRNA and protein often begins or ends precisely at the CTS border of a segment. How such 'on-off' regulation occurs coincident with the passing of the tubule from one segment to the next is unknown. This study examined the segmentation of epididymides in rats and mice. The average adult Sprague-Dawley rat and C57BL/6 mouse caput, corpus and cauda epididymides has seven, two and four, and three, one and two segments, respectively. The apoptosis response of the caput epididymal epithelium to deprivation of lumicrine factors 24 h after efferent duct ligation in rats and the epididymal expression of a marker protein, beta-galactosidase, in mice were segmented precisely. This validated both at a general response and at a specific protein level that many epididymal functions are regulated within segments. Blue dextran (molecular weight 20000) and erythrocine red (molecular weight 880) dyes infused into the interstitial space of specific segments by micropuncture were retained by the CTS of the segments. In similar micropuncture experiments, [(3)H]H(2)O (molecular weight 18) was able to diffuse into an adjacent segment relatively freely whereas [(14)C]polyethylene glycol (molecular weight 4000) could not. These studies indicate that the interstitium of intra-regional segments is organized into different physiological compartments and that these compartments play a role in regulating the epididymal epithelium.

Response of the adult prostate to prepubertal and postpubertal obstruction of the vas deferens in the rat.

OBJECTIVES: To determine whether obstruction of the vas deferens alters several general measures of prostate development during puberty and during prostate maintenance in the adult rat. Previous reports have suggested the possibility that vasectomy results in alterations of prostate function in experimental animals and humans. METHODS: Adult rats and 10-day-old rats were subjected to bilateral sham operations or bilateral vasectomy, and the prostates were extirpated either 14 or 60 days later. The total prostate weight and dorsolateral and ventral lobe total protein per milligram tissue, DNA per milligram tissue, and DNA per milligram protein were determined. Dorsolateral and ventral prostate lobe sections from each group were also stained with hematoxylin-eosin and subjected to histologic examination. RESULTS: The histologic features of the adult rat prostate were not qualitatively altered by vasectomy whether it occurred before puberty or in adult animals with mature prostates. Furthermore, vasectomy did not significantly alter the prostate weight or the protein or DNA content of either the dorsolateral or ventral lobes of the prostate compared with the sham-operated animals of either age. CONCLUSIONS: Vas deferens obstruction does not significantly alter the parameters associated with the development or maintenance of the adult rat prostate measured in this study.

On the regulation of Crisp-1 mRNA expression and protein secretion by luminal factors presented in vivo by microperfusion of the rat proximal caput epididymidis.

Synthesis and secretion of certain epididymal proteins are regulated by lumicrine factors from the testis or from upstream regions of the excurrent ducts. Cysteine-rich secreted protein-1 (Crisp-1) is a major androgen regulated protein in the epididymal lumen fluid of the rat and other species. Previous research has demonstrated that disturbance of the luminal microenvironment through obstruction of the tract reduces Crisp-1 synthesis and secretion. The present study was undertaken to determine the influence of the luminal microenvironment on rat proximal caput epididymal Crisp-1 secretion into lumen fluid and on Crisp-1 gene expression in the same tubules. Western blot analysis demonstrated that Crisp-1 protein concentrations were reduced from control levels by perfusion with artificial caput fluid containing no testicular factors and were not increased by perfusion with fluids containing rete testis fluid proteins. Crisp-1gene expression was also reduced by perfusion with artificial caput fluid and not increased by perfusion with rete testis fluid proteins. Perfusion with artificial caput fluid containing 5alpha-dihydrotestosterone did increase one Crisp-1 transcript. This study demonstrates that intraluminal testicular proteins are not important co-regulators with androgens of Crisp-1gene expression or resulting Crisp-1 secretion into the rat proximal caput tubule lumen in vivo.

Essential role of neutrophils in germ cell-specific apoptosis following ischemia/reperfusion injury of the mouse testis.

This study investigates the role of neutrophils in ischemia-induced aspermatogenesis in the mouse. Previous studies in the rat have demonstrated that ischemia-inducing testicular torsion followed by torsion repair and reperfusion resulted in germ cell-specific apoptosis. This was correlated with an increase in neutrophil adhesion to subtunical venules, an increase in reactive oxygen species, and increased expression of several apoptosis-associated molecules. In the present investigation, wild-type C57BL/6 mice were subjected to various degrees and duration of testicular torsion. A torsion of 720 degrees for 2 h caused disruption of the seminiferous epithelium and significantly reduced testis weight and daily sperm production. An immunohistochemical method specific for apoptotic nuclei indicated that these effects were due to germ cell-specific apoptosis. An increase in myeloperoxidase (MPO) activity and an increase in the number of neutrophils adhering to testicular subtunical venules after torsion repair/reperfusion demonstrated an increase in neutrophil recruitment to the testis. In contrast, E-selectin knockout mice and wild-type mice rendered neutropenic showed a significant decrease in neutrophil recruitment as evidenced by MPO activity and microscopic examination of subtunical venules. Importantly, germ cell-specific apoptosis was also reduced. Thus, germ cell-specific apoptosis is observed after ischemia/reperfusion of the murine testis, and this apoptosis is directly linked to the recruitment of neutrophils to subtunical venules. Endothelial cell adhesion molecules, particularly E-selectin, play an important role in mediating this pathology.

The study of varicocele through the use of animal models.

The pathophysiology of the varicocele has received considerable study, both in humans and in animal models. Mechanistic information is difficult to obtain from human subjects because study designs must not be invasive and the subject population is variable in the status of the varicocele, patient age, fertility or other health-related issues. Because of these limitations, animal models of varicocele have been developed in several species, the most common being the rat. Surgery to establish the varicocele involves partial obstruction of the left renal vein, causing a varicosity of the left spermatic vein, including the pampiniform plexus. Studies using this model have shown that experimental left varicocele induces bilateral increases in testicular blood flow and temperature contemporaneous with decreases in intratesticular testosterone and testicular sperm output. Spermatic vein reflux is not related to the pathophysiological consequences of experimental varicocele. Many questions remain regarding the mechanism by which varicocele induces testicular dysfunction, chief among them being how the unilateral varicocele causes a bilateral testicular response in the first place.

Fluctuations in rat testicular interstitial oxygen tensions are linked to testicular vasomotion: persistence after repair of torsion.

Testicular microvascular blood flow is known to exhibit vasomotion, which has been shown to be significantly altered in the short term following the repair of testicular torsion. This loss of vasomotion may ultimately be responsible for the loss of spermatogenesis observed after testicular torsion in rats. In the present study, testicular vasomotion and interstitial oxygen tensions were simultaneously measured prior to, during, and at various time points after repair of testicular torsion in the rat. Testicular torsion was induced by a 720 degrees rotation of the testis for 1 h. Laser-Doppler flowmetry and an oxygen electrode were used to simultaneously measure vasomotion and interstitial oxygen tensions (PO(2)), respectively. Pretorsion control testes had a mean blood flow of 16.3 +/- 1.3 perfusion units (PU) and displayed vasomotion with a cycle frequency of 12 +/- 0.2 cycles per minute and a mean amplitude of 4.2 +/- 0.3 PU. Mean testicular interstitial PO(2) was 12.5 +/- 2.6 mm Hg, which displayed a cyclical variation of 11.9 +/- 0.4 cycles per minute with a mean amplitude of 2.8 +/- 0.8 mm Hg. During the torsion period, both mean blood flow and interstitial PO(2) decreased to approximately zero. Upon detorsion, mean microvascular blood flow and mean interstitial PO(2) values returned to values that were not significantly different from pretorsion values within 30 min; however, vasomotion and PO(2) cycling did not return, even after 24 h. It was 7 days after the repair of torsion before a regular pattern of vasomotion and PO(2) cycling returned. These results demonstrate for the first time a correlation between testicular vasomotion and interstitial PO(2) cycling, and this correlation persists after the repair of testicular torsion.

Molecular pathway of germ cell apoptosis following ischemia/reperfusion of the rat testis.

The present study investigates the molecular apoptotic pathway in germ cells following acute ischemia of the rat testis. Rats were subjected to ischemia-inducing torsion and testes were harvested after reperfusion. Apoptotic cells were identified with an antibody to single-stranded DNA. Seminiferous tubule RNA was examined by RNase protection assay or by reverse transcriptase-polymerase chain reaction (RT-PCR) for the presence and regulation of apoptotic molecules. Proteins from seminiferous tubules were used for Western blot analysis of cytochrome c. Germ cell apoptosis was maximal at 24 h after repair of torsion. Germ cells in stages II-III of the seminiferous epithelium cycle were the predominant early responders. The RNase protection assays revealed that Bcl-X(L) was the prominent mRNA species. Caspases 1, 2, 3, and Bax mRNA were consistently upregulated; however, the time of upregulation after torsion was variable. The Bcl-X(L) and Bcl-X(S) mRNAs were less consistently upregulated and there was no evidence for upregulation of Fas or Bcl-2. Fas ligand (FasL) was not detected by RNase protection assay, but RT-PCR revealed a significant increase in FasL expression 4 h after the repair of torsion. Western blot analysis for cytochrome c release demonstrated a significant increase 4 h after the repair of torsion. Results suggest that germ cell apoptosis following ischemia/reperfusion of the rat testis is initiated through the mitochondria-associated molecule Bax as well as Fas-FasL interactions.

Postvasectomy alterations in protein synthesis and secretion in the rat caput epididymidis are not repaired after vasovasostomy.

Many men who have undergone vasectomy later request vasovasostomy. Unfortunately, significant numbers of these men remain infertile despite the reestablishment of patent ducts. This report examines the possibility that epididymal function remains compromised after vasovasostomy in the rat by examination of quantifiable, in vivo protein synthesis and secretion in the caput epididymidis. Rats were studied 30 days after vasectomy, 30 days after a vasovasostomy (which was performed 30 days after vasectomy), or after sham operations. Epididymal lumen fluids (LF) were collected by micropuncture after 3 hours' in vivo microperifusion of tubules with 35S-amino acids. Proteins were separated by 2-dimensional electrophoresis and were detected by Coomassie blue staining. Synthesized proteins in tubule extract and synthesized and secreted proteins in LF were detected by autoradiography and image analysis. Specific proteins that appeared to be affected by vasectomy-vasovasostomy were identified by internal sequence analysis. LF contained an average of 87 detectable proteins synthesized and secreted in the control caput. Nineteen of the most prominent LF proteins were selected for more focused study. The most prominent proteins were clusterin, cysteine-rich secretory protein (CRISP)-1, and epididymal retinoic acid-binding protein. Among these, CRISP-1 remained reduced in LF after vasovasostomy. Two more minor proteins that remained reduced after vasovasostomy were identified as prostaglandin D2 synthase and phosphatidylethanolamine-binding protein. All 3 of these proteins occur in the epididymides of multiple species and have been associated with sperm fertilizing capacity.

A blood-prostate barrier restricts cell and molecular movement across the rat ventral prostate epithelium.

INTRODUCTION: Blood-epithelial barriers have been described in the testis and epididymis, but the possibility of such barriers in other regions of the male genitourinary tract has received little investigation. The purpose of this study was to use in vivo micro-puncture to determine if the blood-epithelial barrier exists in the rat ventral prostate. In addition, using a model of prostatic inflammation, we sought to examine the effect of inflammation on the passage of blood borne molecules and leukocytes into the prostatic ductal lumen. MATERIALS AND METHODS: Adult Sprague-Dawley rats were divided into two groups, control and 24-hour lipopolysaccharide (LPS)-induced inflammation. Both groups were subjected to vascular infusion of radiolabeled 3H dextran, 14C urea, and 3H water. Contemporaneous in vivo micropuncture sampling of prostatic ductal fluid (DF) and arterial blood occurred at multiple time points over 120 minutes. Transepithelial movement of radiolabeled compounds at each sampling time point was quantified by the expression of DF isotope concentrations as a percentage of serum isotope concentrations at that time point. Histology of representative specimens of control and inflamed prostates was used to confirm the inflammatory response and to examine for the presence of leukocytes into the ductal lumen. RESULTS: The transepithelial movement of radiolabeled compounds from blood to prostatic lumen varied in direct relationship to the compound's molecular weight. 3H-water (MW = 18) movement into the ductal lumina was relatively rapid plateauing at 70-80% of serum values. 14C urea (MW = 60) achieved intermediate penetration into ductal fluid (50-60% of serum values) and 3H dextran (MW = 2 x 106) was essentially excluded from entry (<2% of serum). These results were not altered by LPS-induced inflammation. Histology revealed a diffuse leukocyte infiltrate in the inflamed prostatic interstitium, but penetration of inflammatory cells into the ductal lumen was very restricted. CONCLUSIONS: Our findings demonstrate a blood-prostate barrier in the rat ventral prostate with characteristics similar to the blood-testis barrier. This blood-prostate barrier is not affected by LPS-induced acute inflammation. Further, this persistent barrier apparently restricts the passage of leukocytes into prostate DF even in the presence of pronounced interstitial inflammation. This observation may help to explain the observation that expressed prostatic secretions in human males are often free of leukocytes in clinical prostatitis.

Effect of inflammation on prostatic protein synthesis and luminal secretion in vivo.

PURPOSE: Inflammation of the prostate, or prostatitis, can be caused by an infectious process or can occur in a reportedly non-bacterial form, the etiology of which is largely unknown. The present study was undertaken to establish a method of studying prostatic protein synthesis and secretion in vivo and determine the effects of lipopolysaccharide (LPS)-induced prostatic inflammation on these processes. MATERIALS AND METHODS: Sprague-Dawley rats were divided into three groups: control, 24 hours LPS-inflammation, and 24 hours LPS + antibody against tumor necrosis factor (anti-TNF). 35S-methionine was perifused in vivo around ventral prostate ducts for 3 hours. Ductal fluid (DF) was collected by micropuncture and ductal extract (DE) was collected by tissue homogenization. DE and DF were then subjected to SDS-PAGE and autoradiography. Densitometric analysis of gels and autoradiograms was used to compare protein synthesis (total DE 35S-proteins) and protein secretion (DF 35S-proteins) among the three groups. RESULTS AND CONCLUSIONS: The method proved to be effective for studying prostatic protein synthesis and secretion in vivo. LPS-induced inflammation caused an increase in total 35S-proteins in both the DE and the DF when compared with controls. There were significant increases in both the total number of proteins produced as well as the densitometric quantity of protein in the inflamed group. Some specific prostatic proteins were also upregulated by inflammation. The addition of anti-TNF did not significantly alter inflammation-induced protein synthesis or secretion at the time/dose studied.

Obstruction of the vas deferens alters protein secretion by the rat caput epididymidal epithelium in vivo.

Obstruction of epididymal lumen fluid flow alters the intraluminal environment and potentially changes epididymal epithelial cell function when those functions are dependent on intraluminal regulatory molecules. This investigation tested the hypothesis that obstruction of the rat vas deferens alters caput epididymidal protein synthesis and secretion In vivo. Adult male rats were subjected to vasal obstruction or sham operation. Fourteen days later, caput epididymides were subjected to in vivo microperfusion with medium containing a [35S]-amino acid mixture. At the end of a 3-hour perifusion, micropuncture was used to obtain caput lumen fluid (LF). Tubule extract (TE) was obtained as supernatant after homogenization and centrifugation of caput tubules. Tubule extract contained all [35S]-proteins synthesized within the 3-hour experiment, and LF contained the secreted [35S]-proteins. Radioactivity of trichloroacetic acid (TCA)-precipitable proteins in LF and TE was determined, and two-dimensional electrophoresis and autoradiography of each sample were carried out. The resultant autoradiograms were evaluated densitometrically. A protein synthesis index calculated from the TCA-precipitable radioactivity data demonstrated that a significant decline in overall protein synthesis was induced by vasal obstruction. Densitometry of autoradiograms demonstrated that the total number of radiolabeled proteins detected in both the LF and TE of obstructed animals was significantly smaller than the same number in control animals (P < 0.05). Autoradiography revealed seven major, consistently appearing gene products in LF, and these were subjected to amino acid sequence analysis. Cysteine-rich secretory protein (CRISP)-1 proteins were significantly reduced in the LF of obstructed animals, which implies that these proteins are dependent on luminal regulatory molecules for their normal production.

p53 independent, region-specific epithelial apoptosis is induced in the rat epididymis by deprivation of luminal factors.

Luminal testicular factors are known to be important for the regulation of the epididymal epithelium. The present study was undertaken to test the hypothesis that complete deprivation of luminal factors by efferent duct ligation (EDL) would induce apoptosis in the epididymal epithelium, as does removal of trophic factors from other cell types. Additionally, experiments were performed to determine whether the apoptosis detected was p53 dependent or independent. Apoptosis detection was by terminal deoxynucleotidyl-mediated deoxyuridine triphosphate-biotin nick-end labeling and by DNA fragmentation studies. EDL caused loss of testicular luminal contribution in zone 1A of the rat epididymis (proximal initial segment) within 6 hr and induced epithelial apoptosis within 12 hr of the efferent duct obstruction. The wave of apoptosis in zone 1A was completed by three days after EDL and was followed by a much smaller wave in zone 1B which peaked three days after EDL. Significant apoptosis was not detected in any epididymal region distal to the initial segment for periods as long as 15 days after EDL. p53, a key apoptotic-pathway molecule in many tissues and conditions was tested by immunohistochemical and Western blot techniques and was not upregulated in the initial segment epithelium within the time cells were undergoing apoptosis and well before the wave of apoptosis was complete. It was concluded that epithelial apoptosis in the initial segment of the rat epididymis is induced by deprivation of luminal testicular factors, is localized to the proximal and middle initial segment, and is p53 independent.

Microvascular blood flow is altered after repair of testicular torsion in the rat.

PURPOSE: Previous studies have shown that experimental testicular torsion with a duration of 1 hr. or longer causes irreversible damage to the rat testis, but that testicular blood flow values are normal 24 hrs. after repair of torsion. More acute evaluation of return blood flow after repair of torsion has not been performed and was the topic of this study. MATERIALS AND METHODS: Laser-Doppler flowmetry was used to evaluate testicular microvascular blood flow before application of 1, 2, or 4 hr., 720 degrees torsion, during torsion, and at several time points after repair of torsion. Experiments were performed in both adult and prepubertal rats. RESULTS: Testicular torsion essentially eliminated blood flow in both adult and prepubertal testes. Considering all the flow data within each group after torsion repair, increasing time of torsion was associated with significantly less return blood flow in both adult and prepubertal animals. Interestingly, only the four hour torsion data was associated with reduced return flow in prepubertal animals while both two and four hour torsion were associated with poor return flow in adult animals. Vasomotion, or pulsatile microvascular flow, often seen before torsion in both adult and prepubertal animals, was never seen after torsion repair. CONCLUSIONS: Increasing times of torsion are associated with lower microvascular blood flow values during the hour following the relief of torsion. Vasomotion is eliminated by torsion during the period studied. Whether vasomotion returns is unknown, but altering this flow pattern might be involved in the mechanism of injury caused by acute torsion.

Rescue of testicular function after acute experimental torsion.

PURPOSE: Spermatic cord torsion results in impairment of testicular function. The mechanism of this injury is unclear; however, intracellular Ca++ influx and reactive oxygen species have been implicated in the testicular damage following torsion and reperfusion. In the present study a model of testicular torsion in the rat was used to determine whether testicular function following torsion can be rescued by the administration of antioxidants and Ca++ channel blockers. MATERIALS AND METHODS: Seventy-two animals were divided into 9 groups. Animals underwent 1 hr. or 2 hrs., 720 degrees experimental torsion. Animals received combinations of superoxide dismutase (SOD), catalase, allopurinol, and verapamil. Drugs were administered intravenously during the last 15 minutes of experimental torsion and the first hour of reperfusion. Bilateral testicular function was determined 60 days after experimental torsion by measuring testis weights, daily sperm production (DSP), and testicular venous testosterone concentrations. Ipsilateral values were compared to both control and contralateral values. RESULTS: SOD + catalase and SOD + catalase+verapamil treatments caused significant rescue of tests function following 1 hr. experimental torsion. Mean +/- s.e. testis weights and DSP in control animals were 1.75 +/- 0.6 g. and 18.4 +/- 0.3 x 10(6) sperm/g./d. The same value for testes experiencing 1 hr. experimental torsion were 0.72 +/- 0.6 g. and 2.3 +/- 0.5 x 10(6) sperm/g./d. The values from testes receiving 1 hr. experimental torsion followed by SOD + catalase were 1.45 +/- 0.17 g. and 9.9 +/- 1.8 x 10(6) sperm/g./d. Neither allopurinol nor verapamil added benefit. No significant rescue was seen in testes undergoing 2 hrs. experimental torsion. CONCLUSIONS: Treatment with oxygen radical scavengers provides significant rescue of testicular function after acute experimental torsion.

Acute testicular ischemia results in germ cell-specific apoptosis in the rat.

Testis torsion-induced aspermatogenesis is not necessarily due to permanent loss of blood flow nor to dysfunctional Leydig cells or Sertoli cells. This investigation was undertaken to gain further insight into the mechanism underlying torsion-induced germ cell loss. Male rats were subjected to 1-h or 2-h ischemia-inducing torsion, and testes were examined at either 1, 2, 4, 24, or 48 h after torsion, depending on the study. Testes were examined for evidence of 1) in situ apoptosis by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labeling (TUNEL) technique, 2) apoptosis by the DNA "laddering" technique, 3) leukocyte margination and diapedesis in testicular vessels by immunocytochemical and histological techniques, and 4) testicular lipid peroxidation by the thiobarbituric acid reactive substances assay. The first TUNEL evidence for torsion-induced apoptosis was at 4 h after repair of 1-h torsion. Induction of apoptosis was confirmed by the electrophoretic laddering of DNA fragments. It was hypothesized that apoptosis was induced by reactive oxygen species arising from reperfusing leukocytes. A significant increase in both leukocyte margination and diapedesis occurred 4 h after torsion repair as did a significant increase in intratesticular lipid peroxidation products. These events were contemporaneous with the first appearance of apoptosis and consistent with the hypothesis that post-torsion, germ cell-specific apoptosis is induced by reactive oxygen species.