Gene expression profile during testicular development in patients with SRY-negative 46,XX testicular disorder of sex development.

OBJECTIVE: To elucidate alternative pathways in testicular development, we attempted to clarify the genetic characteristics of SRY-negative XX testes. MATERIALS AND METHODS: We previously reported 5 cases of SRY-negative 46,XX testicular disorders of sex development and demonstrated that coordinated expression of genes such as SOX9, SOX3, and DAX1 was associated with testicular development. We performed a case-control study between the aforementioned boy with 46,XX testicular disorders of sex development and an age-matched patient with hydrocele testis (46,XY). During their consecutive surgeries, testicular biopsy specimens were obtained. Genes with differential expression compared with XY testis were identified using polymerase chain reaction (PCR)-based subtractive hybridization and sequencing. For validation of differential gene expression, real-time RT-PCR was performed using gene-specific primers. The distribution of candidate proteins in the testicular tissue was clarified by immunohistochemistry in human and rodent specimens. Moreover, in vitro inhibitory assays were performed. RESULTS: We identified 13 upregulated and 7 downregulated genes in XX testis. Among the candidate genes, we focused on ROCK1 (Rho-associated, coiled-coil protein kinase 1) in the upregulated gene group, because high expression in XX testis was validated by real-time RT-PCR. ROCK1 protein was detected in germ cells, Leydig cells, and Sertoli cells by immunohistochemistry. Moreover, the addition of specific ROCK1 inhibitor to Sertoli cells decreased SOX9 gene expression. CONCLUSION: On the basis of in vitro inhibitory assay, it is suggested that ROCK1 phosphorylates and activates SOX9 in Sertoli cells. Testes formation might be initiated by an alternative signaling pathway attributed to ROCK1, not SRY, activation in XX testes.

Measurement of hydroxyproline and nitric oxide, and comparison of sac fluid acidity in different inguinal pathologies.

PURPOSE: To compare hydroxyproline and nitric oxide levels, and fluid acidity between sac tissues from inguinal hernia, hydrocele and undescended testis patients. MATERIAL AND METHOD: Thirty patients were in the inguinal hernia group, 11 patients in the hydrocele group and 5 patients in the undescended testis with inguinal hernia group. Intrasaccular fluid acidity, and nitric oxide and hydroxyproline levels in sac tissues were measured and analyzed statistically between groups. RESULTS: Intrasaccular fluid could be obtained from 24 inguinal hernia and 6 hydrocele patients. Acidity level (pH) was 7.5 ± 0.74 in the hernia group and 7.86 ± 0.63 in the hydrocele group (p < 0.05). All patients in the three groups were evaluated for nitric oxide and hydroxyproline. Results for hydroxyproline (mg/g dry tissue)/nitric oxide (nmol/g wet tissue) were 3.53 ± 0.54/2.02 ± 0.39 for inguinal hernia, 2.89 ± 0.9/1.92 ± 1.05 for hydrocele and 3.02 ± 1.23/2.86 ± 1.09 for undescended testis group (p > 0.05). CONCLUSION: Hydroxyproline and nitric oxide pathways do not seem to be responsible for the different characteristics of inguinal diseases. However, differences in the acidity of sac fluid may be evidence of the different mechanisms of hernia and hydrocele formation.

Expression of anti-Mullerian hormone receptor on the appendix testis in connection with urological disorders.

The female internal sex organs develop from the paramesonephric (Mullerian) duct. In male embryos, the regression of the Mullerian duct is caused by the anti-Mullerian hormone (AMH), which plays an important role in the process of testicular descent. The physiological remnant of the Mullerian duct in males is the appendix testis (AT). In our previous study, we presented evidence for the decreased incidence of AT in cryptorchidism with intraoperative surgery. In this report, the expression of the anti-Mullerian hormone receptor type 2 (AMHR2), the specific receptor of AMH, on the AT was investigated in connection with different urological disorders, such as hernia inguinalis, torsion of AT, cysta epididymis, varicocele, hydrocele testis and various forms of undescended testis. The correlation between the age of the patients and the expression of the AMHR2 was also examined. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the receptor's mRNA and protein levels, respectively. We demonstrate that AMHR2 is expressed in the ATs. Additionally, the presence of this receptor was proven at the mRNA and protein levels. The expression pattern of the receptor correlated with neither the examined urological disorders nor the age of the patients; therefore, the function of the AT remains obscure.

Reactive pseudo-glandular mesothelial hyperplasia in testis tunica vaginalis: a case report.

A case of benign reactive pseudo-glandular mesothelial hyperplasia arising in the context of chronic vaginitis in presented: morphological and immunohistochemical investigations and differential diagnoses are described.

Smooth muscle cell differentiation in the processus vaginalis of children with hernia or hydrocele.

PURPOSE: Incomplete obliteration of the processus vaginalis (PV) in children with inguinal hernia or hydrocele has recently been proposed to relate to smooth muscle cell (SMC) persistence. The aim of this study was to evaluate the diversity and differentiation of smooth muscle phenotypes in sacs associated with inguinal hernia and hydrocele through the expression of alpha-smooth muscle actin (SMA), h-caldesmon, desmin, and vimentin. METHODS: Sacs associated with male hernia (n = 22), female hernia (n = 8), and hydrocele (n = 10) were immunohistochemically evaluated using monoclonal antibodies against SMA, h-caldesmon, desmin, and vimentin. Peritoneal samples (male, 4; female, 3) and obliterated PV (male, 3) obtained from age-matched patients served as controls. Expressions according to the groups were compared through chi-squared test, and P values less than 0.05 were considered to be statistically significant. RESULTS: Immunohistochemistry did not shown the presence of SMCs in control samples. The expression of SMA, desmin, and h-caldesmon did not differ among sacs obtained from patients with inguinal hernia and hydrocele. However, strong expression of vimentin in SMCs within sacs obtained from patients with hydrocele in comparison with sacs from male patients with inguinal hernia were observed. CONCLUSIONS: Our results indicate that sacs from patients with inguinal hernias and especially from male inguinal hernias have fully differentiated SMCs. On the other hand SMCs in sacs obtained from boys with hydrocele are in an intermediate state of differentiation-dedifferentiation. This phenotypic modulation may represent attempted apoptosis of SMCs, since sacs more sensitive to apoptosis appeared to have more dedifferentiated SMCs. It also probably depicts the differing influence of sympathetic and parasympathetic tonuses during the descent of the testis and the obliteration of PV.

Evaluation of extracellular matrix protein composition in sacs associated with undescended testis, hydrocele, inguinal hernia, and peritoneum.

OBJECTIVES: To investigate and compare the distribution and intensity of staining of extracellular matrix proteins--laminin, fibronectin, and types 1 and 4 collagen--in various congenital inguinoscrotal abnormalities and the peritoneum through immunohistochemical staining. METHODS: The sacs associated with undescended testis (n = 28), hydrocele (n = 29), inguinal hernia (n = 31), and parietal peritoneum (n = 28) were stained with antibodies for laminin, fibronectin, and types 1 and 4 collagen. The peritoneum served as the control group. Appropriate tissue sections of each group of samples were identified and compared with regard to the intensity and distribution of staining. RESULTS: The expression of laminin was significantly greater in the sacs associated with undescended testes and inguinal hernia than in the peritoneum. The expression pattern of fibronectin did not differ in the sacs of the different inguinoscrotal abnormalities compared with peritoneum. Type 1 collagen was strongly expressed in the sacs obtained from the hydroceles compared with the other groups and the peritoneum. Expression of type 4 collagen was significantly increased in the sacs associated with hydrocele and inguinal hernia compared with the peritoneum. CONCLUSIONS: The distribution and expression of laminin and types 1 and 4 collagen showed variability in the sacs associated with undescended testis, hydrocele, and inguinal hernia and the peritoneum. The tensile forces that the sacs associated with childhood inguinoscrotal abnormalities are exposed to per se could account for some of the differences seen between the inguinoscrotal tissues and anterior peritoneum. These differences in the extracellular matrix proteins could also reflect congenital properties of various childhood inguinoscrotal abnormalities.

Do alterations in collagen synthesis play an etiologic role in childhood inguinoscrotal pathologies: an immunohistochemical study.

BACKGROUND/PURPOSE: Previous findings have suggested that the development of adult inguinal hernias could be related to alterations in fibrillar collagen synthesis in the hernial sac as a decrease in the ratio of the relative amounts of type I/III collagen. The aim of this study was to investigate whether an alteration in type I and type III collagen synthesis was associated with the development of childhood inguinoscrotal pathologies. METHODS: The authors analyzed sacs from patients with inguinal hernia (n = 20), hydrocele (n = 10) and undescended testis (n = 10) immunohistochemically using monoclonal antibodies against alpha-smooth muscle actin, collagen type I and III. Peritoneal samples (n = 10) obtained from age-matched patients served as controls. Immunostaining was evaluated by semiquantitative scoring and chi2 test. RESULTS: The expression pattern of type I and III collagen did not differ among sacs obtained from patients with inguinal hernia, hydrocele, and undescended testis when compared with that of controls. However, strong expression of type III collagen was observed in the hernial sacs of right-sided male inguinal hernia compared with left side. CONCLUSIONS: Although altered collagen synthesis was reported to play an important role in the development of adult inguinal hernias, our results indicate that a pivotal role in childhood inguinoscrotal pathologies is not likely.

Autonomic nervous system appears to play a role in obliteration of processus vaginalis.

BACKGROUND: Failed obliteration of processus vaginalis (PV) has recently been proposed to be due to persistence of the smooth muscle (SM), which is transiently present to generate the physical force to descend the testis. Sacs from hernia, hydrocele, or undescended testis reveal alterations in Ca(2+) contents. Since Ca(2+) signaling and some regulatory proteins are involved in programmed cell death (PCD), a flow cytometric evaluation was planned to evaluate the expression of inducers or inhibitors of PCD in sacs from different diagnostic sources. METHODS: Sacs associated with male hernia ( n=16), female hernia ( n=11), undescended testis ( n=9), and hydrocele ( n=11) were evaluated for binding of annexin and expressions of Bcl-2, Bax, Fas, Fas-ligand (Fas-L), and Fas+Fas-L. The binding and expressions in cells that express alpha-smooth muscle actin (alpha-SMA) were also determined. Expressions according to the groups were compared through unpaired t-test, and P values less than 0.05 were considered to be significant. RESULTS: Sacs associated with undescended testis that contain the least SM revealed the most annexin binding, and sacs associated with hernia that contain the most SM binded the least annexin. However, expressions of Bcl-2, Bax, Fas, Fas-L and Fas+Fas-L have not revealed a significant difference. On the other hand, Bax and Fas expressions of cells that express alpha-SMA have been significantly higher in sacs associated with undescended testis. CONCLUSIONS: Since increase of Bax and Fas in cells that express alpha-SMA are encountered in sacs with the least SM content, Bax and Fas appear to play roles in the amount of persisting SM. The necessities of initial depletion of Ca(2+) stores through inositol 1,4,5-trisphosphate receptors and subsequent increase of Bax and Fas levels in the mitochondrial pathway of PCD, together with the role of the sympathetic system in maintenance of SM, suggest a determinative role of the autonomic nervous system for obliteration of PV.

Paratesticular mesothelial proliferations.

Varied mesothelial proliferations are found around the testes. Benign reactive mesothelial proliferations in hydrocoeles may be very florid and histologically worrisome, but these can usually be separated from malignant mesotheliomas of the tunica vaginalis because they are confined to a sharply demarcated zone near the luminal surface. In addition, benign inflamed hydrocoeles tend to show a distinct zonation with cellular areas near the luminal surface and more fibrotic areas beneath. The proliferating mesothelial cells in benign reactions often form lines that are parallel to the surface of the hydrocoele. Malignant mesotheliomas of the tunica vaginalis are usually grossly visible as single or multiple nodules. Histologically, they show an epithelial- or mixed epithelial- and sarcomatous-morphologic image, with evidence of stromal invasion. Well-differentiated papillary mesotheliomas are usually solitary exophytic nodules with a distinctive morphologic appearance and benign course, but they must be carefully separated from malignant mesotheliomas with a focally exophytic papillary growth pattern. By definition, well-differentiated papillary mesotheliomas do not exhibit stromal invasion. Peritesticular adenomatoid tumors are also very common; they are benign circumscribed cellular nodules with an appearance comparable to that of adenomatoid tumors in other body locations such as the uterine serosa.

Total calcium content of sacs associated with inguinal hernia, hydrocele or undescended testis reflects differences dictated by programmed cell death.

INTRODUCTION: Inguinal hernia and hydrocele are suggested to result from the persistence of smooth muscle (SM) which should undergo programmed cell death (PCD) after presenting transiently to propel the testis. Since Ca(2+) is involved in PCD, the Ca(2+) contents of the peritoneum and sacs associated with undescended testis, inguinal hernia and hydrocele were determined and compared. MATERIALS AND METHODS: Sacs were obtained from boys with undescended testis (n = 11), inguinal hernia (n = 22) and hydrocele (n = 10), and girls with inguinal hernia (n = 7). The calcium content of the sacs and peritoneal samples (n = 6) was determined through atomic absorption spectrophotometry. Calcium contents were compared according to their sources using the Mann-Whitney U test and p values of <0.05 were considered significant. RESULTS: While revealing similar Ca(2+) contents as the peritoneum, sacs associated with undescended testis and hydrocele contained more Ca(2+) contents than the sacs of boys and girls with inguinal hernia (p < 0.05). CONCLUSIONS: Sacs associated with inguinal hernia, which are known to contain SM all around the mesothelial layer, contain the least Ca(2+). Despite the decrease in SM, sacs associated with hydrocele contain more Ca(2+). Since PCD is associated with Ca(2+) overload and inhibition of Ca(2+) load inhibits PCD, differences in Ca(2+) content may reflect the inhibition of PCD at different stages and for different reasons in inguinal hernia or hydrocele of childhood.

Less calcium in cremaster muscles of boys with undescended testis supports a deficiency in sympathetic innervation.

In addition to an increase in contractility, contracted fibers and small and electron-dense mitochondria have suggested an increase of cytosolic Ca(2+) within the cremaster muscles (CM) associated with undescended testis. Therefore, the Ca(2+) content of CM associated with an undescended testis was determined and compared with the Ca(2+) content associated with inguinal hernia and hydrocele and in internal oblique muscles. CM samples from boys with undescended testis (n = 9), inguinal hernia (n = 15), and hydrocele (n = 7) and from girls with inguinal hernia (n = 8) were obtained. The calcium contents of these samples and of samples from internal oblique muscle (n = 6) were determined by atomic absorption spectrophotometry. While the Ca(2+) contents of CM from boys with undescended testis and from girls with inguinal hernia were similar (p > 0.05), the internal oblique muscle and the CM from boys with inguinal hernia and hydrocele contained more Ca(2+) than CM from boys with undescended testis (p < 0.05). Despite evidence of an increase in cytosolic calcium, the decrease in total calcium content suggests a decrease in the adenylyl cyclase activity, thus inhibition of influx of Ca(2+), but an increase in phospholipase C activity, and generation of more inositol 1,4,5-trisphosphate and mobilization of calcium from internal stores. Since the sympathetic system acts through adenylyl cyclase in striated muscles and is sexually dimorphic, similar Ca(2+) contents as encountered in girls suggest an alteration in the balance of autonomic innervation against the sympathetic system in boys with undescended testis.

Tumor markers in hydrocele fluids of patients with benign and malignant scrotal diseases.

PURPOSE: We evaluated the presence of human chorionic gonadotropin (hCG), alpha-fetoprotein (AFP) and a panel of other tumor markers in the compartment next to the tumor (that is, the malignant hydrocele fluid). MATERIALS AND METHODS: We measured hCG, AFP, neuron-specific enolase, carcinoembryonic antigen and cytokeratin-19 fragments in cubital vein sera and in hydrocele fluids of patients with testicular cancer. Results were compared with those obtained from hydrocele fluids of patients with benign disease. RESULTS: All tumor markers remained under the respective cutoff values in benign hydroceles. In patients with pure seminomas, hCG levels were elevated in 66% of hydroceles but only once in peripheral sera, whereas AFP remained low in both compartments. Furthermore, of 11 cases of nonseminomatous germ cell tumor hydrocele fluids, 3 with negative peripheral tumor marker values had to be reclassified marker positive, of which 2 showed elevated hCG levels and 1 had increased levels of AFP. Significant changes of neuron-specific enolase and carcinoembryonic antigen concentrations could not be observed. However, a cytokeratin-19 fragment measured by Cyfra 21-1 assay was elevated in 2 of 3 seminomatous and in 4 of 8 nonseminomatous hydroceles. CONCLUSIONS: These data give a new insight into the in vivo secretion pattern of testicular germ cell neoplasms, which demonstrates that the term "marker negative" should be restricted to selected cases of testicular cancer. Analysis of tumor markers in hydrocele fluids may be a helpful tool in patients with scrotal swelling if clinical and sonographic results remain uncertain.

Cholesterol crystals in hydroceles: sonographic detection and possible significance.

OBJECTIVE: The aim of this study of seven patients who had elective surgical repairs of a hydrocele was to try to differentiate by microscopy and chemical analysis hyperechoic hydrocele fluid from the more typical anechoic hydrocele fluid. CONCLUSION: Hyperechoic hydrocele fluid is related to the presence of cholesterol crystals. Cholesterol crystals were noted on microscopic examination in all three patients with hyperechoic hydrocele fluid. No cholesterol crystals were evident in the four patients with typical anechoic hydrocele fluid.

Metallothionein in human seminal plasma.

Metallothionein (MT) concentrations were measured in the seminal plasma of 4 fertile and 35 infertile men and in the hydrocele and spermatocele fluids. The relationship between MT content and sperm density, total number of sperm per ejaculate, sperm motility and abnormal form rates, leukocyte count and zinc levels in seminal plasma, as well as the relationship between MT and serum follicle-stimulating hormone, luteinizing hormone, testosterone, and prolactin were examined. MT was not detected in the hydrocele and spermatocele fluids. MT levels were related to zinc levels and to the leukocyte count in seminal plasma, but there was no correlation between MT and the other factors examined. This study supported previous findings that MT was secreted predominantly from the prostate and induced by inflammation of the prostate gland or seminal vesicles; the findings suggest that MT binds mainly to zinc and is one of the zinc-binding proteins in seminal plasma.

Biochemical analysis of tunica vaginalis fluid in patients with or without idiopathic hydroceles.

OBJECTIVE: To establish the differences, if any, between the biochemical composition of idiopathic hydrocele fluid and the fluid normally present in the tunica vaginalis. PATIENTS AND METHODS: Aspiration and sclerotherapy of 37 idiopathic hydroceles from patients who presented to this urology clinic were performed. The biochemical content of the fluid was compared with that of the tunica vaginalis fluid from a small group of controls (n = 8), taken from patients undergoing orchidectomy for carcinoma of the prostate. RESULTS: Differences in several measurements were recorded; in particular, there were significantly higher concentrations of calcium, albumin, total protein and creatine-kinase in the hydrocele group. The levels of potassium, aspartate transaminase, alanine transaminase and alkaline phosphatase were significantly lower in the hydrocele group. CONCLUSION: Whether these differences have a role in the causation of an idiopathic hydrocele is, at this stage, speculative. A similar study on a larger scale would probably be more conclusive.

Human chorionic gonadotropin (hCG) and its free subunits in hydrocele fluids and neoplastic tissue of testicular cancer patients: insights into the in vivo hCG-secretion pattern.

To obtain insight into the secretion pattern of human chorionic gonadotropin (hCG) and its free subunits, hCG alpha and hCG beta, in vivo, we analyzed hydrocele fluids of 13 patients with testicular cancer and correlated the respective values to those of cubital vein and testicular vein serum. As a control population, patients with nonmalignant hydroceles (n = 11) were studied. Analyses were performed with a set of highly sensitive and specific time-resolved fluoroimmunoassays based on our own panel of monoclonal antibodies. In the collective of testicular cancer patients, increased hydrocele levels of either hCG or free hCG alpha or free hCG beta were observed in 77, 54, and 92% of cases; the corresponding percentages for cubital vein serum were 62, 23, and 31%. The cubital vein ratio of hCG:hCG alpha (546:1) and hCG:hCG beta (51:1) decreased to 64:1 and to 7:1 in the hydrocele fluids. Surprisingly, hydrocele fluids of five patients with pure seminoma, who were negative for the three markers in the periphery, revealed an elevation of free hCG beta in all cases, while hCG alpha and holo-hCG were elevated twice. Final proof that hCG beta and hCG alpha are indeed produced by these previously termed "marker negative" seminomas has been achieved by reverse transcriptase-polymerase chain reaction with primers specific for the alpha-subunit and the four most abundantly transcribed hCG beta genes 3, 5, 7, and 8. From these data, we conclude that: (alpha) seminomatous and nonseminomatous testicular cancers, irrespective of histology, secrete hCG and its free subunits; (b) the amount of free subunits being secreted in vivo by these tumors has been underestimated; and (c) the classification in marker-positive and marker-negative testicular cancer should be reconsidered.

Leydig cell tumor: estradiol level at different locations.

The authors report on estradiol levels at different locations in a patient with a Leydig cell tumor. The highest value was found in the testicular vein. The estradiol level was, however, ten times higher in the peritesticular vaginal fluid than in the peripheral veins.

Composition of fluids obtained from human epididymal cysts.

The fluid composition of five epididymal spermatocoeles, one epididymal cyst and a hydrocoele was examined. The fluid obtained from the spermatocoeles was a dilute suspension of mainly immotile spermatozoa. The sperm-free fluid contained less protein, phosphate, glucose, triglyceride and cholesterol than serum but more testosterone and chloride than peripheral blood. It contained no epididymal secretion products. Proteins in the fluid differed from those in serum. From the fluid composition these cysts appeared to be continuous with the rete testis, either dilatations of efferent ducts or Haller's superior aberrant duct (vas aberrans of the rete testis). Fluid from an epididymal cyst containing no spermatozoa was mainly of similar composition. In contrast, hydrocoele fluid resembles blood serum.

The absorption of chlortetracycline following transscrotal instillation for the treatment of primary hydrocele testis.

We describe the absorption of chlortetracycline from the emptied hydrocele sac, which was instilled to treat a primary hydrocele of the testis. The study included 7 patients and 2 control subjects who were given 500 mg. chlortetracycline. Plasma concentration was determined at 0, 1/2, 1, 2, 4, 6, 12 and 24 hours after instillation, and in 2 patients plasma levels also were determined at 48 and 72 hours. The area under the plasma concentration-time curve (AUC1) was calculated in each case by using the trapezoidal rule. In the control group the plasma level was determined at 0, 1, 2, 4 and 6 hours after instillation. The area under the plasma concentration-time curve (AUC2) was calculated by the formula AUC2 = Co/Kc. The fraction F of absorption can be expressed as F = AUC1/AUC2; median F = 0.67 (range 0.41 to 0.92). We conclude that chlortetracycline passes readily and almost completely through the tunica vaginalis lining the hydrocele sac.

Transforming growth factor beta-like activity in human hydrocele fluid.

In this study transforming growth factor beta (TGF-beta)-like activity in human hydrocele fluid was investigated. Inhibition of DNA synthesis of adult rat hepatocytes in primary culture and stimulation of colony formation of normal rat kidney (NRK) fibroblasts, clone 49F in soft agar were observed in all acidified hydrocele fluids and these activities were neutralized by the specific antibody raised against human native TGF-beta. In samples obtained from recurrent cases of hydrocele, TGF beta-like activity was observed in its active form (without acidification). These results suggest that human hydrocele fluid contains TGF beta-like activity and that the active form of TGF-beta in recurrent hydrocele fluid may be responsible for the recurrence of the disease even after repeated aspiration.