Luiz E.M. Cardoso

Testicular Migration: Remodeling of Connective Tissue and Muscle Cells in Human Gubernaculum Testis

PURPOSE We present the main morphological modifications in the human gubernaculum during testicular migration in humans. MATERIALS AND METHODS We obtained 12 gubernacula from fresh, macroscopically normal human fetuses at 15 to 29 weeks of gestation. Collagen was evidenced using trichrome and Sirius red staining procedures, while Weigerts resorcinol-fuchsin and anti-human elastin antibody were used to reveal elastic system fibers. Smooth muscle cells were detected by anti-human smooth muscle alpha-actin antibody. RESULTS When the testes were still located in the abdomen at 15 to 16 weeks of gestation, collagen fibers were sparse and embedded in a loose extracellular matrix. The amount of fibers then gradually increased with age and at 28 weeks of gestation the gubernaculum was mostly collagenous in composition. Elastic fibers had a similar growth pattern, although they were located mainly at the distal end of the gubernaculum. Fibroblasts largely predominated over other cell types and decreased in number with gestational age, whereas smooth muscle cells were restricted to the walls of blood vessels. Striated muscle cells were detected at the scrotal end of the gubernaculum, where they were disposed as isolated and scattered bundles running in various directions. Like fibroblasts, their number also decreased with age. CONCLUSIONS During testicular migration gubernacular connective tissue undergoes extensive remodeling and ultimately becomes an essentially fibrous structure rich in collagen and elastic fibers. Such changes should decrease the size of the gubernaculum and, thus, contribute to other forces that cause the testes to move toward the scrotum. In fact, because of the lack of smooth muscle cells, and the amount and organization of striated muscle cells, active contraction of the gubernaculum is less likely to be an important factor in testicular descent.

EXTRACELLULAR MATRIX CHANGES IN URETHRAL STRICTURE DISEASE

PURPOSE Glycosaminoglycans (GAGs) and collagen are major components of the extracellular matrix and they have key roles in fibrotic diseases. Little is known about the molecular environment in urethral stricture and the majority of the studies available focused on collagen analysis. However, to our knowledge there are no data on GAG composition in urethral stricture disease. MATERIALS AND METHODS Bulbar urethral strictured segments were obtained from 10 patients 18 to 61 years old (mean age 41.8) who underwent end-to-end anastomotic urethroplasty. GAGs in dry tissue samples were extracted by papain digestion and cetylpyridinium chloride/ethanol precipitation. The concentration of total GAGs was assessed by hexuronic acid assay and expressed in microg. hexuronic acid per mg. dry tissue, while the proportion of sulfated GAGs was determined by agarose gel electrophoresis. The concentration of hyaluronic acid was determined by ion exchange chromatography and total tissue collagen was estimated as its hydroxyproline content. The control group consisted of 10 bulbar urethras obtained from fresh normal cadavers 22 to 53 years old (mean age 32.8). RESULTS Mean total GAG concentration plus or minus standard deviation in the stricture group was 1.09 +/- 0.13, which was significantly lower than in controls (p <0.05). While the predominant GAG in normal bulbar urethras was hyaluronic acid, dermatan sulfate predominated in strictured urethras (mean 44.1% +/- 8.4 and 45.6% +/- 7.7%, respectively). Hyaluronic acid decreased 49.9% and dermatan sulfate increased 68.3%. There were no significant changes in the concentration of heparan sulfate or chondroitin sulfate in normal and strictured bulbar urethras. Mean total collagen significantly increased 32.3% (p <0.05). CONCLUSIONS Composition changes in GAGs in strictured urethras could contribute to the noncompliant nature of urethral scar tissue and cause functional changes. These results may be useful for defining new targets for therapy for urethral stricture disease.

Platelet-derived growth factor differentially regulates the expression and post-translational modification of versican by arterial smooth muscle cells through distinct protein kinase C and extracellular signal-regulated kinase pathways

The synthesis of proteoglycans involves steps that regulate both protein and glycosaminoglycan (GAG) synthesis, but it is unclear whether these two pathways are regulated by the same or different signaling pathways. We therefore investigated signaling pathways involved in platelet-derived growth factor (PDGF)-mediated increases in versican core protein and GAG chain synthesis in arterial smooth muscle cells (ASMCs). PDGF treatment of ASMCs resulted in increased versican core protein synthesis and elongation of GAG chains attached to the versican core protein. The effects of PDGF on versican mRNA were blocked by inhibiting either protein kinase C (PKC) or the ERK pathways, whereas the GAG elongation effect of PDGF was blocked by PKC inhibition but not by ERK inhibition. Interestingly, blocking protein synthesis in the presence of cycloheximide abolished the PDGF effect, but not in the presence of xyloside, indicating that GAG synthesis that results from PKC activation is independent from de novo protein synthesis. PDGF also stimulated an increase in the chondroitin-6-sulfate to chondroitin-4-sulfate ratio of GAG chains on versican, and this effect was blocked by PKC inhibitors. These data show that PKC activation is sufficient to cause GAG chain elongation, but both PKC and ERK activation are required for versican mRNA core protein expression. These results indicate that different signaling pathways control different aspects of PDGF-stimulated versican biosynthesis by ASMCs. These data will be useful in designing strategies to interfere with the synthesis of this proteoglycan in various disease states.

PROXIMAL INSERTION OF GUBERNACULUM TESTIS IN NORMAL HUMAN FETUSES AND IN BOYS WITH CRYPTORCHIDISM

PURPOSE We determine how the proximal gubernaculum testis is attached to the testis and epididymis in human fetuses, and compare these data with findings in boys who had undergone surgery for cryptorchidism. MATERIALS AND METHODS We analyzed 280 testes and epididymides with the gubernacula of 140 well preserved, fresh human fetuses ranging from 10 to 35 weeks after conception with no detectable congenital malformations and 36 undescended testes of 28 boys 2 to 15 years old (mean age 6.8) who had undergone surgery for cryptorchidism. In both groups the different conformations of the relationship among the proximal gubernaculum, testis and epididymis were classified according to a system used for patients with cryptorchidism. In group A the gubernaculum is attached to the testis and epididymis, in group B the gubernaculum is attached only to the testis with a tail disjunction epididymal anomaly, in group C the gubernaculum is attached only to the testis with total disjunction of the epididymis, in group D the gubernaculum is attached only to the epididymal tail and in group E there are no attachments among gubernaculum, testis and epididymis. RESULTS Of the 280 fetal testes studied 194 (69.2%) were in the abdomen, 38 (13. 57%) in the inguinal canal and 48 (17.14%) in the scrotum. There were 277 cases (98.9%) in group A and 3 (1.1%) in group B. Of the 36 undescended testes analyzed 2 (5.6%) were abdominal and 34 (94.4%) were inguinal. There were 26 cases (72.2%) in group A, 8 (22.2%) in group B and 2 in group D. CONCLUSIONS In fetuses without congenital malformations or epididymal alterations, such as tail disjunction or elongated epididymis, the proximal portion of the gubernaculum was attached to the testis and epididymis in all cases. In undescended testes there was an increased incidence of paratesticular structure malformations accompanied by gubernacular attachment anomalies compared to the testes in normal fetuses.

REGIONAL DIFFERENCES IN THE EXTRACELLULAR MATRIX OF THE HUMAN SPONGY URETHRA AS EVIDENCED BY THE COMPOSITION OF GLYCOSAMINOGLYCANS

PURPOSE Despite the concept that the spongy urethra is a unique entity clinical evidence suggests the existence of segmental structural differences. The spongy urethra has a vascular nature, its cells may express different phenotypes and the extracellular matrix that they synthesize should reflect these differences. Glycosaminoglycans are components of the extracellular matrix that have key roles in the normal physiology and pathology of several tissues. Although total collagen content of the urethra was determined, we also analyzed urethral glycosaminoglycans (GAGs). MATERIALS AND METHODS Fresh, macroscopically normal cadaveric urethral samples were obtained from 15 men who died at a mean age of 25.4 years. The urethra was divided into glanular, penile and bulbar segments, which were then analyzed separately. Total GAG concentration was assessed by hexuronic acid assay and expressed as microg. hexuronic acid per mg. dry tissue, while the proportions of sulfated GAGs were determined by agarose gel electrophoresis. Hyaluronan concentration was determined by ion exchange chromatography and total tissue collagen was estimated as hydroxyproline content. RESULTS Total GAG concentration was heterogeneous along the spongy urethra (p <0.001). Mean values plus or minus standard deviation in the glanular, penile and bulbar segments were 2.53 +/- 0.42, 2.11 +/- 0.47 and 1.47 +/- 0.4 microg./mg., respectively. The most predominant GAG was hyaluronan and its highest mean concentration of 50.1% +/- 3.7% was found in the glanular urethra. The most predominant sulfated GAG in the male urethra was dermatan sulfate, followed by chondroitin sulfate and heparan sulfate. Total collagen content and the GAG-to-collagen ratio varied along the spongy urethra and were lowest in the bulbar segment. CONCLUSIONS The extracellular matrix of the human spongy urethra shows regional differences, as evidenced by biochemical analysis of GAG and collagen. This heterogeneity implies functional adaptations in the various segments and may affect the physiology and segmental incidence of urethral diseases.

Effects of castration and hormone replacement in the urinary bladder of rats: structural, ultrastructural, and biochemical analysis.

We evaluated, by qualitative and quantitative methods, the structural alterations in the bladder wall of rats submitted to surgical castration, as well as the role of hormone replacement in reversing the possible structural alterations. Twenty-four 12-week-old male Sprague-Dawley rats were used. The animals were divided into 3 groups comprising 8 animals each and treated as follows. Members of group CONTR (control) underwent a sham operation only and were sacrificed after 2 months. Members of group ORCH (orchiectomy) underwent bilateral orchiectomy and were sacrificed after 2 months. Members of group ORCH+TEST (testosterone) underwent orchiectomy, received testosterone replacement after 1 month, and were sacrificed 1 month later. We performed a qualitative and quantitative analysis of collagen by light microscopy, scanning electron microscopy, biochemistry, and a histomorphometric analysis of smooth muscle and elastic fibers in the 3 groups. The results showed a significant decrease in absolute values of elastic fibers in the castrated group. The histomorphometric analysis of epithelial height did not show differences among the groups. There was no statistical difference in quantitative analysis of collagen, either by histomorphometry or by biochemistry. Also, there was no difference in the smooth muscle cells. However, the qualitative analysis revealed differences in collagen (castrated group) when compared with controls and with rats submitted to hormone replacement. Hormone replacement with testosterone was able to revert the alterations observed. The findings suggest that hormone replacement, even when instituted at a late stage, is effective in reversing the bladder wall alterations produced by secondary hypogonadism.

Collagen and Elastic Fibers in the Penis of Human Fetuses at 28 Weeks Postconception

Objectives: The extracellular matrix is a key element in penile function and pathology, yet little is known of its development. Herein we investigated the morphological organization of collagen and elastin in the corpora cavernosa and tunica albuginea of human fetuses. Material and Methods: The penises from 5 fresh human fetuses at 28 weeks postconception (WPC) were routinely fixed and embedded, and all staining procedures were carried out on paraffin sections. Collagen was evidenced by staining with: (1) Gomori’s trichrome; (2) sirius red, followed by observation under polarized light, and (3) an antihuman collagen type-III antibody. Elastin and the whole elastic system were revealed using an antihuman elastin antibody and Weigert’s resorcin fucsin, respectively. Results: At this stage of fetal development, the albuginea is formed predominantly by dense bundles of collagen. Near the corpora cavernosa, the presence of type-III collagen was also observed. Weigert staining showed numerous fibers of the elastic system in the albuginea. Type-III collagen was found to be strongly positive in the cavernous trabeculae and in the connective sheath surrounding the central artery. Using Weigert staining and an immunolabeling method with primary antibody against human elastin, we found an important quantity of elastic system fibers in the trabeculae of the corpora cavernosa. Conclusion: In fetuses at 28 WPC the albuginea is formed predominantly by dense bundles of collagen. The trabecular structures of the corpora cavernosa present a significant quantity of type-III collagen and elastic system fibers.

The penis in diabetes: structural analysis of connective tissue and smooth muscle alterations in a rabbit model.

Study Type – Aetiology (case control)

Stereological and biochemical analysis of muscular and connective tissue components in the penile corpus cavernosum adjacent to the fibrous plaque of Peyronie's disease.

To investigate the structural organization of the connective tissue in the corpus cavernosum (CC) adjacent to the fibrous plaque in Peyronie’s disease (PD) using stereological and biochemical techniques, as most studies on PD have focused on the analysis of the fibrous plaque that forms in the tunica albuginea (TA). Because this fibrotic reaction is mediated by various inflammatory soluble factors, adjacent connective tissues might also be affected and this secondary effect might explain, for example, the erectile dysfunction that occurs in PD.

Does prolonged pneumoperitoneum affect the kidney? Oxidative stress, stereological and electron microscopy study in a rat model

PURPOSE Pneumoperitoneum (Pp) at 12 to 15 mmHg in rats is associated with kidney damage. However, Pp at 8 mmHg is now known to best correlate to working pressures used in humans. Thus the aim of this work was to study the kidney of rats submitted to prolonged Pp at 8 mmHg. MATERIALS AND METHODS Rats were divided into a Sham group (n = 14), submitted to anesthesia, and a Pp group (n = 14), submitted to Pp at 8 mmHg, followed by deflation. In both groups, 7 animals were immediately killed and their kidneys were used for oxidative stress analyses. The remaining 7 rats in each group were evaluated after 6 weeks for the number of glomeruli and podocyte morphology. RESULTS For all analyzed parameters Sham and Pp groups presented no statistical difference. CONCLUSION When submitted to adequate Pp pressures (8 mmHg), no kidney damage occurs in rats.