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Normal and varicocele testis in adolescents

G. Santoro1, C. Romeo2

1Department of Biomorphology and Biotechnologies, 2Department of Medical and Surgical Paediatric Sciences, University of Messina, 98125  Messina, Italy

Asian J Androl  2001 Dec; 3: 259-262


Keywords:  varicocele;  testis; seminiferous tubules; adolescence
Abstract
The authors reviewed the results of their research on the structure and composition of normal and varicocele seminiferous tubules in adolescents. They give new evidences of normal structure of adolescent testis and demonstrate, for the first time, the ultrastructural and immunohistochemical modifications of the lamina propria and basal lamina in the adolescent varicocele patients, which are similar to those observed in adults, but less severe,  and of the adherence junctions in seminiferous tubules. They also report the presence of oxidative stress in adolescents limited to testis and not generalised as in the adults. These data are well correlated to different clinical studies that support the hypothesis of a progressive course of varicocele and the need for surgical treatment in adolescent varicocele patients.

The adolescent age plays a key role in the development of testicular diseases, since the lesions observed are often progressive. For this reason we have recently focused our attention on the structure and composition of normal and varicocele seminiferous tubules in the adolescents. The varicocele is a common disease of the testis and represents one of the most common causes of male infertility[1].

The human seminiferous tubules are formed by a very complex stratified epithelium containing spermatogenic cells in different stages of development, and supporting cells, the Sertoli cells. The epithelium is surrounded by a lamina propria composed of a basal layer and 5-7 external cellular layers. The cellular layers are constituted of 3-5 inner layers of myofibroblasts and one or more outer layers of fibroblasts. The basal layer, composed of extracellular matrix (ECM) components, is formed by two layers: the inner electron-transparent layer, the lamina lucida or rara, is situated close to the plasma membrane of the germinal epithelium, and the outer electron-dense layer, the lamina densa. The external cellular layers are separated by laminae of ECM consisting of glycosaminoglycans (GAGs), proteoglycans and collagen fibers. The myofibroblasts are individual flat cells with a diameter of 40-60 m; they do not form continuous cell layers and are not completely covered by a basal lamina. In the angular interstices between various seminiferous tubules, there are Leydig cells, which represent the endocrine component of the testis[2-5].

The lamina propria of normal adolescent seminiferous tubule showed a maximum thickness of 10 m and a surface area between 3970 and 4472 m2, while the tubular diameter was between 158 and 200 m2. In adolescent affected by varicocele, we showed different degrees of thickening of the lamina propria till a maximum value of 35 m. It was the result of an increased deposition of extracellular components, starting from the innermost layer of collagen fibers extending to the outer extracellular layer[2]. This condition was responsible for the formation of deep invaginations facing the germinal epithelium, which, in other testicular pathologies, were ascribed to tubular damage caused by a blockage in the mediation of the lamina propria between the interstitium and the germinal epithelium[6].The increased thickness of the lamina propria corresponded to a progressive increase in its surface area and a reduction in the tubular diameter[2]. This damage was not as severe as that described in adult varicocele, in which a diffuse sclerosis of the lamina propria was observed[7].

Despite the modifications of the extracellular layers, the myofibroblasts still maintained their morphological features with only mild alterations[3]. Moreover, using -smooth muscle isoactin as specific marker of myofibroblasts, we do not observe any myofibroblast transformation into fibroblast, as already demonstrated in adult varicocele, that corresponds to the reported progressive sclerosis of the lamina propria[7]. The myofibroblasts are responsible for the contraction of the seminiferous tubules necessary for the transport of testicular spermatozoa and fluid and take part in the regulation of spermatogenesis and the creation of the blood testis barrier[8, 9]. The integrity of myofibroblasts may be an important feature of adolescent varicocele.

In adolescent varicocele patients, the peritubular basal lamina also showed ultrastructural changes characterized by an uneven profile with a variable thickness[10]. Similar observations have been reported in adult varicocele[7] and in other testicular pathology[11,12]. The peritubular basal lamina was also altered in two of its major components: laminin and collagen type IV[10]. In normal testis laminin is localized in the lamina lucida, while collagen type IV in both layers of the peritubular basal lamina; both are present as a continuous and uniform line[5]. These two molecules, apart from a mechanical linkage, play a key role in the regulation of major biological cellular functions[13-15]. Through the relationship with the adhesion receptors of the integrin family localized in the cellular membrane, and consequently with the intracellular actin-associated proteins, messages are transmitted from the ECM to the nuclear compartment, thus controlling the ubiquitous process of differentiation, proliferation, adhesion, migration, gene expression[16,17] and spermatogenesis[18,19]. In adolescent varicocele patients, laminin immunereaction displayed an irregular line with a wavy profile that sometimes appeared to be interrupted. Collagen type IV showed areas of annular thickening of the immunereaction alternating with areas of interrupted and reduced immunopositivity. Moreover, its immunofluorescence distribution appeared to be irregular and wavy, following the morphology of the basal lamina observed by transmission electron microscopy along the deep invaginations[4]. The observed modification in the morphology and composition of the peritubular basal lamina could represent one of the mechanisms responsible for the lesions characteristic of varicocele.

Another pivotal role in the testicular function is played by two actin-associated proteins, i.e., vinculin and talin; they are important in cell activity as previously described[16-19].

Vinculin and talin have been identified in different tissues and cell types. Particularly, vinculin is localized at cell-cell and cell-ECM adhesion sites, while talin is present only at cell-ECM junction[20-22]. They frequently co-localize within cells[20] taking part in the formation of the adherens junction. Miyamoto et al[23] have demonstrated that talin,-actinin and vinculin represent a unique subset of three cytoskeletal proteins whose membrane accumulation requires both integrin aggregation and ECM occupancy, but not the tyrosine-kinase activity needed for many other integrin-responsive proteins. Alterations of one of these components of the adherens junction could negatively influence the others. We have demonstrated, for the first time, that in normal human adolescent testes, these two actin-associated proteins had the same pattern as that observed in other tissues. Particularly, in Sertoli and Leydig cells vinculin was expressed at the cell-cell and the cell-ECM adherence junctions, while talin was present only at the cell-ECM adherence junctions level[24]. In the adolescent varicocele patients, there were slight alterations of vinculin and talin pattern only in the Sertoli cells, while the Leydig cells presented an expression and distribution similar to the normal testes. Occasionally, in the same sample, altered seminiferous tubules were found adjacent to normal ones. These evidences could represent a not well consolidated and generalized tubular damage[24]. On the basis of our results, we speculate that the modifications in two components of the adherence junction, i. e., basal lamina and actin-associated protein, could negatively influence the spermatogenesis in varicocele patients. In this regard, Pelletier et al have reported an altered peritubular basal lamina and changes in Sertoli cell junctions in tubular regions where germ cells were depleted[25].

Recently nitric oxide (NO) has been identified in the testis where it should play certain roles. NO has been reported to be important in the regulation of male reproductive function and fertility, Leydig cell function, myofibroblast contraction and hence tubular peristalsis[26,27]. NO acts also on the peritubular lamina propria modulating its permeability and regulates the activity of muscle cells and pericytes of testicular vessels[28]. Nevertheless experimental studies have shown that NO at supra-physiological levels can be harmful for both testicular and sperm function[29,30]. In normal testis, two isoforms of NO synthases, i.e., endothelial and inducible nitric oxide synthase (eNOS and iNOS) were demonstrated by immunohistochemistry and western blot analysis[31]. In adolescent varicocele patients, the two isoforms were also identified but iNOS appeared to be up-regulated[31]. This important finding could explain the significant increase of NO recorded in the spermatic veins of adolescent varicocele patients. It is possible that varicocele causes an up-regulation of the iNOS that produces increased amount of NO, causing an oxidative stress, which if long continued will result in testis and sperm dysfunction[32].

In conclusion, we gave new evidences of normal structure of adolescent testis.  Moreover, in adolescent varicocele patients we have demonstrated, for the first time, ultrastructural and immunohistochemical modifications of the lamina propria and basal lamina similar to but less severe than those observed in adults, and of the adherence junctions in seminiferous tubules[2,3,10,24]. We also demonstrated a condition of oxidative stress in the adolescents, which is limited to the testis[31] but not generalised as in the adults[36]. Our data are well correlated to different clinical studies that support the hypothesis of a progressive course of varicocele. In fact, hypotrophy has been demonstrated only in the left testis in intermediate grade varicocele patients[33] and a follow-up study has been performed after varicocele repair[34,35]. In contrast, the clinical data available do not support a similar progressive effect of varicocele in adulthood[1]. Taken together, all these data support the need for surgical treatment in adolescent varicocele patients to interrupt the vicious cycle that causes blood stasis, NO overproduction and ultrastructural modifications.

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Correspondence to: Dr. Giuseppe Santoro, Department of Biomorphology and Biotechnologies, 1st Floor Torre Biologica, Policlinico Universitario, Viale Gazzi, 98125 Messina, Italy.
Tel: +39-90-221 3637      Fax: +39-90-692 449
E-mail: santorof@unime.it
Received 2001-09-29      Accepted 2001-11-22