Peritubular Wall Research Articles

Identification of Catecholamine and Drug Target α2A-Adrenoceptor in Human Testis and Human Testicular Peritubular Cells.

Background: Clonidine has been used in clinical medicine, e.g., to treat high blood pressure and other conditions. Animal studies have linked its use to impairments of male reproductive functions, and although only a few reports exist for the human species, such actions may exist in man as well. The underlying reasons and, specifically, possible actions of clonidine at the level of the testis are not known. Introduction: Clonidine is an agonist at the α2A-adrenoceptor (ADRA2A), which, as data bank mining indicated, is expressed by several cells of the human testis. The human testis and most of its cells are, however, not readily accessible to experimental testing. Cells from the peritubular wall compartment (human testicular peritubular cells; HTPCs) are the exception. Methods and Results: As shown by immunohistochemical/immunocytochemical and PCR techniques these cells express ADRA2A and retain expression upon isolation and culture. When tested over a concentration range (1-1000 µM) and 24 h, clonidine did not visibly affect HTPC morphology but significantly stimulated IL6 mRNA levels in a concentration-dependent manner. ELISA measurements of cell culture supernatants confirmed a stimulatory action of clonidine (10 µM) on secreted IL6. When examined in collagen gel contraction assays of HTPCs, clonidine (10 µM) exerted a slight relaxing action, while a proteomic study revealed that clonidine (10 µM) did not significantly change cellular protein abundance of HTPCs after 24 h (data available via ProteomeXchange with identifier PXD052220). Conclusion: Thus, ADRA2A-bearing cells in the human testis are targets for catecholamines and drugs such as clonidine. The results of this HTPCs-focused study only show the tip of the iceberg. It is likely that catecholamines/catecholaminergic drugs have the potential to interfere with human testicular functions.

Pleiotropic actions of melatonin in testicular peritubular myoid cells of immature Syrian hamsters

BackgroundPeritubular myoid cells are emerging as key regulators of testicular function in adulthood. However, little is known about the role of testicular peritubular myoid cells (TPMCs) in the development of the male gonad. We found that, compared to testes of young adult hamsters, gonads of 21 day-old animals show increased melatonin concentration, seminiferous tubular wall thickening and a heterogeneous packaging of its collagen fibers thus raising the question whether melatonin may be involved in the regulation of TPMCs. MethodsWe established primary cultures of TPMCs from immature hamsters (ihaTPMCs), which we found express melatonergic receptors. ResultsExogeneous melatonin decreased the levels of inflammatory markers (NLRP3 inflammasome, IL1β) but increased the expression of cyclooxygenase 2 (COX2, key enzyme mediating prostaglandin synthesis) and of the glial cell line-derived neurotrophic factor (GDNF) in ihaTPMCs. Melatonin also stimulated ihaTPMCs proliferation and the expression of extracellular matrix proteins such as collagen type I and IV. Furthermore, collagen gel contraction assays revealed an enhanced ability of ihaTPMCs to contract in the presence of melatonin. ConclusionMelatonin regulates immune and inflammatory functions as well as contractile phenotype of the peritubular wall in the hamster testis. General significanceIf transferable to the in vivo situation, melatonin-dependent induction of ihaTPMCs to produce factors known to exert paracrine effects in other somatic cell populations of the gonad suggests that the influence of melatonin may go beyond the peritubular wall and indicates its contribution to testicular development and the establishment of a normal and sustainable spermatogenesis.

The cell\u2013cell junctions of mammalian testes. III. Absence of an endothelial cell layer covering the peritubular wall of the seminiferous tubules\u2014an immunocytochemical correction of a 50-year-old error in the literature

In the molecular biological and ultrastructural studies of the peritubular wall cells encasing the seminiferous tubules of mammalian testes, we found it necessary to characterize the outermost cell layer bordering on the interstitial space in detail. For half a century, the extremely thin cells of this monolayer have in the literature been regarded as part of a lymphatic endothelium, in particular in rodents. However, our double-label immunofluorescence microscopical results have shown that in all six mammalian species examined, including three rodent ones (rat, mouse, guinea pig), this classification is not correct: the very attenuated cells of this monolayer are not of lymphatic endothelial nature as they do not contain established endothelial marker molecules. In particular, they do not contain claudin-5-positive tight junctions, VE-cadherin-positive adherens junctions, “lymph vessel endothelium hyaluronan receptor 1” (LYVE-1), podoplanin, protein myozap and “von Willebrand Factor” (vWF). By contrast and as controls, all these established marker molecules for the lymphatic endothelial cell type are found in the endothelia of the lymph and—partly also—blood vessels located nearby in the interstitial space. Thus, our results provide evidence that the monolayer cells covering the peritubular wall do not contain endothelial marker molecules and hence are not endothelial cells. We discuss possible methodological reasons for the maintenance of this incorrect cell type classification in the literature and emphasize the value of molecular analyses using multiple cell type–specific markers, also with respect to physiology and medical sciences.

The cell\u2013cell junctions of mammalian testes: II. The lamellar smooth muscle monolayer cells of the peritubular wall are laterally connected by vertical adherens junctions\u2014a novel architectonic cell\u2013cell junction system

The testes of sexually mature males of six mammalian species (men, bulls, boars, rats, mice, guinea pigs) have been studied using biochemical as well as light and electron microscopical techniques, in particular immunolocalizations. In these tissues, the peritubular walls represent lamellar encasement structures wrapped around the seminiferous tubules as a bandage system of extracellular matrix layers, alternating with monolayers of very flat polyhedral “lamellar smooth muscle cells” (LSMCs), the number of which varies in different species from 1 to 5 or 6. These LSMCs are complete SMCs containing smooth muscle α-actin (SMA), myosin light and heavy chains, α-actinin, tropomyosin, smoothelin, intermediate-sized filament proteins desmin and/or vimentin, filamin, talin, dystrophin, caldesmon, calponin, and protein SM22α, often also cytokeratins 8 and 18. In the monolayers, the LSMCs are connected by adherens junctions (AJs) based on cadherin-11, in some species also with P-cadherin and/or E-cadherin, which are anchored in cytoplasmic plaques containing β-catenin and other armadillo proteins, in some species also striatin family proteins, protein myozap and/or LUMA. The LSMC cytoplasm is rich in myofilament bundles, which in many regions are packed in paracrystalline arrays, as well as in “dense bodies,” “focal adhesions,” and caveolae. In addition to some AJ-like end-on-end contacts, the LSMCs are laterally connected by numerous vertical AJ-like junctions located in variously sized and variously shaped, overlapping (alter super alterum) lamelliform cell protrusions. Consequently, the LSMCs of the peritubular wall monolayers are SMCs sensu stricto which are laterally connected by a novel architectonic system of arrays of vertical AJs located in overlapping cell protrusions.

Changes of myoid and endothelial cells in the peritubular wall during contraction of the seminiferous tubule.

The wall of the seminiferous tubule in rodents consists of an inner layer of myoid cells covered by an outer layer of endothelial cells. Myoid cells are a type of smooth muscle cell containing α-actin filaments arranged in two independent layers that contract when stimulated by endothelin-1. The irregular surface relief of the tubular wall is often considered a hallmark of contraction induced by a variety of stimuli. We examine morphological changes of the rat seminiferous tubule wall during contraction by a combination of light, confocal, transmission and scanning electron microscopy. During ET-1-induced contraction, myoid cells changed from a flat to a conical shape, but their actin filaments remained in independent layers. As a consequence of myoid cell contraction, the basement membrane became wavy, orientation of collagen fibers in the extracellular matrix was altered and the endothelial cell layer became folded. To observe the basement of the myoid cell cone, the endothelial cell monolayer was removed by collagenase digestion prior to SEM study. In contracted tubules, it is possible to distinguish cell relief: myoid cells have large folds on the external surface oriented parallel to the tubular axis, whereas endothelial cells have numerous cytoplasmic projections facing the interstitium. The myoid cell cytoskeleton is unusual in that the actin filaments are arranged in two orthogonal layers, which adopt differing shapes during contraction with myoid cells becoming cone-shaped. This arrangement impacts on other components of the seminiferous tubule wall and affects the propulsion of the tubular contents to the rete testis.

Human testicular peritubular cells exert anti-angiogenetic action: A role for pigment-epithelium derived factor (PEDF) in the maintenance of avascular seminiferous tubules, the blood-testis barrier and the spermatogonial stem cell niche in man?

Male fertility depends on spermatogenesis, which takes place in the seminiferous tubules. This compartment is devoid of blood vessels, yet the peritubular capillary network is an important part of the spermatogonial stem cell niche, as suggested by studies in mouse. Our proteomic studies of cultured human testicular peritubular cells (HTPCs) revealed that they constitutively secrete pro-angiogenetic factors (e.g. VEGF) and the anti-angiogenetic factor, pigment epithelium derived factor, PEDF. This factor has not previously been reported in the human testis. Immunohistochemistry performed with human testicular samples revealed that it is present in vivo, in the peritubular wall. Cellular co-culture studies, using HTPCs and human endothelial cells (HUVEC), and migration analysis showed that HUVECs are repulsed by HTPCs. The factor involved is likely PEDF and its actions were abolished by a PEDF-antiserum. Heat-inactivation of the antiserum reverted this action. Gene expression analysis revealed that PEDF is expressed in the testis of a non-human primate before puberty suggesting involvement in the establishment and maintenance of the distinct vascular pattern of seminiferous tubules and of its overall avascular nature. Addition of dihydrotestosterone to HTPCs increased PEDF (qPCR), indicating that it is subject to hormonal modulation. Thus, testicular peritubular cells prevent vascularization of human seminiferous tubules by secreting the anti-angiogenetic protein PEDF. However, fine-tuning of its production (e.g. by androgens) may bring about local differences in the control of testicular interstitial microvessels and thereby possibly may influence the spermatogonial stem cell niche. (Supported by DFG MA1080/20 – 1; FOR1041)

Dentin decalcification during lithium treatment: case report

Severe dental decay and changes in tooth structure have been reported in association with the use of lithium in Psychiatry, but lithium effects on tooth inorganic composition remain unknown. A 30-year-old woman with bipolar disorder, treated with lithium carbonate presented severe dental decay. Dentin samples from lithium and healthy volunteers were collected and submitted to ionic and ultrastructural analysis. Samples from the lithium patient exhibited irregular peritubular walls and the mineral crystals were irregularly arranged in the intertubular dentin. In addition, a decrease in Mg/P/Ca and an increase of Zn concentrations were detected. These data suggest that the severe dental decay and changes in the tooth structure observed for the lithium-treated patient are related to dentin mineral loss and that this pathological condition is different from caries lesions.

High levels of the extracellular matrix proteoglycan decorin are associated with inhibition of testicular function

Decorin (DCN), a component of the extracellular matrix of the peritubular wall and the interstitial areas of the human testis, can interact with growth factor (GF) signalling, thereby blocking downstream actions of GFs. In the present study the expression and regulation of DCN using both human testes and two experimental animal models, namely the rhesus monkey and mouse, were examined. DCN protein was present in peritubular and interstitial areas of adult human and monkey testes, while it was almost undetectable in adult wild type mice. Interestingly, the levels and sites of testicular DCN expression in the monkeys were inversely correlated with testicular maturation markers. A strong DCN expression associated with the abundant connective tissue of the interstitial areas in the postnatal through pre-pubertal phases was observed. In adult and old monkeys the DCN pattern was similar to the one in normal human testes, presenting strong expression at the peritubular region. In the testes of both infertile men and in a mouse model of inflammation associated infertility (aromatase-overexpressing transgenic mice), the fibrotic changes and increased numbers of tumour necrosis factor (TNF)-α-producing immune cells were shown to be associated with increased production of DCN. Furthermore, studies with human testicular peritubular cells isolated from fibrotic testis indicated that TNF-α significantly increased DCN production. The data, thus, show that an increased DCN level is associated with impaired testicular function, supporting our hypothesis that DCN interferes with paracrine signalling of the testis in health and disease.

15-Deoxy-Δ12-14-Prostaglandin-J2 Induces Hypertrophy and Loss of Contractility in Human Testicular Peritubular Cells: Implications for Human Male Fertility

The wall of the seminiferous tubules contains contractile smooth-muscle-like peritubular cells, thought to be important for sperm transport. Impaired spermatogenesis in men typically involves remodeling of this wall, and we now found that smooth muscle cell (SMC) markers, namely myosin heavy chain (MYH11) and smooth muscle actin (SMA) are often lost or diminished in peritubular cells of testes of men with impaired spermatogenesis. This suggests reduced contractility of the peritubular wall, which may contribute to sub- or infertility. In these cases, testicular expression of cyclooxygenase-2 (COX-2) implies formation of prostaglandins (PGs). When screening different PGs for their ability to target human testicular peritubular cells (HTPCs), only a PG metabolite, 15-deoxy-Delta(12-14)-prostaglandin-J2 (15dPGJ2), was effective. In primary cultures of HTPCs, 15dPGJ2 increased cell size in a reversible manner. Importantly, 15dPGJ2 treatment resulted in a loss of typical differentiation markers for SMCs, namely MYH11, calponin, and SMA, whereas fibroblast markers were unchanged. Collagen gel contraction assays revealed that this loss correlates with a reduced ability to contract. Experiments with an antagonist (bisphenol A diglycidyl ether) and agonist (troglitazone) for a cognate 15dPGJ2 receptor (i.e. peroxisome proliferator-activated receptor-gamma) indicated that peroxisome proliferator-activated receptor-gamma is not directly involved. Rather, the mode of action of 15dPGJ2 involves reactive oxygen species. The antioxidant N-acetylcysteine not only blocked ROS formation but also prevented the increase in cell size and the loss of contractility in HTPCs challenged with 15dPGJ2. We conclude that 15dPGJ2, via reactive oxygen species, influences SMC phenotype and contractility of human peritubular cells and possibly is involved in the development of human male sub-/infertility.

Immunophenotypical characterization of contractile cells in caput epididymidis of men affected by congenital or post-inflammatory obstructive azoospermia

Myoid cells of the human caput epididymidis are replaced by large cells with ultrastructural features of smooth muscle cells (SMC) in chronic obstruction of the male genital tract. To evaluate whether these cellular changes are associated with different functional phenotypes we analysed the immunohistochemical expression of myosin heavy chain isoforms and of extracellular matrix (EM) components in the human caput epididymidis contractile cells in normal and in obstructed epididymides. Normal caput epididymidis myoid cells expressed a scattered immunostaining for SM2, marker of differentiated contractile SMC, while no staining was detected for SMemb (the non-muscle-type myosin heavy chain isoform) and for its transcription factor BTEB2, markers of undifferentiated proliferating SMC. A faint immunoreaction (IR) for EM was observed in the peritubular wall of the normal caput. In the contractile wall of the obstructed caput epididymidis a strong IR was detected for all myosin heavy chain isoforms as well as for collagen type IV and for fibronectin, markers for a secretory function of SMC. These findings, unknown in other models of SMC pathophysiology, suggest that myoid cells resume the molecular machinery of both mature SMC and of differentiating/secretory cells in the chronic obstruction of the human caput. Contractile cells of the epididymal duct represent a unique model to study the plasticity of SMC.

Mechanism of alcoholic testicular damage

To observe the mechanism of alcoholic testicular damage, in a previous experiment we used weanling male SD rats aged 45 days, weighing about 200 g, and fed a liquid diet (Lieber's) containing 5% ethanol. The latter accounted for 36% of total caloric intake for 7 weeks, but did not result in testicular atrophy. In a later experiment, we used a liquid diet in which ethanol accounted for 46% of the total calorie count. It provided a high-fat, low-protein content which simulated the nutritional background of patients with alcoholic liver diseases. This diet resulted in testicular atrophy. Histological and biochemical changes accompanying this experimental testicular atrophy included the following: 1) The testes of alcohol-fed animals contained smaller seminiferous tubules with reduced numbers of total cells, but no degeneration was seen in the spermatids. 2) In the peritubular wall of the seminiferous tubules, we observed curvature, irregularities, infolding of the basement membrane, and lamellation of the lamina densa, as well as hyperplasia of collagen fibers in the tunica propria. 3) In the cytoplasm of the Sertoli cells, deposits of gigantic fat droplets and stratification of the mitochondria were observed. The permeability of the Sertoli cell tight junction was confirmed using the Lanthanum method. 4) Testosterone levels in both the serum and testes declined. 5) Lactate dehydrogenase-X (LDH-X) activity in the testes declined. 6) Low Km alcohol dehydrogenase (ADH) activity localized in the testicular interstitial tissue was increased. These results indicate that the composition of three major nutritional elements as well as alcohol concentration are important in the mechanism of alcoholic testicular damage, and this damage affects both the testicular interstitial cell and the seminiferous tubules, particularly the Sertoli cells and peritubular wall of the latter. In addition, the findings suggest that ADH is involved in alcohol metabolism in the interstitial cells of the testes.

Effects of stress on the reproductive system of male rats and mice.

Male rats were immobilized for 2 hr/day, 5 days a week, for varying periods of time (7-31 days) to assess the effects of stress on the reproductive system. Mice were immobilized for 25-38 days. The effectiveness of the stress was demonstrated by assessing the adrenal and body weight. Adrenal weights were significantly increased (p less than .05--t-test) compared to controls. Body weights were decreased compared to controls. No consistent change was noticed in the testicular weight and progressive motility of the spermatozoa drawn from the cauda epididymis and vas deferens. There were no deleterious effects of immobilization stress on the morphology of the differentiating germ cells, peritubular wall, and the interstitial tissues of the testis.

Testicular Biopsy of Secretory Azoospermia: Electron and Light Microscopic Analysis

The histological and ultrastructural features of testicular cells were examined in testes with secretory azoospermia in seven patients with germinal cell arrest, two with Sertoli-cell-only; and one with tubular hyalinization. Germinal cell arrest was characterized by the presence of spermatogonia AD, AP, B, and some primary spermatocytes: these cells had similar ultrastructural features typical of the adult normal testis. Sertoli cells contained large number of various lipid inclusions and lipofuscin bodies. The multilayered peritubular wall presented increased collagen fibers. The cytoplasm of Leydig cells was filled with dilated vesicles of smooth endoplasmic reticulum and precursors of Reinke crystalloids. A blockage in the seminiferous tubules occurred after puberty, when tubular components, peritubular wall and Leydig cells had reached maturity.

睾丸機能の研究

Electron microscopic observation of the peritubular wall of the seminiferous tubules was made on 15 clinical cases, including one normal adult, one infant, 11 of hypospermatogenesis (oligospermia 7; azoospermia 3; hematospermia 1), 2 of Klinefelter's syndrome. The conclusion was as follows:1) The peritubular wall of the seminiferous tubules in the normal adult and infant showed the three main layers: (1) the basement membrane; (2) the non-cellular layer, containing mainly collagen fibers and very fine filaments; (3) the cellular layer, consisting of myofibroblast like cells and a few collagen fibers. The last two layers were composed of the so-called tunica propria.2) The basement membrane of the seminiferous tubules in hypospermatogenesis revealed the various changes such as “Infolding”, “Lamellation”, “Thickening”, and “Pleat”. These changes were essentially similar to the changes due to X-irradiation, local heat, inflammation, chemical stimulation and hypophysectomy in rat and mouse testis.3) The protrusion of the basement membrane into seminiferous epithelium has been called “Infolding”. The infoldings showed a spherical and columnar shape, and in part irregular projections. Two types of the infolding were recognized.Type 1: The basement membrane protruded into the intercellular clefts between the spermatogonias or between the spermatogonia and Sertoli cell. This type of the infolding which was variously crooked showed a columnar, club like or sometimes labyrynthic shape. Its surface was irregularly uneven and showed in part projections. The size ranged from about 1-2μ to about 6-10μ. In some cases, the top of the giant infolding reached as far as the height of the spermatogonia. This type was recognized mainly in hypospermatogenesis.Type 2: The basement membrane protruded directly into the cytoplasma of the Sertoli cell and occasionally spermatogonia. The shape appeared spherical and oval, and the size was about 1-2μ. This type was recognized mainly in Sertoli tubules.4) The findings of the basement membrane was classified into four major types. (Cf. Fig. 21)Type A: The basement membrane was thin, about 0.1μ in thickness, and was seen to be a monoor di-lamellar structure. Small infoldings (almost Type 1) were rarely present. This type was recognized in normal and in mild hypospermatogenesis.Type B: The basement membrane showed a polylamellar structure. The infoldings, which were mainly Type 2, were frequently present. The pleats of the basement membrane were regularly seen. This type was recognized in mild, moderate, severe hypospermatogenesis.Type C: The infoldings, lamellation, and pleats were frequently observed. Type of the infolding was almost Type 2. This type C was recognized in Sertoli tubules.Type D: The basement membrane became thick, but the lamellar structure was obscure. Type of the infolding was almost Type 2. The pleats were not observed. This Type D was recognized in Sertoli tubules.Moreover, the basement membrane-like structure could not been observed in the hyalinized seminiferous tubules, and the hyalinized space was occupied by a great number of collagen fibers. By electron microscope, the basement membrane of Sertoli tubules were divided into two different types, that is Type C and Type D, although light microscope could not distinguish between both typse.5) The striking change of the tunica propria was a marked increase of collagen fibers in hypospermatogenesis. In such a case, collagen fibers were increased not only in the non-cellular layer, but also between peritubular cells in the cellular layer.6) The degree of the changes in the basement membrane and tunica propria was almost proportional to the histological classification of spermatogenic failure. The more testicular damage, the more marked the changes of infolding, lamellation, thickening, pleat in basement membrane.

Bacterial progresssion in carious dentine: the effect of the calcified peritubular wall

Bacterial progresssion in carious dentine: the effect of the calcified peritubular wall