The cells responsible for production of the male sex hormone testosterone, the Leydig cells of the testis, are post-mitotic cells with neuroendocrine characteristics. Their origin during ontogeny and regeneration processes is still a matter of debate. Here, we show that cells of testicular blood vessels, namely vascular smooth muscle cells and pericytes, are the progenitors of Leydig cells. Resembling stem cells of the nervous system, the Leydig cell progenitors are characterized by the expression of nestin. Using an in vivo model to induce and monitor the synchronized generation of a completely new Leydig cell population in adult rats, we demonstrate specific proliferation of vascular progenitors and their subsequent transdifferentiation into steroidogenic Leydig cells which, in addition, rapidly acquire neuronal and glial properties. These findings, shown to be representative also for ontogenetic Leydig cell formation and for the human testis, provide further evidence that cellular components of blood vessels can act as progenitor cells for organogenesis and repair.

Alterations of the nitric oxide receptor, soluble guanylate cyclase (sGC) may contribute to the pathophysiology of pulmonary arterial hypertension (PAH). In the present study, the expression of sGC in explanted lung tissue of PAH patients was studied and the effects of the sGC stimulator BAY 63-2521 on enzyme activity, and haemodynamics and vascular remodelling were investigated in two independent animal models of PAH.Strong upregulation of sGC in pulmonary arterial vessels in the idiopathic PAH lungs compared with healthy donor lungs was demonstrated by immunohistochemistry. Upregulation of sGC was detected, similarly to humans, in the structurally remodelled smooth muscle layer in chronic hypoxic mouse lungs and lungs from monocrotaline (MCT)-injected rats. BAY 63-2521 is a novel, orally available compound that directly stimulates sGC and sensitises it to its physiological stimulator, nitric oxide. Chronic treatment of hypoxic mice and MCT-injected rats, with fully established PAH, with BAY 63-2521 (10 mg?kg) partially reversed the PAH, the right heart hypertrophy and the structural remodelling of the lung vasculature.Upregulation of soluble guanylate cyclase in pulmonary arterial smooth muscle cells was noted in human idiopathic pulmonary arterial hypertension lungs and lungs from animal models of pulmonary arterial hypertension. Stimulation of soluble guanylate cyclase reversed right heart hypertrophy and structural lung vascular remodelling. Soluble guanylate cyclase may thus offer a new target for therapeutic intervention in pulmonary arterial hypertension.KEYWORDS: BAY 63-2521, cardiovascular diseases, nitric oxide, pharmacology, pulmonary arterial hypertension, smooth muscle P ulmonary arterial hypertension (PAH) is a disabling disease, with high mortality, characterised by sustained elevation in pulmonary arterial pressure (Ppa) and pulmonary vascular remodelling due to proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) [1]. Imbalance of vasodilatory and vasoconstrictive mediators has been implicated in these changes. Reduced urinary excretion of prostaglandin (PG)I 2 and augmented excretion of thromboxane metabolites were found in patients with idiopathic PAH (IPAH) [2], and immunohistological studies have shown reduced expression of PGI 2 synthase in the pulmonary vessels originating from those patients [3]. Another important mediator in the regulation of vascular tone is nitric oxide (NO), which is synthesised by NO synthases. Local NO production from endothelium and epithelium regulates pulmonary perfusion, depending on alveolar ventilation to assure optimised ventilation/perfusion distribution [4][5][6]. In patients with IPAH, it has been reported that the expression of endothelial NO synthase is downregulated [7], while other reports show an upregulation in plexiform lesions of IPAH patients [8]. However, little is known about the expression and regulation of soluble guanylate cyclase (sGC) which operates as a receptor for NO. Typically, sGC is found as a hetero...

Cyclic nucleotide-gated (CNG) channels are key elements of cGMP- and cAMP-signaling pathways in vertebrate photoreceptor cells and in olfactory sensory neurons, respectively. These channels form heterooligomeric complexes composed of at least two distinct subunits (α and β). The α subunit of cone photoreceptors is also present in mammalian sperm. Here we identify one short and several long less abundant transcripts of β subunits in testis. The α and β subunits are expressed in a characteristic temporal and spatial pattern in sperm and precursor cells. In mature sperm, the α subunit is observed along the entire flagellum, whereas the short β subunit is restricted to the principal piece of the flagellum. These findings suggest that different forms of CNG channels coexist in the flagellum. Confocal microscopy in conjunction with the Ca2+ indicator Fluo-3 shows that the CNG channels serve as a Ca2+ entry pathway that responds more sensitively to cGMP than to cAMP. Assuming that CNG channel subtypes differ in their Ca2+ permeability, dissimilar localization of α and β subunits may give rise to a pattern of Ca2+ microdomains along the flagellum, thereby providing the structural basis for control of flagellar bending waves.

TRPV6 [transient receptor potential vanilloid 6] is a calcium ion (Ca²+)-selective channel originally identified in the duodenal epithelium and in placenta; replacement of a negatively charged aspartate in the pore-forming region with an uncharged alanine (D541A) renders heterologously expressed TRPV6 channels nonfunctional. We found that male, but not female, mice homozygous for this mutation (Trpv6(D541A/D541A)) showed severely impaired fertility. The motility and fertilization capacity of sperm were markedly reduced, despite intact spermatogenesis. Trpv6 was expressed in epididymal epithelium where the protein was detected in the apical membrane, whereas it was not expressed in spermatozoa or the germinal epithelium. The Ca²+ concentration of the fluid in the cauda epididymis of Trpv6(D541A/D541A) males was 10 times higher than that of wild-type mice, which was accompanied by a seven- to eightfold decrease in Ca²+ absorption through the epididymal epithelium and was associated with reduced sperm viability. Thus, appropriate Ca²+ absorption and a consequent TRPV6-mediated decrease in the extracellular Ca²+ concentration toward the distal segments of the epididymal duct are essential for the acquisition of basic functions and the survival of spermatozoa.

A number of marker substances for neuronal and neuroendocrine cells have been demonstrated in the cytoplasm of the interstitial Leydig cells of human testes using basic immunocytochemical methods and some of their modifications. We were able to reveal immunoreactivity for enzymes involved in the synthesis of the catecholamines dopamine and noradrenaline (tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine-beta-hydroxylase), for the indolamine 5-hydroxytryptamine (serotonin), as well as for a number of well-known neuronal markers such as the neurofilament protein 200, synaptophysin, chromogranin A + B, the neural cell-adhesion molecule (N-CAM), the microtubule-associated protein (MAP-2), and the calcium-binding proteins: S-100, calbindin and parvalbumin. Immunoreactivity for these substances was found in the majority of the interstitial cells although differences in the staining intensity among the individual Leydig cells and among Leydig cells from different patients were observed. At the electron-microscopic level the Leydig cell cytoplasm was seen to contain microtubules, intermediate- and microfilaments as well as clear (40-60 nm) and dense-core (100-300 nm) vesicles, providing a morphological correlate for some of the immunocytochemical results. Although individual marker substances are not absolutely specific for nerve and neuroendocrine cells, the results obtained, together with the already established neuron-specific enolase-, substance P-, methionine-enkephalin- and proopiomelanocortin (POMC)-derived peptide-like immunoreactivity, provide strong evidence for the neuroendocrine (paraneuronal, APUD-like) nature of the Leydig cells of the human testis.

Previous studies have demonstrated that nitric oxide (NO) influences Leydig cell function. Here we provide evidence for NO production and activity in seminiferous tubules and blood vessels of the human testis. By immunohistochemistry, the soluble guanylyl cyclase (sGC), the intracellular NO receptor, and the second messenger, cyclic guanosine monophosphate (cGMP), were detected in myofibroblasts of the peritubular lamina propria in Sertoli cells, as well as in endothelial and smooth muscle cells of testicular blood vessels. Performed with isolated tubules and blood vessels, the biological activity of sGC could be proved by cGMP generation in response to treatments with the NO donor, sodium nitroprusside. The endothelial and neuronal subtypes of NO synthase (NOS) were localized immunohistochemically to the same cell types that express sGC and cGMP. In isolated tubules and vessels, the presence of endothelial NOS and neuronal NOS was confirmed by immunoblotting, and NOS activity was demonstrated by decreased cGMP production upon incubation with the NOS inhibitor L-nitro arginine methylester. These findings show that peritubular cells, Sertoli cells, and testicular blood vessels may be sites of NO production and activity, possibly involved in relaxation of seminiferous tubules and blood vessels to modulate sperm transport and testicular blood flow, respectively.

Background: The TRPV6D541A pore mutation abrogates epididymal Ca 2ϩ absorption causing hypofertility in mice, raising the possibility of residual TRPV6 D541A channel activity. Results: Trpv6 deletion reduces fertility parameters to the same extent as the D541A pore mutation. Conclusion:The D541A pore mutation leads to complete inactivation of TRPV6 channels in epididymal epithelium. Significance: Targeted mutations in mice help to understand the function of TRPV6 proteins in native systems.

By means of immunocytochemical methods, immunoreactivity for the brain isoform of nitric oxide synthase (NOS-I) was recognized in numerous Leydig cells of the human testis as well as in MA-10 tumor and TM3 non-tumor mouse Leydig cell lines. Within the Leydig cell cytoplasm, immunocytochemical results suggested the occurrence of factors known to activate NOS-I such as glutamate and aspartate, as well as molecules involved in the regulation of the NOS-I activity such as calmodulin and Ca2+/calmodulin-dependent protein kinase II. Leydig cells, Sertoli cells, some endothelial cells of the testis, MA-10- and TM3 mouse Leydig cell lines exhibited a relatively strong NADPH-diaphorase enzyme activity as well. Double sequential immunostainings provided evidence that NOS-like immunoreactivity of the testicular Leydig cells is colocalized with testosterone, calmodulin, aspartate, glutamate, and Ca2+/calmodulin-dependent protein kinase II. Sodium nitro-prusside treatment did not result in increased cGMP formation by MA-10- or TM3 mouse Leydig cells, suggesting that NO produced by these cells acts primarily in a paracrine fashion. The NO produced by NOS-I immunoreactive Leydig cells may act as a messenger: 1) between neighbouring NOS-I positive and/or negative Leydig cells as well as to mediate the action of numerous intracellular and extracellular neuroactive substances and growth factors; 2) between Leydig cells and the muscle cells or pericytes of blood vessels to regulate local blood flow and permeability; and 3) between Leydig cells and pertibular myofibroblasts to influence their contraction and the permeability of the lamina propria.