Ralf Middendorff

Progenitor cells of the testosterone-producing Leydig cells revealed

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.

The Leydig cell of the human testis- a new member of the diffuse neuroendocrine system

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 (tryosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine-β-hydroxylase), for the indolamine 5-hydroxytryptamine (serotonin), as well as for a number of wellknown 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 neuronspecific enolase-, substance P-, methionine-enkephalinand proopiomelanocortin (POMC)-derived peptide-like immunoreactivity, provide strong evidence for the neuroendocrine (paraneuronal, APUD-like) nature of the Leydig cells of the human testis.

Nitric oxide/cGMP pathway components in the Leydig cells of the human testis

Abstract.In this study we sought to determine whether the main components of the nitric oxide (NO) pathway are localized within the Leydig cells of the human testis and whether the soluble guanylyl cyclase (sGC), the enzyme that accounts for NO effects, is functionally active in these cells. Using an amplified immunocytochemical technique, immunoreactivity for nitric oxide synthase (NOS-I), sGC and cyclic guanosine monophosphate (cGMP) was detected within the cytoplasm of human Leydig cells. Distinct differences in staining intensity were found between individual Leydig cells, between cell groups and between Leydig cells of different patients. By means of a specific cGMP-RIA, a concentration-dependent increase in the quantity of cGMP was measured in primary cultures of human Leydig cells following exposure to the NO donor sodium nitroprusside. In addition, NOS-I immunoreactivity was seen in Sertoli cells, whereas cGMP and sGC immunoreactivity was found in Sertoli cells, some apically situated spermatids and residual bodies of seminiferous tubules. Dual-labelling studies and the staining of consecutive sections showed that there are several populations of Leydig cells in the human testis. Most cells were immunoreactive for NOS-I, sGC and cGMP, but smaller numbers of cells were unlabelled by any of the antibodies used, or labelled for NOS-I or cGMP alone, for sGC and cGMP, or for NOS-I and sGC. These results show that the Leydig cells possess both the enzyme by which NO is produced and the active enzyme which mediates the NO effects. There are different Leydig cell populations that probably reflect variations in their functional (steroidogenic) activity.

Male fertility depends on Ca2+ absorption by TRPV6 in epididymal epithelia

Production of functional spermatozoa requires regulation of the Ca2+ concentration in epididymal fluid by TRPV6. A Good Environment Is Crucial Male infertility can result from decreased production of or functional deficits in sperm. Ca2+ signaling plays a crucial role in sperm function, and, here, Weissgerber et al. uncovered a role for the Ca2+-selective TRPV6 channel in regulating Ca2+ concentration in the lumen of the epididymis as well as sperm motility and survival. Sperm maturation—including the acquisition of motility—occurs in the epididymis, after their exit from the testis. Male transgenic mice bearing an inactive form of TRPV6 showed decreased fertility, and the motility, viability, and in vitro capacity to fertilize eggs of sperm isolated from their caudal epididymides were impaired. TRPV6 was present in epididymal epithelial cells but not in the sperm themselves, and the Ca2+ concentration in the lumens of the epididymides of transgenic mice was 10 times higher than in wild-type mice. Moreover, sperm exposed to comparable extracellular Ca2+ concentrations showed an increase in intracellular Ca2+ concentration. The authors thus conclude that TRPV6 channels function to decrease the Ca2+ concentration of the intraluminal fluid in the epididymis and propose that the impaired function and survival of sperm in the transgenic mice results from the disturbed microenvironment in the epididymal fluid. 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 (Trpv6D541A/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 Trpv6D541A/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.

Neuroendocrine marker substances in human Leydig cells — changes by disturbances of testicular function

Summary. A number of neuroendocrine and neuronal markers were demonstrated in Leydig cells of the testes of 18 men aged between 20 and 81 years. Tissue sections were divided into five groups, i.e. carcinoma of the prostate (control cases; n = 4), seminoma (n = 8), anti‐androgen therapy (n = 3), oestradiol therapy (n = 2) and cryptorchidism (n = 1). The following substances were immunocytochemically tested: the monoamine synthesizing enzymes tyrosine hydroxylase, aromatic L‐amino acid decarboxylase, dopamine‐β‐hydroxylase and phenylethanolamine‐N‐methyltransferase, the indolamine serotonin, the calcium‐binding proteins parvalbumin, calbindin and S‐100 protein, the microtubule associated protein‐2, as well as neurofilament protein 200, synaptophysin, neuron specific enolase, substance P and chromogranin A + B. All these substances were found in Leydig cells of all sections independently of the pathological changes of the testes. Compared with the control cases, all the other groups showed a significantly weaker immunore‐activity for all markers. The uniformity of staining among the different antibodies allows the deduction that these neuroactive peptides may belong to a basic equipment of Leydig cells probably stabilizing their function in an autocrine manner. On the other hand, Leydig cells themselves seem to be a stable structural component of the testis, which are not essentially involved in the pathogenesis of the disturbances mentioned above.

The expression of neurotrophins and their receptors in the prenatal and adult human testis: evidence for functions in Leydig cells

Previous studies have demonstrated local functions for neurotrophins in the developing and mature testis of rodents. To examine whether these signaling molecules are present and also potentially active in the human testis, we characterized immunohistochemically the expression and cellular localization of the known neurotrophins and their receptors during prenatal testicular development as well as in the adult human testis. Results obtained revealed the presence of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 and 4, as well as neurotrophin receptors p75NTR, TrkA, TrkB, and TrkC during testis morphogenesis. These proteins were also detectable in the adult human testis, and their local expression could be confirmed largely by immunoblot and RT-PCR analyses. Remarkably, the Leydig cells were found to represent the predominant neurotrophin/receptor expression sites within both fetal and adult human testes. Functional assays performed with a mouse tumor Leydig cell line revealed that NGF exposure increases cellular steroid production, indicating a role in differentiation processes. These findings support previously-recognized neuronal characteristics of Leydig cells, provide additional evidence for potential roles of neurotrophins during testis morphogenesis and in the mature testis, and demonstrate for the first time a neurotrophin-induced functional activity in Leydig cells.

Pituitary Adenylate Cyclase‐Activating Peptide and Vasoactive Intestinal Peptide Receptor Expression in Immortalized LHRH Neurons

The regulation of LHRH secretion is extraordinarily multifarious. To no small extent, this insight has been gained through studies using the immortalized hypothalamic LHRH neuronal line, GT1‐7. In the present study, we examined these cells for potential expression of the receptors for the related peptides PACAP and VIP. By means of reverse transcription‐polymerase chain reaction (RT‐PCR) with PACAP receptor‐specific primers, in combination with restriction enzyme analysis and cDNA sequencing, we were able to identify all PACAP‐specific receptor splice variant forms with variable degrees of expression. Of the two nonselective VIP/PACAP receptors (i.e. VIP‐R type I and II) only the latter isoform was detected by RT‐PCR. In view of these results, we sought to establish whether PACAP and VIP receptors are functional in GT1‐7 cells. Cyclic AMP (cAMP) accumulation after addition of PACAP‐38 (or PACAP‐27) was dose‐dependent with maximal 3‐fold increases. VIP also elevated cAMP with a similar potency. Phosphatidylinositol (PI) turnover was unaffected by either PACAP or VIP. Acute LHRH secretion was stimulated equally by nanomolar concentrations of both PACAP and VIP. These results point to PACAP and VIP having direct actions via the VIP2R on cAMP signalling and LHRH release, in addition to the known effects of these peptides on pituitary functions.

The neuroendocrine leydig cells and their stem cell progenitors, the pericytes

The Leydig cells of the testis represent the main source of androgens. The idea of Leydig cells as endocrine cells has been the leading characteristic of this interesting cell population till now. Our studies of the last 2 decades allowed us to reveal a new important feature of Leydig cells that is their obvious similarity with structures of the central and peripheral nervous system. This includes the expression of neurohormones, neurotransmitters, neuropeptides and glial cell antigens. In this way, it became evident that in addition to the well established control by steroids and systemic hormones, important local auto- and paracrine control me chanisms of testicular functions exist. Thus, the Leydig cells represent a specialized cell population with both endocrine and neuroendocrine properties. The discovery of the neuroendocrine features of Leydig cells gave rise to the hypothesis of a potential neuroectodermal and/or neural crest origin of testicular Leydig cells. In an experimental animal model we revealed that adult Leydig cells originate by transdifferentiation from stem/progenitor cells (pericytes and smooth muscle cells), underlying the close relationship of Leydig cells with testis microvasculature. This and the supporting data from the literature provided the basis for revealing the pericytes as a common adult stem cell type of mammalian species. Distributed by the microvasculature through the entire body, the pericyte, acting as a resting early pluripotent adult stem cell, provides an ingenious system to assure the maintenance, physiological repair and regeneration of organs, each under the influence of specific local environmental factors.

The Effect of NO‐Donors in Bovine and Rat Pineal Cells: Stimulation of cGMP and cGMP‐Independent Inhibition of Melatonin Synthesis

The presence of soluble guanylate cyclase in the pineal and its regulation by adrenergic pathways has been well documented. Recent evidence points to adrenergically stimulated nitric oxide generation as a mechanism for coupling this pathway. To what extent nitric oxide (NO) signalling can influence adrenergically stimulated melatonin synthesis has not been investigated. Cyclic guanosine 3′,5′‐monophospate (cGMP) signal transduction in the bovine pineal has also received little attention. We describe in the present report: 1) a dose‐dependent elevation of cGMP in response to the nitrovasodilators, sodium nitroprusside (SNP) and 3‐morpholino‐sydnonimine (SIN‐l), 2) a dose‐dependent inhibition of melatonin synthesis by SNP and SIN‐1, but not by 8‐Br‐cGMP in both bovine and rat pineal cell cultures, which is not due to cytotoxicity as judged by two different approaches, and 3) immunohistochemical evidence for the presence of nitric oxide synthase (NOS) (EC 1.14.23.‐) in the intact bovine pineal gland and in cultured bovine pinealocytes. These data support the view that NOS is a component of the cGMP‐generating system in mammalian pinealocytes. Although NO‐donor molecules are also potent activators of cGMP accumulation, they may have other important actions in the pineal, namely the inhibition of adrenergic‐stimulated melatonin synthesis. As SNP and SIN‐1 exerted this inhibitory effect on cells regardless of whether they were stimulated by isoproterenol, forskolin or 8‐Br‐CAMP it would appear that NO‐donors can act ‘downstream’ from the receptor/adenylate cyclase level.

Soluble polysialylated NCAM: a novel player of the innate immune system in the lung

Posttranslational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is well studied in the nervous system and described as a dynamic modulator of plastic processes like precursor cell migration, axon fasciculation, and synaptic plasticity. Here, we describe a novel function of polysialylated NCAM (polySia-NCAM) in innate immunity of the lung. In mature lung tissue of healthy donors, polySia was exclusively attached to the transmembrane isoform NCAM-140 and located to intracellular compartments of epithelial cells. In patients with chronic obstructive pulmonary disease, however, increased polySia levels and processing of the NCAM carrier were observed. Processing of polysialylated NCAM was reproduced in a mouse model by bleomycin administration leading to an activation of the inflammasome and secretion of interleukin (IL)-1β. As shown in a cell culture model, polySia-NCAM-140 was kept in the late trans-Golgi apparatus of lung epithelial cells and stimulation by IL-1β or lipopolysaccharide induced metalloprotease-mediated ectodomain shedding, resulting in the secretion of soluble polySia-NCAM. Interestingly, polySia chains of secreted NCAM neutralized the cytotoxic activity of extracellular histones as well as DNA/histone-network-containing “neutrophil extracellular traps”, which are formed during invasion of microorganisms. Thus, shedding of polySia-NCAM by lung epithelial cells may provide a host-protective mechanism to reduce tissue damage during inflammatory processes.