Evidence for production and functional activity of nitric oxide in seminiferous tubules and blood vessels 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.

Natriuretic peptides in the human testis: evidence for a potential role of C-type natriuretic peptide in Leydig cells.

Functional studies indicate that natriuretic peptides have direct effects on Leydig cells of the testis. In this report, we demonstrate local synthesis of one member of the natriuretic peptide family, C-type natriuretic peptide (CNP), in Leydig cells of human testes. Using RT-PCR assays, messenger RNA (mRNA) for the CNP precursor was detected in human testis and found to be prominently expressed in Leydig cells. Immunohistochemical analyses revealed CNP to be almost exclusively associated with Leydig cells. Distinct differences in the staining intensity-including cells without detectable staining-suggest a heterogeneity of CNP expression within the Leydig cells. Moreover, the presence of transcripts for the CNP receptor, a particulate guanylate cyclase, termed GC-B, was demonstrated by RT-PCR in human testis and in isolated Leydig cells. The expression of this receptor in human testis membranes could be confirmed by affinity labeling with 125I-labeled CNP. These findings demonstrate, for the first time, the production of a natriuretic peptide in human Leydig cells. The occurrence of CNP and its receptor in the human testis points to a local role of the peptide, presumably acting in an auto- or paracrine manner to modulate organ-specific functions.

Synthesis of C-type natriuretic peptide (CNP) by immortalized LHRH cells.

Former studies have indicated an influence of natriuretic peptides on LHRH secretion. In this report we demonstrate local synthesis of CNP in immortalized LHRH neurons (GT1-7 cells). Using reverse transcription-polymerase chain reaction and RNase protection assays a transcript for the CNP precursor was identified in these cells. Immunocytochemical data revealed the presence of the peptide CNP in GT1 cells, using a specific polyclonal antiserum against CNP. Electron microscopic immunohistochemical investigations also showed the strongest CNP-immunoreactivity in some small vesicles, providing initial evidence for the potential secretion of this peptide by immortalized LHRH neurons. Subsequent experiments demonstrated also that CNP elevates LHRH production in static cultures of GT1 cells. These data show for the first time the co-production of the functionally relevant natriuretic peptide, CNP, by immortalized LHRH neurons. Together with the recent demonstration of CNP receptor expression by these cells, we suggest that CNP may represent a novel autocrine regulator of LHRH neuronal activity. It remains to be elucidated, however, to what extent CNP expression in immortalized LHRH neurons reflects a co-localization in situ of CNP and LHRH peptides.

Substance P modulating effect on the binding capacity of hamster Leydig cell LH receptors.

The influence of Substance P was studied on the binding characteristics of LH receptors in purified Leydig cells collected from golden hamsters kept under natural long or short days. Substance P exerted a differential effect on the binding capacity of LH receptors. A significant increase in Bmax was estimated in Leydig cells obtained from young hamsters living under long days. In contrast, Substance P reduced the number of the LH binding sites in Leydig cell cultures prepared from adult hamsters housed under short-day conditions.

New aspects of Leydig cell function.

Previous studies indicated that the Leydig cells of the human testes show similarities to neuroendocrine cells. In this context, the local synthesis of two neuroactive signaling molecules, namely nitric oxide (NO) and C-type natriuretic peptide (CNP), both acting via the second messenger, cyclic guanosine monophosphate (cGMP), might be of physiological relevance. By immunoblotting, immunohistochemical analyses and affinity crosslinking experiments, respectively, the presence of soluble guanylate cyclase (sGC), the NO receptor, and of guanylate cyclase B (GC-B), representing the CNP receptor, was demonstrated in Leydig cells, seminiferous tubules and blood vessels of the human testis. Moreover, cGMP and its binding protein cGMP-dependent protein kinase type I (GK I) were found in these structures. The functional activity of the two receptors was proved by generation of cGMP in response to treatments with the NO donor, sodium nitroprusside (SNP), and with CNP, respectively. As indicated by immunohistochemical analyses and by treatments of cells with either SNP or CNP, human Leydig tumour cells and MA10 cells, representing a mouse Leydig tumour cell line, were found to be distinguished by a reduced expression of the receptors for NO and CNP. Furthermore, expression levels of the components of the two cGMP-generating systems were found to be widely unchanged in Leydig cells during different ontogenetic stages. Though cGMP has been shown to influence testosterone release, the constant developmental expression patterns of NO and CNP apparently independent of differences in androgen production, the down-regulation of their receptors in tumorous cells, and the presence of GK I, may point to additional autocrine functions of these factors and of cGMP in Leydig cells. Moreover, possible paracrine actions of NO and CNP may include relaxation of seminiferous tubules and blood vessels in order to modulate sperm transport and testicular blood flow, respectively. These findings suggest that Leydig cell-derived factors may exert activities different from or in addition to those involved in the regulation of testosterone production.

Reinvestigation of the transitional epithelium (urothelium) of the human ureter.

The transitional epithelia (urothelia) of the ureters of 30 patients of different ages were studied by means of light and electron microscopical, histochemical and immunocytochemical methods. A great variability of the normal structural appearance of the urothelium was established. Structural features and the uptake of exogenous peroxidase by the surface epithelial cells provided high endocytotic activity. Urothelial cells take up many low and high molecular weight substances from the urine and further metabolize and transport these toward the subepithelial connective tissue. Lymphocytes, macrophages, monocytes, plasma cells and rarely polymorphonuclear leukocytes are distributed intra- and extraepithelially and are involved in the immunological response to agents which enter the intercellular spaces of the epithelium. These cells are also responsible for the elimination of aged and degenerating superficial squamous cells. The present investigation establishes the existence of immunological defence mechanisms in the adult human urothelium. The results obtained suggest that the human ureter contains three functional barriers directed against aggressive components of the urine: the first represented by structures of the superficial squamous cells, the second by the upper cells of the ureteric intermediate layer and the third comprising epithelial and immunological cells involved in immune defence mechanisms.

Immunocytochemistry--possibilities for detection of different tissue antigens and establishment of the functional role of cells.

The present review underlines briefly the wide distribution and application of the main immunocytochemical methods for the detection and localization of numerous antigens in tissue sections. For example, a lot of neuroactive substances have been revealed in structures of the central and peripheral nervous system by means of immunocytochemical techniques. The results obtained serve for the better understanding of the main physiological role and the interrelationships of different neuronal classes. Furthermore, some important problems accompanying an immunocytochemical staining connected with the quality of the antibodies used, with the resemblance of the chemical composition of different antigens, and with the coexistence of more neuroactive substances in a nerve cell are pointed out.

Coexistence of GABA- and choline acetyltransferase (ChAT)-like immunoreactivity in the hypoglossal nucleus of the rat.

Single and sequential double immunocytochemical techniques were applied to localize gamma-aminobutyric acid (GABA)- and choline acetyltransferase (ChAT)- like immunoreactivity (-LI) in the hypoglossal nucleus of the rat. After subsequential double staining a relatively high number of hypoglossal motor neurons showed the coexistence of both ChAT- and GABA-LI. Coexistence of both substances was also revealed in the axons of the hypoglossal nerve situated within the medulla oblongata. Cells showing only ChAT- or GABA-LI were also observed. Differences in immunostaining between the different cell groups of the hypoglossal nucleus were established. Following axotomy of the right hypoglossal nerve, a decrease or loss of the immunoreactivity for both ChAT and GABA in the motor neurons was established until the 3rd week after the operation. The results obtained do not give evidence on the origin of the GABA-like immunoreactive material and its functional significance in the cholinergic neurons. It can be only speculated that the GABA-like material is either taken up from the intercellular space or is synthesized by the ChAT-LI nerve cells. Functionally, the importance of GABA for the synthesis of gamma-hydroxybutyrate (a novel neurotransmitter candidate) and its postsynaptic transmitter action or presynaptic regulatory action (through autoreceptors in the membrane of the nerve endings) on the release of acetylcholine (ACh) should be taken into consideration.

Age-related changes in serotonin-immunoreactivity in the telencephalon and diencephalon of rats.

Comparison of the appearance and density of the serotoninergic fibers and terminals in some tel- and diencephalon areas of young adult (3 months) and aged (28 months) rats was made immunohistochemically using an antibody to serotonin. In young adult rats a characteristic arrangement of the serotonin-immunoreactive (SER-IR) fibers and terminals in the tel- and diencephalon was observed. In aged rats the distribution pattern was the same but changes in the appearance and density of the SER-IR fibers were demonstrated. Many swollen and folded fibers forming cluster-like structures as well as reduced fiber networks were seen in the neocortex, hippocampal formation and striatum of aged rats. Aberrant fibers and decreased fiber density were also observed in the bed nucleus of the stria terminalis, lateral septum, nucleus accumbens and thalamic nuclei. Reduced fine granular immunostaining and scattered swollen varicosities were found in some amygdaloid and hypothalamic nuclei of aged rats. These results provide further morphological evidence for changes in the serotoninergic innervation during aging.

Acetylcholinesterase activity and type C synapses in the hypoglossal, facial and spinal-cord motor nuclei of rats. An electron-microscope study.

Using the electron-microscope technique of Lewis and Shute, we studied the localization of the acetylcholinesterase (AChE) activity in the hypoglossal, facial and spinal-cord motor nuclei of rats. The technique used selectively detects synapses with subsynaptic cisterns (type C synapses) as well as heavy deposits of reaction products in the rough endoplasmic reticulum, in fragments of the nuclear envelope, in some Golgi zones and on parts of the pericaryal plasma membrane, the axolemma and the dendritic membrane. In C synapses, AChE activity was located in the synaptic cleft and on the membrane of presynaptic boutons. Some C synapses exhibited distinct synaptic specialization in the form of multiple 'active zones'. These zones were characterized by dense presynaptic projections, short dilations of the synaptic cleft, and postsynaptic densities localized between the postsynaptic membrane and the outer membrane of the subsynaptic cistern. Within the postsynaptic densities, rows of rod- or channel-like structures were observed. The subsynaptic cisterns were continuous with the positive rough endoplasmic reticulum. The results are discussed in terms of the possible role of C synapses in the regulation of AChE synthesis in postsynaptic cholinergic neurons and/or in the regulation of AChE release into the extracellular space as well as in the establishment of new synaptic contacts.

The vegetative network in the thoracolumbar spinal cord of the guinea pig: a comparison of the distribution of AChE-enzyme activity and choline acetyltransferase-like immunoreactivity.

Histochemically the acetylcholinesterase (AChE) enzyme activity and immunocytochemically the choline acetyltransferase-like immunoreactivity (ChAT-LI) were located in the components of the vegetative network of the thoracolumbar spinal cord of the guinea pig. Both reaction products showed an identical distribution among the preganglionic sympathetic cells and the processes of the vegetative network, namely: cells of the nucleus intermediolateralis pars principalis (ILp), the nucleus intermediolateralis pars funicularis (ILf), the nucleus intercalatus spinalis (IC) and the nucleus intercalatus paraependymalis (ICpe; terminology according to Petras and Cummings 1972). In longitudinal horizontal sections through the intermediate zone of the thoracolumbar spinal cord both AChE-positive- and ChAT-like immunoreactive nerve fibers were organized into two longitudinal lateral fascicles (FLL), two longitudinal medial fascicles (FLM) as well as oblique and transverse bundles that interconnect repeatedly the autonomic cell groups of this zone along the spinal cord and contribute to the ladder-like shape of the vegetative network. The ChAT-like immunostaining of the vegetative network showed that the dendrites of the ILp cells are oriented mainly in a rostrocaudal, but also in a mediolateral direction. Similar orientation of the dendrites was observed for the ICpe cell groups of the thoracolumbar intermediate zone. Thus it is evident that ILp cell bodies and dendrites are involved in the formation of the FLL, whereas the ICpe cells and their dendrites--of the FLM. The IC cells send their dendrites towards both the ILp and ICpe cells and build up together with dendrites of the ILp and ICpe cells the transverse and oblique interconnecting bundles. The vegetative network is strongly developed within the intermediate zone of T1-T4 (mostly T3) and T7-T8 segments of the spinal cord. In these segments a greater variety of interconnections between the preganglionic sympathetic cell groups which are constituents of the network are also revealed. In the remaining segments the vegetative network is more poor developed. In the first two lumbar segments the distance between the interconnecting bundles in the rostrocaudal direction diminishes to 100 microns in contrast to 300-500 microns within the upper segments. The results obtained reveal that the cholinergic preganglionic sympathetic nuclei of the intermediate zone of the thoracolumbar spinal cord together with their dendrites represent the basis (frame) of the ladder-like vegetative network to which join in addition different peptidergic fibers of supraspinal, peripheral and propriospinal origin.

Localization of some neuropeptide- and serotonin-like immunoreactivities in the vegetative network of guinea pig spinal cord.

The localization of Substance P(SP)-, Methionine-Enkephalin(met-Enk)-, Somatostatin(SOM)- Serotonin(SER)-, Cholecystokinin(CCK)-, and Vasoactive intestinal polypeptide (VIP)-like immunoreactivity (-LIR) has been determined immunocytochemically in the thoracic spinal cord intermediate zone of male and female guinea pigs. All neuroactive substances studied are exclusively localized in nerve fibre varicosities and terminals building up the vegetative network of the thoracic spinal cord intermediate zone. This network is situated dorsally to the central canal as a longitudinal plate of approximate thickness of 90-100 microns. Immunoreactive fibres are observed in the two Fasciculi longitudinales laterales and the two Fasciculi longitudinales mediales which are interconnected by transverse and oblique peptide-containing bundles (the terminology used by Petras and Cummings 1972; Galabov and Davidoff 1976). All these bundles interconnect the nuclei intermediolaterales principales and funiculares, the nuclei intercalates spinales and the nuclei intercalates paraependimales in ipsi- and contralateral as well as in rostral and caudal direction. The neurones of these nuclei are surrounded by immunoreactive varicosities and terminals. The quantity of the immunoreactive structures and intensity of the staining varied for the different neuroactive substances. As to the origin of the vegetative network immunoreactive fibres three main possibilities exists: a). From primary afferent neurones situated in the dorsal root ganglia, which send their axons via the dorsal roots (mainly for SP and perhaps for CCK); b). From supraspinal neurones which send their axons descending in the white matter funiculi and in the fasciculi longitudinales laterales and mediales and c). From intrinsic spinal cord neurones, which send their neurites in ascending and descending directions, ipsi- and contralaterally and interconnect the spinal cord segments. The different origin of the vegetative network immunoreactive fibres as well as the complex innervation of the preganglionic sympathetic nerve cells in the intermediate zone of the spinal cord suggests that this network may play an important role in the integration of the central and peripheral vegetative nervous system as well as probably in the integration of the somatic and the vegetative nervous system.

In vitro effects of substance P and arginine-vasopressin on testosterone production in Leydig cells of short and long photoperiodic hamsters.

The aim of the present study was to determine the interaction between substance P (SP) and arginine-vasopressin (AVP) on basal and LH-stimulated testosterone production by Leydig cells isolated from hamsters kept under long or short days (LD-hamsters, SD-hamsters, respectively). SP inhibited the testosterone production of Leydig cells, its effect being more pronounced in the case of LH-stimulated steroidogenesis in LD-hamsters. Similarly, the addition of AVP to the culture medium resulted in a diminution of basal, as well as LH-stimulated testosterone secretions. When Leydig cells were co-incubated with SP (10(-7) M) and AVP (10(-7) M), a strong inhibition of the testosterone production by 50-60% was established in LD-animals. However, even within the experimental circumstances in SD-hamsters, the modulation of testosterone production by SP and AVP was evident. The reported results suggest that there is an interference of two regulatory pathways, namely photoperiodic dependence and paracrine control of testicular steroidogenesis in hamsters.

The fate of newly synthesized proteins in nerve and glial cells following nerve injury as shown by electron microscope radioautography.

Following peripheral crush of hypoglossal nerve the fate of newly synthesizing proteins in corresponding nerve and glial cells was traced out using light and high resolution radioautography after intraventricular injection of 3H-leucine. Radioautographic analysis was applied on the normal and chromatolytic neurons and their glial surroundings 45 min after injection of labelled precursor. Both chromatolytic neurons and surrounding glia showed higher incorporation of labelled proteins than those of control nerve and glial cells. Relative specific radioactivity (RSR) of ergastoplasm and lysosomes were increased in the chromatolytic neurons, whereas RSR of nuclei, mitochondria and Golgi apparatus were decreased. Contrary to neurons, RSR of corresponding compartments in experimental glial cells except lysosomes showed reciprocal values in comparison with control glial cells. The higher RSR of mitochondria and Golgi apparatus in experimental glial cells depended mostly on the astrocytes, whereas those of nuclei and lysosomes were related first of all with microglial cells. These findings are correlated with present concepts of the complex interdependency between neurons and glia in which mutual regulatory controls and influences are exerted.

Are Leydig cells of neural origin? Substance P-like immunoreactivity in human testicular tissue.

Using the immunocytochemical peroxidase-antiperoxidase method we observed strong and selective staining of Leydig cells after incubation of human testicular tissue with an antiserum against substance P and a slightly weaker immunoreactivity against methionine-enkephalin. These results indicate that the embryologic origin of Leydig cells may require reconsideration and also offer a new perspective for research upon the local control mechanisms of spermatogenesis.

Substance P-induced inhibition of Leydig cell steroidogenesis in primary culture.

The effect of substance P (SP) on hamster Leydig cell steroidogenesis in primary culture was investigated. Purified Leydig cells were cultured with or without SP for 24 h. The levels of testosterone and progesterone in the culture media were 174 +/- 20 pg ml-1 and 105 +/- 12 pg ml-1, respectively. In the presence of SP (10(-7) mol l-1), testosterone concentration significantly decreased to 123 +/- 19 pg ml-1, e.g. with 29.3%. By contrast, at the concentration used, SP had no effect on progesterone secretion. The possible molecular mechanism of the SP action on Leydig cell function was discussed. The reported results indicate that SP can modulate Leydig cell steroidogenesis in culture.