Contractility of the epididymal duct: function, regulation and potential drug effects

During their transit through the epididymis, spermatozoa mature and acquire motility and fertilizing capacity. The smooth muscle cells (SMCs) of the epididymal duct are thought to be responsible for the adequate transport of spermatozoa. Thus, precise regulation of SMC function also represents a prerequisite for sperm maturation thereby contributing to male fertility. In this review, we would like to highlight various aspects of epididymal SMC function and discuss several angles with respect to regulation of contraction and relaxation. Different to the vas deferens, where disturbed SMC pathways resulting in male infertility could be defined, comparable information is missing in the epididymis. We therefore include some vas deferens data which could also be useful for a better understanding of epididymal SMC function. Furthermore, we would like to draw attention to drugs used in clinical practice and their potential (side) effects on contractions in the epididymis.

Epididymitis: ascending infection restricted by segmental boundaries.

STUDY QUESTION: Is the regionalization of epididymitis related to epididymal segmentation? SUMMARY ANSWER: We show for the first time that luminal ascent of bacteria is strictly gated by epididymal segment boundaries, involving ductal constriction adjacent to the infected area. WHAT IS KNOWN ALREADY: The epididymal duct is a continuous, unbranched tube, coiled into segments that are divided by connective tissue septa. Sonographic analysis indicates that swelling associated with epididymitis is predominant in the cauda region. Epididymal segmentation has never been investigated in the context of pathological alterations. STUDY DESIGN, SIZE, AND DURATION: We analyzed segment-specific changes in the epididymal duct in a mouse model and in men. In the mouse epididymitis model (3 days post-infection, injection of bacteria into the lumen of the vas deferens), two Escherichia coli strains were tested: a uropathogenic strain CFT073 (UPEC, n = 7) and a fecal non-pathogenic strain NPEC470 (NPEC, n = 5). Two control groups: phosphate-buffered saline, sham-treated animals (n = 4) and untreated mice (n = 8). In addition, segmentation was verified by ex vivo injection of dye into the interstitial spaces of untreated mouse epididymides. Histological findings were compared with specimens from epididymitis patients (n = 10, age range 14-78, median 60 years) who underwent surgical intervention; control: samples from patients without epididymitis (n = 16, age range 38-87, median 73 years). PARTICIPANTS/MATERIALS, SETTING, AND METHODS: We investigated the ascending infections by detailed histological analysis in correlation with local infection status in a mouse epididymitis model. As a proof of concept, rare patient material from two archives was analyzed: epididymides from patients who underwent surgical intervention for persisting epididymitis, and for control, histologically normal epididymides from men who underwent orchiectomy for therapy of prostatic carcinoma. MAIN RESULTS AND THE ROLE OF CHANCE: Luminal ascent of E. coli in mice was strictly gated by epididymal segment boundaries. In the mouse model, both strains of E. coli were detected exclusively in the distal cauda segment associated with damage of the epithelium and muscle layer. Ductal constriction occurred in the non-infected upstream segments of infected area, putatively blocking further luminal ascent of bacteria in UPEC-infected animals. Corresponding histological and morphological changes were found in epididymitis patients. The caput region was found to be unaffected in patients and the mouse model. LIMITATIONS, REASONS FOR CAUTION: Patient samples represented advanced cases of epididymitis that made surgical intervention necessary. WIDER IMPLICATIONS OF THE FINDINGS: Our data demonstrate the impact of epididymal segmentation, presumably a protective response mechanism against infectious invasion and bacterial ascent, during epididymitis and affirm the importance of rapid intervention. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from the State of Hessen (LOEWE-MIBIE) and the DFG (KFO 181). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: No clinical trial involved.

Disturbed spermatogenesis associated with thickened lamina propria of seminiferous tubules is not caused by dedifferentiation of myofibroblasts.

BACKGROUND: In the human testis, myofibroblasts are the main cellular components of the lamina propria (LP) of seminiferous tubules. Thickened ('fibrotic') LP and dilated tubules are found in a large number of infertile patients, and myofibroblast dedifferentiation has been described in elderly men. It is not known, however, whether dedifferentiation of myofibroblasts is responsible for disturbed spermatogenesis associated with LP alterations. METHODS: The LP of testicular tissue from infertile men (n = 35) was investigated by new histological and morphometric approaches, RT-PCR after laser microdissection and western blotting. RESULTS: Myofibroblasts were found in the LP of all seminiferous tubules. On the basis of LP morphology, each tubule could be assigned to one of the four groups, which showed increasing pathology: intact LP (Group 1), increased extracellular matrix (ECM) in-between the network of myofibroblasts (Group 2), two layers of myofibroblasts engulfing thickened ECM (Group 3) and LP additionally lacking an inner myofibroblast layer (Group 4). All myofibroblasts of all groups and of dilated tubules were fully differentiated, as could be shown by the expression of α-smooth muscle actin, myosin heavy chain, calponin 1 as well as relaxation-mediating cGMP-dependent protein kinase I and phosphodiesterase 5. Independently of the clinical background, the same patterns of thickened LP were detectable. There was a gradual decrease in intact spermatogenesis and in diameter/LP ratio from Groups 1 to 4, indicating that patterns of LP alterations reflect the quality of spermatogenesis. The thickness of myofibroblast layers increased towards Group 4 without cell proliferation, but CD34(+) cells, marking cells of haematopoetic lineage and progenitor cells (in lung fibrosis), were found in close proximity to tubules. CONCLUSIONS: Data indicate that dedifferentiation of myofibroblasts is not responsible for disturbed spermatogenesis associated with LP alterations. Thus, myofibroblasts, presumably newly developed in part, might contribute to disturbed spermatogenesis as key players during development of fibrotic LP alterations but not by contractile dysfunction.

Cyclic nucleotide-gated channels on the flagellum control Ca2+ entry into sperm.

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 (alpha and beta). The alpha subunit of cone photoreceptors is also present in mammalian sperm. Here we identify one short and several long less abundant transcripts of beta subunits in testis. The alpha and beta subunits are expressed in a characteristic temporal and spatial pattern in sperm and precursor cells. In mature sperm, the alpha subunit is observed along the entire flagellum, whereas the short beta 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 alpha and beta subunits may give rise to a pattern of Ca2+ microdomains along the flagellum, thereby providing the structural basis for control of flagellar bending waves.

Expression and function of soluble guanylate cyclase in pulmonary arterial hypertension.

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 x kg(-1) x day(-1)) 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.

C-type natriuretic peptide (CNP) in the pituitary: is CNP an autocrine regulator of gonadotropes?

Natriuretic peptides act via receptors with intrinsic guanylate cyclase activity to stimulate cGMP production and are thought to be important regulators of neuroendocrine systems. C-Type natriuretic peptide (CNP) is of particular interest in this regard because the highest tissue concentrations of CNP occur in the anterior pituitary, where it is a highly potent stimulator of cGMP production. Here we show that pituitaries of rats and mice contain abundant CNP prohormone messenger RNA (mRNA), but no atrial natriuretic peptide or B-type natriuretic peptide prohormone mRNAs. Using reverse transcriptase-polymerase chain reaction, both A- and B-type natriuretic peptide receptor (GC-A and GC-B, respectively) transcripts were detected in rat and mouse pituitaries, although only the GC-B mRNA was measurable by Northern blotting. Immunohistochemistry revealed CNP-positive cells in the anterior, but not posterior, pituitaries of rats, and the vast majority of these cells were identified as gonadotropes by colocalization of CNP and LH immunoreactivities. Targeted toxicity using GnRH conjugated to the ricin-A chain was used to test whether gonadotropes are also direct targets for GnRH action. The conjugate dose dependently inhibited the proliferation of alpha T3-1 cells (gonadotrope-derived cells with GnRH receptors), but had no such effect on GH3 cells (which do not have GnRH receptors). Culture of rat pituitary cells with the conjugate caused comparable reductions in CNP-stimulated cGMP production, GnRH-stimulated LH release, and CA2+ ionophore (A23187)-stimulated LH release, but did not measurably alter cAMP production in response to pituitary adenylate cyclase-activating polypeptide. We conclude that CNP is synthesized in the pituitary, where it is located predominantly in gonadotropes, and GC-B receptors expressed in the pituitary mediate the direct effects of CNP in gonadotropes. Together with the recent demonstration of CNP synthesis and action in alpha T3-1 cells, the data suggest CNP to be a novel autocrine regulator of gonadotropes.

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.

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.

A fusca gene homologue in mammalian tissues: Developmental regulation in the rat testes.

By differential screening of a rat pineal cDNA library we identified earlier a novel transcript having a 57% nucleotide homology and a 45% amino acid identity with a plant fusca-gene (fus6) to which a corresponding human sequence (gps1) has recently been reported. Expression of this mammalian fusca homologue (mfh) was seen in a variety of mammalian tissues, including kidney, pineal and retina, but it was particularly strong in the testes. Northern blot analysis demonstrated that the rat testicular mfh message increases markedly from day 28 onwards. Additionally, by in situ hybridization, mfh was localized primarily to the seminiferous tubules with a stage-dependent distribution pattern, a result which was confirmed by immunohistochemistry with antibodies raised against a synthetic MFH oligopeptide. Western blotting also revealed strong signals of the expected molecular weight in testicular extracts from several species. In view of its homology to fus6, a plant gene known to be involved in repressing photomorphogenesis in darkness, the conservation of mfh in mammals suggests a potential function for MFH in signaling pathways involved in the regulation of mammalian differentiation and development.

Guanylyl cyclase-B represents the predominant natriuretic peptide receptor expressed at exceptionally high levels in the pineal gland.

The generation and function(s) of the signalling molecule cyclic GMP (cGMP) in brain are still poorly understood. One mechanism to raise intracellular cGMP levels is binding of C-type natriuretic peptide (CNP) to a membrane guanylyl cyclase (GC), termed GC-B. Here, we demonstrate an exceptionally strong expression of GC-B in the pineal gland. Crosslinking experiments performed with 125I-Tyr(0)-CNP and membranes from various rat tissues identified the receptor as a 130-kDa protein, expressed at highest levels in pineal membranes. Receptor autoradiography on brain sections confirmed a striking density of CNP binding sites in pineal tissue, whereas binding sites for the related atrial natriuretic peptide (ANP) predominate in other regions of the brain. Incubations of freshly dissected whole pineal glands in either the absence or presence of natriuretic peptides followed by immunohistochemical analyses of cGMP revealed strong accumulations of cGMP in response to CNP but not to ANP in the majority of pinealocytes. Stimulation of soluble GC (sGC) activity by use of sodium nitroprusside (SNP) resulted in a very similar pattern of cGMP immunostaining, indicating a co-expression at high levels of particulate and soluble forms of GC. These findings point to a major role of cGMP signalling in pinealocytes and suggest an important regulatory function for CNP.

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-1), 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.

Natriuretic peptides stimulate cyclic GMP production in an immortalized LHRH neuronal cell line.

The role of cyclic 3',5'-guanosine monophosphate (cGMP) as a second messenger in LHRH neurons is not well understood. Recent studies involving nitric oxide, a direct activator of soluble guanylate cyclase (GC), have implicated cGMP in the regulation of LHRH secretion both in vivo and in vitro. Evidence for the membrane-bound form of GC in LHRH neurons has thus far not been reported. In polymerase chain reaction screening of various cell lines for the natriuretic peptide receptors--which represent GCs--we identified both GC-A and GC-B cDNAs by southern blot hybridization in reverse transcribed and amplified extracts of the GT1-7 cell line, an immortalized LHRH neuronal cell line. Subsequent experiments demonstrated that all of the natriuretic peptides elevated cGMP production with a rank order of potency: CNP > ANP > BNP. Time course studies revealed a rapid intracellular accumulation of cGMP following exposure to CNP with a peak at 2.5 min. CNP was some 200-fold more potent than the NO donor, sodium nitroprusside, in stimulating cGMP accumulation in these cells. These data show for the first time the presence of functional mGCs on LHRH cells, and suggest that the natriuretic peptides may also participate in the regulation of LHRH activity.

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.

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.

Expression and regulation of mPer1 in immortalized GnRH neurons.

Hypothalamic GnRH (gonadotropin-releasing hormone) neurons play a critical role in the initiation and maintenance of reproduction competence. Using the mouse GnRH neuronal cell line, GT1-7, we have characterized the expression of the gene mPer1, a recognized key element of the mammalian circadian clockwork. Both mPer1 transcripts and the 136 kDa mPER1 gene product could be detected in these cells. Immunocytochemical analysis also confirmed expression of mPER1 both in vitro and in vivo in GnRH neurons. Activation of cyclic AMP signalling pathways in vitro elevated GnRH secretion as well as mPer1 expression and nuclear mPER1 immunoreactivity. As mPER1 is known to feedback on transcriptional activities in many cell models, the data presented here point to a role for mPER1 in the regulation of gene expression in GnRH neurons, and thus in the control of neuroendocrine activities.

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.

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

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-beta-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 immunoreactivity 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.