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Biomedical subjects

R Middendorff

Publications and source records attributed to R Middendorff.

At least 19 recordsLinked to original sources
PubMed2003

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.

Animals↗
PubMed2001

Developmental changes of nitric oxide-sensitive guanylyl cyclase expression in pulmonary arteries.

Inhaled nitric oxide (NO) is known to influence the contractile state of pulmonary arteries most likely by activation of soluble guanylyl cyclase (sGC) in smooth muscle cells. However, the cellular distribution of sGC has not been determined empirically, due to a lack of specific antibodies. Here, we describe a novel antibody directed against the beta1 subunit of sGC to study the cellular distribution of sGC in lung during development. Using the novel antibody, the enzyme was demonstrated in fetal, neonatal, and adult lungs by Western blot, showing maximum expression in neonatal lung. These data were confirmed by measurements of sGC activity. In pulmonary arteries of fetal lung sGC-beta1 immunoreactivity was present in smooth muscle cells and absent in endothelial cells. With postnatal development an increase in immunoreactivity in endothelial cells and a reciprocal decrease in smooth muscle cells was apparent. The reported changes in sGC expression likely contribute to the known age-dependent differences in response to inhaled NO.

Aging↗
PubMed2001

Glial cell line-derived neurotrophic factor (GDNF) and its receptors GFRalpha-1 and GFRalpha-2 in the human testis.

Glial cell line-derived neurotrophic factor (GDNF) and its receptors GFRalpha-1 and GFRalpha-2 were found in the human testis during fetal development (15-34 weeks of gestation) and in adult men (51-86 years of age) by means of RT-PCR, immunohistochemistry and Western blot techniques. Gene expression of GDNF could be established in the human testis and immunoreactivity (IR) for GDNF was detectable in Leydig cells, Sertoli cells, some spermatocytes and round spermatids as well as in smooth muscle cells of the wall of arterioles and small arteries. In the adult human testis, Sertoli and Leydig cells showed GFRalpha-1-IR, whereas GFRalpha-2-IR was located exclusively in Leydig cells. Different to man, in the rat GDNF-IR in Sertoli cells was detectable only until postnatal day10, providing evidence for species related variability in the expression of GDNF. These findings suggest a critical role for GDNF during the differentiation of testicular structures and provide evidence for an additional important function in the adult human and rodent testis.

Aged↗
PubMed2000

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.

Animals↗
PubMed2000

Generation of cyclic guanosine monophosphate by heme oxygenases in the human testis--a regulatory role for carbon monoxide in Sertoli cells?

Previous studies have demonstrated that cGMP is produced by nitric oxide-mediated activation of soluble guanylyl cyclase (sGC) in seminiferous tubules of the human testis. It is not known, however, whether carbon monoxide (CO), another activator of sGC, is also involved in testicular function. To address this issue, testicular probes from 65- to 75-yr-old men have been examined. The CO-generating enzyme, heme oxygenase-1 (HO-1), could be localized by immunohistochemical and immunoblot analyses to Sertoli cells. In these cells, HO-1 is detectable in adluminal cell compartments, whereas sGC immunoreactivity is distributed exclusively in basal compartments. Treatments of isolated tubules with either sodium arsenite, known to induce HO-1, or hematin, an HO substrate, resulted in 4.4- and 1.8-fold, respectively, increases in cGMP levels. ODQ, a specific sGC inhibitor, inhibited completely the sodium arsenite-stimulated cGMP production. Moreover, the HO inhibitor zinc protoporphyrin-IX and the CO scavenger hemoglobin both significantly reduced (77% or 46% of control, respectively) tubular cGMP generation. These findings, demonstrating for the first time a link between HO-1 activity in Sertoli cells and sGC-dependent cGMP production in seminiferous tubules, suggest a functional role of CO in the human testis.

Aged↗
PubMed2000

Multiple roles of the messenger molecule cGMP in testicular function.

The messenger molecule cyclic guanosine monophosphate (cGMP) is produced by different isoforms of the enzyme guanylate cyclase (GC). Natriuretic peptides (ANP and CNP) bind to and activate particulate GCs, whereas NO and CO activate a soluble form of GC. The specific relevance of the cGMP system for reproductive functions has been recently demonstrated by the successful use of sildenafil (Viagra), an inhibitor of cGMP-specific phosphodiesterase type 5, for the treatment of erectile dysfunction. In the testis, cGMP signal transduction pathways are involved in a variety of local functions, based on autocrine or paracrine effects. In particular, cGMP has been suggested to influence motility in spermatozoa, development of testicular germ cells, relaxation of peritubular lamina propria cells, testosterone synthesis in Leydig cells and dilatation of testicular blood vessels. The physiological significance of cGMP accumulation in Scrtoli cells is not yet clear. Taken as a whole, the evidence suggests that cGMP-mediated processes might influence both the potentia coeundi within the penis and the potentia generandi at various levels within the testis.

Animals↗
PubMed1999

Sertoli and Leydig cells of the human testis express neurofilament triplet proteins.

Using RT-PCR, western blot and enzyme and fluorescence immunocytochemical techniques, the three isoforms of neurofilament proteins (NFPs), namely NF-L (NFP-68 kDa), NF-M (NFP-160 kDa) and NF-H (NFP-200 kDa) were found in Sertoli and Leydig cells of human testes. RT-PCR showed specific for the three NFP fragments in testicular tissue, in isolated seminiferous tubules and in isolated Leydig cells. In protein preparations from the same testicular components, western blot analysis detected bands with molecular weights characteristic for NF-H, NF-M and NF-L. Application of immunofluorescence and immunoenzyme methods on cryostat and paraffin sections resulted in differences in the staining pattern in Sertoli cells and Leydig cells. In these cells, the NFPs showed predominantly a perinuclear location from which bundles emerge that were directed towards the basal, apical and lateral extensions of the Sertoli cells as well as the periphery of Leydig cells. NF-H coexists with vimentin-type filaments as seen by dual staining and staining of consecutive serial sections of material embedded in paraffin. In Sertoli cells, vimentin and NF-H showed distinct dynamic changes depending on the stage of spermatogenesis and some structural variations of seminiferous tubules. Although in some tubules both vimentin and NF-H immunoreactivity was present at high levels, in the Sertoli cells from most individuals an inverse relationship in the staining intensity of vimentin and NF-H was observed. The strongest NF-H immunoreactivity was detected in Sertoli cells associated with stage 3 spermatids, whereas vimentin immunoreactivity was most abundant in association with stage 5 spermatids. The leydig cells did not show functional changes of the NFP immunoreactivity. The results obtained provide new evidence for the heterogeneous phenotype of human Sertoli cells and raise the question of their exact nature and origin.

Aged↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1997

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.

Adult↗
PubMed1997

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

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.

Adult↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Alternative Splicing↗
PubMed1997

Melatonin synthesis in the bovine pineal gland is regulated by type II cyclic AMP-dependent protein kinase.

We investigated the expression of regulatory (R) and catalytic (C) subunits of cyclic AMP-dependent protein kinase (cAK; ATP:protein phosphotransferase; EC 2.7.1.37) in the bovine pineal gland. In total RNA extracts of bovine pineal glands moderate levels of RI alpha/RII beta and high levels of C alpha and C beta mRNA were found. We were able to detect a strong signal for RII and C subunit at the protein level, whereas RI was apparently absent. Probing sections of the intact bovine pineal gland with RI and RII antibodies stained only RII in pinealocytes. Pairs of cyclic AMP analogues complementing each other in activation of type II cAK, but not cAKI-directed analogue pairs, showed synergistic stimulation of melatonin synthesis. Moreover, melatonin synthesis stimulated by the physiological activator norepinephrine in pineal cell cultures was inhibited by cAK antagonists. Taken together these results show the presence of RII regulatory and both C alpha and C beta catalytic subunits and thus cAKII holoenzyme in the bovine pineal gland. The almost complete inhibition of norepinephrine-mediated melatonin synthesis by the cAK antagonists emphasizes the dominant role of cyclic AMP as the second messenger and cAK as the transducer in bovine pineal signal transduction.

Animals↗
PubMed1997

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.

Adult↗