Second harmonic generation microscopy of fetal membranes under deformation: normal and altered morphology.

INTRODUCTION: Insight into the microstructure of fetal membrane and its response to deformation is important for understanding causes of preterm premature rupture of the membrane. However, the microstructure of fetal membranes under deformation has not been visualized yet. Second harmonic generation microscopy, combined with an in-situ stretching device, can provide this valuable information. METHODS: Eight fetal membranes were marked over the cervix with methylene blue during elective caesarean section. One sample per membrane of reflected tissue, between the placenta and the cervical region, was cyclically stretched with a custom built inflation device. Samples were mounted on an in-situ stretching device and imaged with a multiphoton microscope at different deformation levels. Microstructural parameters such as thickness and collagen orientation were determined. Image entropy was evaluated for the spongy layer. RESULTS: The spongy layer consistently shows an altered collagen structure in the cervical and cycled tissue compared with the reflected membrane, corresponding to a significantly higher image entropy. An increased thickness of collagenous layers was found in cervical and stretched samples in comparison to the reflected tissue. Significant collagen fibre alignment was found to occur already at moderate deformation in all samples. CONCLUSIONS: For the first time, second harmonic generation microscopy has been used to visualize the microstructure of fetal membranes. Repeated mechanical loading was shown to affect the integrity of the amnion-chorion interface which might indicate an increased risk of premature rupture of fetal membrane. Moreover, mechanical loading might contribute to morphological alterations of the fetal membrane over the cervical region.

Establishment of a protocol for large-scale gene expression analyses of laser capture microdissected bladder tissue.

PURPOSE: Lower urinary tract symptoms (LUTS) can be caused by structural and functional changes in different compartments of the bladder. To enable extensive investigations of individual regions even in small bladder biopsies, we established a combination protocol consisting of three molecular techniques: laser capture microdissection microscopy (LCM), RNA preamplification and quantitative polymerase chain reaction (qPCR). METHODS: Urinary bladders of ten mice were resected and frozen immediately or after a delay of 15 min. Cryosections were obtained and smooth muscle was isolated using the LCM technique. Then, RNA was extracted, including protocols with and without DNase digestion as well as with and without the addition of carrier RNA. Extracted RNA was either used for reverse transcriptase (RT)-PCR plus qPCR or for a combination of RNA preamplification and qPCR. RESULTS: Our data showed that with RNA preamplification, 10 µg cDNA can be regularly generated from 2.5 ng RNA. Depending on expression levels, this is sufficient for hundreds of pPCR reactions. The efficiency of preamplification, however, was gene-dependent. DNase digestion before preamplification lead to lower threshold cycles in qPCR. The use of partly degraded RNA for RNA preamplification did not change the results of the following qPCR. CONCLUSIONS: RNA preamplification strongly enlarges the spectrum of genes to be analyzed in distinct bladder compartments by qPCR. It is an easy and reliable method that can be realized with standard laboratory equipment. Our protocol may lead in near future to a better understanding of the pathomechanisms in LUTS.

mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR.

mRNA profiling is a promising new method for the identification of body fluids from biological stains. Major advantages of mRNA profiling are the possibility of detecting several body fluids in one multiplex reaction and of simultaneously isolating DNA without loss of material. A reverse transcription endpoint polymerase chain reaction (PCR) method and a realtime PCR assay were established for the identification of blood, saliva, semen, vaginal secretions and menstrual blood, and were compared to conventional enzymatic and immunologic tests. The results for specificity, sensitivity and suitability to biological stains were satisfying and mRNA stability was demonstrated for up to 2-year-old stains. Two novel multiplex assays were created with the endpoint PCR primers: multiplex 1 amplifies two markers for each of the above mentioned body fluids and is suited for screening; multiplex 2 was designed for the detection of blood, vaginal secretions and menstrual blood. The results demonstrate that both endpoint PCR and realtime PCR are suitable for the identification of body fluids in forensic stains and represent an effective alternative to conventional enzymatic and immunologic tests.

Histomorphological analysis of the urogenital diaphragm in elderly women: a cadaver study.

The objective of this study was to describe the histomorphological structure of the urogenital diaphragm in elderly women using a modern morphometric procedure. Biopsies were taken from the posterior margin of the urogenital diaphragm of 22 female cadavers (mean age, 87 years) using a 60-mm punch. Hematoxylin/eosin and Goldner sections were analyzed with the Cavalieri estimator. The mean thickness of the urogenital diaphragm was 5.5 mm. The main component was connective tissue. All biopsies contained smooth muscle. Eighteen biopsies contained more smooth muscle than striated muscle. In six of 22 biopsies, no striated muscle was found. The ratio of striated to smooth muscle to connective tissue was 1:2.3:13.3. Muscle fibers were dispersed in all parts of the urogenital diaphragm. The urogenital diaphragm of elderly women mainly consists of connective tissue. Smooth muscle was also found but to a lesser extent. The frequently used English term "perineal membrane" for the urogenital diaphragm is justified and well describes our findings in elderly women.

Expression of smoothelin in the normal and the overactive human bladder.

PURPOSE: We established the expression pattern of smoothelin, a marker protein for contractile smooth muscle cells, in the human detrusor and investigated its possible impact on bladder overactivity. MATERIALS AND METHODS: Detrusor samples of 13 overactive bladders (sensory urge and detrusor instability) were obtained before botulinum toxin injection and compared to those of 8 normally contractile, nonobstructed bladders obtained during radical cystectomy. Smoothelin mRNA expression patterns were investigated by Northern blot and variant specific reverse transcriptase-polymerase chain reaction as well as by quantitative reverse transcriptase-polymerase chain reaction on laser capture, microdissected smooth muscle. At the protein level smoothelin was investigated by standard and quantitative immunohistochemistry. RESULTS: The bladder muscularis expressed vascular and visceral smoothelin isoforms, and 2 of the known splice variants. In the smooth muscle of patients with detrusor instability and sensory urge a significant 2.4 and 2.2-fold increase, respectively, in smoothelin variant 1 mRNA was observed in comparison to that of normal controls. Analyses at the smoothelin protein level confirmed significant up-regulation in these bladder dysfunctions by a factor of 2.3 and 1.8, respectively. No significant difference in smoothelin expression was observed between detrusor instability and sensory urge. CONCLUSIONS: Increased expression of smoothelin in patients with detrusor instability and sensory urge implies that the etiology of these dysfunctions includes changes in myogenic parameters. In addition, our data support the new classification of the International Continence Society for overactive bladder proposing that sensory urge and detrusor instability represent a single clinical entity.

Effects of insulin--like growth factor-I treatment on the endocrine pancreas of hypophysectomized rats: comparison with growth hormone replacement.

BACKGROUND: In GH-deficient humans, GH and IGF-I treatment cause opposite effects on serum insulin concentrations and insulin sensitivity. This finding contrasts with the somatomedin hypothesis that IGF-I mediates GH action, as postulated for skeletal growth, and raises the question whether GH-induced IGF-I acts on the endocrine pancreas in the same way as administered IGF-I. OBJECTIVE: To compare the effects of the two hormones on the endocrine pancreas of hypophysectomized rats. METHODS: Animals were infused for 2 days, via miniosmotic pumps, with IGF-I (300 microg/day), GH (200 mU/day) or vehicle. We measured (i) glucose, IGF-I, insulin, C-peptide and glucagon in serum and (ii) IGF-I, insulin and glucagon mRNAs and peptides in the pancreas by radioimmunoassay, immunohistochemistry and northern analysis. RESULTS: Both GH and IGF-I treatment increased serum and pancreatic IGF-I but, unlike GH, IGF-I treatment strongly reduced serum insulin and C-peptide (and, to a lesser extent, serum glucagon). Nevertheless, the animals did not become hyperglycaemic. GH, but not IGF-I, increased pancreatic insulin and glucagon content, as also indicated by immunohistochemistry, and increased IGF-I mRNA. Neither GH nor IGF-I caused significant changes in insulin and glucagon mRNA. CONCLUSIONS: The decrease in serum insulin and C-peptide by IGF-I treatment without significant changes in insulin gene expression and pancreatic insulin content suggests inhibition of insulin secretion. Within this setting, the absence of hyperglycaemia points to enhanced insulin sensitivity, although an insulin-like action of infused IGF-I may have partially compensated for the decreased insulin concentrations. GH-induced circulating or pancreatic IGF-I, or both, does not mimic the pancreatic effects of infused IGF-I in the absence of GH, suggesting that GH may counteract the action of GH-induced IGF-I on the endocrine pancreas.

The effect of seminal vesicle-sparing radical prostatectomy on serum prostate-specific antigen level.

OBJECTIVE: To investigate if the remaining seminal vesicle tips can affect serum levels of prostate-specific antigen (PSA) in patients after seminal vesicle-sparing radical prostatectomy (SVRP). PATIENTS AND METHODS: Thirty-six patients were treated by either radical retropubic prostatovesiculectomy (23) or SVRP (13). Serum PSA was monitored in all patients before surgery, and at 6 weeks and 30 months afterward. Samples of normal seminal vesicles from radical cystectomies (six) were also snap-frozen and either processed for semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using primers against PSA and alpha-actin (for normalization) or for PSA immunohistochemistry. RESULTS: RT-PCR and sequencing showed that the seminal vesicles synthesise PSA mRNA. Furthermore, PSA peptide was detectable in the glandular epithelium of the seminal vesicle using immunohistochemical methods. There was no significant difference in serum PSA levels after standard or SVRP, with median (range) values (ng/mL) at 6 weeks of 0.04 (0.04-0.9) and 0.04 (0.04-0.66) and at 30 months of 0.17 (0.04-3.8) and 0.22 (0.04-58.2), respectively. CONCLUSION: Although the seminal vesicles produce PSA, the PSA derived from the remaining seminal vesicle tips after SVRP has no effect on the oncological follow-up of these patients.

[Gap junctions in the human urinary bladder]. / Gap junctions in der menschlichen Harnblase.

PURPOSE: Gap junctions are intercellular contacts important for the synchronization of muscle cell activity through electrical coupling. Since the role of gap junctions for the function of smooth bladder muscle is still a matter of debate, we investigated the occurrence of gap junctions and the gap junctional protein connexin (Cx) 45 in the detrusor of the nonobstructed stable human bladder. MATERIALS AND METHODS: Detrusor biopsies from 6 patients aged 64 (55-72) years with stable nonobstructed bladders were investigated for the occurrence of gap junctions by electron microscopy, molecular biological techniques and immunohistochemistry. RESULTS: Transmission electron microscopy and freeze fracture showed the presence of gap junction at plasma membranes of detrusor smooth muscle cells. By reverse transcriptase (RT) polymerase chain reaction (PCR) and in situ hybridization, we found an expression of Cx45 in the detrusor. These data were confirmed by immunolocalization of Cx45 on smooth muscle cells. CONCLUSIONS: This study provides morphological as well as molecular biological and immunohistochemical evidence that bladder smooth muscle cells are electrically coupled.

Immunological alterations in the ejaculate of chronic prostatitis patients: clues for autoimmunity.

The aim of this prospective study was to observe immunophenotypic patterns in the ejaculate of patients with noninflammatory chronic pelvic pain syndrome (Cat IIIB CPPS) and to test for a possible autoimmune aetiology. Thirty-five patients of a total of 88 patients with chronic prostatitis Cat IIIB were consecutively selected. Monthly ejaculate testing was carried out for IgG, IgA, IgM, IL-1alpha, sIL-2R and IL-6. The control group for ejaculate analysis was composed of 96 normal ejaculates (according to the WHO criteria). Immunohistochemical detection of CD3 cells (T lymphocytes) and CD20 cells (B lymphocytes) was performed in 71 biopsy cylinders of Cat IIIB CPPS patients and in 25 prostate biopsy cylinders of subjects without symptoms or obstruction. Intra-acinar T-lymphocytic infiltrates were dominated by T-cytotoxic cells (P = 0.05). Ejaculate IL-6 and ejaculate IgA increased significantly and dropped again, correlating with a release of clinical symptoms. Inflammatory ejaculate interleukin concentrations correlated with the immunohistochemical findings with presence of large numbers of T cells (all P-values < or = 0.01). Immunomodulation was performed in a pilot series of three patients by five monthly cycles of IgG (Sandoglobulin), 1 g kg-1 body weight. Immunomodulation with IgG decreased pain moderately and did not change ejaculate interleukin and immunoglobulin concentrations. In summary, interleukin and immunoglobulin determinations in the ejaculate revealed an inflammatory process even in Cat IIIB CPPS. The findings of intra-acinar T-cell rich infiltrates and the associated inflammatory reaction may indicate a possible autoimmune component in the aetiology of CPPS. Exact origin and role of interleukin changes in the ejaculate of CPPS patients need to be further evaluated. Unfortunately, pilot series with immunomodulation with IgG do not seem to provide clear clinical benefit.

Ontogeny of neurohormonal peptides, serotonin, and nitric oxide synthase in the gastrointestinal neuroendocrine system of the axolotl (Ambystoma mexicanum): an immunohistochemical analysis.

The ontogeny of the neurohormonal peptides vasoactive intestinal polypeptide (VIP), neurotensin (NT), substance P (SP), calcitonin gene-related peptide (CGRP), gastrin/cholecystokinin (GAS/CCK), and somatostatin (SOM) as well as serotonin (SER) and nitric oxide synthase (NOS) was investigated in the gastrointestinal tract of the urodele Ambystoma mexicanum, the axolotl, using immunohistochemical techniques. The first regulatory substances to appear were SP, SOM, and SER that could be immunohistochemically detected up from stage 1. At early stage 2, VIP immunoreactivity was observed infrequently in enteric nerve fibers. With the onset of external feeding at late stage 2, SP-immunoreactive (IR) and SER-IR endocrine cells and VIP-IR nerve fibers were present throughout the gastrointestinal tract. Furthermore, in the small intestine NT-IR and GAS/CCK-IR endocrine cells appeared. At stage 3, SER immunoreactivity was observed not only in endocrine cells but also in nerve fibers. CGRP-IR and SP-IR nerve fibers were detectable at stage 4 and stage 5, respectively. From stage 5 on, a minority of the CGRP immunoreactivity occurred in SP-IR nerve fibers. NOS immunoreactivity did not appear before stage 6 when it was found infrequently in nerve fibers. Thus, several phases of development can be distinguished: (1) at the yolk sac stages only few regulatory substances are present. (2) At the onset of external feeding, all endocrine cell types investigated were readily detectable. Thus, the onset of external feeding seems to trigger the development of the gastrointestinal endocrine system. (3) The endocrine cells are first found in the proximal part of the gastrointestinal tract and later in higher numbers in the distal parts. (4) The dually distributed neurohormonal peptides and SER first appear in endocrine cells and later additionally in nerve fibers. Thus, the nerve fibers likely set up the fine regulation of gastrointestinal blood flow and motility.

Evidence of trigonal denervation and reinnervation after radical retropubic prostatectomy.

PURPOSE: Urinary incontinence continues to be a major consequence of radical prostatectomy. To understand the pathophysiology of this dysfunction we studied the impact of autonomic innervation of the superficial trigone on postoperative urinary continence. MATERIALS AND METHODS: To investigate nerve fiber density biopsies of the superficial trigone were obtained in 34 patients preoperatively as well as 6 weeks and 6 months postoperatively in 15 and 19, respectively. Specimens were Bouin fixed, paraffin embedded and processed for light microscopic immunohistochemical evaluation using an antibody against protein gene product 9.5, a general neuronal marker protein. In parallel we performed a comprehensive urodynamic evaluation, including determination of maximal urethral closure pressure and posterior urethral sensory threshold. RESULTS: Postoperatively protein gene product 9.5 immunoreactive nerve fiber density was generally decreased. However, nerve fiber density after 6 weeks of incontinence in 12 of 15 patients was only 7%, while 3 of 15 who were continent preserved 36% of initial nerve fiber density. After 6 months nerve fiber density in 19 patients increased in 3 with incontinence to 20% and in 16 with continence to 44% of intraoperative density. Urinary incontinence was associated with decreased trigonal innervation, a high sensory threshold and low maximal urethral closure pressure. CONCLUSIONS: Protein gene product 9.5 immunoreactive nerve fiber density corresponds with posterior urethral sensory threshold and urinary continence. Thus, preserving trigonal innervation and postoperative reinnervation may be important factors for achieving early postoperative urinary continence after radical prostatectomy.

Neurohormonal peptides, serotonin, and nitric oxide synthase in the enteric nervous system and endocrine cells of the gastrointestinal tract of neotenic and thyroid hormone-treated axolotls (Ambystoma mexicanum).

Immunoreactivity against vasoactive intestinal polypeptide (VIP), neurotensin (NT), substance P (SP), calcitonin gene-related peptide (CGRP), gastrin/cholecystokinin (GAS/CCK), somatostatin (SOM), serotonin (SER), and nitric oxide synthase (NOS) was investigated in the gastrointestinal tract of the urodele Ambystoma mexicanum, the axolotl, by the use of immunohistochemical techniques. The study also compares the distribution patterns and frequencies of the neurohormones, and NOS in neotenic and thyroxine-treated (metamorphosed) individuals. GAS/CCK, SP, NT, SOM, and SER immunoreactivities occurred in endocrine mucosal cells and VIP, SP, CGRP, NTSER, SER, and NOS immunoreactivities in the enteric nervous system. The GAS/CCK-immunoreactive (-IR) cells were restricted to the upper small intestine. NT-IR and SP-IR endocrine cells were found in the entire gastrointestinal tract and were most prominent in the distal large intestine. The density of the SOM-IR cells decreased from the stomach toward the large intestine. SER-IR endocrine cells were found throughout the gastrointestinal tract, with particularly high densities in the stomach and distal large intestine. The VIP-IR enteric nerve fibers were the most prominent ones, present in all layers of the entire gastrointestinal tract, and supplied the smooth muscle and the vasculature. The SER-IR fibers exhibited similar distribution patterns but were less numerous. Very few NT-IR but many SP-IR fibers were found in the muscle and submucosal layers. The NT-IR fibers mainly supplied blood vessels, while the SP-IR fibers were also in contact with the smooth muscle. In the muscle and submucosal layers, CGRP-IR fibers were associated to the vasculature; CGRP immunoreactivity occurred also in a minority of SP-IR fibers. NOS-IR nerve fibers were in contact with submucosal arteries but were the least frequent. After metamorphosis provoked by exogenous thyroxine, the number of SOM-IR endocrine cells in the stomach mucosa was increased as well as the density of VIP-IR, SER-IR, and SP-IR nerve fibers in the gastrointestinal tract. It is proposed that the observed increases may reflect refinements of the neurohormonal system after metamorphosis.

Enhanced proteolytic activity directed against the N-terminal of IGF-I in diabetic rats.

We have recently identified in serum an acid protease which is capable of generating des(1-3)IGF-I from intact IGF-I. Here we have utilized a synthetic substrate with the sequence, biotin-G-P-E-T-L-C-BSA which contains the N-terminal sequence of IGF-I, to investigate the levels of this protease activity in streptozotocin-diabetic rats. Protease activity, quantified in terms of the amount of the biotin label lost, was determined in serum and hepatic extracts from normal control rats, diabetic rats and insulin-treated diabetic rats. Both the serum protease activity and protease activity in hepatic extracts were significantly increased in diabetic rats compared with control rats (P<0.02 and P<0.005). Following acute administration of insulin, a rapid and marked reduction in serum protease activity was observed; with an approximately 50% reduction apparent at 30 min (P<0.001). Chronic insulin treatment of diabetic rats also significantly reduced the serum and hepatic protease activity to the levels seen in control rats. A positive correlation between protease activity and serum glucose level was observed (r=0.58, P<0.005). The abundance of Spi 2.1 mRNA, a serine protease inhibitor, capable of inhibiting the IGF-I protease activity in vitro, was significantly decreased in the liver of diabetic rats and insulin treatment of diabetic rats did not normalize Spi 2.1 mRNA levels. These data suggest that the conversion of IGF-I to the more active des(1-3)IGF-I variant may be enhanced in diabetic animals. Since serum IGF-I levels are reduced in diabetic rats, increased des(1-3)IGF-I-generating protease activity would enhance the functional activity of the circulating IGF-I.

An immunohistochemical and morphometric analysis of insulin, insulin-like growth factor I, glucagon, somatostatin, and PP in the development of the gastro-entero-pancreatic system of Xenopus laevis.

The ontogeny of the classical islet hormones insulin (INS), glucagon (GLUC), somatostatin (SOM), and pancreatic polypeptide (PP) as well as insulin-like growth factor I (IGF-I) in the gastro-entero-pancreatic (GEP) system of Xenopus laevis (stages 41-66) was studied using double immunofluorescence and morphometric analysis. As early as stage 41, clustered INS-immunoreactive (-IR) and isolated GLUC-IR cells occurred in the pancreas. The first SOM-IR cells appeared at stage 43, followed by PP-IR cells at stage 46. About 79% of the PP immunoreactivity was confined to a subpopulation of the GLUC-IR cells. Both the GLUC/PP-IR cells and the PP-IR cells were located in a distinct area of the pancreas. The first islets occurred in premetamorphosis (around stage 50) and comprised mainly INS-IR and GLUC-IR cells. The majority of SOM-IR, PP-IR, and GLUC/PP-IR cells was dispersed. The numbers of hormone cells remained quite constant until the end of prometamorphosis (stage 58). Around stages 60-62, the islets were partly disintegrated and the numbers of islet cells slightly decreased. At stage 63, the cell number began to increase and reached the levels typical for the adult around stage 66. After metamorphic climax, the islets were reformed. In the gastrointestinal tract, transient INS-IR cells occurred prior to the adaptation of the gastrointestinal tract to feeding (stages 41-44) and during metamorphosis when there is remodeling of the gastrointestinal tract (stages 60-63). Therefore, INS released from the transient mucosal INS-IR cells may be involved in the temporary proliferation of mucosal epithelial cells. The first GLUC-IR and SOM-IR cells were seen at stage 41. PP-IR cells followed at stage 46. In contrast to the islets, GLUC-IR and PP-IR cells constituted different cell populations. Around stage 46, the first IGF-I immunoreactions appeared in the GEP-system. In pancreas, IGF-I immunoreactivity was found in the GLUC/PP-IR, cells (85-99%) but was absent from INS-IR, GLUC-IR, and SOM-IR cells. The IGF-I-IR gastro-entero-endocrine cells, however, seemed to contain none of the classical islet hormones.

The growth hormone dependent serine protease inhibitor, Spi 2.1 inhibits the des (1-3) insulin-like growth factor-I generating protease.

The conversion of insulin-like growth factor-I (IGF-I) to the biologically more active des (1-3) IGF-I variant is catalyzed by a ubiquitous protease. This proteolytic activity is inhibited by human alpha1-antitrypsin and soy-bean trypsin inhibitor and is up-regulated in serum and tissue extracts of hypophysectomized rats. These observations lead us to investigate whether the growth hormone regulated, serine protease inhibitor, Spi 2.1 was able to inhibit the des (1-3) IGF-I generating protease. Dihydrofolate reductase deficient Chinese hamster ovary (CHO(dhfr-ve)) cells were transfected with a rat Spi 2.1 expression vector containing the dhfr and neomycin resistance gene. Stable transfectants were selected using G418 and amplified using methotrexate. Conditioned medium from Spi 2.1 transfected CHO cells potently inhibited proteolytic activity directed against a synthetic hexa-peptide with a sequence identical to the N-terminal of IGF-I. In contrast conditioned medium from wild-type CHO cells had little effect. Based upon these observations we suggest that our previous finding of enhanced des (1-3) IGF-I generating protease activity in growth hormone deficient rats may be, at least partly explained by reduced levels of Spi 2.1. Furthermore, we propose that the regulation of the generation of des (1-3) IGF-I may be an additional potential site of growth hormone regulation of IGF-I action.

Immunohistochemical localization of insulin-like growth factor I and II in the endocrine pancreas of birds, reptiles, and amphibia.

Immunoreactive insulin-like growth factors I and II (IGF-I, IGF-II) were sought in the endocrine pancreas of representative birds, reptiles, and amphibia using antisera specific for mammalian IGF-I and IGF-II and the classical islet hormones insulin (INS), glucagon (GLUC), somatostatin (SOM), and pancreatic polypeptide (PP) in double immunofluorescence. Both IGF-I and IGF-II immunoreactivities were present in the endocrine pancreas of all species. IGF-II immunoreactivity was exclusively found in INS-immunoreactive (-IR) cells, indicating evolutionary conservation of the islet IGF-II system. In contrast, IGF-I immunoreactivity was distributed differently among the species and never occurred in INS-IR cells. In the anuran Xenopus laevis, IGF-I immunoreactivity was present in islet cells showing coexistence of GLUC and PP immunoreactivities. In reptiles, the lizards (Lacerta viridis, Scincus officinalis) exhibited IGF-I immunoreactivity in PP-IR and SOM-IR cells and the snakes (Psamophis leniolatum, Coluber ravergieri) in SOM-IR and GLUC-IR cells. In birds, IGF-I immunoreactivity was located either in SOM-IR cells only (Gallus g. domesticus, Streptopelia roseogrisea) or in PP-IR and SOM-IR cells (Coturnix c. japonica). Thus, the distribution patterns of islet IGF-I immunoreactivities in birds, reptiles, and amphibia are equivalent to those in mammals and most bony fish. They differ, however, from those found in cartilaginous fish, cyclostomes, and protochordates, where a total or partial coexistence of IGF-I and INS immunoreactivities has been obtained. Therefore, the divergence of IGF-I and INS seems to have occurred early in vertebrate phylogeny. Furthermore, the existence of IGF-I immunoreactivity likely is common in the islets of all vertebrates. Finally, no phylogenetic trend to concentrate IGF-I immunoreactivity in a particular islet cell type is apparent.

IGF-2-like peptides are present in insulin cells of the elasmobranchian endocrine pancreas: an immunohistochemical and chromatographic study.

Evidence for the presence of peptides, related to insulin-like growth factor 2 (IGF-2), has been obtained in the endocrine pancreas of the elasmobranchian species Raja clavata, the sting ray. By radioimmunoassay, IGF-2-like immunoreactivity was detected in Raja pancreas extract. Further characterization of this activity by acid gel chromatography revealed two distinct peaks of IGF-2-like immunoreactivity with apparent molecular weights of approximately 8.2 kDa and 4.5 kDa. Using the same IGF-2 antibody as well as antisera specific for mammalian IGF-1, insulin, glucagon, somatostatin and pancreatic polypeptide in double immunofluorescence studies, IGF-2-like immunoreactivity was located exclusively in insulin-immunoreactive cells. In contrast, IGF-1-like immunoreactivity was mainly observed in somatostatin- and glucagon-immunoreactive cells. A varying proportion (0-70%) of insulin-immunoreactive cells, however, displayed both IGF-1- and IGF-2-like immunoreactivity. Absorption studies indicated that the IGF-2-like peptides in Raja are different from mammalian and submammalian insulin and mammalian IGF-1, but similar to mammalian IGF-2. Thus, IGF-2-like peptides seem to occur during evolution as early as the phylogenetic development of the elasmobranchians. Furthermore, the results indicate a particularly conservative evolution of the islet IGF-2 system.

The branching of insulin-like growth factor 1 and insulin: an immunohistochemical analysis during phylogeny.

The co-existence of insulin-like growth factor 1 (IGF-1) with the classical islet hormones insulin (INS), glucagon (GLUC), somatostatin (SOM) and pancreatic polypeptide (PP) in the endocrine pancreas of representative species of cyclostomes (Myxine glutinosa), cartilaginous fish (Raja clavata, Squalus acanthias) and bony fish (Cottus scorpius, Carassius auratus, Cyprinus carpio, Anguilla anguilla) was studied by the use of monoclonal and polyclonal antisera and the double immunofluorescence technique. In all species investigated, IGF-1-like-immunoreactive cells were found in the endocrine pancreas, however, in varying localization. In Myxine glutinosa, all INS-immunoreactive cells and some of the SOM-immunoreactive cells contained IGF-1-like-immunoreactivity. In Raja and Squalus, only a minority of the INS-immunoreactive cells also displayed IGF-1-like-immunoreactivity. The majority of the IGF-1-like-immunoreactivity was observed in SOM- and in GLUC-immunoreactive cells. Different results were obtained in bony fish. In Cottus, in the Brockmann bodies and the small islets IGF-1-like- and INS-immunoreactivities co-existed to 100%. In contrast, in the other bony fish studied IGF-1-like-immunoreactivity was not observed in INS-immunoreactive cells: in Cyprinus, IGF-1-like-immunoreactivity was found in GLUC-, PP- and SOM-immunoreactive cells and in Carassius and Anguilla, in SOM-immunoreactive cells only. Thus, in all bony fish species with the exception of Cottus, IGF-1 and insulin display a distinct cellular distribution, similar to that of mammals. The present results, thus, may indicate that the branching of IGF-1 and insulin has occurred at the phylogenetic level of bony fish.

Immunohistochemical localization of insulin-like growth factor 1 and 2 in the endocrine pancreas of rat, dog, and man, and their coexistence with classical islet hormones.

Immunohistochemical techniques were used to study the occurrence and distribution of insulin-like growth factor 1 (IGF-1) and IGF-2 in the pancreas of man, dog, and rat and their possible coexistence with insulin (INS), glucagon (GLUC), somatostatin (SOM) and pancreatic polypeptide (PP). All control experiments, including pre-absorption of the antisera with synthetic peptide hormones, indicated the specificity of the immunoreactions obtained. In all species investigated, IGF-2-immunoreactivity occurred exclusively in INS-immunoreactive cells as was found by the use of consecutive sections and double immunofluorescence on identical sections. In contrast, IGF-1-immunoreactivity co-existed with GLUC-immunoreactivity. In man, singular SOM-immunoreactive cells also contained IGF-1-immunoreactivity. Thus, IGF-1 and IGF-2 can be localized by means of immunohistochemistry in the mammalian pancreas, and can be shown to occur in different islet cell populations. It is presumed that IGF-1 derived from A-cells and/or D-cells acts on the B-cells in a paracrine manner. The co-existence of IGF-2-immunoreactivity and INS-immunoreactivity in the human, rat, and dog endocrine pancreas indicates that mammalian IGF-2 and INS genes are regulated simultaneously.

A phylogenetic survey of pancreastatin and chromogranin immunoreactivity in chromaffin (TH-, DBH-, and PNMT-immunoreactive) cells of the adrenal organ of vertebrates.

Region-specific antisera raised against different amino acid sequences of pancreastatin (Pst) (Pst-1-6, Pst-1-17, Pst-14-49 and Pst-33-49) and two antisera towards chromogranin (Cg) A and CgA/B were applied in immunofluorescence to examine the occurrence and distribution of Pst-immunoreactive (-IR) and Cg-IR cells in adrenal organs of several mammals, birds, reptiles, amphibia, and bony fish. The catecholamine-containing cells were identified using antisera against enzymes of catecholamine synthesis (tyrosine-hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyl-transferase). No animal showed any Pst-IR or Cg-IR cells in the adrenal cortex or in its homolog, the interrenal. All antisera reacted with chromaffin cells in porcine adrenal medulla. Both adrenaline (A)- and noradrenaline (NA)-containing cells displayed Pst- and Cg-immunoreactivity. Pst- and CgA-immunoreactivities were observed in coexistence using double immunofluorescence. However, strongly reacting Pst-IR cells showed only low CgA immunoreactivity and vice versa. This inverse relationship between Pst- and CgA-immunoreactivities might reflect different levels of processing of the likely Pst-precursor CgA. In all nonmammalian vertebrates studied, Pst- and Cg-immunoreactivities were also found in both A- and NA-containing adrenal cells. However, the chromaffin cells reacted only with the antisera Pst-1-6, Pst-1-17, Pst-33-49, and CgAB. The adrenal chromaffin cells of nonmammalian vertebrates appear to contain Pst-/Cg-like peptides akin to those of the enteroendocrine cells but different from those of their endocrine pancreas. Since no immunoreactions were obtained with antiserum CgA, nonmammalian Pst may be derived from a precursor different from mammalian CgA.