No evidence for the presence of oogonia in the human ovary after their final clearance during the first two years of life.

BACKGROUND: Conflicting results of studies on mouse and human have either verified or refuted the presence of oogonia/primordial germ cells in the post-natal ovary. The aim of this study was to trace whether oogonia recognized by immunohistochemical methods in the first trimester human ovary were present also in peri- and post-natal ovaries. METHODS: For this study, 82 human ovaries were collected: 25 from embryos from 5 to 10 weeks post conception (wpc), 2 at 18 wpc, 32 from 32 wpc to 2 years and 23 from 2 to 32 years. Of these, 80 ovaries were fixed and paraffin-embedded and 2 (8 year-old) ovaries were processed for plastic sections. Serial sections were prepared for immunohistochemical detection of markers for oogonia: tyrosine kinase receptor for stem cell factor (SCF)(C-KIT), stage-specific embryonic antigen-4 (SSEA4), homeobox gene transcription factor (NANOG), octamer binding transcription factor 4 (OCT4) and melanoma antigen-4 (Mage-A4), while noting that C-KIT also stains diplotene oocytes. RESULTS: Almost all oogonia exclusively stained for SSEA4, NANOG, OCT4 and C-KIT, whereas MAGE-A4 only stained a small fraction. At birth only a few oogonia were stained. These disappeared before 2 years, leaving only diplotene oocytes stained for C-KIT. From 18 wpc to 2 years, the medulla contained conglomerates of healthy and degenerating oogonia and small follicles, waste baskets (WBs) and oogonia enclosed in growing follicles (FWB). Medulla of older ovaries contained groups of primordial, healthy follicles. CONCLUSIONS: We found no evidence for the presence of oogonia in the human ovary after their final clearing during the first 2 years. We suggest that perinatal medullary WB and FWB give rise to the groups of small, healthy follicles in the medulla.

Human primordial germ cells migrate along nerve fibers and Schwann cells from the dorsal hind gut mesentery to the gonadal ridge.

The aim of this study was to investigate the spatiotemporal development of autonomic nerve fibers and primordial germ cells (PGCs) along their migratory route from the dorsal mesentery to the gonadal ridges in human embryos using immunohistochemical markers and electron microscopy. Autonomic nerve fibers in the dorsal mesentery, the pre-aortic and para-aortic plexuses and in the gonadal ridge were stained for beta III tubulin, neuron specific enolase and the glia fibrillary acidic protein. Electron microscopy demonstrated the presence of neurofilaments and neurotubules in these nerve fibers and their intimate contact with PGCs. PGCs expressed GAGE, MAGE-A4, OCT4 and c-Kit. Serial paraffin sections showed that most PGCs were located inside bundles of autonomic nerve fibers with the majority adjacent to the most peripheral fibers (close to Schwann cells). We also show that both nerve fibers and PGCs arrive at the gonadal ridge between 29 and 33 days pc. In conclusion, our data suggest that PGCs in human embryos preferentially migrate along autonomic nerve fibers from the dorsal mesentery to the developing gonad where they are delivered via a fine nerve plexus.

Cryopreservation of intact testicular tissue from boys with cryptorchidism.

BACKGROUND: Boys with cryptorchidism often face fertility problems in adult life despite having orchiopexy performed at a very young age. During this operation, a biopsy of the testis is normally taken in order to evaluate their infertility potential and the presence of malignant cells. This study evaluated the morphology and functional capacity of cryopreserved testes biopsies and their possible use in fertility preservation. METHODS: Biopsies from 11 testes (eight boys) were obtained. Each biopsy was subdivided into six pieces and two pieces were frozen in each of two different cryoprotectants. One fresh and two cryopreserved pieces were cultured for 2 weeks. All pieces were prepared for histology. Used culture media were analysed for testosterone and inhibin B concentrations. RESULTS: The morphology of the fresh and frozen-thawed samples was similar, with well-preserved seminiferous tubules and interstitial cells. A similar picture appeared after 2 weeks of culture, but a few of the cultured biopsies contained small necrotic areas. The presence of spermatogonia was verified by c-kit-positive immunostaining. Production of testosterone and inhibin B (ng/mm(3) testis tissue) in the frozen-thawed pieces was on average similar to that of the fresh samples. CONCLUSIONS: Intact testicular tissue from young boys with non-descended testes tolerates cryopreservation with surviving spermatogonia and without significant loss of the ability to produce testis-specific hormones in vitro. It may be an option to freeze part of the testis biopsy, which is routinely removed during the operation for cryptorchidism, for fertility preservation in adult life.

Fetuin and fetuin messenger RNA in granulosa cells of the rat ovary.

The hardening reaction that occurs in the zona pellucida to block polyspermy can be overcome in oocyte cultures in the presence of fetal serum or the serum component fetuin. Fetuin may also prevent precocious zona hardening by inhibiting a ZP2 proteinase released spontaneously by cortical granules during maturation of the oocyte. We demonstrated fetuin mRNA in the rat ovary by reverse transcriptase-polymerase chain reaction and localized it by in situ hybridization. Fetuin mRNA was present in all granulosa cells of growing and large follicles. Immunohistochemical analysis revealed that the fetuin protein was only present in some of the small, growing follicles. In large, healthy follicles, fetuin protein was confined to cumulus cells and granulosa cells bordering the antrum. Fetuin was present in atretic follicles, but the staining pattern differed from that of healthy follicles. The follicular antrum contained a substantial amount of fetuin, but whether granulosa cells secreted it or it originated in the ovarian blood supply could not be confirmed. We concluded that at least a portion of the fetuin is produced by granulosa cells of growing and large follicles, suggesting that fetuin may function in a paracrine manner to maintain the zona pellucida in a penetrable state for fertilization.

Glucose and pyruvate metabolism during mouse gonadal sex differentiation.

In the mouse, gonadal sex differentiation starts around E12 and meiosis begins in the ovary shortly after E13. In the search for metabolic changes that might be correlated to gonadal sex differentiation and/or possibly the onset of meiosis, we investigated the metabolism of glucose and pyruvate in the developing mouse ovary before (E11.5-E12.5), during (E14.5-16.5), and after meiosis (E18.5), and in fetal testes without meiosis. Gonads were cultured with 14C-labeled glucose (0.02 and 5.58 mM) and 14C-pyruvate (0.17 mM). The oxidation expressed as 14CO2 production and the organification expressed as retention of 14C in the tissues were measured and correlated to the protein content of the gonads. Using 0.02 mM glucose, a decline in oxidation and organification was found in ovaries as well as in testes, which is probably related to starvation. Using 5.58 mM glucose, a continuous decline in oxidation was seen only in the testis. Organification of 0.17 mM pyruvate increased at E12.5 and E14.5 in the ovary but not in the testis. This was in despite of an exponential increase of protein content in the testes compared to only a moderate increase in the ovary. The CO2 production from 5.58 mM glucose was equal to that from 0.17 mM pyruvate in gonads of both sexes. In conclusion, an increased metabolism of 5.58 mM glucose and 0.17 mM pyruvate in the ovaries as compared to the testes is related to sex differences during gonadal formation and onset of meiosis in the ovaries. J. Exp. Zool. 288:130-138, 2001.

Octylphenol does not mimic diethylstilbestrol-induced oestrogen receptor-alpha expression in the newborn mouse uterine epithelium after prenatal exposure.

This study examined whether the endocrine disruptor octylphenol (OP) mimics the synthetic oestrogen diethylstilbestrol (DES) in ability to induce oestrogen receptor-alpha (ER-alpha) expression in the newborn mouse uterine epithelium after prenatal exposure. Pregnant mice were given daily s.c. injections with DES (10 or 100 microgram DES/kg maternal wt) or OP (100 or 250 mg/kg maternal wt) or with vehicle alone from day 11.5 to 16.5 of pregnancy. ER-alpha expression was evaluated on histological sections by detecting ER-alpha mRNA with the in situ hybridization technique and ER-alpha protein using immunohistochemistry. The immunostaining was quantitated using a microspectrophotometer. Oestrogen-like activity of the DES and OP batches used for in vivo exposure was confirmed in an in vitro assay based on transient gene expression of an oestrogen-dependent reporter plasmid. In mice exposed prenatally to vehicle alone, the uterine epithelium did not express either ER-alpha mRNA or protein, while both were highly expressed in the stroma. Exposure to either DES dose induced the expression of both ER-alpha mRNA and protein in the epithelium, whereas it was unchanged in the stroma. In contrast, neither OP dose induced the expression of ER-alpha mRNA or protein in the epithelium and expression was unchanged in the stroma. Our data stress the importance of in vivo studies when investigating endocrine disruptors.

Ontogeny of oestrogen receptor alpha in gonads and sex ducts of fetal and newborn mice.

The distribution of nuclear oestrogen receptor alpha (ER-alpha) in the sex organs of fetal and newborn mice has been investigated immunohistochemically. There was no visible ER-alpha immunoreaction in the sexually undifferentiated gonads, whereas a faint immunoreaction was detected in a few cells surrounding the sex ducts, the Müllerian and Wolffian ducts. After sex differentiation, the immunoreaction of ER-alpha was observed in various cell types, with the exception of both the male and female germ cells. In the fetal ovary, immunoreaction was restricted to the surface epithelium and a few stroma cells without any preferential localization. In the testis, the number of ER-alpha-immunopositive cells, identified as Leydig and peritubular cells, increased with age. Immediately after sex differentiation, cells surrounding the sex ducts were ER-alpha-immunopositive, but no immunoreaction was detected in the epithelium in either sex. During development, the epithelium of the sex ducts attained a topographic difference in ER-alpha immunoreaction. In females, immunoreaction was detected in the epithelium of the oviduct, but not in the uterus. In males, the immunoreaction of ER-alpha was intense in the epithelium of the efferent ducts, weak in the epididymis and absent in the vas deferens. ER-alpha immunoreaction in the cells surrounding the sex duct differed between the sexes, being high in all these cells in females, but of varying intensity in males. ER-alpha may not play an important role in the development and function of ovarian cells, but may be involved in the development of Leydig and peritubular cells. Furthermore, substances that react with ER-alpha may influence the male germ cells indirectly through the ER-alpha-immunopositive peritubular cells. In addition, in both sexes, ER-alpha-immunopositive cells surrounding the sex ducts may be involved in the mediation of growth and functional differentiation of the ductal epithelium.

Binding properties of the galactose-detecting lectin Pseudomonas aeruginosa agglutinin (PA-IL) to skeletal muscle fibres. Quantitative precipitation and precipitation inhibition assays.

Pseudomonas aeruginosa agglutinin (PA-IL) staining and the influence of various carbohydrates on lectin binding to muscle sections were investigated quantitatively using a scanning and integrating microspectrophotometre. A strong dose-dependent inhibition of PA-IL staining in the sections was recorded with galabiose (Galalpha1-4Gal) while lactose (Galbeta1-4Glc) had no inhibitory effect. The affinity of PA-IL to Galalpha1 carbohydrates was studied by ELISA using immobilized glycoconjugates in which Galalpha1 glycans were attached to bovine serum albumin or ceramide. PA-IL exhibited strong binding to both simple glycoconjugates having a single Galalpha moiety and to di- and trisaccharides with terminal Galalpha1 at the non-reducing end. In all cross-sectioned muscle fibres incubated with PA-IL, the staining was present as a honeycomb-shaped network through the entire cytoplasm. Further, a dense punctuate staining could be shown in most fibres. A similar staining pattern was noticed after incubation with a monoclonal antibody against ryanodine receptors and with biotinylated ryanodine suggesting that the network could represent the sarcoplasmic reticulum. Further, Western blots of a sarcoplasmic reticulum preparation showed multiple bands after incubation with PA-IL. It may therefore be proposed that glycoconjugates carrying terminal Galalpha1 show affinity for PA-IL and are located to the sarcoplasmic reticulum.

Immunohistochemical quantification of heparan sulfate proteoglycan and collagen IV in skeletal muscle capillary basement membranes of patients with diabetic nephropathy.

In IDDM patients, an increased permeability of the glomerular capillaries has been associated with a general loss of negatively charged heparan sulfate proteoglycans (HSPGs) within basement membranes (BMs). In the present study, we used immunohistochemical staining to quantify heparan sulfate (HS), HSPG core protein, and collagen IV in capillary basement membranes of skeletal muscle biopsies taken from 9 healthy control subjects (C) and 20 IDDM patients: 7 with normal albumin excretion rate (<30 mg/24 h) (D0), 5 with incipient nephropathy (albumin excretion rate 30-300 mg/24 h) (D1), and 8 with clinical nephropathy (albumin excretion rate >300 mg/24 h) (D2). In the capillaries, staining was measured by a scanning and integrating microspectrophotometer. A significant difference in the absorbance of HS was found among the four subgroups (means +/- SD): 0.477 +/- 0.082 (C), 0.627 +/- 0.031 (D0), 0.542 +/- 0.098 (D1), and 0.371 +/- 0.118 (D2) (P = 0.006). Similarly, an overall significant difference in the absorbance of collagen IV was demonstrated (means +/- SD): 0.836 +/- 0.111 (C), 0.838 +/- 0.300 (D0), 0.970 +/- 0.173 (D1), and 0.512 +/- 0.248 (D2) (P = 0.02). No statistical difference in the absorbance of core protein was demonstrated among the groups. Within the diabetic groups, HS was inversely correlated to albuminuria (r = -0.76, P = 0.003) and albuminuria corrected for creatinine clearance (r = -0.69, P = 0.008). Because, in IDDM patients with albuminuria, alterations of the content of HS and collagen IV within the capillary BM have been demonstrated immunohistochemically, not only in the glomerular filtration barrier, but also in the skeletal muscle capillary BM, we suggest that these changes reflect universal quantitative or qualitative alterations within the capillary filtration barrier.

The impact of fixatives on the binding of lectins to N-acetyl-glucosamine residues of human syncytiotrophoblast: a quantitative histochemical study.

We describe a quantitative histochemical method for demonstration of five N-acetyl-glucosamine binding lectins in the syncytiotrophoblast of human term placenta. The method employs biotinylated lectins and alkaline phosphatase-conjugated avidin. The alkaline phosphatase activity is detected by using 5-bromo-4-chloro-indoxyl phosphate as the substrate and nitroblue tetrazolium as the capture agent. The effect of 13 fixative solutions on specific lectin binding and nonspecific background staining was quantified by microspectrophotometry. Acid fixatives or fixatives containing mercuric chloride, e.g., Carnoy's and Zenker's fixatives, gave intense specific lectin binding and low background staining. Glutaraldehyde, carbodiimide, and ethanol resulted in low specific lectin binding and a very high background staining that was mainly due to endogenous placental alkaline phosphatase. Lectin binding to N-acetyl-galactosamine, mannose, galactose, and fucose was also significantly higher in sections from tissues fixed in an acid fixative compared with a neutral buffered fixative. Unfixed cryosections revealed a considerably lower degree of specific lectin binding compared with sections from fixed tissues. The activity of endogenous placental alkaline phosphatase was inhibited dose-dependently by mercuric chloride and decreased with L-phenylalanine concentration over the range of 7.8 x 10(-4) M to 5 x 10(-2) M, after which there was no further inhibition. Calf intestinal-type alkaline phosphatase conjugated to avidin was not inhibited by 5 x 10(-2) M L-phenylalanine. Endogenous placental biotin did not contribute significantly to background staining. Despite the high level of placental alkaline phsophatase, the intestinal-type alkaline phosphatase can be used as a marker enzyme in the sensitive ABC technique, provided that the nonspecific background is measured and substracted. Moreover, it is advisable to use an acid- and/or mercuric chloride-containing fixative and to add L-phenylalanine during incubation steps.

Detection of plasma proteins in CNS neurons: conspicuous influence of tissue-processing parameters and the utilization of serum for blocking nonspecific reactions.

Despite the presence of a blood-brain barrier (BBB), plasma proteins have been detected intraneuronally in regions with axonal projections confined to the CNS. This finding raises the question of whether plasma proteins are taken up from the brain interstitium or whether the results are due to experimental artifact. We examined the effect of various protocols for tissue processing on the intraneuronal distribution of plasma proteins using immunohistochemistry. The detection level of plasma proteins decreased after prolonged fixation, irrespective of the fixative and embedding method employed. In cryostat sections, attempts to block nonspecific staining by serum protein caused considerable nonspecific staining in itself. When nonspecific staining was blocked with a serum-free buffer, specifically labeled neuronal perikarya were found in cryostat sections of brains fixed by perfusion with paraformaldehyde without postfixation. Albumin and IgG occurred predominantly in neurons having projections beyond the BBB but also sparsely in neurons having projections confined to the CNS. Transferrin was evenly distributed within neuronal somata, irrespective of the orientation of projections. The immunoreaction product of the three plasma proteins exhibited a specific intraneuronal localization in the differently projecting neurons. In circumventricular organs, plasma proteins were observed extracellularly and in projecting fibers. In conclusion, plasma proteins are present in neurons with projections confined to the CNS and are probably taken up from the brain interstitium.

Microfluorometric kinetic analysis of cathepsin B activity in single human thyroid follicular epithelial cells using image analysis and continuous monitoring.

The activity of a cysteine proteinase, cathepsin B (EC 3.4.22.1), was determined in unfixed single human thyroid follicular epithelial cells at room temperature using an image analysis system. The formation of the reaction product was monitored every minute by measuring the increasing fluorescence intensity of emitted light from a Schiff-base product formed by the substrate N-CBZ-ala-arg-arg-4-methoxy-2-naphthylamide and the coupling agent 5-nitrosalicylaldehyde. Non-specific fluorescent signals were eliminated by adjusting the video signal to zero using leupeptin, a specific inhibitor of cathepsins B, H and L. The enzyme activity was expressed as fluorescence intensity versus time. After a mean lag period of 5 min (range 4-8 min, n = 4), the enzyme activity increased linearly lasting on average 5 min (range 3-8 min), followed by a marked decrease in reaction rate for 3 min (range 1-4 min), and another linear increase for 11 min (range 8-14 min). The second linear part of the curve was not as steep as the first one. The reaction velocity recorded in individual granules resulted either in a biphasic curve or straight line, suggesting the presence of two distinct organelle compartments with differences in membrane permeability. It is concluded that human thyroid follicular epithelial cells in culture exhibit cathepsin B activity which can be monitored continuously by videomicrofluorometry without interference from non-specific fluorescence.

Immunocytochemical localisation of tumor necrosis factor alpha in thyroid tissues from patients with neoplastic or autoimmune thyroid disorders.

It is disputed to what extent tumor necrosis factor-alpha is present in the thyroid follicular epithelial cells and/or in the interstitial cells in different disorders of the thyroid gland. We describe the immunohistochemical detection of tumor necrosis factor-alpha using formaldehyde fixed and paraffin embedded tissue and a polyclonal anti-serum with high tumor necrosis factor-alpha neutralising activity. We examined the distribution of tumor necrosis factor-alpha in interstitial cells and follicular epithelial cells in thyroid carcinomas, adenomas, non-toxic multinodular goiters and autoimmune thyroid diseases. Tumor necrosis factor-alpha was demonstrated in thyroid follicular epithelial cells, most frequently in non-toxic multinodular goiters (six of seven patients) and less frequently in adenomas (three of nine patients), papillary carcinomas (two of five patients), follicular carcinomas (one of five patients), Hashimoto's disease (one of six patients) and Grave's disease (one of seven patients). Tumor necrosis factor-alpha producing interstitial cells were found in two thirds of patients with all six thyroid diseases.

Immunocytochemical localisation of interleukin-1 alpha and interleukin-6 in thyroid tissues from patients with neoplastic or autoimmune thyroid disorders.

We describe the distribution of interleukin-6 and interleukin-1 alpha in thyroid tissues obtained from patients with autoimmune diseases or neoplastic thyroid disorders employing immunohistochemistry in sections from paraffin embedded tissue blocks. Interleukin-6 was found in thyroid follicular epithelial cells (TFEC) from papillary carcinomas (four of five patients) but not in follicular carcinomas (five patients). Interleukin-6 was also detected in non-toxic multinodular goiters (four of seven patients), in patients with Graves' disease who did not have an early recurrence of hyperthyroidism after surgery (three of four patients), in follicular adenomas (five of nine patients), in Hashimoto's thyroiditis (two out of six patients, both belonging to a group of three with an early stage of the disease), and in paraadenomatous tissues (in three of nine patients). Interleukin-1 alpha positive TFEC were found less frequently than interleukin-6, and only in tissues with interleukin-6 positive TFEC. Only few interleukin-6 and interleukin-1 alpha positive interstitial cells were found, even in the lymphocyte infiltrates (in both the autoimmune, benign or malignant disorders). In conclusion, both interleukin-6 and interleukin-1 alpha could be demonstrated in TFEC from patients with autoimmune diseases, benign neoplasms or papillary carcinoma, whereas follicular cancer tissues were without interleukin-6 and interleukin-1 alpha. In contrast with previous studies, interleukin-6 and interleukin-1 alpha were demonstrated in TFEC from patients with both Graves' disease and Hashimoto's thyroiditis, and the presence of these cytokines was related to the stage of the autoimmune process.

Contribution of glutamate decarboxylase antibodies to the reactivity of islet cell cytoplasmic antibodies.

The contribution of glutamate decarboxylase (Mr 65000) antibodies to the reactivity of islet cell cytoplasmic antibodies with the 'whole' islet staining pattern from patients with newly diagnosed Type I diabetes was investigated. Diluted sera (n = 10) were preincubated with increasing concentrations of purified recombinant human islet glutamate decarboxylase (Mr 65000) and the change in islet cell cytoplasmic antibody binding was evaluated by quantitative immunocytochemistry. Binding to islet cells was partially blocked by glutamate decarboxylase in 9/10 diluted sera; the maximum blocking obtained at high concentrations of glutamate decarboxylase (5 micrograms/ml) was 36% (median, range 24-61%). In contrast, binding to islet cells in three diluted sera (two polyendocrine patients without Type I diabetes and one patient with newly diagnosed Type I diabetes) with the 'selective' islet staining pattern was totally blocked by glutamate decarboxylase. The concentration of glutamate decarboxylase required to achieve maximum blocking was less for the 'whole' islet (0.4-8.0 micrograms/microliters undiluted serum) compared to the 'selective' islet (20-645 micrograms/microliters undiluted serum) positive sera. All sera were positive for glutamate decarboxylase antibodies in an immunoprecipitation assay using 35S-methionine labelled extract of baby hamster kidney cells transfected with glutamate decarboxylase. However, the binding activity of these antibodies was less in the sera positive for the 'whole' islet compared to the 'selective' islet staining pattern. In conclusion, glutamate decarboxylase antibodies contribute partially to the reactivity of islet cell cytoplasmic antibodies of the 'whole' islet staining pattern in the sera of newly diagnosed patients with Type I diabetes, and totally to reactivity of the 'selective' islet staining pattern. The antigens recognized by the other antibodies contributing to the 'whole' islet reactivity remain to be defined.

Elevated GLUT 1 level in crude muscle membranes from diabetic Zucker rats despite a normal GLUT 1 level in perineurial sheaths.

Recently, we demonstrated that approximately 60% of GLUT 1 in a crude membrane fraction of rat skeletal muscle originates from perineurial sheaths. To study the in vivo regulation of GLUT 1 expression in different tissues in muscles, we measured the level of GLUT 1 in crude muscle membranes and in perineurial sheaths in diabetic (fa/fa) Zucker rats and lean controls, with and without metformin treatment. The GLUT 1 concentration in perineurial sheaths was identical in all four groups of rats, both when measured by quantitative immunofluorescence and by immunoblotting and densitometry. In a fraction of crude membranes of soleus muscles GLUT 1 expression was more than two-fold higher in (fa/fa) rats than in lean controls (p < 0.005). Metformin treatment significantly elevated GLUT 1 in control rats (p < 0.05) and tended to decrease GLUT 1 in diabetic rats (p < 0.075). The expressions of GLUT 1 and GLUT 4 in crude muscle membranes were inversely correlated (p < 0.01), and GLUT 1 expression correlated positively with fasting glucose (p < 0.05). In conclusion, GLUT 1 expression in perineurial sheaths is unaffected by alterations in glucose homeostasis and by the genes responsible for obesity and diabetes in the Zucker rat. GLUT 1 expression in a crude membrane fraction of soleus muscle is increased in the diabetic animals, likely due to an increased expression in muscle cells proper.

Metformin ameliorates diabetes but does not normalize the decreased GLUT 4 content in skeletal muscle of obese (fa/fa) Zucker rats.

We studied the expression of the glucose transporter GLUT 4 in the soleus and red gastrocnemius muscles from obese, diabetic (fa/fa) Zucker rats compared to their lean littermates (Fa/-), with and without treatment with the antidiabetic drug metformin. In the untreated groups of rats, the GLUT 4 content in a crude membrane fraction of both the soleus and the red gastrocnemius muscles were significantly lower in the obese (fa/fa) rats (3.46 +/- 0.28 vs. 6.04 +/- 0.41, p < 0.001 and 6.0 +/- 0.24 vs. 9.1 +/- 0.48, p < 0.0001, respectively). Differences in GLUT 4 expression in soleus muscle from the same rats were confirmed by quantitative immunofluorescence microscopy, and the results were significantly correlated with the results obtained from quantitative immunoblotting (rho = 0.70, p < 0.0005). The decreased expression of GLUT 4 in fa/fa rats could contribute to the well-established insulin resistance in skeletal muscle of these animals. After 4 weeks of treatment with metformin, weight gain was not affected in either the diabetic (fa/fa) rats or the lean (Fa/-) rats. Improvement of glucose homeostasis by metformin was not associated with normalization of the GLUT 4 expression in the skeletal muscles studied, indicating (1) that the decreased GLUT 4 expression is not directly related to hyperinsulinaemia and diabetes mellitus and (2) that metformin does not normalize the expression of GLUT 4 in skeletal muscle of the diabetic (fa/fa) Zucker rats.

Dipeptidyl peptidase IV is sorted to the secretory granules in pancreatic islet A-cells.

Dipeptidyl peptidase IV (DP IV:EC 3.4.14.5) was localized in endocrine cells of pig pancreas by immunohistochemical and enzyme histochemical methods. Immunolight microscopy with both monoclonal and polyclonal antibodies demonstrated DP IV immunoreactivity in cells located in the peripheral part of the islets of Langerhans. The antigen is enzymatically active, as shown by enzyme histochemical analysis with a synthetic DP IV substrate. By immunoelectron microscopy (immunogold labeling), the labeling of DP IV in the islets was associated with the secretory granules of the A-cells, as identified by double labeling using a monoclonal glucagon antibody as the second primary antibody. These results show that DP IV is sorted to secretory granules in the pig pancreatic islet A-cells. Furthermore, this secretory granule enzyme, as opposed to intestinal brush border DP IV, is suggested to be a soluble protein, since the gold particles appear all over the granules and are not specifically associated with the granule membrane.

Intracellular pH regulation in human thyrocytes: evidence of both Na+/H+ exchange and Na(+)-dependent Cl-/HCO3- exchange.

The present study was undertaken to investigate the regulation of intracellular pH in human thyroid epithelial cells and to characterize the kinetics of the acid-extruding processes operating in the absence and presence of HCO3-, Na+ and Cl-. A dynamic technique of dual excitation microfluorimetry and the pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein was employed. The intracellular pH was 7.01 +/- 0.27 (n = 29) and 6.94 +/- 0.25 (n = 54) in the absence and presence of HCO3- respectively. Both in the absence and presence of HCO3-, the recovery from intracellular acid loads was not only due to an Na+/H+ exchange, but also to an Na(+)-dependent HCO3-/Cl- exchange. In alkaline conditions caused by NH4Cl pulsing, an HCO3-/Cl- exchange was also found. The cells in HCO3- responded with a wide range of maximal hydrogen efflux rates in experiments where cells were either pretreated with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid or incubated with amiloride. The heterogeneity might be due to subpopulations of thyrocytes in different metabolic states or at different points in the cell cycle. It is concluded that recovery from intracellular acidification in human thyroid cells is due to both Na+/H+ exchange and Na(+)-dependent Cl-/HCO3- exchange even in nominally HCO3(-)-free conditions, and that recovery from intracellular alkalinization is due to a Cl-/HCO3- exchange which needs to be characterized further.

Simultaneous quantification of DNA and RNA in tissue sections. A comparative analysis of the methyl green-pyronin technique with the gallocyanin chromalum and Feulgen procedures using image cytometry.

For simultaneous cytophotometric measurement of DNA and RNA, the standardized Methyl Green-Pyronin Y technique is an obvious choice. It is, however, first necessary to correlate the uptake of Pyronin Y to the staining intensity of RNA. The material consisted of paraffin sections of formalin- or Carnoy-fixed rat liver. The sections were pretreated with water, buffer, deoxyribonuclease, ribonuclease, or both enzymes in sequence, and stained with the standardized Methyl Green-Pyronin Y procedure, Gallocyanin chromalum, or the Feulgen reaction. Sections stained directly without pretreatment served as controls. Staining intensities were measured with an image analyser for cell nuclei, nucleoli and cytoplasm. After deoxyribonuclease treatment, nuclear staining intensity with Methyl Green, Gallocyanin chromalum, and Schiff's reagent dropped nearly to zero. The same was seen for both nucleoli and cytoplasm with Pyronin Y and Gallocyanin chromalum after ribonuclease treatment. Staining intensity of Pyronin Y correlated directly with that of Gallocyanin chromalum for nucleoli and cytoplasm. After ribonuclease treatment, a direct correlation was found between the nuclear staining intensity of Methyl Green and nuclear absorption of Gallocyanin chromalum. We conclude that the standardized Methyl Green-Pyronin Y stain is reliable for the simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine.