Martin Ritzén

Fluorescence microspectrophotometry of cellular catecholamines and 5-hydroxytryptamine.

Abstract The fluorescent products formed from certain biogenic monoamines, when they are treated with formaldehyde according to the method of Falck and Hillarp, have been studied in model systems and in tissue sections. The analysis of cellular monoamine fluorescence has been made possible by a recently developed fluorescence microspectrograph. The results show that when models containing catecholamines or 5-hydroxytryptamine are treated as described for the histochemical method, consistent results are obtained; catecholamines show excitation/emission maxima at 410 470 mμ, and 5-hydroxytryptamine at 385– 415 520 mμ. The same results are found for cells normally containing these amines; adrenergic neurones, rat mast cells and rabbit enterochromaffin cells are selected as examples. Tryptamine and m-tyramine can also be identified by fluorescence microspectrographic methods due to their characteristic fluorescence following formaldehyde treatment. The reaction conditions between the amines and formaldehyde must be carefully controlled since too much humidity in the formaldehyde gas will cause a shift in emission of high concentrations of primary catecholamines from the original blue-green into yellow. In the fluorescence microscope, this might be interpreted as 5-hydroxytryptamine fluorescence. With this in mind, it can be stated that it is generally possible to distinguish between catecholamines and 5-hydroxytryptamine also in an ordinary fluorescence microscope.

Carcinoma in situ of the testis in children with 45,X/46,XY gonadal dysgenesis

The frequency of gonadal tumors in intersex patients with a karyotype including a Y chromosome is very high. In other at-risk groups, testicular germ cell tumors have been shown to be preceded by carcinoma in situ (CIS) changes. We investigated gonadal tissue from four children, aged 1 month to 18 years, with 45,X/46,XY gonadal dysgenesis, and with male or ambiguous genitalia, for the presence of CIS germ cells. Twelve gonadal biopsies and gonadectomy specimens were analyzed by means of conventional histology and densitometric DNA measurements. CIS changes were detected in specimens from all four patients, and aneuploid DNA distributions of the CIS germ cells confirmed the malignant potential of these cells. In one case, electron microscopic analysis revealed the same ultrastructural features of the CIS germ cells as previously described in seminoma cells. These observations indicate that in all patients with 45,X/46,XY gonadal dysgenesis and a male phenotype, gonadal biopsies should be considered as soon as the syndrome is diagnosed. We believe that the finding of CIS warrants gonadectomy.

Quantitative fluorescence microspectrophotometry of catecholamine-formaldehyde products: Model experiments☆

Abstract The relative fluorescence yield of catecholamines after condensation with formaldehyde according to the histochemical technique of Falck and Hillarp has been investigated in solution and in model systems, the latter in order to imitate the situation in freeze-dried tissues. The models consisted of catecholamines dissolved in a sucrose or protein solution, which was sprayed onto object slides to form dried micro droplets. After formaldehyde treatment according to the histochemical technique, the models were examined with respect to absorption and emission characteristics, reaction conditions, and influence of water in the specimens. Special attention was paid to the relation of the fluorescence intensity to the concentration of the fluorophore. It was found that the linear relationship between the two at low concentrations was broken at around 4.5 × 10 −2 moles/1 dried droplet volume. Above this concentration, a further increase in catecholamine concentration was not accompanied by a corresponding increase in fluorescence intensity, possibly due to intermolecular energy transfers between the relatively closely packed molecules. This concentration quenching should be kept in mind when trying to interpret fluorescence intensity as a measure of catecholamine concentration. It is pointed out that a similar concentration quenching is likely to appear also in other cytochemical fluorescence techniques where the concentrations often are much higher than in the commonly used biochemical fluorescence assays. This error is eliminated if the distance between the fluorescent molecules is kept constant.

Hormonal Regulation of Spermatogenesis

Publisher Summary This chapter discusses the hormonal regulation of spermatogenesis. The hormonal regulation of spermatogenesis involves interplay of sex steroids and pituitary gonadotropic hormones acting on specific cells of the testis. The target cells for pituitary hormones and steroid hormones within the testis and to the elucidation of molecular mechanisms for hormone action are identified. The mechanisms involved in the hormonal control of Sertoli cells and the interaction between Sertoli cells and germ cells are also reviewed in the chapter. Androgenic hormones provide a major stimulus for spermatogenesis. Production of testosterone by Leydig cells is regulated by interstitial cell-stimulating hormone (ICSH). Leydig cell development and steroidogenic function may require a direct action of testosterone or dihydrotestosterone. Prolactin and follicle-stimulating hormone (FSH) enhances Leydig cell responsiveness whereas estrogen appears to inhibit Leydig cell response to ICSH with respect to steroidogenesis. Androgens stimulate peritubular cell differentiation and the development of tubular contractions. Androgens and FSH regulate Sertoli cell function.

Quantitative fluorescence microspectrophotometry of 5-hydroxytryptamine-formaldehyde products in models and in mast cells

Abstract The quantitative aspects of the paraformaldehyde condensation method of Falck and Hillarp for histochemical demonstration of 5-hydroxytryptamine (5-HT) have been investigated by fluorescence microspectrographic methods in a model system of dried protein microdroplets and in rat peritoneal mast cells. It was found that the pronounced variations in fluorescence yield depending on the reaction conditions and the way of preparation of the mast cell specimens make it advisable to bring in some sort of reference system with each reaction. The concentration quenching of the fluorescence of the 5-HT-formaldehyde product was not quite as disturbing as that of the fluorescent catecholamine products described elsewhere. Thus, when the concentration of 5-HT in dried protein was increased above a critical value (around 9 × 10−2 M), a moderate quenching of the fluorescence appeared. The possibilities of using the reaction in a quantitative way was also tested in rat peritoneal mast cells, the 5-HT content of which had been made to vary by treatment in vitro with compound 48 80 . The mean fluorescence of groups of mast cells which had been treated with increasing concentrations of 48 80 as measured in the fluorescence microspectrograph was shown to be well correlated to the biochemically determined means of 5-HT per mast cell in the same groups. Thus, in this system, the method of Falck and Hillarp can be used to obtain adequate estimates of the relative content of 5-HT in individual mast cells.

Androgens and fetal growth

Boys are heavier than girls at term birth. Children with a 46,XY karyotype and androgen insensitivity syndrome (clinically complete form and/or proven mutations in the androgen receptor gene) were found to have a birth weight comparable to that of girls. These findings support the hypothesis that the difference in birth weight between boys and girls is generated by androgen action.

Studies on uptake of intraventricularly administered tritiated noradrenaline and 5-hydroxytryptamine with combined fluorescence histochemical and autoradiographic techniques

SummaryTritiated noradrenaline (NA) and 5-hydroxytryptamine (5-HT) (1.5–30 μC) have been injected intraventricularly into normal or reserpine-nialamide pretreated rats 1/2 to 2 hours before the killing. Various parts of the brains were freeze-dried, reacted with formaldehyde gas and embedded in paraffin or Araldite. Before application of the stripping film emulsion many sections were photographed in the fluorescence microscope in order to perform a combined histochemical and autoradiographic study of the monoamine neurons. By such an approach it was possible to demonstrate 1. that the accumulation of radioactivity in cell bodies after 3H-NA and 3H-5-HT injection is localized to catecholamine (CA) and 5-HT cell bodies respectively; 2. that injected 3H-NA and 3H-5-HT in the doses used relatively selectively are taken up into the NA and 5-HT nerve terminals respectively, since the distribution of grains in the sections follow that of the fluorescent terminals; 3. that the accumulation of silver grains only reaches the zone (200–400 μ) close to the ventricles and the ventral part of the subarachnoidal space. By grain counting it was possible to estimate that the degree of concentration of radioactivity in the monoamine cell bodies was up to 4 times that in the immediate surroundings. — The Araldite sections consistently gave a better resolution in the autoradiographic picture than the paraffin sections. It is postulated that freeze-drying and plastic embedding for autoradiography will be a valuable method for the cellular demonstration of certain biogenic amines which are not easily demonstrated by the histochemical fluorescence method and of other biologically active water-soluble compounds, since diffusion will be restricted to a minimum.

Chlorinated contaminants, growth and thyroid function in schoolchildren from the Aral Sea region in Kazakhstan

It has been shown by others that offspring of mothers who had been exposed to dioxins and polychlorinated biphenyls (PCBs) during pregnancy have elevated plasma levels of thyroid‐stimulating hormone (TSH) for at least 3 months after birth and reduced plasma levels of free and total thyroxine during the second week after birth. As elevated levels of dioxins and PCBs can thus alter thyroid hormone status, the relation between the levels of some polychlorinated organic compounds in the blood lipids and growth and thyroid hormone status was studied in 12 hospitalized schoolchildren from the Aral Sea region known to have high exposure to such compounds. Their level of PCBs was two to four times higher than in healthy Stockholm children. Their height was found to be lower than in healthy Swedish children of the same age mean (SDS ‐0.52) and the body mass index (BMI) was inversely correlated to the total concentrations of PCBs and dichlorodi‐phenyltrichloroethane (DDT) and its metabolite dichlorophenyldichloroethylene (DDE) in the blood lipids. As the levels of insulin‐like growth factor‐1 were reduced to the same extent as the BMI it seems likely that PCBs and DDT cause malnutrition as a result of malabsorption. None of the children had any impairment of thyroid function, as revealed by the plasma levels of TSH and thyroxine. Although the concentrations of β‐hexachlorocyclohexane (β‐HCH) and DDE were extremely high in some of the children there was no relation between thyroid hormone status and the blood lipid levels of PCBs, hexachlorocyclohexane and DDT. However, the concentration of dioxins was not analysed.

Efficacy and safety of hormonal treatment of cryptorchidism: current state of the art.

Meta‐analyses of randomised trials using hCG or GnRH for treatment on testicular descent show in most studies overall efficacy of about 20%, less if retractile testes were excluded. In recent years a number of potentially serious side effects have been reported.

Peptides of postulated inhibin activity: Lack of in vitro inhibin activity of a 94-residue peptide isolated from human seminal plasma, and of a synthetic replicate of its C-terminal 28-residue segment

A 94‐residue polypeptide isolated from human seminal plasma and its chemically synthesized C‐terminal 28‐residue segment were studied in an in vitro inhibin bioassay utilizing rat pituitary cell cultures. Both peptides have previously been claimed to have inhibin activities, and the effects on the secretion and cellular content of gonadotrophins (FSH and LH) were now assessed in the in vitro assay. No inhibition was found. After 72 h of culture, both the cellular content and the spontaneous as well as the LHRH‐stimulated release of bioactive or immunoactive FSH and LH remained unaffected. Similarly, no effects were found on the storage and/or release of prolactin, growth hormone, or thyrotropin. We conclude that both the native 94‐residue peptide and the synthetic replicate of its C‐terminal 28‐residue segment, do not influence the pituitary FSH secretion when assessed in this in vitro system.