M. Terlou

Purification of gonadotropic hormone from the pituitary of the African catfish, Clarias gariepinus (Burchell), and the development of a homologous radioimmunoassay

Glycoprotein gonadotropic hormone (GTH) was purified from 2000 pituitaries of male and female African catfish, Clarias gariepinus. The first step was chromatography on concanavalin A-Sepharose followed by filtration on Ultrogel Aca 54, chromatography on DEAE-cellulose, and filtration on Ultrogel Aca 54, respectively. Finally, the purified fractions were analyzed by polyacrylamide gel electrophoresis. The gonadotropic activity in the different fractions was characterized using two tests: the radioimmunoassay for carp gonadotropin-beta subunit was used to quantify the immunoreactive GTH and a cAMP accumulation test was applied to measure the GTH biological activity. The purified glycoprotein GTH was used to raise antibodies and to develop a radioimmunoassay. This resulted in an assay with a variation between assays of approximately 4%, a precision of 4-8%, and an accuracy of 4-8%. GTH levels can be measured over a range of 0.8 to 12.5 ng/ml.

Hypothalamo-hypophysial relations in amphibian larvae

Abstract The results of studies on the differentiation of the preoptic nucleus and of experiments regarding the effects of propylthiouracil and extirpation of the preoptic area demonstrate that in Xenopus laevis tadpoles a thyrotropin-releasing factor (TRF) is formed in peptidergic cells in the dorsal part of the preoptic nucleus. The differentiation of this nucleus depends on thyroid hormones, but at the same time these hormones inhibit the activity of the TRF cells. Young larvae of X. laevis acquire the ability to adapt themselves to a white background simultaneously with the appearance of aminergic neurosecretory centers in the caudal hypothalamus. During prometamorphosis these centers comprise a paraventricular organ (PVO), a nucleus infundibularis, fiber tracts to the telencephalon and to the pars intermedia of the adenohypophysis, and a nucleus in the caudalmost part of the dorsal tuber cinereum. The PVO consists of sensory nerve cells and cells secreting products into the cerebrospinal fluid; the infundibular nucleus contains numerous liquor-contacting neurons. The aminergic neurosecretory cells contain catecholamines such as dopamine. Depletion of these cells by reserpine leads to a dispersion of melanin granules in the skin melanophores. It is believed that among the functions of the aminergic neurosecretory centers is the production of a melanotropin-inhibiting factor. In the ventral tuber cinereum of X. laevis , neurosecretory cells are situated that are neither peptidergic nor aminergic. They are more active in adult animals than in tadpoles.

Quantitative enzyme histochemistry of steroid and glucuronide synthesis in testes and seminal vesicle, and its correlation to plasma gonadotropin level in Clarias gariepinus

In spawning Clarias gariepinus from the Hula Nature Reserve in Northern Israel, the mean value for the seminal vesicle somatic index (SVSI) had decreased as compared with that in prespawning fish, due to loss of seminal vesicle fluid. Mean gonadosomatic index (GSI), however, had increased, probably as a result of hydration, 3α-Hydroxysteroid dehydrogenase (3αHSD), 3βHSD and uridine diphosphoglucose dehydrogenase (UDPGD) reactions were demonstrated in interstitial cells of both testis and seminal vesicle. UDPGD reactions were also found in the epithelium of the seminal vesicle tubules. Quantitative determination of the enzyme reactions with a computerized image analysis system revealed that total activity of all three enzymes in interstitial cells of the seminal vesicle was distinctly stronger in spawning fish. Prespawning and spawning fish did not differ in total UDPGD activity in the epithelium lining the seminal vesicle tubules. In the testes of spawning fish, total 3βHSD activity was stronger and total UDPGD activity weaker than in prespawning fish, but total 3αHSD activity was not different. Plasma gonadotropin (GTH) levels in male catfish were low as compared with females. This level was significantly increased in spawning males and extremely high in ovulated female spawning fish. The findings point to marked changes in the production of steroids and glucuronides in the reproductive organ system during spawning, especially in the seminal vesicle. These changes are accompanied by elevated levels of circulating GTH.

Histophysiological and immunocytochcmical study on the nature of the thyrotrops in the pituitary of immature rainbow trout, Salmo gairdneri

SummaryAlcian blue and periodic acid-Schiff-positive “granular basophils” with electron-dense granules ±160 nm in diameter, and weakly developed irregular cisternae of the granular endoplasmic reticulum (GER) are present in the rostral pars distalis (RPD) and the proximal pars distalis (PPD) of immature rainbow trout. They all react with antisalmon αβ-gonadotropin. However, only granular basophils in the caudal RPD and the rostro-dorsal PPD, often bordering on and sometimes surrounded by neurohypophysial tissue, react with anti-human β-TSH. These cells, considered as the source of thyrotropin, show degranulation and dilatation of the GER-cisternae in fish treated with potassium perchlorate. The thyroids of the goitrogen-treated animals had relatively numerous small follicles with a high epithelium. The remaining granular basophils are gonadotrops.

Accumulation of glycoprotein gonadotropin in the pituitary of juvenile rainbow trout in response to androgens and C21-steroids, including 11-steroids

SummaryEffects of steroids on the accumulation of glycoprotein gonadotropin (GTH) in pituitaries of juvenile trout were investigated by means of scanning cytophotometry applied to immunocytochemical preparations, and with the use of a radioimmunoassay. Effects on other aspects of GTH-cell activity were analyzed by measuring the size of the gonadotrops and their nuclei.Progesterone added to aquarium water and methyltestosterone incorporated into the food showed a pronounced stimulatory effect on the accumulation of GTH. To a lesser extent, treatment with cortisol, cortisone, and desoxycorticosterone acetate administered to aquarium water, and 11β-hydroxy-androstenedione added to the food resulted in an increase of the hypophysial content of GTH. Steroids stimulating the accumulation of GTH in the pituitary also exhibited a positive effect on GTH-cell activity as indicated by an increase in the size of gonadotropic cells. Progesterone incorporated into the food did not influence the GTH-content and the GTH-cell activity. It is suggested that the route of administration of an exogenous steroid is essential for its effect on GTH cells in trout.Comparison of GTH values reveals an excellent correlation between the data from the radioimmunoassay and those from the corresponding densitometric measurements. No correlation was observed between values of morphometrically determined GTH-cell activity and the densitometric values reflecting hypophysial GTH content.

Immunocytochemical devidence for hypophysial projections of the cells of the nucleus lateralis tuberis pars lateralis in the rainbow trout (Salmo gairdneri)

SummaryIn the rainbow trout the pars lateralis is the most prominent part of the nucleus lateralis tuberis (NLT). To demonstrate a morphological relationship between this lateral part of the NLT and the pituitary, immunocytochemistry was applied as a staining method.Experiments were carried out on glutaraldehyde-picric acid-acetic acidfixed brain sections of mature male and female rainbow trout using the peroxidase-anti-peroxidase immune technique with an antiserum against 27-S-methylglucagon as the first antibody.Most of the cells in the NLT/pars lateralis reacted with the antiserum. Axons from these cells enter the pituitary, extending exclusively in the numerous neurohypophysial digitations in the pars intermedia. No immunoreactive neurohypophysial protrusions were found in those parts of the adenohypophysis where the gonadotropic cells are located, indicating that the lateral part of the NLT is not directly involved in the control of gonadotropin secretion.In addition to cells of the NLT/pars lateralis only prolactin cells in the rostral pars distalis of the adenohypophysis reacted with the antiserum used.

Cortisol effects on the testicular androgen synthesizing capacity in common carp, Cyprinus carpio L.

Our previous studies on the effect of stress on pubertal development in carp have shown that repeated temperature changes caused an increase in cortisol levels and a retardation of the first waves of spermatogenesis. Identical effects, accompanied by a decrease in 11-ketotestosterone (11KT) plasma levels and the gonadosomatic index (GSI) were induced by cortisol administration via cortisol containing food pellets. The decrease in plasma 11KT is caused by a direct effect of cortisol on the steroid producing capacity of the testis, independent of luteinizing hormone (LH) levels. However, the mechanism through which cortisol interferes with testicular steroidogenesis is unknown. In the present study, we showed that in vitro physiological levels of cortisol can inhibit the conversion of 11β-hydroxyandrostenedione (OHA) into androstenetrione (OA), which is the precursor of 11KT, possibly by competing for the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD). The same mechanism may occur in vivo. However, our results demonstrate that an elevation of plasma cortisol levels during acute cortisol treatment did not result in lower plasma levels of OA and 11KT, but we did observe an accumulation of OHA. We suggest that the previously observed decrease in 11-oxygenated androgens, as an effect of long-term cortisol treatment, is caused by a retardation of testicular development. This results in a lower steroid synthesizing capacity of the testis as a whole. Although the in vitro observed cortisol inhibition of the conversion of OHA into 11KT plays a role in the accumulation of OHA, it apparently has no effect on the final 11KT plasma concentration.

Structural and Functional Aspects of Two Types of Gomori-Negative Neurosecretory Centres in the Caudal Hypothalamus of Amphibia

Combining light and electron microscopical techniques three types of paired neurosecretory centres can be identified in the hypothalamus of Amphibia. The first comprises the nucleus preopticus which consists of Gomori-positive and Falcknegative peptidergic cells containing elementary granules with a diameter of more than 1000 A. The second type is formed by the paraventricular organ and the nucleus infundibularis dorsalis. In these nuclei the cells are Gomori-negative and Falck-positive, and are therefore labelled aminergic neurosecretory cells. They contain dense-core vesicles with a diameter not seldom less than 1000 A. The third type is represented by the nucleus infundibularis ventralis. Its nerve cells are Gomori- as well as Falck-negative, and contain elementary granules larger than 1000 A in diameter (Peute and van Oordt, 1974). The Gomori-negative centres are discussed in this paper.

Quantitative estimation of pituitary gonadotropin in juvenile rainbow trout with scanning cytophotometry applied to immunocytochemical preparations

SummaryA densitometrical method with computer-controlled scanning cytophotometry has been developed to quantify the amount of gonadotropin (GTH) in immunocytochemically treated paraffin sections of pituitaries of juvenile rainbow trout. Results obtained with this method totally correspond with those established with a radioimmunoassay of pooled pituitaries. Scanningcytophotometry offers the advantage that GTH-content of individual pituitaries of juvenile or small adult fish can be investigated.

Scanning cytophotometry : A method for studying the amount of neurosecretory material in the preoptic nucleus of the trout (Salmo gairdneri)

In order to study the activity of the preoptic nucleus (NPO) during the annual cycle, the amount of neurosecretory material was measured by means of scanning cytophotometry. This objective method implies standardization of the histological method and computerized procedures for the analysis of the data. Preliminary results of such studies on the NPO of sexually mature male rainbow trout, together with cytological information, point to cyclical changes in the activity of the nucleus. An active period was indicated from the beginning of June to January and an inactive period from February to June.