Glycoprotein gonadotropin in the plasma and its cellular origin in the adenohypophysis of sham-operated and ovariectomized rainbow trout,salmo gairdneri

Summary Among the cells of the pituitary generally believed to produce glycoprotein gonadotropin (GTH) five forms were distinguished, based on the amount and the diameter of granules and globules and the appearance of the rough endoplasmic reticulum. In sham-operated trout so-called globular cells predominated, whereas after ovariectomy these were replaced by so-called cisternal cells, suggesting that both belong to one GTH-cell type. In addition, ovariectomy caused a strong increase in plasma GTH-levels. This indicates that the transition from globular to cisternal cells is accompanied by extrusion of GTH, and thus points to a storage of GTH in the granules and globules. It is argued that one of the five forms has the morphological characteristics of thyrotropic cells and may not produce glycoprotein GTH.The authors are indebted to Mr. L.W. van Veenendaal for preparing the illustrations and the photographic layout     

The brain-pituitary-gonadal axis in the rainbow trout,Salmo gairdneri: Gonadal hormones and the maturation of gonadotropic cells

Summary Intact and castrated juvenile male rainbow trout (Salmo gairdneri) were treated with testosterone and gonadotropic hormone (GTH) to determine the maturational effects of these hormones on the GTH-cells. Electron-microscopic studies of the GTH-cells revealed that GTH and testosterone in intact animals, and testosterone in castrated fish, caused GTH-cell maturation: These cells now displayed the same appearance as GTH-cells in adult trout, including the presence of globules, a well-developed Golgi apparatus and rough endoplasmic reticulum, all of which were absent in GTH-cells of control animals. Animals with stimulated GTH-cells also had an increased GTH content of the pituitary; release of GTH could not be demonstrated. Animals treated with GTH exhibited an accelerated development of the testes, resulting in complete gametogenesis and elevated plasma testosterone levels. These results indicate that exogenous steroids as well as endogenous gonadal steroids can stimulate the full development of GTH-cells and accelerate GTH synthesis. The significance of this stimulating effect of the gonadal hormones with respect to the development of the brain-pituitary-gonadal axis and the onset of puberty is discussed.The results were presented as a poster at the 11th conference of the European Society of Comparative Endocrinology, Jerusalem, August 10–14, 1981     

Effect of salmon gonadotropic hormone on sex steroids in male rainbow trout: plasma levels and testicular secretion in vitro

Summary Male rainbow trout were treated with salmon gonadotropic hormone (GTH) at different stages of the circannual reproductive cycle; spawning fish were also treated with an antiserum against salmon GTH. Injection of GTH led to a several-fold increase of plasma sex steroid levels during spermatogenesis and in the spawning season but was without effect at early stages of testicular development. GTH neutralization during the spawning season was followed by a several-fold decrease of plasma sex steroid levels. During spermatogenesis and in the spawning season, both treatment regimes resulted in an increased sensitivity of testicular explants in response to a subsequent stimulation of steroid secretion in vitro. This up-regulatory response may facilitate and maintain the high sex steroid plasma levels observed during the spawning season. It may also be necessary to allow for concomitant peak values of plasma GTH and sex steroids in the spawning season, a situation difficult to understand within the negative feedback concept. The adaptive capacities of the testicular steroidogenic system indicate that it is not only an effector site for GTH but also an active part of the endocrine system controling reproduction.Abbreviations BSA bovine serum albumin - bw body weight - E₂ 17-estradiol - GnRH gonadotropin releasing-hormone - GTH gonadotropic hormone - LH luteinizing hormone - OHT 11-hydroxytestosterone - OT 11-ketotestosterone - 17-20P 17-hydroxy, 20-dihydroprogesterone - PE pituitary extract - raGTH rabbit anti-GTH antiserum - rPS rabbit preimmune serum - T testosterone     

Protein synthesis in tissues cultured from the bovine hoof

Salmon gonadotropin (GTH II) is a heterodimeric glycoprotein hormone ( and II subunits), serving as a maturational GTH, and is produced in a specific gonadotropic cell-type (GTH II-cells) containing small granules and large globules. In trout GTH II-cells, double immunolabeling for the - and II-subunits shows that colocalization of the - and II-immunolabeling is confined to the small granules, indicating storage of functional GTH II. On the other hand, -immunolabeling is absent in the large globules, even though II labeling is abundant throughout the period of seasonal gametogenesis. The -specific antiserum recognizes the intact -subunit as well as the reduced and deglycosylated -subunits by immunoblotting. These results indicate that an accumulation of the II-subunit is specifically generated in the large globules of these cells. In fact, with sexual maturity, the quantity of II-subunits becomes elevated in the trout pituitary due to a marked increase in GTH II-cells containing many large globules. However, the derivation and function of the large globules and the fate of their contained II-subunits remains unknown.     

Differential production and regulation of gonadotropins (GTH I and GTH II) in the pituitary gland of rainbow trout,Oncorhynchus mykiss,during ovarian development

Summary Biosynthesis of salmon gonadotropins, GTH I and GTH II, during ovarian development, were examined by means of in situ hybridization histochemistry and indirect immunocytochemistry. In rainbow trout pituitary glands, expression of GTH I- and II-subunit genes appeared separately in distinct cells (GTH I- and GTH II-cells), whereas the GTH -subunit gene was expressed in both cell-types. In the GTH I-cells, coordinated increases in GTh, and I messenger ribonucleic acids (mRNAs) occurred coincident with the onset of vitellogenesis, indicating active synthesis of GTH I during vitellogenesis. In contrast, in the GTH II-cells, both GTH -and II-mRNA signals markedly increased from a later stage of vitellogenesis and persisted throughout oocyte maturation and ovulation, supporting the idea that GTH II is actively synthesized as a maturational GTH. GTH -mRNA levels in the GTH I-cells selectively decreased prior to final oocyte maturation, although I-mRNA levels remained elevated, thus suggesting a decline of biosynthesis of GTH I after vitellogenesis. These findings clarify how the synthesis of GTH I and GTH II are coordinated in the piscine pituitary, and indicate that the expression of GTH subunit genes during gametogenesis is regulated differentially in a cell-specific manner, both temporally and spatially.     

Effect of 17α-methyltestosterone,estradiol-17β and synthetic LHRH on production of gonadotropic hormone in pituitaries of rainbow trout (organ culture)

Pituitary glands from 6-month-old sexually immature female rainbow trout, Salmo gairdneri, were kept in organ culture for 48 or 72 h. Certain groups of pituitaries were cultivated for 48 h on either control medium or medium with 17 alpha-methyltestosterone (MT), or with estradiol-17 beta (E2) in concentrations of 8.5 X 10(-7) M. Other groups of pituitaries were cultivated for 72 h on control medium, or for 48 h on either control medium or MT-medium or E2-medium, and subsequently for 24 h on medium with synthetic LHRH in concentrations of 8.5 X 10(-7) M and 8.5 X 10(-10) M. Gonadotropic (GTH) cells are identified by Alcian Blue-Periodic Acid Schiff-Orange G staining and the double-antibody immunoenzyme-cytochemical technique using anti-carp beta GTH as the first antibody. A quantitative histological procedure was used to study the nuclear size of the GTH cells in response to the different hormones. Secretory activity was estimated by measuring the gonadotropin (GTH) content in extracts of pituitaries, plasma, and the culture media every 24 h by radioimmunoassay. Cultivation on MT- or E2-enriched medium results in an increase of the total amount of GTH in the pituitary and medium, an accumulation of GTH in GTH-cells (approximately 20 percentage points) and an increase in their nuclear size, indicating a stimulation of GTH synthesis. However, autonomous GTH-release is not affected by these steroids. Subsequent cultivation of the pituitaries for 24 h with LHRH causes stimulation of GTH synthesis (approximately 20 percentage points). Preincubation with steroids increases the GTH synthesis capacity of LHRH only when used in a concentration of 8.5 X 10(-10) M. Moreover, 8.5 X 10(-7) M LHRH causes a stimulation of GTH-release. Preincubation of the pituitaries with steroids increases the responsiveness of GTH-cells to LHRH. It is concluded that GTH-production in pituitaries of immature female rainbow trout can be directly influenced by gonadal steroids and by a hypophysiotropic substance.     

The effects of aromatizable androgens,non-aromatizable androgens,and estrogens on gonadotropin release in castrated African catfish,Clarias gariepinus (Burchell)

Summary To study the feedback mechanism of gonadal hormones on GTH secretion in male African catfish, the effects of castration and steroid replacement on GTH release, pituitary GTH content, and ultrastructural appearance of gonadotropes were investigated.Castration resulted in an increase in plasma GTH levels, a decrease in pituitary GTH content, and a degranulation of many gonadotropes. The aromatizable androgens testosterone and androstenedione were able to abolish the castration-induced increase in plasma GTH. This was accompanied with a restoration of pituitary GTH content and a regranulation of gonadotropes. The non-aromatizable androgens 5-dihydrotestosterone and 11-hydroxyandros tenedione did not have these effects. Replacement with estrone or estradiol resulted in an increase in pituitary GTH, however, without abolishing the elevated plasma GTH levels; ultrastructurally, many gonadotropes showed a welldeveloped granular endoplasmic reticulum together with a regranulation.The results of the present study indicate the significance of androgen aromatization in the feedback mechanism of gonadal steroids on the brain-pituitary axis.     

Both Contest and Scramble Competition Affect the Growth Performance of Brown Trout, Salmo Trutta, Parr of Wild and of Sea-ranched Origins

The effect of contest and scramble competition on the growth performance of wild and sea-ranched juvenile (0+) brown trout, Salmo trutta, originating from the River Dalälven, Sweden was scrutinised. In a mirror image stimulation (MIS) experiment, and in a 35000 1 stream-water aquarium the trout was studied for three weeks (20 individuals in each of four replicates). Activity in MIS was correlated with swimming activity in the stream-water aquarium. The MIS results could not be used for predicting any social behaviour patterns or the growth performance of a fish. No behavioural differences between the two strains were noted. However, the sea-ranched strain grew faster than the wild one, both in regard to the RNA/DNA ratio and the weight-specific growth rate. Because the strains had the same genetic background and prior to the experiments were raised under similar hatchery condition, the results of this study suggest that the sea-ranching process selects for faster juvenile growth in brown trout. The ultimate mechanisms underlying the faster growth by the domesticated strain probably involves both contest and scramble competition.     

Increase of steroid-producing cells in interrenal tissue and masculinization of gonads after long-term treatment of juvenile rainbow trout with cyanoketone

Summary Cyanoketone administered via the food (0.1, 0.2 and 2 mg/g) for 8 weeks from the first feeding (day 46 after fertilization) or via the aquarium water (3 and 30 mg/100 l) for 4 weeks from day 41 does not influence the activity of 3-hydroxysteroid dehydrogenase (3-HSD) in the interstitial cells of the gonads or interrenal cells of juvenile trout in vivo. However, the number of 3-HSD-positive interrenal cells was strongly increased by administration of the highest dose of cyanoketone via both routes. These high doses furthermore affect the sex ratio in favor of males. It is concluded that interrenal tissue is responsible for the masculinizing effect of cyanoketone via increased production of androgens and/or corticosteroids. Cyanoketone at concentrations of 0.01 to 100 g/ml causes a dose-response inhibition of 3-HSD activity in the interrenal cells, when the substance is administered to an incubation medium for demonstration of this enzyme in tissue sections. The controversial in-vivo and in-vitro effects of cyanoketone on 3-HSD activity are discussed.     

Immunocytochemistry of gonadotropic cells and identification of cell types in ultrathin cryosections of the pituitary of the rainbow trout,Salmo gairdneri

Summary The most frequently occurring cell types in the pars distalis of the pituitary gland of the rainbow trout, (i) the lactotropic, (ii) the gonadotropic, and (iii) the somatotropic cells, were identified in cryosections. Their morphological characteristics were compared with those of Epon-embedded material. Cell location, cell form, position of the nucleus, arrangement of rough endoplasmic reticulum and sizes of secretory granules proved to be useful parameters for identification. The size distribution of secretory granules of corresponding cells in cryosections and Epon sections proved to be similar. Additionally, both the immunoferritin and the unlabeled antibody enzyme method were applied for the immunocytochemical labeling of gonadotropic hormone-producing cells in cryosections. Anti-salmon-GTH as well as anti-carp-GTH serum showed the presence of GTH in both the smaller and the larger granules of the classical GTH cells, but also produced a reaction in TSH cells. Labelling of TSH cells was absent when using anti--carp-GTH. Specificity of the reaction depended upon the degree of dilution of the anti-GTH serum. Results with dilutions of 14,000 and 18,000 in the unlabeled antibody enzyme method, and of 18,000 up to 132,000 in the immunoferritin technique were optimal. Acid phosphatase activity in the smaller granules was demonstrated by enzyme cytochemistry in Epon sections. The relationship of the presence of hormone in these granules is discussed. The high sensitivity of the immunocytochemical labeling procedure is discussed with respect to cryo-ultramicrotomy.     

A daily periodicity in pituitary gonadotropin in White-crowned Sparrows

Summary Values of pituitary gonadotropic hormone (GTH) were determined throughout a 24 hour period in adult and first-year male White-crowned Sparrows (Zonotrichia leucophrys gambelii), maintained on a photoperiod of 20 hours light, 4 hours darkness. Samples taken every two hours revealed four peaks (Fig. 1) in pituitary GTH (06:00, 12:00–14:00, 18:00, 22:00). During the remaining time, GTH values were at lower levels (the minima approximately 9.0 g/gland in first-year males and 11.8 g/gland in adult males). Maximal values in adult males were from 50–125% above minimal levels, and maxima in first-year males reached 50–200% above minimal levels.Supported by NIH Predoctoral Fellowships GM-33,458 (to M.H.S.) and GM-36,159 (to J.E.E.) and by NIH Research Grant NB-06187 to Donald S. Farner. The hormones used in this investigation were generously supplied by the Endocrinology Study Section of the National Institutes of Health.     

Swimming response of goldfish,Carassius auratus,and the tetra,Hemigrammus caudovittatus,larvae to individual food items and patches

Synopsis Swimming speed and swimming path of goldfish and tetra larvae were studied in aquaria containing food patches composed of decapsulated cysts and immobilized nauplii of Artemia salina or sparsely distributed prey. The mean swimming speed of starved larvae in the medium without food was about four times higher than the speed of larvae feeding in a patch. Satiated larvae swam about 1.5 times slower than hungry fish. Consumption of single prey items by starved larvae caused the following sequence of swimming responses: handling pause (cessation of swimming), slow swimming in a restricted area, and fast swimming (approximately twice as fast as hungry larvae before encountering food) accompanied by a widening of the area searched (area increased searching). Mean swimming speed was constant over a broad range (10¹–10³ ind·1^–1 of food density, although at extreme (high or low) values of food density it depended on swimming responses of the predator. Frequency of visits to the different parts of the aquarium strongly depended on encounters of hungry fish with food particles or patches.     

Microbial transformation of sterols to C19-steroids by Rhodococcus equi

A wild-type strain of Rhodococcus equi, isolated from soil, degraded cholesterol, -sitosterol, stigmasterol and mixed sterois to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD). A definite preference for a relatively simply structured cholesterol side chain was observed. Highest specific cholesterol side-chain cleavage was associated with active growth of the culture. Maximum yield of ADD was obtained when sodium acetate and cholesterol were incorporated together in the medium. Specific side-chain cleavage required the presence of 2,2-dipyridyl, an inhibitor of ring cleavage.S. Ahmad and B.N. Johri are with the Department of Microbiology, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantriagar 263 145, Nainital, UP, India. P.K. Roy, A.W. Khan and S.K. Basu are at Fermentation Technology Division, Central Drug Research Institute, Lucknow, India.     

Changes in nucleolar structure,number and size in cellular activation and inactivation

In a cytophysiological study it was investigated whether in juvenile trout gonadal steroids stimulate the gonadotropic (GTH)-cells directly or indirectly via the brain. Pituitaries of donor animals were transplanted into the caudal musculature of testosterone-treated and non-testosterone-treated host fish. Testosterone treatment caused an increase in GTH-content in the in situ pituitaries and in the grafts. Accordingly, the gonadotrops displayed ultrastructural changes such as the appearance of well-developed Golgi systems and large globules. The stimulation of the morphological development of gonadotrops and of synthesis and storage of GTH in the allografted pituitaries indicates that testosterone affects the GTH-cells directly. In untreated juvenile trout the gonadotropin content of the pituitary and the gonadotropin concentration in the plasma vary with the time of year. This variation and the role of testosterone and gonadotropin-releasing hormone on the release of GTH are discussed.     

Effects of Fish-farm Activity on the Limnetic Community Structure of Brown Trout, Salmo trutta, and Arctic Charr, Salvelinus alpinus

Establishment of four fish-farms during the period 1971 to 1994 in the oligotrophic lake Skogseidvatnet affected Arctic charr, Salvelinus alpinus, but not brown trout, Salmo trutta. From 1971 to 1987, an increase in mean individual size of Arctic charr was recorded, while the mean individual size of brown trout remained stable. Arctic charr were found to use deeper benthic areas than brown trout. Approximately 8% of the Arctic charr population (>26cm), were found to switch to waste food from fish-farms, resulting in a novel feeding habitat for the species. They were, however, found in gillnets distant from the fish farm cages, indicating high mobility. The habitat segregation between the two species can most likely be explained by selective differences and asymmetric competition with brown trout as the dominant species. Based on the present results, changes in the Arctic charr population may be due to increased food availability and due to a new habitat use as a waste food feeder. The reason for the brown trout population to have remained stable with respect to mean size, growth pattern and habitat use, may be due to a different diet choice than Arctic charr in this lake. Brown trout were found to feed mainly on terrestrial insects, while Arctic charr fed mainly on zooplankton and on waste food.     

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

A 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.     

Subcellular fractionation of rainbow trout gonads with emphasis on microsomal enzymes involved in steroid metabolism

Summary Rainbow trout gonads were subfractionated by differential centrifugation with emphasis on obtaining preparations suitable for the study of steroid-metabolizing enzymes. A fractionation scheme was evaluated for the mature testis and for 3 ovarian developmental stages. The distribution of cell organelles among the fractions was determined using enzyme-markers and electron microscopy. The fractionation scheme was found to be suitable for separating mitochondria and microsomes which were recovered at similar yields to those that had been reported for other extraheptic fish tissues. Fractionation of the mature ovary was fraught with problems probably because a large yolk protein cytosole fraction interfered with the recovery of microsomes. However, no difference in the specific activity of microsomal NADPH-cytochrome c-reductase between the various organ preparations was evident. The testis microsomes contained detectable amounts of cytochrome P450, whereas its content in the various ovary microsomes was too low to be detected. Progesterone 17-hydroxylase was detected in microsomes from testes and early developing ovaries, and microsomal aromatase activity was present in microsomes from early developing, mature and postovulatory ovaries. Furthermore, the testis microsomes contained a highly active UDP glucuronosyltransferase with testosterone used as a substrate.     

Comparative Analysis of Content of Omega-3 Polyunsaturated Fatty Acids in Food and Muscle Tissue of Fish from Aquaculture and Natural Habitats

Two fish species reared in aquaculture (pink salmon Oncorhynchus gorbuscha and whitefish Coregonus lavaretus) and ten fish species from natural habitats (whitefish C. lavaretus, tugun Coregonus tugun, broad whitefish Coregonus nasus, least cisco Coregonus sardinella, vendace Coregonus albula, boganid charr Salvelinus boganidae, charr Salvelinus alpinus complex, northern pike Esox lucius, sharp-snouted lenok Brachymystax lenok, and taimen Hucho taimen) have been studied. The content of two long-chain polyunsaturated omega-3 fatty acids (PUFAs), eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), in the muscle tissue of the fish and in their food (intestine contents) are compared. In the aquacultures of whitefish and pink salmon, the total content of EPA and DHA is significantly higher in feed than in the muscle tissue of the fish, which indicates losses of PUFA in the two-link food chain of the aquaculture during their transfer to the upper trophic level. EPA and DHA losses in aquaculture, which are confirmed by numerous literature data, mean an inefficient usage of the available sources of PUFAs and the aggravation of the global deficit of these biochemicals in the human diet. A study of natural fish populations reveals the accumulation of EPA and DHA in their biomass compared to food in many cases, although opposite phenomena are also observed. An assumption on the presence of an optimal, physiologically adequate species-specific level of PUFA in the fish muscle tissue has been made based on our data and literature data. If the level of PUFAs in the muscles is lower than optimal, their accumulation (bioaccumulation) from food and/or de novo synthesis are observed. When the optimal level is exceeded, the content of EPA and DHA in biomass approaches maximum species-specific values; however, part of these PUFAs entering from food is not digested or is catabolized. According to the obtained data, the species of the order Salmoniformes have an optimal level of 2 to 6 mg/g of wet weight. It has been found that in aquaculture approaching to maximum values of EPA + DHA content was accompanied by their losses (scattering) in the food chains, while in natural ecosystems the maximum values of PUFA content in the fish biomass are achieved by their accumulation from the lower trophic level. Boganid charr S. boganidae had the highest content of EPA + DHA in the muscle tissue among all known fish species (32.78 mg/g of wet weight).     

Hormonal Induction of Male Courtship Behavior in the Japanese Newt, Cynops pyrrhogaster

In the breeding season, the sexually mature male newt, Cynops pyrrhogaster, vibrates the tail in front of the female at an early stage of courtship. Effects of prolactin (PRL), gonadotropin (GTH), and sex steroids on this behavior were investigated in the male paired with the female receiving PRL and GTH. The behavior was elicited in the sexually inert male by injections of PRL of bovine, ovine, or bullfrog origin and human chorionic gonadotropin or bullfrog LH and FSH in combination. The effect of PRL or GTH alone was less marked than that of PRL plus GTH, especially in terms of frequency of the behavior. In the hypophysectomized male, combination of PRL and GTH significantly increased both the incidence and frequency of the behavior. However, PRL alone was not effective, and the effect of GTH alone was less pronounced than that in the intact animal receiving GTH injections. The effect of GTH was nullified by castration. In the PRL-treated castrated animal, testosterone or dihydrotestosterone, but not estradiol, was effective in inducing the behavior.     

Optimal foraging: food patch depletion by ruddy ducks

Summary I studied the foraging behavior of ruddy ducks (Oxyura jamaicensis) feeding on patchily distributed prey in a large (5-m long, 2-m wide, and up to 2-m deep) aquarium. The substrate consisted of a 4x4 array of wooden trays (1.0-m long, 0.5-m wide, and 0.1-m deep) which contained 6 cm of sand. Any tray could be removed from the aquarium and loaded with a known number of prey. One bird foraged in the aquarium at a time; thus, by removing a food tray after a trial ended and counting the remaining prey, I calculated the number of prey consumed by the bird. I designed several experiments to determine if ruddy ducks abandoned a food patch in a manner consistent with the predictions of a simple, deterministic, patch depletion model. This model is based on the premise that a predator should maximize its rate of net energy intake while foraging. To accomplish this, a predator should only remain in a food patch as long as its rate of energy intake from that patch exceeds the average rate of intake from the environment. In the majority of comparisons, the number of food items consumed by the ruddy ducks in these experiments was consistent with the predictions of the foraging model. When the birds did not forage as predicted by the model, they stayed in the patch longer and consumed more prey than predicted by the model. An examination of the relation between rate of net energy intake and time spent foraging in the food patch indicated that by staying in a patch longer than predicted, the ruddy ducks experienced only a small deviation from maximum rate of net energy intake. These results provided quantitative support for the prediction that ruddy ducks maximize their rate of net energy intake while foraging.