Role of the sertoli cell in spermatogenesis: TheSqualus testis model

In the dogfish sharkSqualus acanthias different germ cell stages are topographically segregated within the testis. Using this species we have developed methods for the isolation and culture of Sertoli cells from premeiotic, meiotic and post-meiotic stages of spermatogenesis and present preliminary evidence for stage-dependent variations in cell morphology and behavior, thymidine incorporation, protein synthesis and steroidogenesis. The goal of future studies is to determine how maturational changes are regulated in Sertoli cells and, in turn, to elucidate Sertoli cell-germ cell interactions.     

On the blood volume of the Mediterranean dogfish,Scyliorhinus canicula

The blood volume of the Mediterranean dogfish,Scyliorhinus canicula was determined to be 4.06±0.92 ml/100 g body weight. This result is discussed in relation with the dye dilution technique used, the degree of dye binding with circulating plasma proteins and the values and techniques used by other authors.     

Stimulatory and inhibitory regulation of DNA synthesis during spermatogenesis: studies in Squalus acanthias

Spermatogenesis is a unique developmental sequence involving multiple cell to cell interactions and several categories of regulatory molecules. In contrast to conventional mammalian models in which testicular organization is highly complex, the testis of the dogfish shark Squalus acanthias is technically advantageous for elucidating stage-dependent structural and functional charactericsics and for in vitro regulatory studies. Using incorporation of [³H]thymidine into acid-insoluble molecules as a measure of DNA synthesis by spermatocysts (germ cell/Sertoli cell units) of premeiotic stages, we obtained evidence of a growth inhibitory bioactivity (chalone) within the testis. This activity is differentially distributed (postmeiotic > meiotic > premeiotic), suggesting that more advanced developmental stages, which are upstream in the vascular pathway within the testis, may control the size of the proliferating spermatocyst population and, hence, the advance of less mature stages. These data provide direct evidence for humoral communication between stages of spermatogenesis.     

Reducing elasmobranch bycatch: Laboratory investigation of rare earth metal and magnetic deterrents with spiny dogfish and Pacific halibut

Spiny dogfish (Squalus acanthius) comprises a significant unwanted bycatch on demersal longlines set for halibut and cod in shelf waters of the east and west coasts of North America. In this laboratory study, attacks on baits were tested in the presence of two different rare earth materials (neodymium–iron–boride magnets and cerium mischmetal) believed to deter elasmobranch catch. Experiments were made with spiny dogfish and with Pacific halibut (Hippoglossus stenolepis) in pairwise tests of the rare earth materials and inert metal controls. Dogfish attacked and consumed baits tested with cerium mischmetal at a lower frequency than controls. Times to attack the baits were significantly higher in the presence of mischmetal, as were numbers of approaches before first attack. The time differential between mischmetal and control treatments and the number of baits consumed converged with increasing food deprivation (1 h, 2 d, and 4 d), but treatment differences were always significant. Cerium mischmetal appeared to be irritating to dogfish and may disrupt their bait detection and orientation abilities. Magnets also appeared to irritate dogfish but provided no protection for baits in feeding trials. Pacific halibut showed no reaction whatsoever to the rare earth magnets or cerium mischmetal. Mischmetal, therefore, may be useful in reducing spiny dogfish bycatch in the halibut fishery. Disadvantages in using mischmetal in commercial operations are expense, hazardous nature, and relatively rapid hydrolysis in seawater.     

Stimulation of gonadal and germ cell development in larval and juvenile carp (Cyprinus carpio L.) by homologous pituitary extract

The data presented show that in larval carp, gonadal size has increased distinctly after treatment with homologous pituitary extract (PE). Moreover, the precocious onset of primordial germ cell proliferation and of sex differentiation into male and female gonads was induced. Body weight of treated and control specimens did not differ significantly from each other throughout the experiment. Treatment of juvenile carp with homologous PE led also to an increase in gonadal size without concomitant increase in body weight. Induction of precocious spermatogenesis was observed too. GTH-levels in control and PE-treated carp were monitored by means of a homologous radioimmunoassay, showing that in PE-treated carp the GTH-level was distinctly elevated. The possible role of pituitary hormones in larval and juvenile gonadal development is discussed.     

Ontogenetic changes of alpha-melanocyte-stimulating hormone (α-MSH)-like immunoreactivity in the brain and hypophysis of a scyliorhinid dogfish (Scyliorhinus torazame): An immunohistochemical study

Ontogenetic development of α-MSH-IR (alpha-melanocyte-stimulating hormone-like immunoreactivity) in the brain and hypophysis of a scyliorhinid dogfish (Scyliorhinus torazame) was studied immunohistochemically. α-MSH-IR first appeared in the hypothalamus of the brain in embryos at the 43-mm stage. Thereafter, α-MSH-positive cells and fibers gradually increased in number in mainly the tuberculum posterius of the hypothalamus during the embyonic periods. In the post-hatching juveniles at the 95-mm and 125-mm stages, α-MSH-positive structures in the brain were comparable to those in adult fish, although the distributional range of α-MSH-IR structures in the brain was less extensive than those in adult fish. In the hypophysis, α-MSH-positive cells first appeared in the caudal part of the adenohypophysis at the 54-mm stage, approximately coincident with the onset of pigmentation in the skin. During ontogenesis, the MSH-producing cells increased in number straightforwardly in the adenohypophysis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Existence of APGWamide in the testis and its induction of spermiation in Haliotis asinina Linnaeus

Immunolocalization by using rabbit polyclonal antibody against APGWamide demonstrated that there was APGWamide immunoreactivity (APGW-ir) in the varicosed nerve fibers in the capsule, connective tissue and trabeculae of the testis whereas there was no APGW-ir in the ovary of sexually mature broodstocks of Haliotis asinina. In vivo bioassay, by injecting APGWamide into foot muscle of mature males and females (age over 24 months), showed that most males (87.50%) injected with 200 μl of 10^− 3 M APGWamide (170 μg APGWamide/animal) could be induced to spermiate whereas fewer males (43.75%) injected with 200 μl of 10^− 6 M APGWamide (0.17 μg APGWamide/animal) could spermiate. Following injection with 10^− 3 M, 53% of males spawned within 3 to 4 h, while the remaining males spawned within 2 to 3 h. The former tended to release more sperm at the range of 1.04 to 2 × 10⁹, and the latter at the range 3.62 × 10⁷ to 2.07 × 10⁸ cells, respectively, and that the sperm of the former group appeared to be more motile. Following the injection of 10^− 6 M, all inducible males spawned within 2–3 h, and 43% of spawned males have sperm around 2 × 10⁹, while 57% have fewer sperm number ranging from 2.75 × 10⁷ to 9.94 × 10⁸ cells. In contrast to males, none of the mature females with ripen ovary could be induced to ovulate. These data suggest that APGWamide is an important signaling molecule that plays a role in the male reproductive process that could be used to induce spermiation.     

Bilirubin-binding protein in the serum of spawning-migrating chum salmon,Oncorhynchus keta: Its identity with carotenoid-carrying lipoprotein

The serum carotenoid level gradually increased during the spawning migration of chum salmon (Oncorhynchus keta). Zeaxanthin, a yellowish carotenoid, was detected in the serum of spent males, while astaxanthin was a main carotenoid in the serum of males and females at other developmental stages. A high bilirubin level was found in the serum of spent males; the serum of spent male was yellow in colour due to the presence of zeaxanthin and bilirubin, although the serum of the other fish was bright orange.Three types of carotenoid-carrying lipoprotein such as low density lipoprotein, high density lipoprotein (HDL) and very high density lipoprotein fractions were present in the serum. Carotenoid-carrying lipoprotein from the HDL fraction became a main component during spawning migration. The serum bilirubin from the spent male salmon was distributed in the HDL fraction. Because bilirubin, as well as carotenoids, were present in the HDL fraction, the carotenoid-carrying lipoprotein from the HDL fraction was identical with bilirubin-binding protein in the serum of spent males. This carrier lipoprotein had two subunits (molecular weight 24,000 and 12,000); no disulfide bond was detected between the two subunits.     

Thyroxine-induced alterations in the testis and seminal vesicles of air-breathing catfish, Clarias gariepinus

Effect of experimentally induced thyroxine overdose on the testis and seminal vesicles was studied in the air-breathing catfish, Clarias gariepinus during the preparatory and the pre-spawning phase. The present study revealed a marked reduction in testosterone level in serum, testis and seminal vesicles (SV). Histological examination showed a considerable reduction in the number of spermatozoa/spermatids in the seminiferous tubular lumen as well as depletion of fluid in the loculi of SV. SDS-PAGE analysis of SV fluid proteins demonstrated a significant decrease in the level of a ~27 kDa protein in thyroxine treated fishes. Evidences are presented here to indicate that thyroid hormone plays a role in regulating testis and SV function in catfish. T.N. Jacob and J.P. Pandey contributed equally     

Active metabolism in larval and juvenile fish: ontogenetic changes,effect of water temperature and fasting

Oxygen consumption, ammonia excretion and fish swimming speed were measured in fish induced to swim by optomotor reaction in a circular metabolism chamber. The relationship between the swimming speed and fish metabolism described by exponential equations allowed the extrapolation to the standard metabolism, i.e. at zero swimming speed. The partitioning of the catabolised protein in the energy supply was estimated based on AQ (volume of ammonia/ volume of oxygen) values. Weight specific standard metabolism, as expressed by the ammonia excretion rate, decreased by one order of magnitude in coregonids as the fish grew from 20 to 780 mg body weight. The slope of the relationship between oxygen uptake and swimming speed decreased in coregonid ontogenesis. In salmon, after 12 days of fasting 28% of energy used was derived from protein, whilst coregonid juveniles utilized mostly lipid. Active swimming in fasted juveniles of coregonid, as well as in salmon, led to the accelerated utilization of protein as a source of energy, based on AQ coefficients. In juveniles acclimated to a range of water temperatures from 14 to 26°C, the changes in standard or active metabolic rate (expressed as oxygen uptake or ammonia excretion) were described by Q₁₀ coefficients. They were generally higher for the ammonia excretion rate than for the oxygen uptake rate and for active metabolism than for standard metabolism. Utilization of protein as energy for swimming differed significantly between the species, being in general one order of magnitude higher in coregonids than in salmon. The use of protein for swimming activity tended to decrease during coregonid ontogenesis.     

Growth and feed utilisation of rainbow trout subjected to changes in feed lipid concentrations

Rainbow trout Oncorhynchus mykiss grew from 44 to 326 g in 96days when held at 12 °C. Fish were fed to satiation twice dailywith either high (L₁: 30.8%, L₂:31.4%) or lower-lipid feeds (C₁: 18.8%,C₂: 21.8%). Four feeding treatments were studied.Group C₁C₂ received feed C₁ for 43 days(days 0–43) and C₂ thereafter (days 44–96).Groups L₁L₂, L₁C₂ andC₁L₂ were subjected to dietary changes asindicated by the feed designations. After a short period of feedadaptation, fish ingested similar amounts of feed energy i.e., they ateless by weight of the lipid-rich (L) feeds. Feed lipid content did notaffect growth but fish fed L-feed had reduced feed conversion ratio(FCR) compared to fish fed C-feed (0.731 vs. 0.773) during days0–43 (P < 0.01). After 96 days,L₁L₂-fish were lower in body protein(15.8%) than the C₁C₂-fish (16.8%)(P < 0.01). L-feeds also tended to increase percentage lipidand reduce percentage whole body moisture and ash. A higher net proteinutilisation (NPU) was recorded in fish fed L-feeds (43.6%)compared to fish fed C-feeds (38.8%) in days 0–43(P < 0.05). This seemed to be the result of a lower proteinintake rather than a protein-sparing effect of feed lipid. Above athreshold value of approximately 6.5 mg protein eaten·g bodywt_{minus 1}·day_–1, NPU decreased.     

Tissue concentrations of RNA and protein for juvenile brook trout (Salvelinus fontinalis): lagged responses to fluctuations in food availability

To be useful as short-term indices of nutritional status when food availability varies, wet weight-at-length and tissue concentrations of nucleic acids and protein must covary closely with a fish's recent feeding history. We measured changes in these indices for young brook trout (Salvelinus fontinalis) (fork length: 20–34 mm) reared under alternating, 4 to 5 d periods of food provisioning and food deprivation. Weight-at-length corresponded closely with current feeding conditions, being higher when the trout were fed than when they were deprived. Concentrations of RNA and protein, however, did not correspond closely with current feeding conditions. Instead, there were significant carry-over effects whereby responses to feeding conditions experienced in one treatment period were not exhibited until the following treatment period. Food provisioning had positive carry-over effects on concentrations of RNA and protein while food deprivation had negative carry-over effects. Consequently, food-deprived trout sometimes had higher concentrations of RNA and protein than well fed trout. Since wild fish may experience short-term fluctuations in food availability, lagged responses in concentrations of nucleic acids or protein like those reported here could hamper attempts to use these biochemical measures to assess the nutritional status of juvenile fish in the field.Author to whom all correspondence should be addressed.     

Stress-induced changes in the affinity and abundance of cytosolic cortisol-binding sites in the liver of rainbow trout,Oncorhynchus mykiss (Walbaum), are not accompanied by changes in measurable nuclear binding

Plasma cortisol levels and the number (N_max) and affinity (K_d) of specific hepatic cortisol-binding sites were determined in rainbow trout subjected to chronic confinement stress for 14 days. Confinement significantly elevated plasma cortisol levels to 47.3 ± 13.5 ng ml^–1 within 24h and although levels declined to 8.0 ± 3.0 ng ml^–1 after 14 days, they were significantly higher throughout than levels in unstressed control fish (< 2.0 ng ml^–1). There was a 60% reduction in cytosolic N_max in stressed fish during the first 24h of confinement (35.8 ± 7.9 cf. 129.0 ± 15.2 fmol mg^–1 protein), a decline which was sustained at 7 days after the onset of stress but, although numbers of binding sites in the liver of stressed fish were still lower than in unstressed fish, the difference was no longer significant after 14 days of confinement. There was an accompanying significant rise in the K_d of cortisol binding in stressed fish during confinement, from 4.0 ± 0.6 nM at time 0 to 8.4 ± 0.8 nM after 24 h confinement. This increment in K_d was sustained at a level significantly higher than in control fish throughout the 14 day confinement period, despite marked reductions in cortisol levels and increases in N_max in stressed fish. Throughout the study, specific binding of cortisol could not be consistently detected in high-salt nuclear extracts from stressed or unstressed fish, suggesting either that high-affinity binding sites for cortisol were absent from these preparations, that receptors were present but unable to interact with ligand because they were occupied, or that receptors were present but not being extracted. These possibilities were investigated using a range of extraction procedures, by varying the temperature of incubation, by employing dexamethasone as ligand and by examining binding in purified, intact, nuclei. Estradiol was employed as a methodological control throughout and substantial amounts of specific estradiol binding were detected in all compartments and preparations. Specific cortisol-binding sites were detected in intact nuclei of both stressed and unstressed fish, at levels an order of magnitude lower than estradiol binding in the same preparations. These data demonstrate that activation of the pituitary-interrenal axis leads to significant changes in the nature of target-tissue binding of cortisol in rainbow trout, and reveal a clear difference in the subcellular distribution of binding-sites for estradiol and cortisol, which reflects the situation in mammalian tissues.     

Use of SDS-page in the assessment of protein hydrolysis by fish digestive enzymes

Inthe field of fish nutrition, the preliminary evaluation of feedstuffs usingin vitro techniques may be an alternative to invivo assays. Degradation of the protein fraction in feedstuffs byseabream (Sparus aurata) digestive enzymes was studiedunder different conditions simulating either acid, alkaline, or acid + alkalinedigestion using a modified pH-stat technique. In addition, a sequential sodiumdodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the releasedproducts was used both to visualise and to quantify the level of proteinhydrolysis. Gels were analysed by optical densitometry and results wereexpressed as a Coefficient of Protein Degradation (CPD). Values of CPD showedclear differences related to the type of protein digested or the steps followedin the digestion. CDP index provides information based on the enzymaticbreakdown of the proteins visualised in SDS-PAGE gels. The pH-stat evaluation(Degree of Hydrolysis) supplies measurement of the total number of peptidebondsbroken down in a given protein. CDP index mainly focuses on the action ofendoproteases, whereas degree of hydrolysis (DH) includes both endo- andexoproteases. CPD and DH are two complementary indexes capable of measuring theprotein hydrolysis by fish enzymes of a given feedstuffs or diet. Both methodswere in agreement with respect to assessing the hydrolysis of protein. Thesignificance of the SDS-PAGE techniques in the assessment of aquafeeds by fishdigestive enzymes is discussed.     

Effects of experimental infection by Caligus rogercresseyi (Copepoda: Caligidae) on the common jollytail, Galaxias maculatus (Osmeriforme: Galaxiidae) (Jenyns, 1842) in Chile

Galaxias maculatus, the common jollytail, is a native smelt fish with a lacustrine or diadromous life cycle. In Chile, the rearing cycle of this fish includes a freshwater and a marine phase. Several diseases and parasites reported for the freshwater phase could be avoided by rearing the fish in salty or brackish water. Nonetheless, this alternative could result in Caligus rogercresseyi infections. The objective of this study was to evaluate experimentally the capacity of C. rogercresseyi to infect the common jollytail and the effects this parasite may cause to the fish. Prevalence and intensity of experimental C. rogercresseyi infections on G. maculatus were estimated and the effects of this parasitosis on the fish were evaluated on fish survival, weight loss, swimming behavior, and skin damage. Two experiments were carried out with adult fish taken from the Maullín Estuary. Fish were acclimatized to a salinity of 32 ppt and divided into two groups of 20 fish each. The experimental group was infected with copepodid stages of C. rogercresseyi and the other was used as a control. The fish were kept in individual tanks until observing ovigerous female copepods. C. rogercresseyi recognizes G. maculatus as a host under experimental conditions and optimal salinity for the parasite. Results show that under experimental condition copepodid stage can successfully infect the fish, developing to the adult stage, mating, and producing eggs (females). C. rogercresseyi caused alterations in fish behavior, superficial body damage, weight loss, and mortality. Rearing the common jollytail in salty water should take into consideration measures to control these infections as they can damage fish and affect surrounding wild fish populations. There are no records of this parasite in natural common jollytail populations; however, our understanding of the ecological role played by the common jollytail in C. rogercresseyi transmissions among sympatric host species could be greatly improved through further research related to host preferences and epidemiological data for different sympatric host species.     

The effects of hydrostatic pressure on signal transduction in brain membranes of deep-sea fishes of the genus Coryphaenoides

To investigate the effects of increased hydrostatic pressure on transmembrane signaling in deep-living marine species, the A₁ adenosine receptor – inhibitory G protein (G_i) – adenylyl cyclase signaling complex was examined in brain membrane preparations from four teleost fish species of the deep-sea family Macrouridae. The combined depth ranges of the adults of these species, Coryphaenoides armatus, C. cinereus, C. filifer, C. pectoralis, span several hundred meters to 5400 m. Basal adenylyl cyclase activity, determined at 5 °C, was inhibited by increased hydrostatic pressure in all four species. At the highest pressure tested, 476 atm, adenylyl cyclase activity was inhibited 60 to 70% relative to the atmospheric pressure values. Pressure inhibition did not result from denaturation or loss of protein components from the membrane due to pressure-induced shedding. Despite the pressure-inhibition of basal adenylyl cyclase activity, the responsiveness of adenylyl cyclase activity to modulation by N⁶-cyclopentyladenosine, an A₁ adenosine receptor agonist, was retained at elevated pressures. Because the accumulation of the second messenger cAMP depends on the summation of modulatory inputs, these results indicate that the transmembrane signaling in these deep-living species is insensitive to hydrostatic pressure changes.     

Mitochondria-rich cells in the branchial epithelium of the teleost,Oreochromis mossambicus,acclimated to various hypotonic environments

Branchial mitochondria-rich (MR) cells were examined on the afferent side of gill filaments in tilapia (Oreochromis mossambicus) acclimated to different hypotonic environments, local fresh water (LFW), hard fresh water (HFW) and 5 salt water (SW). Scanning electron micrographs (SEM) identified three types of apical surfaces of the MR cells, wavy convex, shallow basin and deep hole. In spite of the different types of apical surfaces, light microscopic (LM) and transmission electron microscopic (TEM) studies suggested that these cells were MR cells. The relative abundance of these 3 types of branchial MR cells varied with external hypotonic milieus. Wavy-convexed MR cells were dominant in the gills of fish adapted to HFW, whereas shallow-basined MR cells were evident in LFW-adapted fish. In SW-adapted fish, most of the MR cells were deep holes. Experiments on adaptation to various hypotonic milieus revealed that the changes of the branchial MR cells were reversible and occurred within 24 hours following transfer. The morphological alterations of the MR cells correlated with ionic changes in different milieus, indicating that these distinct types of MR cells may play key roles for osmoregulation in hypotonic media.     

Effects of protein and lipid concentrations in broodstock diets on growth, spawning performance and egg quality of yellowfin sea bream (Acanthopagrus latus)

A study was conducted to determine the effects of dietary protein and lipid concentrations on growth, spawning performance and egg quality of yellowfin sea bream, Acanthopagrus latus. Nine diets representing a combination of three protein concentrations (40, 50 and 60%) and three lipid concentrations (15, 20 and 25%) were tested with three replicates. Each replicate was stocked with 10 fish with a sex ratio of 1:1 and the average weights were 415 g and 236 g for females and males, respectively. Fish were fed to satiation twice daily. The weight gain of A. latus broodstock was not significantly (P > 0.05) different among fish fed diets with the various protein and lipid concentrations. Except for relative fecundity and survival rate of 3-day-post-hatch (3DPH) larvae, spawning performance of A. latus was not significantly affected by different concentrations of dietary protein or lipid. Relative fecundity was found to be significantly elevated by dietary lipid at 20% concentration. However, survival rate of 3DPH larvae was highest from broodstock fed diets with 40% protein and 25% lipid, 50% protein and 15% lipid and 60% protein and 15% lipid concentrations. Egg diameter generally increased with increasing dietary protein from 40 to 60% at each lipid concentration. Total length of hatchlings followed an inverse relationship with dietary protein regardless of the lipid concentration. The oil globule diameter (OGD) of hatchlings significantly increased with decreasing dietary protein from 60 to 40%. Yolk sac length (YSL) and OGD of hatchlings was generally greatest at 15% dietary lipid concentration irrespective of protein concentrations. Total length of 3DPH larvae was greatest from broodstock fed diets with 50% protein and 20% lipid, respectively. Body protein content of broodstock was not significantly affected by dietary protein and lipid concentrations. However, increasing dietary lipid generally resulted in increasing body lipid content regardless of the dietary protein concentration. Protein and lipid contents of eggs, hatchlings and 3DPH larvae were significantly affected by dietary protein and lipid concentrations of the broodstock diets. The present study revealed that the best reproductive performance of A. latus broodstock was achieved at 40% dietary protein and 20% dietary lipid concentrations based on relative fecundity, fertilization rate, hatchability and survival of 3DPH larvae.