Effects of (D-Ala6, Pro9N ethylamide) — LHRH on plasma levels of gonadotropin, 17α,20β-dihydroxy-4-pregnen-3-one and testosterone in male goldeye (hiodon alosoides Rafineque)

Male goldeye were treated with (D-Ala⁶, Pro⁹-N ethylamide) — LHRH (LHRH-A) in saline or a silastic pellet (100 µg.kg^–1 body weight) and changes in plasma gonadotropin (GtH), 17,20-dihydroxy-4-pregnen-3-one (17,20P) and testosterone (T) levels were determined by radioimmunoassay. LHRH-A increased plasma GtH levels, with the response to LHRH-A in saline being of much greater magnitude and duration than the response to silastic pellet implants. Seasonal differences were found in the response to LHRH-A. Spermiated fish were the most responsive, recrudescing fish the least, and regressed fish displayed an intermediate response. Plasma 17,20P levels were elevated in response to LHRHA in fish of all sexual stages although the magnitude of the increase was not related to the magnitude of the increase in GtH levels. Treatment with LHRH-A also resulted in a transitory increase in plasma T levels. The endocrine control of GtH and steroid secretion in goldeye is discussed in relation to studies in cyprinids and salmonids. Address for reprint requests: Department of Zoology, University of Alberta, Edmonton, Alberta T6G 2E9 Canada.     

The role of prostaglandins in the control of ovulation in yellow perch,Perca flavescens

Previous studies have shown that 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P) can induce both germinal vesicle breakdown and ovulationin vitro of yellow perch (Perca flavescens) oocytes. The stimulation of ovulation can be blocked by indomethacin and restored by the subsequent addition of several primary prostaglandins (Goetz and Theofan 1979). In the present investigation, medium levels of prostaglandin F (PGF) and E (PGE) were measured by radioimmunoassay duringin vitro 17α,20β-P-induced ovulation of perch oocytes. PGF levels increased significantly (compared to controls) from 30 to 36h of incubation. Hourly samples taken through the time of ovulation revealed that the increase in PGF was very closely correlated to the time of ovulation though it did not preceed it. Cortisol, testosterone, estradiol-17β, 17α,20α-dihydroxy-4-pregnen-3-one and 17α-hydorxyprogesterone did not increase PGF levels by 48h of incubation, however, several other progestational steroids including 20β-dihydroprogesterone (20β-P) and progesterone did. 17α,20β-P, 20β-P and progesterone also stimulated an increase in PGF in spontaneously ovulating oocytes (in which all oocytes ovulated including controls), indicating that the increase in PGF was not merely a result of the physical process of ovulation but was related to the presence of the steroid. Based on work supported by the National Science Foundation under grant DCB-8517718 and DCB-8718178.     

鲤脑垂体匀浆液和人绒毛膜促性腺激_省略_性腺激素及性类固醇激素含量的影响

雄鳗注射５－６次、雌鳗注射９－１０次鲤脑体匀浆液（ＣＰＥ）＋人绒毛膜促性腺激素（ＨＣＧ）能分别诱导精巢和卵巢发育成熟。在雌雄鳗鲡,注射ＣＰＥ＋ＨＣＧ可显著增加端脑、间脑、中脑和下丘脑ｍＧｎＲＨ的含量,而对后脑和延髓ｍＧｎＲＨ的影响较小;注射ＣＰＥ＋ＨＣＧ增加雄鳗后脑和延髓ｃＧｎＲＨ－Ⅱ含量,对雌鳗脑区ｃＧｎＲＨ－Ⅱ则无显著影响。雌雄鳗鲡每次注射ＣＰＥ＋ＨＣＧ后１天,血清促性腺激素（ＧｔＨ）急剧上升     

Monoaminergic substances in the teleost brain: Catecholamine levels in male and female winter flounder,Pseudopleuronectes americanus Walbaum,associated with gonadal recrudescence

High performance liquid chromatography with electrochemical detection (HPLC-EC) was used to quantitate catecholamine (CA) levels in the winter flounder brain following perchloric acid extraction/alumina purification of CNS tissues. Greater concentrations of norepinephrine (NE) and dopamine (DA) were present in the hypothalamus compared with the CA levels in whole brain. A seasonal study of CA brain levels in reproductively active male and female flounder demonstrated that monoamine levels reach their maxima in October in association with the rapid increases in gonadosomatic index. When perchloric acid extracts of the teleost and rat hypothalamus were submitted to direct HPLC-EC analysis, without alumina purification of CA neurotransmitters, similar hypothalamic profiles were obtained indicating the presence of identifiable biogenic amine neurotransmitters substances including NE, DA and serotonin (5-HT).     

Seasonal changes in reproductive condition and plasma levels of sex steroids in the blue cod,Parapercis colias (Bloch and Schneider) (Mugiloididae)

Gonad and plasma samples were taken from blue cod captured throughout the reproductive cycle, gonad condition was assessed, and plasma levels of 17-hydroxyprogesterone (17OHP), 17,20-dihydroxy-4-pregnen-3-one (17,20P), testosterone (T), 17-estradiol (E₂) and estrone (E₁) were measured by radioimmunoassay. It was confirmed that spawning occurred over an extended period in late winter and spring, with individual fish being involved in multiple spawning events. Plasma levels of T were bimodal in both sexes with peaks (maximum of 6.0 ng.ml^–1) occurring 2 months prior to, and also during the early part of the spawning period. 17,20P was elevated in males (2.1 ng.ml^–1) in mid-spermatogenesis coinciding with the first T peak (4.9 ng.m.^–1). 17,20P was detectable but not significantly elevated (0.6–1.2 ng.ml^–1) at any sample time in females. E₂ was elevated in mature females (1.0 ng.ml^–1) early in the spawning period but remained at assay detection limits (0.3 ng.ml^–1) at all other sample times. Neither 17OHP nor E₁ were detectable in the plasma of either sex. It is suggested that bimodal increases in sex steroids prior to spawning may be a feature of species with rapid recrudescence.     

Capture and handling stress affects the endocrine and ovulatory response to exogenous hormone treatment in snapper, Pagrus auratus (Bloch & Schneider)

Sexually mature female hatchery‐reared snapper, Pagrus auratus (Bloch & Schneider) were captured from sea cages by handline and injected at first capture (control) or 24 h after capture, transport and subsequent confinement (delayed injection) with either saline, luteinizing hormone releasing hormone analogue, human chorionic gonadotropin, or 17α‐hydroxyprogesterone. Blood was sampled before hormone treatment and again after 168 h, and fish were checked daily for ovulation. Plasma levels of 17β‐estradiol (E2), testosterone (T), 17α, 20β dihydroxy‐4‐pregnen‐3‐one (17, 20βP) and cortisol were determined by radioimmunoassay. The ovulatory response was assessed from the proportion of fish ovulating, ovulation volume, egg quality and fertility. A delay in injection resulted in significantly lower plasma E2 and T levels in response to hormone treatment, smaller ovulation volumes, and poorer egg quality than in control fish. The results are consistent with the generally inhibitory effects of stress on reproduction in fish, and confirm the requirement to treat fish with hormones designed to induce ovulation, as soon as possible after capture and disturbance.     

Lipid transport in female Fundulus heteroclitus during the reproductive season

Lipid transport during the reproductive cycle of seasonally spawning teleosts has been well described. However, there is little information on lipid transport in a multiple spawning teleost. We examined lipid transport during the reproductive cycle in Fundulus heteroclitus, a teleost that spawns in a semilunar fashion. Plasma levels of triglycerides more than double prior to spawning events and then drop off sharply around the time of spawning. Plasma levels of total cholesterol also changed throughout the spawning cycle, but those changes were not so pronounced. The fraction of lipid carried by very low density lipoprotein (VLDL) and low density lipoprotein (LDL) was greatest prior to spawning events, while high density lipoprotein (HDL) carried the greatest portion of lipid during the spawning period. Vitellogenin (Vtg) was detected prior to spawning events, but it never represented more than 5% of total plasma lipid, making it very unlikely that Vtg is responsible for the changes in plasma lipids that we observed. It is also unlikely that Vtg is solely responsible for transport of lipid to growing ovaries because it carries only a very small portion of plasma lipid. This is the first study we are aware of, that tracks changes in plasma lipids of a multiple spawning teleost with a great enough sampling frequency to resolve single spawning events. This revised version was published online in August 2006 with corrections to the Cover Date.     

The GnRH systems in the brain and pituitary of normal and hCG treated European silver eels

The distribution of immunoreactive GnRH was studied in the brain and pituitary gland of normal and human chorionic gonadotrophin (hCG) injected silver eels. It was found that the general organization of GnRH systems in this species is similar to that reported in other teleosts. Cell bodies were present in the olfactory bulbs, ventral telencephalon, periventricular hypothalamus and dorsal tegmentum. No positive perikarya could be detected in the preoptic region. Only scarce fibers were observed in the proximal neurohypophysis. Treatment with hCG does not modify the distribution of GnRH but it increases the density of positive structures, in particular at the level of the pituitary. The results are discussed in relation with the present status of knowledge of the mechanisms underlying the blockage of sexual maturation in the European eel at the silver stage.     

硬骨鱼免疫相关microRNA研究进展

MicroRNA(miRNA)是一类长度为18～25 nt的非编码RNA,在硬骨鱼免疫应答中起重要作用.近年来,借助高通量测序与荧光定量PCR(RT-PCR)技术,筛选到大量免疫相关的差异表达miRNA(DE miRNA).通过比较多种硬骨鱼在不同病毒/细菌感染条件的miRNA表达谱,筛选出相同DE miRNA,并认为...     

LH-β and FSH-β mRNA expression in nesting and post-breeding three-spined stickleback, Gasterosteus aculeatus, and effects of castration on expression of LH-β, FSH-β and spiggin mRNA

The mRNA expression of the LH- and FSH- subunits were measured in nesting and post-breeding male three-spined sticklebacks, Gasterosteus aculetaus, as well as in castrated and sham-operated nesting males. Furthermore, expression of an androgen induced kidney protein, spiggin, and 11-ketotestosterone (11KT) levels, were measured in the castrated and sham-operated males. Nesting males had significantly higher levels of both LH- and FSH- mRNA expression compared to post-breeding males. Furthermore, sham-operated males had significantly higher levels of LH- mRNA and spiggin mRNA expression than the castrated fish. Expression of FSH-, on the other hand, did not differ between castrated and sham-operated males. There were strong positive individual correlations between circulating levels of 11KT on the one hand and expressions of LH- and spiggin mRNA, whereas the correlation between 11KT levels and FSH- mRNA was weak. The negative effect of castration on -LH mRNA indicates that gonadal hormones stimulate this expression, whereas this was not the case for -FSH. The observed decline in -LH expression after the end of the breeding season may be the result of cessation of the gonadal stimulation of the pituitary. On the other hand, it is not likely that this can explain the decline in FSH- expression.     

Central regulation of reproduction in teleosts

As in other vertebrates, reproduction in teleosts depends upon interactions taking place along the brain-pituitary-gonads axis. At the central level, these interactions involve at least three types of factors:A gonadotrophin-releasing factor which has recently been isolated from chum salmon brain extracts. This decapeptide, whose structure is (Trp⁷-Leu⁸)-LHRH, appears to have a widespread distribution among teleosts, and is less active that LHRH or LHRH analogues in releasing gonadotrophin from the teleost pituitary. Immunohistochemical and quantitative studies have demonstrated that Gn-RH neurons are mainly located in the ventral telencephalon and the preoptic area, while projections are found in the entire brain and the pituitary gland.A gonadotrophin release-inhibiting factor has been demonstrated in the anterior preoptic region of the goldfish and a large set of data suggests that dopamine has GRIF activity in goldfish, and in other teleost species, by direct action on the gonadotrophs. Accordingly, a dopaminergic preoptico-hypophyseal pathway could be demonstrated in the goldfish brain.Sex steroids exert, depending on the dosages, either a negative feedback in sexually mature fish or a positive feedback in immature fish. Such a positive feedback is caused by estrogens and aromatizable androgens. Accordingly, the brain of teleosts contains high levels of aromatase activity in particular in the telencephalon and anterior hypothalamus. The distribution of estrogens concentrating cells within the brain is consistent with possible interactions with Gn-RH or catecholaminergic neurons at the level of certain brain territories.These data are discussed in relation with the functional significance of different brain areas where interactions between these different factors possibly take place, in particular the terminal nerve, the ventral telencephalon, the preoptic area and nucleus lateralis tuberis.     

Endocrinology and fish culture

In the current practice of fish culture the use of hormones is mainly limited to the field of reproduction, and more precisely to induce or synchronize ovulation and stimulate spermiation. The practice of pituitary homogenates injection (called hypophysation) which started in the early 1930's has allowed spectacular developments in the culture of some cyprinid species, especially in China, India and Europe. HCG has been used successfully in a limited number of species and LHRH-A, often associated with antidopaminergic compounds, started to be used in some species, especially cyprinids, on a commercial scale. Sex steroids are now commonly used to reverse the sex of some species in salmonids, cyprinids, tilapias. Due to legal restriction and consumer opposition, the tendency is to limit the use of steroids, especially in fish which are later offered to consumption. GH, which significantly stimulates growth and reduces the food conversion rate in laboratory experiments, will possibly be used on a large scale in fish farms. Endocrinology has considerably increased the knowledge of the mechanisms controlling some physiological functions; this has allowed the identification of sensitive phases in fish in captivity, and helps in the management and the process of domestication (most of the cultured fish species have been taken form the wild recently).     

Regulation of the interrenal of fishes: non-classical control mechanisms

The regulation of the interrenal of teleostean fishes is reviewed from the perspective of non-classical control mechanisms and new evidence is presented suggesting gonadotropic control of the interrenal. Cortisol secretion by the interrenal, in addition to regulation by ACTH, appears to be mediated by other hormones. Physiologically relevant, direct control of interrenal function by hydromineral factors is unclear.In vitro experiments with interrenals of coho salmon (Oncorhynchus kisutch) indicate that salmon gonadotropin is extremely corticotropic and both ACTH and gonadotropin stimulate the secretion of large quantities of androstenedione from the interrenal.     

A comparison of sex steroid binding protein (SBP) in four species of teleost fish

A sex steroid binding protein (SBP) binding E₂ with high affinity has been detected in a pleuronectid (greenback flounder Rhombosolea tapirina), two sparids (black bream Acanthopagrus butcheri and snapper Pagrus auratus), and its presence has been confirmed in a salmonid (rainbow trout Oncorhynchus mykiss). SBP binding characteristics were measured using a hot saturation assay for trout, bream and snapper, and a cold saturation assay for flounder. Bound and unbound steroid were separated by incubation with dextran-coated charcoal (DCC). Affinity for E₂ was highest in trout (k_D=0.44 nM), followed by bream (k_D=3.39 nM) and snapper (k_D=10.7 nM). The lowest affinity was found in flounder (k_D=84.7 nM). Binding capacity, however, was greatest in flounder (B_max=164 nM), followed by trout (B_max=92 nM), and then bream and snapper (B_max=50 and 39 nM, respectively). Binding of E₂ to SBP had a very rapid rate of association, and most dissociation occurred within 5 min. To confirm that the plasma protein measured here was SBP, the relative binding affinities of SBP for a range of steroids were measured. In trout, bream and snapper, SBP bound E₂ with the highest affinity, followed by T. In contrast, the relative affinity of T for flounder SBP was more than twice that of E₂. The rank orders of affinity of binding indicate the importance of an unhindered 17-hydroxyl group, and a 3-hydroxyl or 3-ketone group for high affinity binding to SBP. These requirements for high affinity binding are present in most animals possessing SBP and indicate conservation of the SBP molecule through evolution.     

Steroidogenic capacity of coho salmon ovarian follicles throughout the periovulatory period

Coho salmon follicles obtained at various times throughout the periovulatory period were incubatedin vitro with graded amounts of partially purified salmon gonadotropin (SG-G100) for 24 h and the amounts of 17β-estradiol, testosterone and 17α20β dihydroxy-4-pregnen-3-one (17α20βP) released into the media determined by radioimmunoassay. By this approach, the pattern of steroid secretion by ovarian follicles was shown to change in relation to the developmental status of the oocyte. Full-grown immature follicles produced large amounts of 17β-estradiol but negligible amounts of testosterone and 17α20βP. Both basal and gonadotropin-stimulated 17β-estradiol production was subsequently reduced with advancing oocyte development. In contrast, the production of testosterone and 17α20βP increased during the course of ovarian development with testosterone production highest in follicles with a peripheral germinal vesicle and 17α20βP production highest in matured and postovulatory follicles. These data are discussed in relation to information on the preovulatory changes in circulating levels of steriod hormones in salmonids.     

Relationship between metabolic and reproductive hormones in salmonid fish

Circulating concentrations of estradiol (E₂), vitellogenin (VTG), thyroxine (T₄), triiodothyronine (T₃) and insulin were measured in reproductively maturing four and five year-old Atlantic salmon. Blood samples were collected from the fish in seawater for one year prior to their spawning in November in fresh water. In females, E₂ and VTG were low but detectable from December to July, and then increased to peak levels in September and October. Plasma levels of T₄ and T₃ were relatively constant in winter and spring, and decreased in July. Plasma concentration of T₄ increased in November when the fish returned to fresh water. Plasma T₃ levels remained low during the autumn. Both T₄ and T₃ levels tended to be higher in males than in females during September through November. Plasma insulin concentrations increased during the spring to peak values in May, and then decreased in June and July in fish of both sexes. There was a significant elevation of plasma insulin in males during October, and the levels in males tended to be higher than those found in females during final maturation.     

Arginine vasotocin and fish osmoregulation

Pituitary arginine vasotocin (AVT) secretion is sensitive to the osmotic challenge associated with transfer of euryhaline teleosts between sea water (SW) and fresh water (FW). Pituitary AVT content in FW-adapted flounders greatly exceeds that in SW-adapted fish. Plasma AVT concentrations are in the range 10⁻¹²−10⁻¹¹ M (1–100 pg/ml). In euryhaline species, like the eel, flounder and trout, there were no consistent, marked differences in plasma AVT concentrations between FW- and SW-adapted fish. In SW- but not FW-adapted flounders plasma AVT and sodium concentrations are correlated. During the initial period of acclimation from FW to SW eels, show a transitory rise in plasma AVT concentration which is associated with a transitory increase in plasma Angiotensin II. In view of the range of plasma AVT concentration observed in SW- and FW-adapted fish, it is evident that of the described dose-dependent effects of AVT on urine production, only the antidiuretic responses are likely to be of physiological significance. In addition to the presence of a V₁-type vascular receptor for AVT, the nephron also possesses a V₂-type receptor, coupled to adenylate cyclase. In the gill tissue AVT receptors are also present, but in this tissue receptor occupancy leads to inhibition of cAMP production rather than the stimulation observed in renal tissue. The functional significance of the gill AVT receptor remains to be established.

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Résumé La sécrétion d'arginine vasotocine (AVT) hypophysaire est sensible aux modifications de la pression osmotique observées lors du transfert d'eau douce en eau de mer de téléostéens euryhalins. Les contenus hypophysaires en AVT chez des flets adaptés à l'eau douce sont largement supérieurs à ceux des poissons adaptés à l'eau de mer. Les concentrations plasmatiques en AVT se situent entre 10⁻¹² et 10⁻¹¹ M. Chez les espèces euryhalines comme l'anguille, le flet et la truite, iln'y a pas de différences marquées dans les concentrations plasmatiques d'AVT en eau douce et en eau de mer. Chez des poissons adaptés à l'eau de mer, les niveaux plasmatiques d'AVT et de sodium sont corrélés ce qui n'est pas le cas chez les poissons adaptés à l'eau douce. Pendant la période initiale d'aclimatation à l'eau de mer, une augmentation transitoire des niveaux plasmatiques d'AVT est observée chez l'anguille, associée à une augmentation de l'angiotensine II plasmatique. Compte tenues des concentrations plasmatiques d'AVT mesurées chez des poissons adaptés à l'eau douce ou à l'eau de mer, il est evident que parmi les effets dose-dépendants de l'AVT sur la production d'urine décrits dans la littérature, seules les réponses antidiurétiques ont vraissemblablement une signification physiologique. En plus de la présence de récepteurs vasculaires pour l'AVT de type V₁, le nephron possède aussi des récepteurs de type V₂ couplés à l'adénylate cyclase. Dans les branchies, les récepteurs à l'AVT sont aussi présents, mais dans ce tissu, l'occupation des récepteurs conduit à une inhibition de la production d'AMPc contrairement à la stimulation observée dans le tissu rénal. La signification fonctionnelle du récepteur branchial à l'AVT reste à établir.