Deduced primary structures of three types of vitellogenin in mosquitofish (Gambusia affinis), a viviparous fish

Three distinct forms of vitellogenin (Vg), 600 kDa VgA and VgB and 400 kDa Vg, were discovered biochemically in estrogen treated female plasma. By sequencing of the three Vg cDNAs, the VgA and VgB were recognized as complete Vgs having all yolk protein (YP) domains, and the 400 kDa Vg was thought to be phosvitinless (Pvl) Vg lacking phosvitin (Pv) domain.     

Vitellogenesis with special emphasis on Indian fishes

Oocyte growth in most oviparous vertebrates including fish is due to the formation of yolk, and eggshell proteins (zona radiata proteins). Zonagenesis leads to the formation of zona radiata proteins in oocytes, which play an important role during oogenesis, whereas vitellogenesis leads to the formation of yolk in oocytes through a series of events during which the yolk precursor protein vitellogenin (Vg) is synthesized and secreted from liver into blood from where it is sequestered into the developing oocytes and thereafter proteolytically cleaved to form yolk proteins (YPs) and finally deposited in the ooplasm. Much research has been done in many fish species with respect to the number and nature of Vg and YPs and their probable functions during fish reproduction. Recent findings of multiplicity of Vg molecules in fishes reject the earlier view of a single-Vg model and have led scientists to explore the functions of individual Vg and their YP derivatives, lipovitellin, phosvitin, and β′-component. Two distinct types of Vg or Vg genes, containing or encoding the three YPs, have been detected in many teleosts. A third unusual, incomplete, phosvitin-poor Vg has been described recently in many fishes. In comparison to much of the information on vitellogenesis in many fishes very little is known for Indian fishes. In India research has been done in a few species such as the catfish, Heteropneustes fossilis and Clarias batrachus, the murrel, Channa punctatus and the Indian major carps, Labeo rohita and Cirrhinus mrigala. Immunological and biochemical analyses suggest the occurrence of multiple forms of Vg and their YP derivatives. The synthesis and incorporation of Vg are regulated by gonadotropin (GTH) and estradiol-17β (E₂). A differential role between estrone (E₁₎ and estriol (E₃) has been demonstrated for Vg synthesis. Enzyme-linked immunosorbent assays (ELISAs) for Vg have been developed to measure plasma Vg. Finally the different roles of Vg1 (HAI) and Vg2 (HAII) on vitellogenesis have been demonstrated. However, more research remains to be carried out in other fish species with respect to the number and nature of Vg and YPs and their genes in order to describe their reproductive functions.     

Fishery characteristics of barfin flounder Verasper moseri in southern Tohoku,the major spawning ground,after the start of large-scale stock enhancement in Hokkaido,Japan

Commercial landings of a rare pleuronectid flatfish, barfin flounder Verasper moseri, recovered drastically after large-scale stock enhancement in Hokkaido conducted since 2006. This study investigated commercial landings, fishing grounds, size distributions, and sex and age compositions of barfin flounder during 2007–2011 in southern Tohoku, their major spawning ground, which is over 700 km south of Hokkaido. Landings, mostly comprising stocked fish, increased drastically in southern Tohoku: from 2.0 tons in 2007 to 20.8 tons in 2010. Over 98 % of them were landed during January–April, with the peak period during February–March. Fishing logbook data of offshore bottom-trawl vessels during 2007–2010 revealed the upper continental slope off southernmost Tohoku as the main fishing ground (35°40′–36°50′N, annual weighted mean depth 267–299 m). Two size modes in the landings consisted of males (ca. 40 cm) and females (ca. 60 cm). The main age at fishery recruitment in southern Tohoku was younger for males (age 2+) than for females (ages 3+ and 4+), the result of which was that the female percentage was lowest at 1.5 % in 2009 and highest at 7.9 % in 2011. Our results showed clearly how the effectiveness of large-scale stock enhancement in Hokkaido extended to southern Tohoku in association with spawning migration of stocked barfin flounder.     

Involvement of Gonadotropin-Releasing Hormone in Thyroxine Release in Three different forms of Teleost Fish: Barfin Founder, Masu Salmon and Goldfish

Effects of gonadotropin-releasing hormone (GnRH) on thyroxine (T₄) release in vivo and in vitro were studied in barfin flounder Verasper moseri, masu salmon Oncorhynchus masou and goldfish Carassius auratus. Seabream GnRH (sbGnRH) at a dose of 200 ng/50 g body weight (BW) significantly increased plasma T₄ levels 1 h after the in vivo injection in the barfin flounder, but thereafter the levels normalized. Salmon GnRH (sGnRH) significantly increased plasma T₄ levels l h after the injection with a significant return to initial levels in male masu salmon and male goldfish. In contrast, sGnRH and cGnRH-II in barfin flounder, and cGnRH-II in male masu salmon and male goldfish were not effective in stimulating T₄ release. To clarify direct involvement of GnRH in T₄ release, dissected lower jaw including scattered thyroid follicles was incubated with sbGnRH (1 μg/well) in barfin flounder, and with two doses (0.1 and 1 μg/well) of sGnRH in masu salmon and goldfish in vitro. T₄ concentrations of control were stable during 24 h. Incubation of lower jaw with high dose (1 μg/well) of GnRH significantly (P<0.05) increased T₄ concentrations of incubation medium at 1 h in all experimental fishes. These results indicate that direct stimulation of T₄ secretion by GnRH occurs widely in teleost fish.     

Yolk mobilization in perch, Perca fluviatilis L., embryos

During embryogenesis of the perch, Perca fluviatilis L., a gradual proteolysis of yolk proteins was revealed by SDS-PAGE and immunoblotting and was comparable to other teleosts. A 118 kDa polypeptide was processed in two 96 and 18 kDa descendants; further proteolysis produced two peptides of 64 and 31 kDa. Ultrastructure and immunocytochemistry were used to compare electrophoresis patterns with morphological changes in the developing embryo and to localise yolk proteins in embryonic cell layers. In contrast to most other freshwater teleost, in spawned eggs of the perch yolk spheres fused together forming a homogenous yolk mass; lipid droplets formed a single oil drop on top of the egg. Combination of electrophoresis data and light and electron microscopical observations suggested a specific and parallel mobilization of yolk and lipid. In addition, yolk absorption by the yolk syncytial layer was observed. Numerous yolk platelets were found in the yolk syncytial layer and, in later stages of embryogenesis, also in the inner cell mass, but never in the cells of the enveloping layer.     

Purification and characterization of lipovitellin from Pacific saury Cololabis saira

ABSTRACT:   Lipovitellin (Lv), the major yolk protein derived from vitellogenin (Vg), was purified from vitellogenic ovaries of Pacific saury Cololabis saira using hydroxylapatite column chromatography followed by gel filtration. The apparent native mass of purified Lv was approximately 420 kDa, while the tertiary structure of Lv revealed by sodium dodecylsulfate–polyacrylamide gel electrophoresis was typical of teleost Lvs, consisting of a heavy chain (∼99 kDa) and a light chain (∼34 kDa). Western blot analysis using rabbit antiserum raised against Pacific saury Lv revealed a specific reaction with a polypeptide (∼194 kDa) that is present in serum from female Pacific saury but not in male serum, suggesting the approximately 194-kDa polypeptide to be the Vg monomer. This study describes the first step toward the development of specific immunoassays for Pacific saury Vg, which will be an effective tool for monitoring the reproductive development of this species.     

Gonadal maturation and spawning migration of stocked female barfin flounder Verasper moseri off the Pacific coast of northern Japan

Commercial catches of barfin flounder Verasper moseri, an important target flatfish for stock enhancement programs in northern Japan, have recovered remarkably since the large-scale release of seedlings from Hokkaido in 2006. However, their reproductive ecology remains unclear. Our study of 2008–2012 investigated seasonal changes in the maturity of stocked female barfin flounder (n = 4,123) off the Pacific coast from Hokkaido to Tohoku (35.7°–43.4°N). Histological observation of ovaries revealed that fish undergoing vitellogenesis were observed mainly off Hokkaido and partially off northern Tohoku during August–January (40.2°–43.4°N). However, fully matured or spawning fish were not found near those areas. They were observed only in southern Tohoku. Ongoing spawning was observed off southernmost Tohoku (35.8°–37.5°N around 300 m depth) from early February through late April, when commercial landings had decreased drastically off Hokkaido. Spent fish were caught only rarely off southern Tohoku, but were observed frequently off Hokkaido during April–June. These results demonstrate that spawning grounds form at the upper continental slope off southernmost Tohoku during February–April. Relations between maturation traits and seasonal landings strongly suggest that female barfin flounder repeatedly migrate more than 700 km from feeding grounds off Hokkaido to spawning grounds off southern Tohoku.     

Purification and development of ELISAs for two forms of vitellogenin in Indian walking catfish, <Emphasis Type="Italic">Clarias batrachus</Emphasis> (L.)

Two forms of vitellogenin (Vg: Vg1 and Vg2) were purified from the plasma of estradiol-17β (E₂)-treated Indian walking catfish, Clarias batrachus, by gel filtration and adsorption chromatography. Native Vg1 and Vg2 had apparent molecular masses of 375 and 450 kDa, respectively, and both Vgs resolved into two similar major bands (95 and 67 kDa) in SDS-PAGE under reducing condition. Polyclonal antisera raised against each form of Vg were absorbed with a combination of hypophysectomized male catfish serum proteins and alternate Vg to ensure specificity. Immunological analyses verified the presence of Vg1 and Vg2 in the plasma of female catfish. Homologous ELISAs were developed for Vg1 and Vg2 using their respective harvested antisera, which exhibited the detection limit of 100 ng ml^?1 for Vg1 and 40 ng ml^?1 for Vg2, and low level of cross-reactivity (not parallel to the standard) was found with alternate Vg in each assay. Treatment of male catfish with E₂ induced both Vgs showing a proportionate ratio of Vg1 to Vg2 at 5.6:1. Plasma concentrations of both Vgs measured by ELISAs at different reproductive phases of field collected female catfish increased in accordance with the ovarian development, keeping the proportionate ratio of Vg1 to Vg2 at about 2:1 in fish undergoing vitellogenesis during prespawning period and 1:20 during spawning period, suggesting that Vg1 may be the major Vg to contribute in yolk formation, whereas Vg2, besides its role in yolk formation, may facilitate other physiological functions. The present study, thus, demonstrates the occurrence of two unequally synthesized Vgs in the catfish.     

Enzyme-linked immunosorbent assay (ELISA) measurement of vitellogenin in plasma and liver histopathology in barfin plaice <Emphasis Type="Italic">Liopsetta pinnifasciata</Emphasis> from Amursky Bay,Sea of Japan

Vitellogenin (Vg) of the barfin plaice Liopsetta pinnifasciata was isolated and purified. In native polyacrylamide gel electrophoresis, Vg appeared as one band. After being subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), Vg fraction produced several polypeptides with molecular masses of 180, 98, 70, 52, 41 and 37 kDa. MALDI–TOF mass spectrometry (MS) of the 180- and 98-kDa Vg polypeptides from the SDS–PAGE gel and de novo sequencing of their four peptide fragments based on MS/MS analysis confirmed that the purified proteins were vitellogenins, which shared high similarity with the Vgs of the barfin flounder Verasper moseri and Atlantic halibut Hippoglossus hippoglossus. The most part of the predicted sequences obtained from the L. pinnifasciata 180-kDa polypeptide has previously been found in the V. moseri vitellogenin type B, the sequences obtained from the 98-kDa polypeptide were found in V. moseri vitellogenin type A, so these findings allow us to propose that L. pinnifasciata has at least two different forms of Vg. Rabbit polyclonal antibodies against Vg were produced, and a quantitative enzyme-linked immunosorbent assay was developed. The concentration of Vg in barfin plaice from the moderately contaminated area of Amursky Bay in the Sea of Japan was detected based on the maturity stage of their gonads. In November 2008, the Vg concentration in the plasma of females with advanced oogenesis varied from 5.295 to 28.367 mg/ml (mean 16.38 ± 6.73 mg/ml, CV = 41.1%); in the plasma of males, the concentration ranged from non-detectable to 0.957 mg/ml (0.29 ± 0.42 mg/ml, CV = 127.9%). In October 2009, the Vg concentration in female plasma was lower than in November 2008 (2.21–13.87 mg/ml). High individual variability of plasma Vg was characteristic for maturing males (CV = 200.3%) and immature females (CV = 255.5%), and there was no significant difference between plasma Vg concentrations in males captured in November 2008 and October 2009 or in maturing males and immature females. Vacuolisation of hepatocytes was more typical for males with low plasma Vg concentrations and females with high plasma Vg concentrations. Necrosis and pyknosis of hepatocyte nuclei were more frequent in males with high Vg concentrations and in females with low plasma Vg concentrations.     

剑尾鱼 2 种卵黄蛋白原全长 cDNA 的克隆及序列分析

卵黄蛋白原是环境雌激素效应研究的重要生物标志物,广泛应用于水环境内分泌干扰物污染的评价。本研究利用RT-PCR和cDNA末端快速扩增法(RACE)克隆获得了剑尾鱼(Xiphophorus helleri)的2种卵黄蛋白原基因—VgA和VgB,并对其基因结构和系统进化进行分析。VgA基因cDNA序列全长5159bp,开放阅读框(ORF)5058bp,编码1685个氨基酸。VgB基因cDNA序列全长5349bp,开放阅读框(ORF)5067bp,编码1688个氨基酸。2种cDNA序列均具有与已发现的卵黄蛋白原分子相似的主要特征和功能域,包括Vitellogenin结构域、VWFD结构域和多聚丝氨酸区域,且与已知其他鱼类卵黄蛋白原基因有较高的同源性。     

Stress inhibits insulin release induced by feeding and arginine injection in barfin flounder Verasper moseri

We evaluated the effect of stress and adrenaline injection on insulin release in barfin flounder Verasper moseri. Flounders were fed to satiation and then exposed to 30 min of stress (chasing using a blunt stick), beginning 15 min after feeding. Plasma insulin concentrations were measured using a time-resolved fluoroimmunoassay for barfin flounder insulin. Peak insulin concentrations in the stressed flounders (3.4 ± 1.0 ng/ml, mean ± SEM) were lower than in the control group (8.3 ± 1.7 ng/ml). Similarly, in flounders injected with l-arginine (Arg; 3.5 mmol/kg body weight), the most potent insulinotropic factor for flounders, insulin levels were lower for up to 5 h in individuals exposed to chasing stress. Furthermore, plasma insulin levels did not increase after injection with Arg followed by exposure to 10 min of stress. This was also true for flounders co-injected with Arg (20 nmol/kg body weight) and adrenalin instead of the chasing stress. We conclude that stress induces a powerful and extended inhibition of insulin release in flounders, even when the stress is acute. We hypothesize that this effect is mediated partly by adrenaline release. Our results provide insight into the deleterious effects of stress on growth.     

Vitellogenin of the Chilean flounder <Emphasis Type="Italic">Paralichthys adspersus</Emphasis> as a biomarker of endocrine disruption along the marine coast of the South Pacific. Part I: induction,purification, and identification

We sought to provide a useful indicator of the presence of endocrine-disrupting contaminants along the marine coast of the South Pacific using Chilean flounder (Paralichthys adspersus). In light of the lack of information on vitellogenin for this species, we induced, purified, and identified the plasma vitellogenin of Chilean flounder inhabiting the Chilean coast. Vitellogenin (Vg) from Chilean flounder was purified by size exclusion and ion-exchange chromatography using plasma from juvenile males induced by injecting 17β-estradiol. The Vg was detected by SDS–PAGE and Western blot analyses using an antibody against turbot (Scophthalmus maximus) vitellogenin. These analyses revealed a protein band of 205 kDa and three minor bands of 120, 90, and 68 kDa. These proteins were identified as Vg by means of mass spectrometry (LCQ Duo ESI-IT-MS), matching sequences of tryptic peptides to known sequences for several other fish species. The matches showed the presence of vitellogenin (VgI, VgII, Vg A and Vg B) in Chilean flounder, similar to species such as mummichog (Fundulus heteroclitus), Japanese medaka (Oryzias latipes), and white perch (Morone americana). These results are discussed in terms of identifying Vg in Paralichthys adspersus with the antibody to turbot Vg. Moreover, we compare the molecular size of Vg from Chilean flounder (large) with that of other flatfish species. Finally, we discuss the potential use of this molecule as a biomarker for the presence of xeno-estrogenic compounds along the Chilean coastline.     

雌核发育与正常二倍体条斑星鲽胚胎发育比较研究

在用牙鲆精子诱导条斑星鲽雌核发育的同时,对条斑星鲽单倍体、雌核发育胚胎发育过程进行了观察,并与普通胚胎发育作了比较.观察结果表明,(1)单倍体胚胎,卵裂开始至64细胞期,发育速度较普通胚胎发育发育速度明显缓慢,至多细胞期发育速度相同,历时14 h;到达高囊胚期比普通二倍体慢1.5 h,到达低囊胚期发育速度相同,历时31 h;到达原肠末期、神经胚期、晶体形成期历时相同,其他发育时期一直处于缓慢状态;孵化仔鱼具有明显的单倍体综合症.(2)雌核发育与普通胚胎的发育的特征相比无明显区别,在卵裂期发育速度明显迟缓,但在囊胚期以后,所用时间基本相同;孵化时间比普通胚胎长约1 h.(3)与普通胚胎相比,雌核发育胚胎、单倍体胚胎孵化率较低,仔鱼畸形率高.     

Effect of different avian egg yolk types on fertilization ability of cryopreserved common carp (Cyprinus carpio) spermatozoa

In spite of the fact that egg yolk from different avian species has successfully been used as an additive for the cryopreservation of sperm in mammalian species, its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Therefore, the present study was carried out to determine the effect of egg yolks from different avian species, namely domestic chicken (Gallus gallus domesticus), turkey (Meleagris gallopavo) and quail (Coturnix coturnix), on post-thaw motility and fertilization ability of cryopreserved common carp spermatozoa. Egg yolks from chicken, turkey and quail were analysed for moisture, total fat, protein, cholesterol and phospholipid profile. Total fat and cholesterol contents of the turkey egg yolk were higher than chicken and quail egg yolks (p < 0.05). Semen was frozen according to conventional slow freezing procedure. The extender contained 350 mM glucose, 30 mM Tris and 5 % glycerol supplemented with different ratios of avian egg yolk (10, 15 and 20 %). Semen was equilibrated at 4 °C for 15 min and placed into 0.25-ml straws and frozen in liquid nitrogen vapour (for 10 min at ?120 °C) and finally stored in liquid nitrogen (?196 °C) tank. The frozen spermatozoa were thawed in a water bath at 35 °C for 30 s. Fertilization was conducted using a ratio of 1 × 10⁵ spermatozoa/egg. Cryopreservation experiments resulted in higher post-thaw motility and fertilization rates. Mean post-thaw motility of cryopreserved spermatozoa was between 45 and 80 %, and fertilization rates, expressed as the percentage of eyed embryos, ranged from 70 to 95 %. In conclusion, the present study showed that turkey and quail egg yolks are suitable alternatives to the chicken egg yolk for the cryopreservation of common carp spermatozoa.     

Role of cathepsins B and D in proteolysis of yolk in the catfish Clarias gariepinus

Fish Physiology and Biochemistry - Yolk processing pathways vary in the oocytes of benthophil and pelagophil teleosts. The present study investigated the yolk processing pattern in the oocytes of...     

Aquaculture performance of triploid barfin flounder Verasper moseri

ABSTRACT:   To evaluate the aquaculture performance of triploid barfin flounder Verasper moseri , the sex ratio, maturation, growth and the relative proportion of body parts were examined. The sex ratio of triploids was similar to diploids under communal rearing conditions, but the proportion of female diploids was higher than that of triploids under separate rearing conditions. The gonadosomatic index of triploid females was very low even during the spawning season, and the ovaries were rudimentary. These results suggest that triploid barfin flounder females were sterile. In addition, triploid males produced a small quantity of milt containing very few spermatozoa with abnormal shapes. Spermatozoa obtained from triploids were aneuploidies. When normal eggs were fertilized with sperm from triploid males, no fry developed. These results suggest that triploid barfin flounder males were functionally sterile. Triploid males grew more slowly than diploid males, and triploid females showed similar or slower growth than diploid females, whether reared separately (23 months) or communally (35 months). The ratios of visceral weight to the edible parts for triploid males were similar to those for diploid males, but ratios for triploid females were higher than for diploid females during the spawning period. In conclusion, a significant improvement of growth was not found in triploid barfin flounders.     

Biological properties of Indian walking catfish (Clarias batrachus) (L.) gonadotropins in female reproduction

The biological activities of catfish LH-like (semi-purified: s200a and purified Qa) and FSH-like (semi-purified: s200b and purified: Qb) were compared in intact and hypophysectomized female catfish, Clarias batrachus, during preparatory and the pre-spawning periods on vitellogenesis and ovarian maintenance, as well as in vitro final maturation of oocytes, germinal vesicle breakdown (GVBD). During preparatory period, in intact catfish, semi-purified FSH-like induced complete vitellogenesis through the production of estradiol-17β (E₂) and vitellogenin (Vg) accompanied by the formation of SIII yolky oocytes. On the other hand, semi-purified LH-like had induced the formation of only SII (characterized by the appearance of cortical alveoli in cytoplasm) oocytes, which indicates the initiation of vitellogenesis. In hypophysectomized female catfish, purified LH-like but not FSH-like induced the formation of SII oocytes in the ovaries. Treatment with semi-purified LH- and FSH-like at the dose level of 5 µg/fish/day for 7 days significantly maintained the yolky oocytes in gravid catfish after hypophysectomy with a significant reduction in plasma Vg, but not E₂ levels, indicating some unknown GtH-induced factor doing the job. In in vitro oocytes culture, both LH- and FSH-like induced GVBD, but the response was significantly more with LH-like than FSH-like. All these findings revealed that both LH-like and FSH-like have overlapping physiological functions, but their responses differ depending on the physiological status of the catfish.     

Effects of photoperiod on gonadotropin-releasing hormone levels in the brain and pituitary of underyearling male barfin flounder

ABSTRACT:   A pleuronectiform fish, the barfin flounder Verasper moseri , expresses three gonadotropin-releasing hormone (GnRH) forms in the brain: salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and seabream GnRH (sbGnRH). To clarify the effects of photoperiod on GnRH systems, changes in brain and pituitary GnRH peptide levels were examined using time-resolved fluoroimmunoassays. In experiment 1, 5-month-old male barfin flounder (mean total length 9.0 cm, body weight 11.0 g) were divided into short (8:16 h light : dark [L:D] cycle; lights on 08.00–16.00 hours) and long photoperiod (16:8 h L:D cycle; lights on 04.00–20.00 hours) groups in mid September and maintained until November under natural water temperature (19.3–15.2°C). Brain sGnRH concentrations were significantly higher in the 16:8 h L:D group than in the 8:16 h L:D group, whereas no significant differences were observed in total length, body weight, plasma testosterone concentration, brain cGnRH-II concentration and pituitary sbGnRH content. In experiment 2, 7-month-old male barfin flounder (mean total length 16.5 cm, body weight 76.8 g) were divided into short and long photoperiod groups in mid December and maintained until February under natural water temperature (12.5–6.6°C). Total length, body weight and condition factor were significantly greater in the 16:8 h L:D group than in the 8:16 h L:D group, whereas no significant differences were observed in plasma testosterone concentration and GnRH levels in the brain and pituitary. These results indicate that levels of sGnRH in barfin flounder are influenced by photoperiodic treatment dependent on water temperature and/or body size.