Comparison of in vitro vitellogenin synthesis among different nonylphenol products using primary cultures of tilapia hepatocytes

Effect of nonylphenol (NP) products from different companies on in vitro vitellogenin (VTG) synthesis was compared using tilapia ( Oreochromis mossambicus ) hepatocytes cultures. Addition of NP at a concentration of 10⁻³ M to the medium caused death of hepatocytes (NP-A and NP-B) and delay of monolayer formation (NP-C). No cell death was observed at a concentration of 10⁻⁴ M (NP-A, -B and -C) but cell adhesion was slower than control. These results suggest that high concentration of NP is toxic against tilapia hepatocytes. When effects of estradiol-17β (E₂, 10⁻⁷ M) and NP (10⁻⁴ M) on in vitro VTG synthesis were examined, addition of E₂ and NP-A and NP-B to the media resulted in elevation of VTG. NP-C did not induce VTG in the medium. Co-treatment of NP-B and tamoxifen, a non-steroidal anti-estrogen, reduced VTG synthesis. These results suggest that NP has estrogenic potential in primary cultures of tilapia hepatocytes and acts through binding to the estrogen receptor, and that there is a difference in the induction level of VTG among different NP products.     

Purification,characterization and immunoassay of striped bass (Morone saxatilis) vitellogenin

The egg yolk precursor, vitellogenin (VTG), was purified from blood plasma of striped bass by chromatography on hydroxylapatite or DEAE-agarose. The fish were first implanted with estradiol-17β (E₂), which induced vitellogenesis. A rabbit antiserum (a-FSPP) raised against plasma from mature female striped bass, and then adsorbed with mature male plasma, was used to detect female-specific plasma protein (FSPP) in the chromatography fractions. Striped bass VTG (s-VTG) was collected from the peak fraction that was induced by E₂, reacted with a-FSPP, and contained all detectable phosphoprotein. It appeared as a single band (M_r ≂ 170,000) in SDS-PAGE or Western blots using a-FSPP, and as a pair of closely-spaced phospholipoprotein bands in native gradient-PAGE, suggesting that there is more than one circulating form of s-VTG. The relationship of s-VTG to the yolk proteins was verified using a-FSPP. The antiserum reacted with the main peak from gel filtration of saline ovary extracts, and it specifically immunostained the two main bands in Western blots of the extracts and the yolk granules of mature oocytes. The amino acid composition of s-VTG was similar to that of VTG from other fish and Xenopus. A radial immunodiffusion assay for s-VTG was developed using a-FSPP and purified s-VTG as standard. The s-VTG was not detected in blood plasma of males, immature females, or regressed adult females, but plasma s-VTG levels were highly correlated with plasma E₂ and testosterone levels, and oocyte growth, in maturing females. The results indicate that the maturational status of female striped bass can be identified by s-VTG immunoassay.     

Endocrine changes during the annual reproductive cycle of the red porgy, Pagrus pagrus (Teleostei, Sparidae)

Changes in 17-estradiol (E2), estrone (E1), testosterone (T), 11-ketotestosterone (11KT), and 17,20-dihydroxy-4-pregnen-3-one (17,20-P) levels were correlated to changes in gonadosomatic index (GSI), vitellogenin concentration (Vg), ovarian and testicular histology during the annual reproductive cycle of the red porgy, Pagrus pagrus. The production of E2, E1, T and 17,20-P was confirmed by analysis of the steroidogenic activity of ovaries. In females, the average concentration of E2 was lower than 2 ng ml^–1. E2 values first increased significantly at the stage of endogenous vitellogenesis and remained high during exogenous vitellogenesis. E1 levels were lower values than E2 (less than 300 pg ml^–1), but they increased at the beginning of exogenous vitellogenesis. Estrogens concentrations followed similar pattern to Vg and were significantly correlated. Mean levels of T were mostly lower than 1 ng ml^–1. They followed a pattern similar to that of E2 except for a further increase observed at the stage of final maturation. T and E2 levels were significantly correlated. The concentration of 11KT did not change significantly. The levels of 17,20-P ranged between 0.22 and 1.22 ng ml^–1 but changes were not related to gametogenesis. In males, the concentrations of T and 11KT fluctuated significantly during the sexual maturity stages, showing a similar pattern and were significantly correlated to GSI changes. T levels increased during spermiogenesis and spermiation stages to reach about 3 ng ml^–1. 11KT levels stayed about half those of T. The levels of estrogens showed no significant changes. Level of 17,20-P showed no significant variation related to male maturity. Results are discussed in relation to changes in plasma steroid levels during gametogenesis of other multiple spawner species.     

Reproductive performance of common dentex, Dentex dentex, broodstock held under different photoperiod and constant temperature conditions

The effect of photoperiod and temperature on the timing and the quality of spawning, and on associated endocrine changes in circulating 17β-oestradiol, 11-ketotestoserone (11-KT) and vitellogenin (Vtg) were investigated in common dentex, Dentex dentex, undergoing their second reproductive cycle. The possibility was also explored of using the measurement of steroids in the culture water of broodstock tanks, rather than in individual blood plasma samples, as a potentially useful tool for assessing the physiological state of a fish without disturbing them. One group of fish was exposed to a simulated natural seasonal cycle and ambient temperature (CONTROL). The other two groups were exposed to simulated seasonal photoperiod cycles (12 month-long) but which were phase-shifted either three months before (ADVANCED) or after (DELAYED) the natural cycle. Temperature was kept at 19.4±0.9 °C all-year-round. In the CONTROL, spawning started in mid-April and lasted until mid-June, while in the ADVANCED group, spawning started 4 months earlier and in the DELAYED group 2 months later than the CONTROL. The total egg production, egg quality, hatching rate, relative fecundity, and spawning index of the experimental groups were similar to the controls. The differences in spawning time induced by photothermal manipulation were associated with a difference in the timing of peak concentrations of plasma E₂, 11-KT and Vtg. In all three groups, the amounts of conjugated 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and free and sulfated 11-KT which could be extracted from the water during the spawning period were significantly higher than those found in the preovulatory period. However, the differences were mostly less than 2-fold suggesting that, at least under the conditions employed in this study, the method was of limited use for non-intrusive detection of gonadogenesis and spawning (as had been hoped). The observed differences in spawning time and in the seasonal changes of sex steroids and Vtg confirm and extend the findings on marine fish. This revised version was published online in August 2006 with corrections to the Cover Date.     

Atlantic halibut (Hippoglossus hippoglossus) vitellogenin: induction,isolation and partial characterization

Vitellogenin (VTG) synthesis was induced by repeated injections of estradiol-17 in juvenile Atlantic halibut (Hippoglossus hippoglossus). VTG eluted as a large, phosphoprotein containing peak on DEAE-Sephacel chromatography of plasma from estradiol-17 treated juvenile and mature female, but not mature male halibut. A purification procedure for Atlantic halibut VTG was developed, where VTG was precipitated with MgCl₂, EDTA and distilled water, and the precipitated protein submitted to anion exchange chromatography on DEAE-Sephacel. Precipitated VTG eluted as a broad, partly dissociated peak on DEAE-Sephacel, when chromatography was run at 4°C, but the protein appeared intact when analysed both by SDS PAGE and native PAGE. DEAE-Sephacel chromatography at room temperature resulted in an irregular elution pattern and a dissociated protein fraction, as analysed by SDS PAGE. Biochemical characterization of VTG showed that the molecular mass of the monomer was ca 160 kDa, as estimated by SDS-PAGE. The total lipid content was 19.8% w/w, with 64%, or 12.7% of the total weight, as phospholipid. Protein bound phosphorus constituted 0.62% w/w of halibut VTG. Plasma dilution curves from mature and maturing female halibut were parallel with a dilution curve from halibut egg yolk homogenate in an homologous RIA. Plasma from mature male, but not juvenile halibut crossreacted with the VTG antiserum.     

Photoperiodic manipulations of the reproductive cycle of sea bass (Dicentrarchus labrax) and their effects on gonadal development, and plasma 17ß-estradiol and vitellogenin levels

The annual profile of plasma vitellogenin (VTG) and 17ß-estradiol (E₂) levels, as well as gonadal development and spawning characteristics were investigated in captive female sea bass (Dicentrarchus labrax). Endocrine and gonadal changes were studied in fish reared under natural conditions or exposed to manipulated photothermal cycles. In natural conditions of photoperiod and temperature, sea bass spawned from February through March (East coast of Spain, 40°N 0°E). One or two months of constant long-days (15L/9D) in a constant short-day photoperiod regime (9L/15D) all-year-round, given early in the year (March and March–April), advanced spawning by 3 months. The same treatment applied later in the year (September–October) delayed spawning by 1 month, compared to controls.In all groups, changes in plasma VTG levels were correlated with E₂ levels, oocyte growth and spawning time. In control females, VTG was low (<100 ng ml^-1) during the summer, until its first surge in plasma 4 months before the beginning of spawning. The VTG (3.1 ± 0.3 mg ml^-1) and E₂ (4.1 ± 0.5 ng ml^-1) levels showed a single annual peak during late vitellogenesis, the time of the highest proportion of vitellogenic oocytes in the ovary. Constant high levels of VTG (1–1.4 mg ml^-1) and E₂ (1.6–1.9ng ml^-1) were maintained during the entire spawning time, together with the presence of vitellogenic oocytes, suggesting the existence of several waves of oocyte growth in the ovary and thus, several spawns per female. Endocrine profiles and oocyte development in fish exposed to constant photoperiods were similar to controls, but were shifted in time in relation to the displacement of the spawning time. In the fish showing advanced spawns, the duration of the gametogenic proces was compressed when compared to controls. The differences observed in the evolution of the reproductive-related factors in the advanced groups, which were exposed to a reduction in temperature to 15°C, suggest an influence of the temperature in the early stages of the reproductive cycle in sea bass.     

印染污水对银鲫卵黄蛋白原和金属硫蛋白水平的影响

分析了不同浓度印染污水对银鲫（Carassiu sauratus）的生殖相关指标和金属硫蛋白（MT）的影响。设计1个对照组和2个试验组,对照组采用天然的水库水,试验组分别用浓度为0.1%和0.2%的印染污水养殖银鲫,每组3个平行,每个平行随机放入50尾银鲫,试验期为4周。结果表明,养殖4周后,0.1%印染污水组银鲫的肝体指数和血清卵黄蛋白原（VTG）水平显著上升（P〈0.05）,0.2%印染污水组银鲫的血清VTG水平显著低于对照组（P〈0.05）,印染污水对银鲫性腺指数没有影响。0.1%印染污水组银鲫在第2、3、4周,肝脏MT水平均显著高于对照组（P〈0.05）,0.2%印染污水组银鲫肝脏MT水平在第3周时显著升高（P〈0.05）,印染污水对银鲫肾脏MT水平没有影响（P〉0.05）;表明印染污水能够影响银鲫繁殖功能和组织MT的水平。     

Effects of temperature and photoperiod on hemolymph vitellogenin levels during spawning events of the blue crab,Callinectes sapidus,in captivity

Vitellogenesis is the process of production and uptake of vitellogenin (VtG), the precursor of vitellin (VT = yolk protein), for ovarian development. In Callinectes sapidus, hemolymph VtG levels are positively correlated with ovarian development. We aimed to develop an invasive tool to monitor ovarian development in order to predict spawning. The present study determined the effects of photoperiod and temperature conditions on levels of hemolymph VtG of the females in captivity kept in a closed recirculating system. Hemolymph VtG levels measured using a competitive ELISA correlates positively with temperature (21° versus 11°C) but are negatively correlated with photoperiod (0L:24D and 8L:16D versus 16L:8D and 24L:0D). Also, heavier (larger) females contain more VtG in their hemolymph and produce greater numbers of larvae. At 21°C, the VtG levels show high individual variations and continuously change over time, indicating that VtG production and uptake by ovary may be continuous and dynamic over multiple spawning events in C. sapidus. Microsatellite analysis of these females reveals a great diversity within and between the batches, implying that resident C. sapidus populations in the Chesapeake Bay are genetically dynamic. Such a great genetic diversity could be responsible for the high variation in VtG levels in these females.