Environmental control of fish reproduction: a different perspective

Nearly all studies on the environmental control of fish reproduction have, to date, focused on the roles played by various parameters of the natural environment, such as changes in water temperature and photoperiod. While these factors undoubtedly still play significant roles, other factors, which have arisen as a consequence of man's activities, also play major roles in at least some aquatic environments. One such factor is pollution; whereas attention is usually focused primarily on severe pollution and the fish mortality that can result, recently some emphasis has been given to the more subtle, but potentially serious, consequences of less severe pollution. Many freshwater habitats, particularly rivers in urban areas, now receive substantial volumes of effluent from various sources (both industrial and domestic). This effluent can contain chemicals capable of mimicking endogenous hormones, and hence has the potential to disrupt endocrine-mediated processes such as reproduction. This paper is concerned with the identity of these endocrine-disrupting chemicals, their mechanisms of action, and the effects they have on reproduction, both in wild fish and in fish maintained in the laboratory.     

Biochemical changes in the liver of the Indian freshwater murrel, Channa punctatus (Bloch) during estradiol-induced vitellogenin synthesis

Estrogen administration in C. punctatus increases RNA: protein and RNA: DNA ratios and depletes glycogen in the liver. Increase in glucose-6-phosphatase activity accounts for glycogen depletion whereas high activity of pyruvate kinase suggests stimulation of the glycolytic pathway at the pyruvate step to generate ATP and to provide carbon skeleton for the vitellogenin molecule. Five-fold increase in the activity of NADP⁺-dependant malate dehydrogenase generates NADPH possibly for fatty acid synthesis. Additional energy requirement (NADPH) is met by stimulation of hexose monophosphate pathway as well as NADP⁺-dependant isocitrate dehydrogenase. Increase in the activity of NADPH-cytochrome c reductase in microsomal fraction suggests stimulation of electron transport chain. Increase in the activity of glutamate pyruvate transaminase can be directly related to the synthesis to specific amino acids needed for the formation of vitellogenin whereas reduction in glutamate oxaloacetate suggests extra hepatic source of amino acids. Reduction in the activity of glutamate dehydrogenase may be due to catabolism.     

Evaluation of a 40 day Assay for Testing Endocrine Disrupters: Effects of an Anti-Estrogen and an Aromatase Inhibitor on Sex Ratio and Vitellogenin Concentrations in Juvenile Zebrafish (Danio rerio)

In the present study, the effects of the anti-estrogen ZM 189,156 and the aromatase inhibitor fadrozole were evaluated in a 40-day juvenile assay developed for screening of endocrine disrupting chemicals. Juvenile zebrafish were exposed from 20 to 60 days post hatch (dph) to ZM 189, 154 (100 μg l⁻¹ and 200 μg l⁻¹) and fadrozole (10, 32, and 100 μg l⁻¹). VTG concentrations were measured at 38 dph by the use of a direct non-competitive sandwich ELISA and sex ratios were determined at 60 dph by histological examination of the gonads. A small but significant increase in VTG concentrations was observed in fish exposed to ZM 189, 156 (100 and 200 μg l⁻¹) and fadrozole (10 and 100 μg l⁻¹) compared to control groups. In fish exposed to ZM 189, 156 and fadrozole, the percentage of females declined and the number of undifferentiated fish increased. These findings show that exposure of juvenile zebrafish to an aromatase inhibitor or an anti-estrogen during early development inhibits differentiation and development of female gonads. The data presented, furthermore, show that the 40-day juvenile assay may be suitable for screening endocrine disrupting chemicals acting as anti-estrogens and aromatase inhibitors.     

The development of a radioimmunoassay for carp,Cyprinus carpio,vitellogenin

The development of an easily — performed and robust radioimmunoassay (RIA) to carp, Cyprinus carpio, vitellogenin (c-VTG) is described. Purified c-VTG was iodinated using Iodogen. The resulting c-VTG label was useful for up to 60 days. High titre antibodies were raised in rabbits to the purified c-VTG. The practical operating range of the c-VTG RIA was between 2 and 200 ng. ml^-1. VTG was detected in plasma from all female mirror carp investigated, and all plasmas diluted parallel to the standard. The plasma VTG level in female carp increased concomitantly with the GSI; levels increasing from the ng. ml^-1 level in juveniles to a maximum of 1 mg. ml^-1 in fully mature females. VTG level was a far more sensitive index of the degree of sexual development than was GSI. In males, blood levels of VTG were always undetectable. Vitellogenic plasma from all subspecies of C. carpio (e.g. mirror, common, Koi) diluted parallel to the standard, as did blood from most other female cyprinids, such as the roach (Rutilis rutilis), bream (Abramis brama), and dace (Leuciscus leuciscus), but not all. These results suggest that the structure of VTG is highly conserved within this family. However, plasma from vitellogenic salmonids did not cross-react in the RIA. The relationship between plasma VTG and calcium levels was studied in both carp and rainbow trout. In rainbow trout it was found that plasma calcium levels do not rise above basal levels until the VTG level exceeds about 1 mg. ml^-1, and therefore in this species it is only useful as an indicator of the degree of ovarian development in the later stages of the reproductive cycle. In the carp, however, and probably other cyprinids, blood VTG levels do not appear to naturally exceed about 1 mg.ml^-1, and plasma calcium levels are not suitable as an indirect measure of the VTG level.     

Characterization of vitellogenin induced by estradiol-17β in protandrous black porgy,Acanthopagrus schlegeli

The objectives of this study were to isolate and characterize vitellogenin from plasma of estradiol-treated protandrous black porgy,Acanthopagrus schlegeli. Vitellogenin concentrations in plasma measured by a validated enzyme-linked immunosorbent assay (ELISA) were also compared. Two-year-old black porgy (n=20) were fed with estradiol-17 (4 mg kg^–1 of feed). Plasma was collected for purification of vitellogenin. Two forms of vitellogenin were found in plasma after chromatography on Sepharose CL-2B column and hydroxylapatite, and polyacrylamide gel electrophoresis. Black porgy vitellogenins are phospho-lipo-glycoproteins based on their chemical staining properties. The apparent molecular weights of the two forms of vitellogenin were 636 kDa and 321 kDa, respectively. The amino acid composition of purified vitellogenin was also analyzed after acid hydrolysis. The presence of immunoreactive vitellogenin was identified in the plasma and mucus extract from control and estradiol-induced females on the basis of Western blotting. Serial dilution of the plasma and mucus extract taken from estradiol-induced black porgy showed reactivity to an antiserum against lipovitellin in the ELISA, whereas mucus extract and plasma from male fish did not. Significantly higher concentrations of plasma vitellogenin were detected in estradiol-stimulated black porgy than in the control males.     

口虾蛄卵黄蛋白原(Vg)基因的初步研究

为研究口虾蛄Oratosquilla oratoria卵黄蛋白原(vitellogenin,Vg)基因,利用c DNA末端快速扩增(RACE)技术克隆口虾蛄Vg基因c DNA全长序列(Gen Bank登陆号:KR422400),并应用相对实时荧光定量PCR技术检测雌雄口虾蛄不同组织、不同发育时期卵巢Vg mRNA的表达量。结果表明:口虾蛄Vg基因全长7727 bp,其中5'端非编码区为36 bp,开放阅读框(ORF)为7521 bp,3'端非编码区为170 bp,共编码2506个氨基酸;预测的氨基酸序列中存在1个长度为20个氨基酸的信号肽,1个类似于枯草蛋白酶的内切蛋白酶识别位点(RSKR),3个潜在的N-连接糖基化位点,1个在无脊椎和脊椎动物卵黄蛋白原中都比较保守的KALGNVG基序;对其结构域分析表明,口虾蛄Vg蛋白含有4种保守结构域,分别为卵黄蛋白原N端结构域(Vitellogenin_N)、血管性血友病因子(v WFD)和两种未知功能结构域(domain of unknown function)DUF1943与DUF1081;经NCBI BLASTP同源性比较,口虾蛄Vg氨基酸序列与其他甲壳类的相似性为46%~52%;系统发育分析结果显示,口虾蛄Vg单独聚为一支;实时定量PCR结果显示,口虾蛄Vg基因在性腺和肝胰腺中均有表达,在肌肉中均不表达,卵巢中Vg mRNA的表达量显著高于其他组织(P0.05),且口虾蛄Vg mRNA的相对表达量随着卵巢的发育不断增加(P0.05),在成熟期时达到最高值,随后显著下降,Vg mRNA表达量在卵巢中的变化规律可以作为了解口虾蛄性腺发育的有效指标。本研究结果为口虾蛄Vg蛋白的功能研究奠定了基础。     

Improved vitellogenesis,gonad development and egg diameter in catfish (Pangasianodon hypophthalmus) supplemented with turmeric (Curcuma longa) powder

An experiment was conducted to study the effect of supplementing catfish (Pangasianodon hypophthalmus) with turmeric (Curcuma longa) on the vitellogenic capacity of the liver as a new method to improve reproductive performance in oviparous animals. The experimental catfish were assigned to a completely randomized design consisting of four doses of supplemental turmeric (0, 1.2, 2.4 and 4.8 g/kg feed) with 10 catfish with body weights ranging from 2 to 4 kg in each group. Blood samples were collected on days 0, 14, 28, 42 and 56 of turmeric supplementation to measure the concentrations of plasma oestradiol‐17ß and vitellogenin. The weights of the body, gonads and liver were measured on days 0, 28 and 56. The fecundities of the catfish were measured on days 28 and 56 of turmeric supplementation, and egg diameters were measured on days 28 and 42. The results showed that catfish supplemented with turmeric at a dose of 2.4 or 4.8 g/kg feed had higher growth rates, higher plasma oestradiol‐17β and vitellogenin concentrations, and higher gonad somatic index values and egg diameters. This is the first report demonstrating that the hepatoprotective activity of the curcumin in turmeric could be used to improve both vitellogenin synthesis, which improves nutrient deposition in the ovulating eggs, and the reproductive performance of teleost fish and oviparous animals.     

Effects of dietary soy isoflavones on estrogenic activity,cortisol level,health and growth in rainbow trout,Oncorhynchus mykiss

Soy isoflavones (the phytoestrogens genistein, daidzein and glycitein) may act as estrogen receptor agonists or antagonists. The aim of this study was to examine the effect of dietary isoflavones on growth, reproduction and health in rainbow trout (Oncorhynchus mykiss). Rainbow trout fed three experimental diets containing different concentrations of isoflavones (0, 500 and 1,500 ppm). Growth, estrogenic activity, plasma estradiol levels, gonadal development, state of stress and histological changes in selected tissues were evaluated at the end of 70 days. Neither growth performance nor the relative mRNA levels of Insulin Growth Factor I (igf‐I) in the liver were influenced by different levels of dietary isoflavones. Plasma and liver vitellogenin (VTG) protein levels and plasma 17‐β‐estradiol (E₂) were unaffected by treatments, although the correlation between plasma levels of E₂ and VTG densitometry values was significant (p < .05). The fish gonadosomatic index (GSI) did not significantly differ among the three experimental groups but correlated with plasma VTG densitometry values (p < .05). Plasma, muscle and fin cortisol concentrations fell within the normal welfare range and were not correlated with isoflavone levels. Histologically, the distal intestine showed a normal morphology with well‐differentiated enterocytes and in the liver hepatocytes were also normal. A supranuclear accumulation of lipid droplets in enterocytes and some lipid droplets in hepatocytes were observed in all tested groups, suggesting an impact of basal dietary lipid on transport/metabolism of fat in the fish. Overall, the present results suggest that the doses of isoflavones tested do not compromise rainbow trout reproduction, growth and health.