Effects of pituitary glycoprotein hormones and thyroid hormones on in-vitro vitellogenin incorporation into organ-cultured oocytes in the Japanese eel, Anguilla japonica

The objectives of the present study were to establish a long-term culture system for previtellogenic ovarian fragments of the Japanese eel (Anguilla japonica) and to identify the effects of salmon pituitary glycoprotein fraction (SPG), thyroxine (T4), and 3,5,3'-triiodothyronine (T3) on the uptake of vitellogenin (VTG) by cultured ovarian fragments evidenced by the appearance of yolk globules (YGs) within the oocytes. Yolk globules first appeared in the oocytes incubated in media containing only VTG (VTG-only group) after 9 days, whereas YGs began to accumulate in the oocytes of ovarian fragments cultured in media containing VTG+SPG (SPG group) following only 3 days of incubation. Furthermore, the occurrence of vitellogenic oocytes (%VO) and proportion of YGs within oocytes (%YG area) were significantly higher in follicles cultured in 30 ng/ml SPG throughout the culture period. No such stimulatory effects of T4 on VTG uptake were observed. Incubation of ovarian fragments with VTG and T3 (T3 group; 50 ng/ml) resulted in an increased %VO compared to follicles in the VTG group by day 9 of culture, and from day 10 onwards, both %VO and %YG area became significantly higher in follicles of the T3 group. Interestingly, SPG stimulated VTG incorporation and YG accumulation even in small oocytes (approximately 150 microm), whereas T3 showed these effects only in larger sized oocytes (> 180 microm). These results suggest that both SPG and T3 can accelerate VTG incorporation, but the mechanisms whereby this is achieved may differ between these hormone preparations.     

Potentiation of epinephrine-induced lipolysis in fat cells from estrogen-treated rats

We investigated concomitantly the effects of ethinyl-estradiol (EE₂) at low dose (1.2 μg/animal for 10 days) and high dose (120 μg/animal for 3 days) on body weight, weight of fat stores, triglyceridemia and fat cell lipoprotein lipase and hormone-sensitive lipase activities in female rats. At low dose, EE₂ increased triglyceridemia and lipoprotein lipase activity, whilst high dose decreased both parameters. At low and high doses, and regardless of whether triglyceridemia and lipoprotein lipase activity were increased and decreased, EE₂ caused depletion in fat stores. Fat cells isolated from depleted fat tissue elicited marked increases in the response of hormone-sensitivelipase to epinephrine. Taken together, the data suggest that the potentiation of epinephrine-induced lipolysis in fat cells is likely to represent a major cause to estrogen-induced fat depletion.     

Effects of exogenous estradiol-17β on early growth and gonadal development of diploid and triploid female rainbow trout (Oncorhyncus mykiss)

Triploidy is a viable condition in teleosts. However, in many salmonids, the triploid condition in the female results in sterility as gametogenesis appears to be disrupted. Although the underlying mechanisms regulating the gonadal development of teleosts have not been clearly elucidated, the reversal of phenotypic sex by the administration of the appropriate exogenous steroid during early development supports the argument that gonadal steroids play a pivotal role in sexual differentiation and subsequent gonad development in these fish. To determine whether the failure of normal ovarian development in triploid female rainbow trout (Oncorhynchus mykiss) is due to an absence or reduction of endogenous sex steroids, ovarian morphology was compared between diploid and triploid juvenile rainbow trout treated with exogenous estradiol-17β (E₂). The ovaries of both untreated and E₂ treated diploid fish, at 145 days post-fertilization, contained synchronously developing oocytes in the perinucleolar stage, whereas ovaries from untreated and estradiol-treated triploid fish of the same age were considerably smaller and devoid of developing oocytes. No differences in the ovaries of triploid untreated fish and triploid fish treated with E₂ were observed. It is reported that exposure to exogenous E₂ during the period of gonadal differentiation is not sufficient to induce oocyte development in triploid rainbow trout. © 1996 Wiley-Liss, Inc.     

埋植雌二醇对养殖中华鲟雌性幼鱼血清生理指标的影响

用硅橡胶作载体,将剂量30 mg/kg的雌二醇(estradiol)埋植入人工养殖11龄、平均体重46.00 kg的雌性中华鲟(Acipenser sinensis)的背部肌肉内。埋植前及埋植后的第30、120、200天从尾部取血,测定血清中总蛋白、白蛋白、球蛋白、甘油三酯、总胆固醇、血糖和卵黄蛋白原的含量。结果显示,与对照组相比,除血糖含量无显著变化外,实验组鱼血清中总蛋白、白蛋白、球蛋白、甘油三酯、总胆固醇和卵黄蛋白原的含量显著升高。此结果为进一步探索外源物质促进中华鲟卵巢发育的可能途径奠定前期基础。     

Ovarian development and serum sex steroid and vitellogenin profiles in the female cultured sturgeon hybrid, the bester

Changes in serum levels of estradiol-17/J (E₂), testosterone (T), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and vitellogenin (VTG), in cultured adult female bester were examined in relation to ovarian development during a 1-year sampling period. Considerable variations in oocyte development were found among fish. Oocytes at previtellogenic stage (≥0–6mm in diameter) generally started to develop concomitantly with the degeneration of the first batch of oocytes. In vitellogenic individuals, ovaries were comprised of more advanced oocytes with diameter ranging from 0–6 to 2–6 mm and in the post-vitellogenic class, oocytes attained their largest size (>2–6mm) while the germinal vesicle was migrating towards the animal pole. Oocytes with a migrating nucleus were maintained during the winter period and massive degeneration started in April–May without germinal vesicle breakdown (GVBD) or ovulation occurring. Seasonal changes in E₂, T and VTG levels were well correlated with the advancement of oogenesis. Their levels increased during vitellogenesis, whereas in the post-vitellogenic (migratory nucleus) stage the levels of E₂ declined from 2–4 ng ml ^?1± to 1–2 ng ml ^?1 and VTG from 4–10 mg ml ^?1 to 0.–0.5 mg ml ^?1 while T levels remained high (50–60 ng ml ^?1). In contrast, serum levels of 17,20?-P were constantly low (less than 0.2 ng ml^?L) throughout the reproductive cycle. These results indicate that the time appropriate for induction of artificial reproduction would be from October–November to April–May when the oocytes are in the late Stages of the development.     

Immunochemical detection of precursor proteins of yolk and vitelline envelope, and their annual changes in the blood of Diodon holocanthus

Two distinct groups of female-specific proteins, vitellogenin (VTG) and vitelline envelope proteins (VEP), were detected in the blood of the porcupine fish Diodon holocanthus , and annual changes in concentration were measured immunochemically. Using antisera against yolk proteins (ab.a-E) and VEP (ab. a-VEP), VTG and VEP could be detected in the blood of maturing female fish and oestradiol-17β (E₂)-treated fish. Neither protein was detected in the blood of male fish. Immunohistochemistry showed that yolk globules and the vitelline envelope enclosing developing oocytes stained with ab.a-E. The vitelline envelope was stained specifically with ab.a-VEP. Hepatocytes from the E2-treated fish had immunoreactivity with both antisera. Thus, VTG and VEP appear to be synthesized in the liver by direct stimulation of E₂, released into the circulation, and incorporated into respective target sites. VTG and VEP in female serum maintained high levels from April until June, suggesting that yolk accumulation, as well as vitelline envelope formation, are occurring actively during these months. Unlike VTG, small amounts of VEP were detected between December and March, suggesting that vitelline envelope formation precedes yolk accumulation and that a slightly different hormonal regulation exists in the synthesis of both proteins in the liver during the early phase of oogenesis.     

Effects of indomethacin and prostaglandins on the spawning behaviour of female goldfish

In goldfish, injection of ovulated eggs (from donor females) through the ovipore and into the ovarian lumen of females with vitellogenic oocytes induces spawning behaviour within several hours. Intraperitoneal (i.p.) injection of indomethacin (IM), 10 μg/g, either 10 h prior to, or coincident with, injection of ovulated eggs, completely inhibits the onset of spawning behaviour. IM injection similarly terminates ongoing spawning behaviour induced by egg injection. PGF_2α (5 μg/g; i.p. injection) restores spawning behaviour of egg-injected, IM-blocked fish; at the same dosage, PGE₂ is marginally effective and PGE₁ is without effect. As PGF_2α and PGE₂ also induce spawning behaviour in females which have not been injected with ovulated eggs, it is suggested that distension of the oviduct following ovulation or egg injection results in the release of PG which then acts in some way to induce spawning behaviour. The ability of PG to induce spawning behaviour is eliminated by hypophysectomy and restored by treatment with salmon gonadotropin: no steroid treatment was effective in restoring PG-induced spawning in fish which had been hypophysectomized for 3–4 months. The possible mode of action of PG in inducing spawning behaviour in female goldfish is discussed.     

Cloning and characterization of vitellogenin cDNA from the common Japanese conger (Conger myriaster) and vitellogenin gene expression during ovarian development

The major yolk protein precursor, vitellogenin (VTG) was detected in plasma from vitellogenic females and estradiol-17β (E₂)-treated immature females, but not in males and immature females by Western blotting in common Japanese conger Conger myriaster. Its molecular mass was approximately 180 kDa under denaturing and reducing conditions. The common Japanese conger VTG cDNA was cloned from the liver of vitellogenic female. It contains 5110 nucleotides including an open reading frame that encodes 1663 amino acids. The deduced amino acid sequence of the common Japanese conger VTG shares 80% identity with that of eel Anguilla japonica VTG-1, and 45–55%, 32–34% and 27–29% identity with the deduced amino acid sequences of other fish, amphibian and avian VTG with polyserin domain, respectively. In female common Japanese conger, VTG gene was highly expressed in the liver of this species similar with other oviparous vertebrates. The expression levels of VTG gene in the liver increased from the oil droplet stage to the tertiary yolk globule stage and were maintained until the migratory nucleus stage.     

Evidence of cylindrospermopsin uptake and clearance in fish (Oreochromis niloticus) under laboratory conditions

Cylindrospermopsin (CYN) is a cyanotoxin that has raised serious concerns about public health in many parts of the world. It can bioaccumulate and affect the health of aquatic organisms, but despite this, few studies have been conducted on CYN uptake and clearance in fish. In this paper, the authors evaluate the uptake and clearance of CYN in the muscle tissue and viscera of juvenile tilapia (Oreochromis niloticus) after exposure to aqueous extracts and whole cells of Cylindrospermopsis raciborskii (CYN-producer). CYN blended with commercial fish food, and three experiments were conducted. In the first trial, fish food, and aqueous extracts containing 0.31 μg CYN g⁻¹ of food per day, was administered to tilapia for 15 days. In the second trial, fish were provided food and intact cells (5.4 μg CYN g⁻¹ of food per day) for 15 days. In the last trial, they were provided fish food and aqueous extracts (0.8 μg CYN g⁻¹ of food per day) for 12 days, and for the next 10 days, the animals were fed food without toxic cell extracts (to simulate a clearance period). The concentration of CYN in muscle tissue and viscera was analysed using ELISA. In the case of juvenile tilapia, the presence of CYN was higher in viscera than in muscle tissue, and the toxin remained in the tissues even after 10 days without the addition of contaminated food. The results suggest that tilapia represents a potential source of CYN transfer through the food web, and this shows the need for a continuous monitoring of this compound in organisms that are used for human and animal consumption.     

Effects of temperature and photoperiod on ovarian recrudescence in the cyprinid fish Mirogrex terrae-sanctae

Mirogrex terrae-sanctae is a commercial, sardine-like cyprinid fish in the Sea of Galilee. The fish spawns from December to the end of March. Females collected at the quiescent phase of the cycle (June) were exposed to four combinations of photoperiod and temperature for 3 months. The highest GSI (2.44 ± 0.22) was found in females exposed to simulated winter conditions (16°C, 9L) while the lowest GSI (0.65 ± 0.04) was found in fish exposed to simulated summer conditions (27°C, 14L). The GSI of fish maintained at the unnatural combination of 16°C, 14L (1.30 ± 0.09) did not differ from that of fish maintained at the other unnatural combination of 27°C, 9L (1.07 ± 0.11). However, histological examinations revealed that vitellogenic oocytes were present only in fish maintained at low temperature regimes, which implies that the growth of the ovarian mass in the 27°C, 9L group was due mainly to oogonial proliferation and that vitellogenesis was inhibited in Mirogrex at a high temperature. Both protein and calcium concentrations increased in the serum of fish following injections of oestradiol-17β. The increase of calcium was evident only in the non-filtrable (bound) fraction, suggesting that the total serum calcium above 13 mg% could be used as an indirect parameter for circulating vitelloprotein. Fish collected at the quiescent phase were injected with oestradiol (50 μg/fish c. 15 g body weight) and subsequently transferred to tanks maintained at either 12°, 20° or 27°C. The total serum calcium increased to 23.0 ± 1.75 mg% in the fish maintained for 8 days at 20°C. This was significantly higher than the concentration of calcium in the serum of fish maintained at either 12°C (7.8 ± 0.52) or 27°C(l1.8 ± 0.77). This implies that the temperature may affect ovarian recrudescence by modulating the response of the liver to oestrogen, allowing the oestrogen-induced vitellogenesis to be relatively high in the cool environment during winter.     

Age-Associated Lipidome Changes in Metaphase II Mouse Oocytes

The quality of mammalian oocytes declines with age, which negatively affects fertilization and developmental potential. The aging process often accompanies damages to macromolecules such as proteins, DNA, and lipids. To investigate if aged oocytes display an altered lipidome compared to young oocytes, we performed a global lipidomic analysis between oocytes from 4-week-old and 42 to 50-week-old mice. Increased oxidative stress is often considered as one of the main causes of cellular aging. Thus, we set up a group of 4-week-old oocytes treated with hydrogen peroxide (H₂O₂), a commonly used oxidative stressor, to compare if similar lipid species are altered between aged and oxidative-stressed oocytes. Between young and aged oocytes, we identified 26 decreased and 6 increased lipids in aged oocytes; and between young and H₂O₂-treated oocytes, we identified 35 decreased and 26 increased lipids in H₂O₂-treated oocytes. The decreased lipid species in these two comparisons were overlapped, whereas the increased lipid species were distinct. Multiple phospholipid classes, phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylserine (LPS) significantly decreased both in H₂O₂-treated and aged oocytes, suggesting that the integrity of plasma membrane is similarly affected under these conditions. In contrast, a dramatic increase in diacylglycerol (DG) was only noted in H₂O₂-treated oocytes, indicating that the acute effect of H₂O₂-caused oxidative stress is distinct from aging-associated lipidome alteration. In H₂O₂-treated oocytes, the expression of lysophosphatidylcholine acyltransferase 1 increased along with increases in phosphatidylcholine. Overall, our data reveal that several classes of phospholipids are affected in aged oocytes, suggesting that the integrity of plasma membrane is associated with maintaining fertilization and developmental potential of mouse oocytes.     

性类固醇激素对胡子鲇性分化的影响

研究采用组织学和荧光实时定量PCR方法,检测17-甲基睾酮（17-MT）和17-雌二醇（17-E2）对胡子鲇（Clarias fuscus）性腺组织学、性别比率以及性分化前后脑型芳香化酶基因（Cyp19a1b）和翼状螺旋/叉头转录因子2（Foxl2）基因表达的影响。结果表明:在出膜后230日龄,17-MT （50、100和200 g/L）浸浴、17-E2（100、200和300 g/g）投喂处理对成活率无显著影响,但中、高剂量（100和200 g/L）17-MT显著抑制卵巢发育,促进精巢发育,卵巢腔出现时间分别推迟4d和6d,初级卵母细胞出现时间分别推迟8d和9d,而初级精母细胞出现时间则分别提前3d和5d,且雄性率分别达70%和76%,显著高于50 g/L组和对照组（P0.05）。相反,中、高剂量17-E2（200和300 g/g）处理使卵巢腔出现时间分别提前2d和3d,初级卵母细胞出现时间分别提前1d和3d,初级精母细胞出现时间分别推迟3d和7d,而雌性率分别达74%和78%,显著高于100 g/g组和对照组（P0.05）。此外,在性腺分化期,17a-MT促进Cyp19a1b但抑制Foxl2的表达,而17-E2促进Cyp19a1b和 Foxl2的表达。结果显示Cyp19a1b不是引起胡子鲇性分化的直接因素,但可能通过下丘脑-垂体-性腺轴对胡子鲇性分化过程产生间接影响;而Foxl2直接参与胡子鲇的性分化,即17a-MT和17-E2分别通过抑制和促进Foxl2的表达来影响雌激素的生物合成,从而调控胡子鲇性分化的方向。       

Vitellogenin Incorporation into Oocytes of Rainbow Trout, Oncorhynchus mykiss, in Vitro: Effect of Hormones on Denuded Oocytes

An in vitro culture procedure to measure vitellogenin (VTG) incorporation into oocytes without follicle cell layers was developed using oocytes of the rainbow trout, Oncorhynchus mykiss . Oocytes incorporated VTG specifically and linearly for up to 24 hr. The maximum incorporation observed was 314 μg/24 hr/oocyte, using vitellogenic (3.6 mm diameter) oocytes.
The effect of hormones was examined by adding insulin, growth hormone, prolactin, gonadotropins (GTH-I, GTH-II), thyroid hormones, testosterone, estradiol-17β, or 17α, 20β-dihydroxy-4-pregnen-3-one to the medium. The results indicated that insulin and thyroxine stimulated uptake of VTG by 13% and 12%, respectively. Insulin specifically stimulated VTG incorporation and did not cause any change in background uptake of insulin. The lack of an effect of gonadotropins hormones on denuded oocytes suggests that the previously observed stimulation of VTG incorporation into follicle cell-enclosed oocytes in vivo and in vitro by GTH-I is most likely mediated by the somatic cells of the ovarian follicle.     

Effects of temperature and fish oil supplementation on ovarian development and foxl2 mRNA expression in spotted scat Scatophagus argus

In this study, the complete foxl2 complementary (c)DNA sequence was isolated by simple modular‐architecture research tool (SMART)er rapid amplification of cDNA ends (RACE). Two year‐old female spotted scat, Scatophagus argus, were reared at different temperatures (23, 26 and 29° C) for 6 weeks, or fed with different concentrations of dietary fish oil (0, 2 or 6%) for 8 weeks. Ovarian development, serum oestradiol‐17β (E₂) levels, as well as ovarian foxl2 expression were measured. At the end of experiment, ovarian foxl2 messenger (m)RNA expression in fish reared at 23 and 26° C was significantly higher than that in fish reared at 29° C, and that in 2 and 6% fish oil groups was also significantly higher than that in control group (P < 0·05). Serum E₂ levels exhibited the same trend with foxl2 mRNA expression in temperature treatment groups and fish oil fed groups. There was a significant positive correlation between stage of oocytes and foxl2 expressions. Results showed that from 23 to 29° C, the optimal temperature for ovarian development in S. argus was 23–26° C, and 6% fish oil supplementation could effectively promote ovarian development. Optimal temperature and fish oil supplement might increase ovarian foxl2 mRNA expressions to promote ovarian development in S. argus.     

Hormonal stimulation of maturation and ovulation of oocytes in Zebrasoma scopas (Acanthuridae)

The experiments were carried out on hormonal stimulation of oocyte maturation in Zebrasoma scopas from the South China Sea, Vietnam. Three variants of surfagon injections were studied: 1—double injections (5 + 20 μg/kg of fish body weight); 2—double injections (2 + 8 μg/kg); and 3—single injections (20 μg/kg). The time interval between two injections comprised 15–24 h. Ovulation of oocytes in variants 1 and 2 was observed in most (67%) females 33–47 h after the first injection. The increase of the time interval between injections I and II was followed by the decrease of the interval between injection II and ovulation. In variant 3, oocytes ripened but ovulation was absent. The oocytes possessed with the competence for maturation are always present in the ovaries because of a continuous type of oogenesis. The morphological changes in oocytes in the process of maturation were observed. Ovulated oocytes could be stored in the ovary cavity no more than 4 h; the number of embryos with normal cleavage decreased during this time from 90 to 53%.     

Combined and Singular Effects of Dietary PrimaLac® and Potassium Diformate (KDF) on Growth Performance and Some Physiological Parameters of Rainbow Trout (Oncorhynchus mykiss)

The present study was conducted to assess the effects of combined and singular dietary administration of PrimaLac® and potassium diformate (KDF) on growth performance, feed utilization, digestive enzymes activity, and some physiological parameters of rainbow trout (Oncorhynchus mykiss) juvenile. Three hundred sixty rainbow trout juveniles (25 ± 1.8 g) were randomly stocked in 300-L tanks (30 fish/tank), and fed three times daily on a basal diet (control), diets incorporated with 12 g kg⁻¹ KDF (FT₁), 1.5 g kg⁻¹ PrimaLac® (FT₂), and combination of 1.5 g kg⁻¹ probiotic and 12 g kg⁻¹ KDF (FT₃) in triplicates, for 8 weeks. At the end of feeding trial, growth performance, body composition, digestive enzymes, liver enzymes, and biochemical parameters were measured. Our results revealed that combined administration of PrimaLac® and KDF (FT₃) exhibited significantly higher weight gain and specific growth rate (SGR) compared to other groups (P < 0.05). Glucose and cortisol levels showed no significant differences between fish fed different test diets (P > 0.05). The highest lipase, protease and amylase activity were observed in group of fish fed FT₃ followed by FT₂ and FT₁. Besides, the diets FT₂ and FT₃ led to significantly lower of ALP, ALT, and AST compared to control group. The present results indicated that combined administration of PrimaLac® and KDF can be considered as a beneficial feed additive and growth promotor for O. mykiss juvenile.

     

Use of deuterium oxide (2H2O) to assess muscle protein synthesis in juvenile red drum (Sciaenops ocellatus) fed complete,and valine-deficient diets

The use of ²H₂O in tank water to assess protein synthesis rates in fish is a relatively novel methodology that could allow for a better understanding of the effects of particular nutritional and environmental variables on rates of protein accretion. As such, this study involved an assessment and comparison of protein synthesis rates in the muscle of juvenile red drum fed a control diet (nutritionally complete) versus a valine (Val)-deficient diet. Six groups of 12 juvenile red drum, initially weighing ~ 4.5 g/fish, were stocked in six separate 38-L aquaria operating as a recirculating system. Fish were acclimatized to experimental conditions for 2 weeks while being fed the control diet. Just prior to initiating the protein synthesis assay, one aquarium of fish was fed the control diet while a second aquarium of fish was fed the Val-deficient diet. Immediately after consuming the experimental diets, each group of fish was moved to an independent aquarium containing ²H₂O, and the fractional synthetic rate (FSR) of protein synthesis was obtained at 12, 24, 36 and 48 h after feeding by collecting two fish per treatment at each time point. This protein synthesis assay procedure was performed in three separate sessions, and considered as replicates over time (n = 3) for fish fed the control or Val-deficient diets immediately before initiating the session. Results indicated that a one-time feeding of a diet deficient in Val significantly reduced protein synthesis rates in the muscle of red drum. In addition, a significant effect of time after feeding was found, where observed FSR values peaked at 12 h after feeding and decreased as time progressed. In conclusion, deuterium methodologies were applicable to red drum, and this approach had the sensitivity to assess differences in protein synthesis rates when dietary perturbations were introduced.

     

Effects of dietary estradiol-17β on feminization,growth and body composition in the Japanese eel (Anguilla japonica)

• 1.1.Juvenile Japanese eels (Anguilla japonica) were fed on a diet supplemented with estradiol-17β (E₂) at doses of 25, 50 and 75 mg/kg. The effects on growth, sex distribution and body composition were investigated in two groups of gonadally undifferentiated stages (early and later juvenile stages).
• 2.2.Feminization (95–100%) was observed in all E₂-treated groups.
• 3.3.The growth rate of fish treated with 25 and 50 mg/kg E₂ diet at the early juvenile stage was significantly increased.
• 4.4.The amount of protein in muscle decreased and that of fat increased in the E₂-treated groups except in the early juvenile stage fed with 25 mg/kg E₂.