The effects of calcitonin on plasma calcium levels and bone metabolism in the fresh water teleost Channa punctatus

Administration of salmon calcitonin (sCT) caused significant reduction in total and ultrafiltrable plasma calcium content in the plasma of a fresh water female teleost Channa punctatus. A time-bound analysis on the effect of sCT showed a highly significant short duration reduction in total and ultrafiltrable plasma calcium content in fish kept in normal tap water and low-calcium water and a moderate hypocalcemia in fish kept in high-calcium water. Sexually immature adult fish showed a greater response than the sexually mature ones. Using tartrate-resistant acid phosphatase (TRACP) and alkaline phosphatase (ALP) activities in plasma and hydroxyproline (HYP) excretion in urine, the effect of sCT on the inhibition of bone calcium resorption were examined. In both sexually mature and immature adult fish, kept in normal tap water, sCT significantly suppressed TRACP and ALP activities in plasma and excretion of HYP in urine within 2-6 h with a maximum at 4 h after injection. Salmon CT treatment to sexually immature adult fish caused significant increase in skeletal bone calcium concentration. Taken together, all this information indicates that CT in a fresh water female teleost is an effective regulator of plasma calcium levels, and its action, at least in part, operates through inhibition of bone calcium resorption.     

Tributyltin inhibits osteoblastic activity and disrupts calcium metabolism through an increase in plasma calcium and calcitonin levels in teleosts

To examine the direct effects of tributyltin acetate (TBTA) on osteoclasts and osteoblasts, teleost scale, which has both osteoclasts and osteoblasts and is similar to mammalian membrane bone, was used in the present study. The activities of tartrate-resistant acid phosphatase and alkaline-phosphatase, as respective indicators of activity in both cells, were used. In freshwater teleost (goldfish) and marine teleosts (nibbler and wrasse), the osteoclastic activity in the scales did not change as a result of TBTA treatment (10(-9) to 10(-5) M). However, the osteoblastic activity decreased in the goldfish, nibbler, and wrasse after 6 h of incubation. In goldfish, even 10(-10) M of TBTA significantly inhibited the osteoblastic activity. The inhibitory activity in goldfish was stronger than that in nibbler and wrasse. Therefore, details of the mechanism were examined using goldfish. The mRNA expressions of the estrogen receptor and insulin-like growth factor-I, which participate in osteoblastic growth and differentiation, decreased in the TBTA-treated scales. However, the mRNA expression of metallothionein (MT), a metal-binding protein that protects the organism from heavy metal, increased much less than those of cadmium and methyl-mercury. Furthermore, we showed that the plasma calcium and hypocalcemic hormone (calcitonin) level increased in goldfish kept in water containing TBTA (10(-10) and 10(-8) M). The current data are the first to demonstrate that, in teleosts, TBTA inhibits osteoblastic activity without affecting osteoclastic activity and disrupts the calcium metabolism, including the calcemic hormone, in goldfish.     

Testosterone and estradiol potentiate the serum gonadotropin response to gonadotropin-releasing hormone in goldfish

The effects of gonadal steroids on gonadosomatic index (GSI; gonad wt/total body wt x 100), pituitary gonadotropin (GTH) content, and serum GTH response to [D-Ala6,Pro9-Net]-luteinizing hormone-releasing hormone (LHRH-A) were investigated throughout the seasonal reproductive cycle of the goldfish. Gonad-intact female fish were implanted i.p. for 5 days with silastic pellets containing no steroid (blank), testosterone (T; 100 micrograms/g), or estradiol (E2; 100 micrograms/g). The serum GTH response at 6 h following i.p. injection of saline or 0.1 microgram/g LHRH-A was assessed. In blank-implanted, saline-injected animals, seasonal variations in GSI, pituitary GTH content, and serum GTH levels were evident; maximal and minimal levels were noted in the spring and summer months, respectively. In blank-implanted fish, LHRH-A effectively stimulated GTH release in females undergoing gonadal recrudescence (late autumn and winter) and in sexually mature (spring) females, but not in sexually regressed (summer and early autumn) females. Implantation of T or E2 raised serum steroid levels to those found during ovulation in goldfish. Steroid treatments did not affect unstimulated serum GTH levels at any time of the year. Testosterone effectively potentiated the serum GTH response to LHRH-A during the entire reproductive cycle, whereas the positive effects of E2 were evident in sexually regressed and post-spawning females only. Both T and E2 potentiated the GTH response to LHRH-A in male fish. To examine the involvement of T aromatization in mediating its actions on induced GTH secretion, male and female fish were implanted with T or the nonaromatizable androgens 5 alpha-dihydroxytestosterone (DHT; 100 micrograms/g) and 11-keto-testosterone (11-KT; 250 micrograms/animal). Testosterone potentiated the GTH response to LHRH-A in both males and females whereas DHT and 11-KT were without effect. Furthermore, the positive action of T on induced GTH secretion was blocked by 2-day pretreatment with the aromatase inhibitor 1,4,6-androstatrien-3,17-dione (100 or 300 micrograms/g). Multiple i.p. injections of hCG (0.2 microgram/g every 3 days for 39 days), probably through stimulation of endogenous T secretion, resulted in potentiation of the GTH response to LHRH-A in mature male goldfish. These results clearly demonstrate that T, through aromatization to E2, can increase pituitary responsiveness to exogenous LHRH-A in gonad-intact male and female goldfish.     

Thyrotropin Releasing Hormone (TRH) in goldfish (Carassius auratus): role in the regulation of feeding and locomotor behaviors and interactions with the orexin system and cocaine- and amphetamine regulated transcript (CART)

TRH is a peptide produced by the hypothalamus which major function in mammals is the regulation of TSH secretion by the pituitary. In fish, TRH does not appear to affect TSH secretion, suggesting that it might regulate other functions. In this study, we assessed the effects of central (intracerebroventricular, icv) injections of TRH on feeding and locomotor behavior in goldfish. TRH at 10 and 100 ng/g, but not 1 ng/g, significantly increased feeding and locomotor behaviors, as indicated by an increase in food intake and in the number of total feeding acts as compared to saline-injected fish. In order to assess possible interactions between TRH and other appetite regulators, we examined the effects of icv injections of TRH on the hypothalamic expression of orexin, orexin receptor and CART. The mRNA expression levels of all three peptides were significantly increased in fish injected with TRH at 100 ng/g as compared to saline-injected fish. Fasting increased TRH, orexin, and orexin receptor hypothalamic mRNA levels and decreased CART hypothalamic mRNA levels. Our results suggest that TRH is involved in the regulation of feeding/locomotor activity in goldfish and that this action is associated with a stimulation of both the orexin and CART systems.     

Relationship between seasonal plasma estradiol-17 beta and testosterone levels and in vitro production by ovarian follicles of amago salmon (Oncorhynchus rhodurus)

Plasma estradiol-17 beta and testosterone levels were assessed by radioimmunoassay during the sexual maturation of female amago salmon (Oncorhynchus rhodurus). Estradiol-17 beta levels gradually increased during vitellogenesis (June to September), reached a peak in September (about 16 ng/ml) and rapidly decreased in mature and ovulated fish (about 3-4 ng/ml) in October. The seasonal pattern of plasma testosterone levels lagged behind and followed that of estradiol-17 beta during vitellogenesis, but levels remained high in mature and ovulated fish (90-110 ng/ml). Estradiol-17 beta levels and the gonadosomatic index (GSI) values correlated well during vitellogenesis: GSI values showed a linear increase, and reached a peak (29.9 +/- 1.4) in October. Values were extremely low in ovulated fish (1.2 +/- 0.2). In vitro production of estradiol-17 beta and testosterone by ovarian follicles in response to partially purified chinook salmon gonadotropin (SG-G100) was examined monthly using 18-h incubations. Throughout the vitellogenic period SG-G100 stimulated both estradiol-17 beta and testosterone production: the steroidogenic response of follicles increased from June (about 2 ng/ml estradiol-17 beta; 0.1 ng/ml testosterone) to September (about 10 and 14 ng/ml, respectively). In October full-grown immature follicles which could be induced to mature in vitro by hormone treatment produced large amounts of testosterone (about 130 ng/ml) but not estradiol-17 beta. Postovulatory follicles also produced testosterone but the values were low (10 ng/ml) compared with full-grown immature follicles. Very low levels of estradiol-17 beta were produced by postovulatory follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ketoconazole Inhibits the Growth and Development of Ichthyophonus sp. (Mesomycetozoa) In Vitro

ABSTRACT. We determined the in vitro effect of the azol-derivative antifungal ketoconazole (KZ) on the morphology, growth, and development of teleost fish parasite Ichthyophonus sp. The KZ was delivered to culture medium using liposomes (L) or a lipid emulsion (E) at five different doses (i.e. 5, 50, 100, 200, and 400 μg/ml) for both L and E formulations. Controls consisted of Eagle's minimum essential medium (MEM) supplemented with 10% foetal bovine serum (MEM-10) alone (C-MEM) or containing amounts of L or E equivalent to those used in the KZ100 and KZ400 treatments (i.e. 100L, 400L, 100E, and 400E, respectively). Morphological alterations, such as a decrease in the number of dividing spores and nuclei, and condensation or even destruction of the cytoplasm, were observed using light and electron microscopy in the MEM-cultured organisms receiving KZ formulations, especially with KZ400L preparations, at both 7- and 14-d postinoculation. The KZ treatments also demonstrated a statistically significant inhibition of Ichthyophonus growth in MEM. These treatments also had an inhibitory effect on subsequent Ichthyophonus germination in Earle's fish saline agar (EFSA) medium, which was more evident for L formulations when the organism was treated for 7 d and for E formulations at 14 d. Our results endorse the potential use of KZ for the treatment for ichthyophonosis and provide support to proceed to in vivo assays.     

Investigation of de novo cholesterol synthetic capacity in the gonads of goldfish (Carassius auratus) exposed to the phytosterol beta-sitosterol

Total and intra-mitochondrial gonadal cholesterol concentrations are decreased in fish exposed to the phytoestrogen beta-sitosterol (beta-sit). The present study examined the potential for beta-sit to disrupt de novo cholesterol synthesis in the gonads of goldfish exposed to 200 microgram/g beta-sit and 10 microgram/g 17beta-estradiol (E2; estrogenic control) by intra-peritoneal Silastic^® implants for 21 days. The de novo cholesterol synthetic capacity was estimated by incubating gonadal tissue with 14C-acetate for a period of 18 hours, followed by chloroform/methanol lipid extraction and thin layer chromatography (TLC) lipid separation. Lipid classes were confirmed using infrared spectroscopy. Plasma testosterone (T) and total cholesterol concentration were measured and gonadosomatic index (GSI) was calculated. Plasma T was significantly reduced in male beta-sit-treated fish compared to control and E2-treated fish (p < 0.001). 14C-Acetate incorporation into cholesterol and cholesterol esters was not significantly different among treatment groups for male and female fish, however, 14C-enrichment was higher than expected in both triglycerides (TG) and free fatty acids (FFA). FFA incorporation was significantly higher in male control fish than either beta-sit or E2 treatments (p = 0.005). Plasma cholesterol concentration was significantly increased in the male beta-sit treatment group compared to controls (p = 0.027). These results indicate gonadal de novo cholesterol biosynthetic capacity is not disrupted by beta-sit or E2 treatment in early recrudescing male or female goldfish, while plasma cholesterol and steroid concentrations are sensitive to beta-sit exposure.     

Opposing effects of prostaglandin E(2)and F(2 alpha) on rat liver-associated natural killer cell activity in vitro

Strain differences in cancer incidence are proposed to be due partly to differences in immune function. As potential cancer-associated immunological regulators, the concentrations of hepatic prostaglandins E(2)(PGE(2 alpha)and F(2 alpha)(PGF(2 alpha)) were compared in 9-week-old male and female F344/N and Sprague-Dawley (SD) rats. There were no strain or gender differences in the concentrations of hepatic PGE(2). No strain difference was found in the concentration of hepatic PGF(2 alpha), but the hepatic PGF(2 alpha)concentration in female rats was two-fold that of the male rat (130 vs 60 ng/g). PGE(2)significantly inhibited hepatic natural-killer cell (NK) activity in vitro compared with untreated cells from both genders and strains (P<0.05), 25 ng PGE(2)/ml inhibited NK activity significantly more than did 10 ng PGE(2)/ml (P<0.05). In contrast, 50 ng PGF(2 alpha)/ml and 100 ng PGF(2 alpha)/ml significantly stimulated hepatic NK activity compared with untreated hepatic cells from both F344/N and SD rats. This study suggests that prostaglandins may have a negligible net effect on NK activity associated with rat liver, and may be unlikely to mediate cancer-related immune function.     

Plasma estrone sulphate (E1S) and estradiol-17beta (E2beta) profiles during pregnancy and their relationship with the relaxation of sacrosciatic ligament, and prediction of calving time in Holstein-Friesian cattle

The objectives of this study were to investigate the plasma E(1)S and E(2)beta profiles during pregnancy and their relationship with the relaxation of sacrosciatic ligament in Holstein-Friesian cattle (n=37) and then to predict the calving time on the basis of E(1)S and E(2)beta profiles and relaxation of the ligament. Blood samples were collected at 4 weeks intervals from days 100 to 190, at 2 weeks intervals from days 190 to 250, every week from days 250 to 270 and thereafter every day from day 270 of gestation until the day after calving. The relaxation in the ligament was measured by using two scales as a distance at a schedule similar to blood sampling plus 5 days postpartum. One scale was kept firm exactly parallel to the ligament between the sacrum and the tuber ischii and other scale was erected perpendicularly to the first scale with the bottom just touching the ligament and the depth was measured in the second scale from the point where it touched the ligament to the point where it touched the first scale. Plasma samples were analyzed for E(1)S and E(2)beta by enzyme immunoassay. E(1)S concentration was low at day 100 (0.8+/-0.3 ng/ml), then increased progressively and drastically to reach the level of 28.4+/-3.6 ng/ml on the day before calving and declined significantly (p<0.05) at 9.5+/-3.1 ng/ml within 1 day postpartum. There was a gradual increase in concentration of E(2)beta from day 100 of gestation (0.1+/-0 ng/ml) until day 4 prepartum (0.6+/-0 ng/ml). Thereafter, it increased drastically and reached the peak level of 1.0+/-0.1 ng/ml (p<0.05) on the day before calving and declined markedly at 0.4+/-0.1 ng/ml within 1 day postpartum (p<0.05). Corresponding to E(1)S and E(2)beta concentrations, a gradual increase in the relaxation of the ligament was observed from day 100 of gestation (8+/-1mm) until day 2 prepartum (24+/-2mm). Thereafter, it showed a significant increase (p<0.05) within 1 day before calving (31+/-2mm) and almost no difference between day 1 prepartum and day 1 postpartum. A marked decrease (p<0.05) was observed thereafter until day 3 postpartum (10+/-2mm) and no significant change between days 3 and 4 as well as 4 and 5 postpartum. The increment of E(2)beta by >or=0.20 ng/ml from the preceding day concentration was 85.2% accurate for predicting calving within 24h in many of the cows (23 of 37) in the herd. The increment in ligament relaxation measurement by >or=5mm from the preceding day measurement was the most efficacious to predict calving within 24h with the highest accuracy (93.9%) in high proportions of cows (31 of 37) in the herd. In conclusion, plasma E(1)S and E(2)beta concentrations and relaxation of sacrosciatic ligament increased gradually as gestation advanced and reached the peak level on the day before calving. The relaxation in the ligament corresponded well to plasma E(2)beta concentrations. Prediction of calving was possible by E(2)beta profile and relaxation in the ligament but not by E(1)S profile. The increment in ligament measurement by >or=5mm from the preceding day measurement was the most useful and accurate in predicting calving within 24h. It is economical and easily applicable in the field condition.     

Effects of nonylphenol and phytoestrogen-enriched diet on plasma vitellogenin, steroid hormone, hepatic cytochrome P450 1A, and glutathione-S-transferase values in goldfish (Carassius auratus)

The effects of nonylphenol (NP) on plasma vitellogenin (VTG) and steroid hormone values, as well as hepatic cytochrome P450 1A (CYP1A) and glutathione-S-transferase (GST) activities, were measured in goldfish (Carassius auratus) fed a diet with a low (formulated diet, FD) or high (commercial diet, CD) content of phytoestrogens, including genistein and daidzein. Male goldfish with secondary sexual characteristics were exposed to nominal NP concentrations of 0.1, 1.0, 10, and 100 microg/L in the water for 28 days while being fed either the FD or CD diet at 1.0% of body weight daily. Plasma VTG concentration in male goldfish exposed to 100 microg of NP/L and fed FD was significantly higher than that in the FD-fed control fish at seven, 21, and 28 days. However, fish of the CD-fed group exposed to 100 microg of NP/ L had significantly higher plasma VTG concentration than did fish of the CD-fed control group at 28 days only. Moreover, plasma VTG concentration in fish of the CD-fed control group was about 100-fold higher than that in fish of the FD-fed control group. Although the estrogenic effects of a phytoestrogen-enriched diet caused a decrease in testosterone and/or 11-ketotestosterone values in the CD-fed fish, there was no dose-response relationship between androgen and amount of NP to which the FD-fed fish were exposed. Nonylphenol does not have appreciable effects on hepatic CYP1A and GST activities in male goldfish at concentrations as low as 100 microg/L. These results suggest that NP has estrogenic activity in male goldfish at the nominal concentration of 100 microg/L, and that phytoestrogens, such as genistein and daidzein, in the CD inhibit an aspect(s) of steroid release and/or synthesis common to testosterone and 11-ketotestosterone. However, results of in vivo screening assays for endocrine-disrupting chemicals may be seriously affected by phytoestrogens in the diet, depending on content or potency of estrogenic activity; therefore, we recommend use in research of a standardized, open-formula diet in which estrogenic substances have been reduced to amounts that do not alter the results of studies that are influenced by exogenous estrogens.     

Modulation of goldfish testicular testosterone production in vitro by tumor necrosis factor alpha, interleukin-1beta, and macrophage conditioned media

The relevance of immune-endocrine interactions to the regulation of testicular steroidogenesis in teleosts is virtually unexplored. The objectives of the present study were: 1) to investigate the effects of murine cytokines, tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta), and trout (Oncorhynchus mykiss) macrophage conditioned media (MCM) on testosterone (T) production by goldfish (Carassius auratus) testis pieces in vitro; and 2) to identify the site(s) of the inhibitory action of TNFalpha on hCG-stimulated T formation. TNFalpha (0-100 ng/ml) affected basal T production differentially depending on the gonadosomatic index (GSI) value of the fish. TNFalpha stimulated basal T of fish with a relatively low GSI (average 1.99), but inhibited T production by testis of fish with a higher GSI (average 5.14). The remaining studies used fish with only high GSI values. IL-1beta (0-10 ng/ml) inhibited basal T production, while MCM (0-25% v/v) had no effect. The cytokines significantly inhibited hCG-stimulated T production at all doses tested, whereas MCM was inhibitory only at the lower doses of 2.5-5% v/v. TNFalpha did not affect basal or hCG-stimulated cAMP levels, but did inhibit forskolin (0.5 microM; adenylate cyclase activator) and 8-bromo-cAMP (0.15 mM; cAMP analog) stimulated T levels. The inhibitory actions of TNFalpha on T production were greatly reduced by treatment of testis with 25-hydroxycholesterol (1 and 10 microg/ml), pregnenolone (50 and 100 ng/ml), and 17 alpha-hydroxypregesterone (50 ng/ml). TNFalpha caused a moderate decrease in pregnenolone (100 ng/ml)-stimulated T production. Together, these data demonstrate that regulatory actions of TNFalpha may occur at multiple sites within the steroid biosynthetic pathway, but the major effect appears to be related to cholesterol availability in the mitochondria. In conclusion, the results of this study implicate macrophage-derived factors in the regulation of teleost testicular androgen biosynthesis.     

Seasonal changes of serum sex steroids concentration and aromatase activity of gonad and brain in red-spotted grouper (Epinephelus akaara)

Levels of serum sex steroids (estradiol-17beta, E2; testosterone, T; 11-ketotestosterone, 11-KT) in male, female and natural sex-reversing red-spotted grouper (Epinephelus akaara), and aromatase activity of gonad and brain in both male and female were investigated throughout an annually reproductive cycle. In females, serum E2 and T peaked during vitellogenesis, but in males and natural sex-reversing fish, 11-KT, T and E2 reached peak during spermatogenesis. In addition, in females, serum 11-KT levels (monthly means: 0.32 +/- 0.03 ng/ml) which were very low did not significantly fluctuate during the annual reproductive cycle. In breeding season, females displayed higher E2 levels than males and sex-reversing fish, while males and sex-reversing fish showed higher 11-KT levels and, to a lesser extent, higher T levels than females. Furthermore, the changing pattern of sex steroids in males was similar to that in natural sex-reversing fish, and a second peak of serum androgens 11-KT and T appeared in December both in male and natural sex-reversing fish; significantly higher serum 11-KT levels were observed in natural sex-reversing fish than that in females from December to April. In females, but not in males, aromatase activity of brain and gonad demonstrated significantly seasonal changes (exhibiting a peak in breeding season); moreover, aromatase activity in females was higher than that in males. Furthermore, significantly lower aromatase activity in testis was observed in breeding season, in contrast to that in ovary. Taken together, the present findings indicated that changes of serum sex steroids levels and aromatase activity in red-spotted grouper were closely associated with sex inversion. In addition, the present results also suggested that sex inversion in red-spotted grouper peaked mainly from December to March.     

Determination of malachite green and leucomalachite green in edible goldfish muscle by liquid chromatography-ion trap mass spectrometry

A liquid chromatography-ion trap mass spectrometry method with three "time segments" has been developed to determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) in edible goldfish muscle. By using the optimized "time segments", MG and LMG as well as the internal standard atrazine-d(5) were analyzed with good sensitivity with positive ESI-MS in a single run. The homogenized fish muscle tissues were extracted with a solution of perchloric acid and acetonitrile, followed by partitioning with dichloromethane. Strata-x polymeric solid-phase extraction column was used for the clean-up process. The determination of MG and LMG was achieved by using a reversed-phase HPLC gradient program coupled with MS/MS in multiple-reaction-monitoring mode. Matrix calibration curves were linear over the ranges of 5-500 ng/ml for MG and 1-100 ng/ml for LMG. Recoveries of the fish tissue extraction at three spiked levels (2, 10 and 30 ng/g for MG as well as 0.4, 2 and 6 ng/g for LMG) were better than 71% and 89%, respectively. Relative standard derivations from six determinations were less than 8%. The method detection limits were 0.13 ng/g for MG and 0.06 ng/g for LMG.     

Essential role of follicle stimulating hormone in the maintenance of caprine preantral follicle viability in vitro

The aims of the present study were to investigate the effects of follicle-stimulating hormone (FSH) on survival, activation and growth of caprine primordial follicles using histological and ultrastructural studies. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM - control medium) supplemented with different concentrations of FSH (0, 10, 50 or 100 ng/ml). Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days in a specific medium were processed for classical histology and transmission electron microscopy (TEM). Additionally, effects of FSH on oocyte and follicle diameter of cultured follicles were evaluated. The results showed that the lowest percentage of normal follicles was observed after 7 days of culture in control medium. After 1 day of culture, a higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FSH. In the presence of 10 and 50 ng/ml of FSH, an increase in diameter of both oocyte and follicle on day 7 of culture was observed. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in MEM plus 50 ng/ml FSH, but did not confirm the integrity of those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that FSH at concentration of 50 ng/ml not only maintains the morphological integrity of 7 days cultured caprine preantral follicles, but also stimulate the activation of primordial follicles and the growth of activated follicles.     

Prolactin inhibits osteoclastic activity in the goldfish scale: a novel direct action of prolactin in teleosts

In teleosts, prolactin is involved in calcium regulation, but its role in scale/bone metabolism is unclear. Using the in-vitro system with goldfish scales developed recently, we explored the effects of teleost prolactin, growth hormone, and somatolactin on osteoclasts and osteoblasts. Addition of prolactin at concentrations of 0.01-100 ng/ml reduced osteoclastic activity, partly via osteoclast apoptosis, after 6-18 h incubation. Conversely, growth hormone and somatolactin at a concentration of 100 ng/ml increased osteoclastic activity after 18 h incubation, indicating the specificity of the inhibitory effect of prolactin on osteoclastic activity. On the other hand, these three hormones promoted osteoblastic activity at concentrations of 10-100 ng/ml. The results from this study are the first demonstration of direct effects of prolactin on scale/bone metabolism and osteoclastic activity in a teleost.     

Somatic lamins in germinal vesicles of goldfish (Carassius auratus) vitellogenic oocytes

In fish and amphibians, B-type lamins are divided into somatic (B1, B2) and oocyte-type (B3) lamins. In this study, we purified nuclear lamins from rainbow trout erythrocytes, raised an anti-lamin monoclonal antibody (L-200) that recognizes goldfish somatic-lamins, and isolated cDNAs encoding goldfish B-type lamins (B1 and B2) from a goldfish cell culture cDNA library. Goldfish B-type lamins are structurally similar to lamins found in other vertebrates with minor amino acid substitutions in the conserved region. Western blot analysis showed that goldfish oocytes contained mainly GV-lamin B3 as well as some somatic lamins. Laser-confocal microscope observations revealed that lamin B3 was present only in GV nuclear lamina, whereas somatic lamins were present in dense fibrillar structures throughout nuclear gels of isolated GVs. Similar nuclear filamentous structures were also observed in GVs of paraffin embedded oocytes. Epitope mapping indicated that L-200 recognized a conserved region containing a short stretch of the alpha-helix coiled-coil rod domain (Y(E/Q)(Q/E)LL). A similar motif is also present in other cytoplasmic intermediate filaments (i.e., vimentin, desmin, peripherin and GFAP). Taken together, these findings suggest that lamins or lamin-related intermediate filaments are an important component of the interior architecture of goldfish vitellogenic oocyte nuclei (GVs).     

注射LHRHa对泥鳅血清性类固醇激素变化研究

研究了注射促黄体激素类似物(LHRHa)后,泥鳅(Misgurnus anguillicaudatus)血清性类同醇激素的变化规律,并探讨在泥鳅繁殖季节时.孕酮(P)、睾酮(T)和雌二醇(E2)对性腺发育的作用及调节机制.实验共分两组,对照组和实验组;对照组只注射生理盐水;实验组注射LHRHa,雌鱼0.2 μg/g,雄鱼减半.注射前尾静脉采血,作为血液样本分析基础水平(Oh),注射药物后分别在7、24、48、72和96h尾静脉采血.测定雌鱼睾酮、雌二醇和孕酮,雄鱼孕酮和睾酬血清浓度.实验结果表明:注射LHRHa,雌鱼血清睾酮和雌二醇浓度显著高于对照组,雄鱼血清睾酮和孕酮显著高于对照组(P<0.05);24h浓度较高.雌鱼孕酬、睾酮和雌二醇分别为(0.710±0.082)ng/mL、(9.00±0.57)ng/mL和(696.4±26.2)pg/mL,雄鱼孕酮和睾酮分别为(0.527±0.121)ng/mL和(9.62±0.62)ng/mL.实验组雌鱼孕酮变化基本规律为,基础水平(0-7h)-逐渐升至最高(7-24h)-逐渐降至基础水平(24-48h)-维持基础水平(48-96h).实验组雌鱼睾酮和雌二醇与雄鱼孕酮和睾酮变化规律基本相似,其规律为,逐渐上升至最高(0-24h)-逐渐降至基础水平(24-72h)-维持基础水(72-96h).24h对照组雌鱼睾酮和雌二醇显著升高,浓度分别为:睾酮(2.20±0.18)ng/mL,雌二醇(269.1±36.6)pg/mL.对照组雄鱼血清孕酮和睾酮浓度实验期间均无显著变化.研究认为:LHRHa能够刺激泥鳅性类同醇激素分泌,特别是睾酮的分泌,显著提高雌鱼性腺指数(GSI).但刺激P的分泌调控能力有限,实验期间处于较低水平,诱导排卵效果差,泥鳅的性类固醇激素可能有特殊的调节机制.雌二醇和睾酮对性腺成熟有重要作用,孕酮对介导卵细胞最终成熟和排卵可能起重要作用,而雌二醇和睾酮无明显效果.     

Some endocrine traits of transgenic rabbits. II. Changes in hormone secretion and response of isolated ovarian tissue to FSH and ghrelin

In the present in vitro experiments we examined FSH- and ghrelin-induced changes in ovarian hormone secretion by transgenic rabbits. Fragments of ovaries isolated from adult transgenic (carrying mammary gland-specific mWAP-hFVIII gene) and non-transgenic rabbits from the same litter were cultured with and without FSH or ghrelin (both at 0, 1, 10 or 100 ng/ml medium). The secretion of progesterone (P4), estradiol (E2) and insulin-like growth factor I (IGF-I) was assessed by RIA. It was observed that ovaries isolated from transgenic rabbits secreted much less P4, E2 and IGF-I than the ovaries of non-transgenic animals. In control animals FSH reduced E2 (at doses 1-100 ng/ml medium) and IGF-I (at 1-100 ng/ml), but not P4 secretion, whereas ghrelin promoted P4 (at 1 ng/ml) and IGF-I (at 100 ng/ml), but not E2 output. In transgenic animals, the effects were reversed: FSH had a stimulatory effect on E2 (at 100 ng/ml) and ghrelin had an inhibitory effect on P4 (at 10 ng/ml). No differences in the pattern of influence of FSH on P4 and IGF-I and of ghrelin on E2 and IGF-I were found between control and transgenic animals. The present observations suggest that 1) both FSH and ghrelin are involved in rabbit ovarian hormone secretion, 2) transgenesis in rabbits is associated with a reduction in ovarian secretory activity, and 3) transgenesis can affect the response of ovarian cells to hormonal regulators.     

Estrogenic modulation of CYP3A38, CYP3A40, and CYP19 in mature male medaka (Oryzias latipes)

We examined cytochrome P450 production and activity and circulating hormone concentrations in male medaka exposed to 17beta-estradiol (E2) or 17alpha-ethinylestradiol (EE2). Intraperitoneal injection of E2 at 1, 10, or 100 microg/g-fish completely suppressed CYP3A38 protein production and suppressed CYP3A40 protein levels by 89%, 52%, or 47%, respectively. CYP3A38 and CYP3A40 mRNA expression was unaltered, and CYP3A enzymatic activity initially increased and then decreased with increasing E2 dose. Males co-cultured with females were exposed to a markedly high concentration (43 ng/L) of E2 secreted by females. CYP3A protein levels in co-cultured males were suppressed. Serum testosterone (TE) and 11keto-testosterone levels in co-cultured males were downregulated to 40% of pre-exposure levels. Serum E2 levels increased in co-cultured males or males exposed to EE2. Testicular CYP19, which converts TE to E2, increased by 9.5 times in males exposed to 50 ng/L EE2 and by 21.5 times in those exposed to 100 ng/L EE2. Male medaka exposed to EE2 showed increased serum Vtg levels. Estrogenic exposure induced Vtg production, suppressed CYP3A protein production, downregulated TE metabolism, and enhanced CYP19 activity. Serum E2 endogenously induced by CYP19 could contribute to Vtg induction in male medaka.