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.     

In vitro induction of vitellogenin by estradiol 17 β in isolated hepatocytes of catfish, Clarias gariepinus

Vitellogenin is a female-specific calcium-binding glycolipophosphoprotein synthesized in the hepatocytes of fishes. Its synthesis can be induced in fishes of either sex by estradiol or by xenoestrogens. To study the in vitro synthesis of vitellogenin, different culture conditions were set up using the hepatocytes of Clarias gariepinus. The present study reports on a non-enzymatic procedure for isolation and culture of hepatocytes from the liver of the catfish Clarias gariepinus, in order to study the effects of estradiol on vitellogenin synthesis in vitro. The procedure employs chelating properties of ethylenediamine tetracetic acid to achieve cell viability in excess of 95%. Equal numbers of isolated cells were incubated in different culture media viz. RPMI F1640, Medium-199, and Williams’ Medium E. At 36 h, cell attachment and monolayer formation is faster in M-199 and Williams’ Medium E than in RPMI. In order to study the effects of estradiol on vitellogenin synthesis, the isolated hepatocytes were seeded in Williams’ Medium E in 24-well cell culture plates. 17 β-estradiol (E₂) was introduced in the culture plates at different concentrations and for different time periods. The media were assayed for vitellogenin using competitive ELISA. Vitellogenin appeared in the medium after 48 h of incubation with 10⁻⁵ M estradiol whereas after 72 h of incubation 5×10⁻⁷ M E₂ could elicit the synthesis.     

In vitro effect of pituitary,interrenal and gonadal hormones on vitellogenin synthesis in primary hepatocyte cultures of Chalcalburnus tarichi

Vitellogenin (Vtg) is an important precursor yolk protein in egg‐laying vertebrates, including fish. The 17β‐oestradiol (E2) plays a crucial role in the Vtg synthesis; moreover, certain hormones can stimulate Vtg synthesis. We investigated the possible role of E2, carp recombinant growth hormone (crGH), insulin (Ins), progesterone (P4) and 11‐deoxycortisol (11‐DOC) hormones in Vtg synthesis on primary juvenile Chalcalburnus tarichi hepatocyte culture. The amount of Vtg in the medium was measured at 2‐day intervals using enzyme‐linked immunosorbent assay (ELISA). The hepatocytes were maintained in culture for more than 2 weeks without the addition of serum components. Vitellogenin localization was visualized with the immunofluorescence method in E2‐supplemented hepatocytes. Among hormones applied to the culture, only E2 had an influence on Vtg synthesis in a time‐dependent manner, while crGH, Ins, P4 and 11‐DOC had no effect. However, in hepatocytes stimulated with E2 in combination with P4, a lower Vtg production was seen compared with Vtg produced when hepatocytes were stimulated with E2 alone. P4 proved to have potentiating effects on co‐treatment with E2‐induced Vtg production. As a result, E2 and P4 are the most important hormones for Vtg synthesis in juvenile C. tarichi hepatocyte culture.     

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.     

Binding and bioactivity of insulin in primary cultures of carp (Cyprinus carpio) hepatocytes

Isolated carp hepatocytes cultured in serum-free, chemically defined medium were used to investigate within the same cell preparation characteristics of the binding of insulin as well as effects of insulin on cellular metabolism. The binding of human [¹²⁵I]-insulin to carp hepatocytes was studied in kinetic, saturation and displacement experiments. A dependency of insulin binding on the collagenase used for cell isolation was demonstrated. Insulin binding decreased during the first 12h of culture but remained constant during the following 12h. The kinetic experiments revealed that [¹²⁵I]-insulin binding reached a steady state within 20–30 min of incubation. The mathematical analysis of the saturation experiments demonstrated the existence of two populations of binding sites, one with high affinity (K_d1 = 5.5 pM) and low capacity (B_max1 = 0.14 fmol/mg protein or 77 binding sites/cell) and one with low affinity (K_d2 = 2.4 nM) and high capacity (B_max2 = 17.6 fmol/mg protein or 9623 binding sites/cell). In competition experiments, 312 pM [¹²⁵I]-insulin was displaced by cold insulin, IGF-I and IGF-II with IC₅₀ values of 2.2, 7.9 and 20.3 nM, respectively. Glucagon was without effect. Binding of insulin to carp hepatocytes resulted in a significant reduction of glucose release and a significant increase of protein synthesis as of de novo fatty acid synthesis. dedicated to Prof. Dr. W. Hanke on the occasion of his 65^th birthday.     

Comparison of the effectiveness of three different growth media for primary isolation of Renibacterium salmoninarum from Atlantic salmon, Salmo salar L., broodfish

Abstract. The effectiveness of three different growth media KDM-2, S-KDM and S-KDM-C for primary isolation of Renibacterium salmoninarum was examined over a 14-week incubation period. Kidney samples were taken from Atlantic salmon, Salmo salar L., broodfish returning to a sea ranch after 2 years at sea. Homogenized samples were inoculated onto two selective media, S-KDM supplemented with lamb serum and S-KDM-C supplemented with activated charcoal. The third medium was the non-selective serum supplemented KDM-2. Renibacterium salmoninarum was isolated from 112 samples on one or more of the media used. Of all positive samples, 91% were positive on S-KDM, 60% on S-KDM-C and 35% on KDM-2. These results demonstrate that selectivity significantly enhances the isolation capacity of the medium and that supplementing with serum is significantly more effective than supplementing with charcoal.     

3种水质调控方式下刺参池塘初级生产力的周年变化

通过对自然纳潮、微孔曝气、养水机池塘不同水层初级生产力及其相关参数的研究,分析养水机对初级生产力的影响。结果表明,3种水质调控方式池塘,初级生产力年均值、P/R值均以养水机池塘最高,微孔曝气池塘次之,自然纳潮池塘最低。养水机池塘、微孔曝气池塘、自然纳潮池塘的初级生产力年均值分别为(6.22±0.54)、(5.37±0.60)、(4.69±0.53) gO₂/(m²·d)。3种水质调控方式下,养水机池塘30～50 cm水层和50～100cm水层初级生产力差异不显著,而微孔曝气池塘和自然纳潮池塘这两水层之间初级生产力差异显著,且养水机池塘50～100 cm的水层初级生产力显著高于微孔曝气和自然纳潮池塘。研究表明,养水机能显著提高刺参池塘50 cm以下水层的初级生产力,缩小上层和下层初级生产力之间的差距,从而提高池塘水体总初级生产力,为刺参饵料和池塘物质快速循环提供基本保障。     

北方寒区2龄和3龄雄性中华绒螯蟹成蟹可食率、色泽及品质比较

为开发中华绒螯蟹(Eriocheir sinensis)新种质并阐述其品质特征, 本研究以北方寒区 2 龄雄性成熟中华绒螯蟹(简称 2 龄蟹)为对照, 探究 3 龄雄性成熟中华绒螯蟹(简称 3 龄蟹)的可食率、色泽、常规营养品质、脂肪酸、游离氨基酸和矿物质元素差异, 为 3 龄蟹新种质开发和利用提供重要的判断依据。结果表明; 9 月 15 日 2 龄蟹肝胰腺指数(HSI)极显著高于 3 龄蟹(P＜0.01), 而出肉率(MY)则显著低于 3 龄蟹(P＜0.05)。2 龄蟹与 3 龄蟹相比, 头胸甲和肝胰腺湿样 L* , 头胸甲和肝胰腺干样 a* 存在显著性差异(P＜0.05)。2 龄蟹肝胰腺中粗脂肪含量显著高于 3 龄蟹 (P＜0.05), 而性腺系统和肌肉中粗蛋白含量则高于 3 龄蟹。就脂肪酸而言, 2 龄蟹肝胰腺、性腺系统和肌肉中 DHA 和 DHA/EPA 含量均高于 3 龄蟹, 而 ARA 则低于 3 龄蟹。肌肉中脂肪酸显著性差异项(5 项)明显高于肝胰腺(1 项) 和性腺系统(1 项)。就游离氨基酸而言, 2 龄蟹肝胰腺、性腺系统和肌肉中总必需氨基酸(ΣEFAA)、总游离氨基酸 (ΣFAA)均高于 3 龄蟹。性腺系统中显著性差异项(6 项)明显高于肝胰腺(0 项)和肌肉(2 项)。2 龄蟹肝胰腺和性腺系统中谷氨酸(Glu)、总鲜味氨基酸(ΣTUV)高于 3 龄蟹, 而肌肉中则低于 3 龄蟹。就矿物质元素而言, 2 龄蟹 3 种可食组织中 Na、K、Ca、Mg 和 Cu 元素含量均低于 3 龄蟹。肝胰腺中矿物质元素显著性差异项(5 项)明显高于性腺系统 (1 项)和肌肉(2 项)。综上所述, 2 龄蟹大部分常规营养成分、脂肪酸、游离氨基酸指标均优于 3 龄蟹, 但可食率参数、大部分矿物质元素含量则明显低于 3 龄蟹。整体来看, 2 龄和 3 龄雄蟹各有优势, 均具有较高的营养价值。     

Induction of fertilizable eggs by conspecific vitellogenin implantation in captive female walking catfish,Clarias batrachus (Linn.)

The synthesis of vitellogenin (Vg) is induced by conspecific Vg (Vg1 and Vg2) and estradiol‐17β (E₂) as demonstrated by the pattern of ³H‐serine incorporation in the liver and plasma proteins. The incorporation studies indicated that the label was first incorporated into the liver after which it appeared in the blood in both E₂‐ and Vg‐treated male catfish. Since Vg was capable of inducing its own synthesis, experiments were conducted in females during preparatory–prespawning period (March–May) to make them gravid by implanting Vg pellets. Two implantations of 4 mg Vg1 pellets into female catfish with an interval of 15 days, followed by laboratory maintenance for 45 days of initial implantation showed a significant increment in ovarian weight with concomitant formation of yolky oocytes through synthesis and incorporation of Vg, whereas Vg2 implantation was not effective in this regard. Histological observation of yolky oocytes in Vg1‐treated group showed the peripheral migration of germinal vesicle (eccentric germinal vesicle), which indicates the onset of maturation. On 45th day, third implantation with 2 mg Vg pellets was performed and after 15 days, fish were hormonally induced with a single injection of hCG (2,000 IU/kg fish). Six groups were considered such as initial control, BSA‐implanted control, Vg1‐implanted, Vg2‐implanted, catfish collected from the field on the last day of the experiment and catfish collected during spawning period in this experiment with 3–7 fish in each group. Each of the experimental fish was sexually mature and the body weight was between 100 and 125 g. The percentage of ovulation and fertilization in the eggs of Vg1‐implanted group was 91% and 78%, respectively, which was almost similar to that of gravid female catfish collected during breeding period (July). The breeding performance in BSA‐ and Vg2‐treated females was very poor. The fertilized eggs were hatched in the laboratory conditions. Thus, in the female catfish, Vg1 not only induces vitellogenesis but also makes the oocytes viable for fertilization.     

Gag (Mycteroperca microlepis) vitellogenin: purification,characterization and use for enzyme-linked immunosorbent assay (ELISA) of female maturity in three species of grouper

Circulating levels of the egg yolk precursor protein, vitellogenin (VTG), can be used as a biochemical indicator of maturation in female fish. Here we report on purification and partial characterization of VTG from a temperate marine serranid, the gag(Mycteroperca microlepis). Development of a competitive, enzyme-linked immunosorbent assay (ELISA) for gag VTG (gVTG) is also described. The gVTG was purified by DEAE-agarose anion exchange chromatography from a pooled plasma sample collected from several juvenile gag after they were injected with 17estradiol. The protein appeared as a major band of M_r183000 after SDS-PAGE ± Western blotting using either a specific rabbit antiserum to gVTG or a universal monoclonal antibody for vertebrate VTGs. Amino acid composition analysis and N-terminal peptide sequencing verified that gVTG is similar in primary structure to VTG from several other teleost species. The purified gVTG and its specific antiserum were used to develop a sensitive, competitive, antibody-capture ELISA for quantifying the protein in blood plasma from maturing females. VTG levels in maturing female gag were highly correlated with oocyte growth and circulating testosterone and 17-estradiol levels, whereas VTG was non-detectable in juveniles, immature females or males. Two size-based maturity schedules for female gag were constructed, one utilizing detection of VTG in their circulation as a marker of maturity and the other relying on histological evidence that their ovaries were in vitellogenic or later stages of maturation. The two schedules were virtually identical. The gVTG ELISA was also used to detect VTG in blood plasma from mature Nassau grouper (Epinephelus striatus) and red hind (E. guttatus). As with gag, the assay was completely reliable for discriminating between reproductively mature females versus males from these two grouper species.     

Dietary fish oil replacement by soybean oil: Effect on plasma vitellogenin,sex steroids and ovarian steroidogenesis in Chinese strip‐necked turtles (Mauremys sinensis)

In order to investigate the effects of dietary fish oil replacement, the turtles (Mauremys sinensis) were fed four experimental diets for 10 months: FO (100% fish oil), FSO (70% fish oil and 30% soybean oil), SFO (30% fish oil and 70% soybean oil) and SO (100% soybean oil), sampled at pre‐vitellogenesis, vitellogenesis and post‐vitellogenesis. The results showed that plasma gonadotropin‐releasing hormone (GnRH) levels were the highest at pre‐vitellogenesis, which promoted the secretion of gonadotropin and sex steroids. Therefore, plasma luteinizing hormone (LH) and estrogen (E₂) levels were significantly increased at post‐vitellogenesis (p < 0.05), while follicle‐stimulating hormone (FSH) levels increased at vitellogenesis (p < 0.05). The FO and FSO groups had significantly higher GnRH and E₂ levels than the other two groups (p < 0.05). In addition, plasma vitellogenin (Vtg) levels significantly increased at vitellogenesis and post‐vitellogenesis (p < 0.05), which were significantly higher in the groups of FO and FSO than SO (p < 0.05). Moreover, the expression levels of hepatic estrogen receptor α (Erα) mRNA were significantly increased at vitellogenesis and post‐vitellogenesis while ovarian Cyp19α1α mRNA were significantly increased at post‐vitellogenesis (p < 0.05), and both were the lowest in SO. Taken together, the replacement of fish oil with 66.7% soybean oil is feasible.     

Comparison of histopathology caused by Vibrio anguillarum and Vibrio ordalii in three species of Pacific salmon*

Abstract. The histopathology associated with naturally acquired vibriosis in chum salmon, Oncorhynchus keta (Walbaum), fingerlings caused by Vibrio anguillarum was compared with that caused by infection with Vibrio ordalii. Pathogenesis of the two forms was found to be different. Bacteraemia caused by V. anguillarum occurred in the early stages with pronounced histopathological changes in blood, loose connective tissue, kidney, spleen, gills and posterior gastrointestinal tract. Bacterial cells appeared uniformly dispersed throughout the affected tissues but were most abundant in the blood. With V. ordalii, bacteraemia developed only in late stages of the disease and the concentration of bacterial cells per ml of blood was less than in the V. anguillarum infection by a factor of 10²–10³. Tissues with most pronounced changes were skeletal and cardiac muscle, anterior and posterior gastrointestinal tract and the gills. Vibrio ordalii observed in the tissues was not evenly dispersed but was present within tissue as colonies or aggregates of cells. The differences in pathology observed in naturally infected chum salmon were produced experimentally with each pathogen by waterborne exposure of chum; coho, Oncorhynchus kisutch (Walbaum); and chinook salmon, Oncorhynchus tshawytscha (Walbaum). Severe decreases in circulating leucocytes accompanied bacteraemia caused by either bacterial species.     

Comparison of NADH-dependent cytochrome b5 reductase activity and in vitro methemoglobin induction by sodium nitrite in Oncorhynchus mykiss, Salmo salar, and Salvelinus fontinalis

Methemoglobin is hemoglobin containing ferric iron. Methemoglobin cannot bind to oxygen and at high concentrations causes tissue hypoxia. Brook trout (Salvelinus fontinalis) develop significantly greater methemoglobinemia than Atlantic salmon (Salmo salar) or rainbow trout (Oncorhynchus mykiss) following general anesthesia with benzocaine or tricaine methanesulfonate. The objective of this study was to compare the activity of the major methemoglobin reducing enzyme, NADH-dependent cytochrome b5 reductase (CB5R), in brook trout erythrocytes to the activity of CB5R in Atlantic salmon and rainbow trout erythrocytes. Methemoglobin levels were compared using co-oximetry following in vitro incubation of erythrocytes with sodium nitrite (NaNO₂). The CB5R activity was measured using a ferricyanide assay. There was significantly greater methemoglobin at time 0 in brook trout erythrocytes than in rainbow trout or Atlantic salmon erythrocytes (2.79 ± 0.29 %, 2.19 ± 0.23 %, 2.08 ± 0.14 %), (P < 0.001). There was significantly greater methemoglobin induction by NaNO₂ in brook trout erythrocytes (33.14 ± 3.32 %) than in rainbow trout or Atlantic salmon erythrocytes (28.73 ± 2.92 % and 24.85 ± 1.40 %, respectively), (P < 0.001). The CB5R activity was significantly less in brook trout erythrocytes (median of 3.05 μmol/min/μl) than in rainbow trout erythrocytes (median of 6.73 μmol/min/μl). The CB5R activity in Atlantic salmon erythrocytes (median 4.09 μmol/min/μl) was not significantly different than in brook or rainbow trout erythrocytes. Total methemoglobin at any one time is a balance between induction by oxidants and reduction by antioxidants. Lower CB5R activity in brook trout erythrocytes may contribute to a species-specific sensitivity to methemoglobin induction; however, there are likely additional factors.     

Comparison of chemical, electrophoretic and in vitro digestion methods for predicting fish meal nutritive quality

Chemical, electrophoretic and in vitro digestion methods were compared with respect to predictions given regarding fish meal (FM) quality. FMs were manufactured by mixing a press-cake, with spray dried stickwater concentrate from the identical raw material, thereby providing samples containing different quantities of water-soluble protein (wsp). A low-temperature-dried FM was employed as a reference. Acquired chemical data for each of the FMs included amino acid analysis and proximal composition (protein, fat, ash, ammonia, titration, salt, moisture). Biological methods in rat (net protein utilization, NPU, biological value, BV, and true digestibility, TD), capillary electrophoresis (sodium dodecyl sulphate-capillary gel electrophoresis, SDS-CGE)) and an in vitro enzymatic assay (trinitrobenzene sulphonic acid-based closed system with rainbow trout enzyme extract) were used for further comparisons with FM wsp content. A high correlation ( R  = 0.97; P  < 0.001) between FM wsp content and titration volume was observed. In contrast to BV and NPU ( R  = 0.98; P  < 0.001), TD ( R  = 0.2; P  = 0.63) did not correlate with FM wsp. The peak area of a 50 kDa signal derived from SDS-CGE showed significant correlation ( R  = 0.98; P  < 0.001) with wsp content. The fish-based in vitro system provided correlations with wsp content with respect to predigestion ( R  = 0.97; P  < 0.0001) and post digestion ( R  = 0.77; P  < 0.03) and for enzymatic liberation of amino groups as post digestion minus predigestion ( R  = 0.97; P  < 0.0001) from the FM examined.     

(鱼免)消化酶与商品酶离体消化蛋白原料的研究

分别应用商品酶及鱼免消化酶两步消化法评定10种蛋白原料的离体消化率.商品酶处理一(CEⅠ)直接在原料中加入胃蛋白酶消化,处理二(CEⅡ)预先将原料悬浊液调节至pH 2.0,再加入胃蛋白酶消化.鱼免消化酶处理一(DEⅠ)的胃消化酶酶活/底物、类胰消化酶酶活/底物分别为250、375 U/g,处理二(DEⅡ)则分别为300、460 U/g.试验结果表明,CEⅡ比CEⅠ部分原料的干物质及蛋白质消化率平均提高5.16%、13.09%(P<0.05),且2种处理的干物质、蛋白质消化率的相关系数分别为0.763(P=0.01)、0.771(P=0.009).DEⅡ比DEⅠ的干物质、蛋白质消化率平均提高12.65%、15.80%(P<0.05),且2种处理的干物质、蛋白质消化率的相关系数分别为0.852(P=0.002)、0.851(P=0.002).CEⅡ与DEⅡ(不包括血粉、阿根廷鱼粉)、CEⅠ与DEⅡ以及CEⅡ与DEⅠ(不包括血粉、阿根廷鱼粉)的蛋白质消化率之间的相关系数分别为0.94(P=0.001)、0.845(P=0.002)、0.823(P=0.012).可以用商品酶替代消化酶评价鱼免对蛋白原料的离体消化率.     

Comparison study of three compounds in Eucommia ulmoides on growth,flesh quality of grass carp (Ctenopharyngodon idella)

This study was conducted to compare the growth‐promoting and flesh quality ‐improving effects of three active compounds in Eucommia ulmoides (EU) on grass carp (Ctenopharyngodon idella). Four iso‐nitrogenous diets supplemented with 400 mg/kg inclusion of geniposidic acid (GA), chlorogenic acid (CGA), geniposide (GP) and their combination (GA:CGA:GP = 1:1:1, the mixture) were prepared and fed to grass carp (47.1 ± 0.6 g) for 75 days. The results indicated that weight gain was increased by 5.22%, and feed conversion ratio decreased by 0.07 by dietary CGA (p < 0.05). In flesh quality, the four supplementations significantly increased muscle fibre density, total collagen and alkaline‐insoluble collagen in skin, and reduced steaming loss of flesh. In addition, dietary CGA, GP and the active compounds mixture further increased total collagen, alkaline‐insoluble collagen and amino acid in flesh. In collagen genes expression, the expression of COL1A1 in muscle and skin was significantly promoted by the supplementation of GA, CGA, GP and their combination (p < 0.05). In conclusion, the supplementation of GA, CGA, GP and their combination improved the flesh quality of grass carp, and the growth was increased by CGA. CGA played more important roles in growth‐promoting and flesh quality‐improving effects than GP and GA.     

镉诱导鲫肝细胞内Ca2+-ATP酶与金属硫蛋白的表达

研究了镉诱发鲫肝细胞相关的胞内游离钙离子变化,以及Ca²⁺-ATP酶及金属硫蛋白表达量的变化。试验分为对照组、5、10、15、20 μmol/L CdCl₂ 5个组。Ca²⁺用Fura-2/AM方法检测,试验后24 h用荧光倒置显微镜观察细胞内游离钙离子变化;分光光度法检测Ca²⁺-ATP酶;石墨炉—原子分光光度法检测了细胞内镉离子浓度;免疫酶联法(ELISA)检测了金属硫蛋白(MT)含量。结果显示,镉可导致细胞存活率下降,具有一定的毒性。镉离子引起胞内Ca²⁺荧光强度和Ca²⁺-ATP酶活性增加(P＜0.01)。随镉浓度升高,处理组Ca²⁺-ATP酶浓度活性分别是对照组的4.52、6.73、6.68、7.19、6.18倍;暴露24 h后各组细胞内镉离子均有上升,其中5 μmol/L组最高,达(2.045±0.322) μmol/L;各处理组金属硫蛋白(MT)含量增高(P＜0.01),且5 μmol/L低浓度组MT增幅最大,达17.15%。结果提示,镉诱导下细胞内Ca²⁺升高,MT表达量上升,且MT可螯合进入细胞内的镉离子,这种螯合可能是降低镉毒理作用的机制之一。