甲基睾酮对雌性斑马鱼性腺发育的抑制作用

用含雄性激素甲基睾酮(MT)的饲料饲喂经产雌性斑马鱼(Danio rerio)来探讨外源激素对其性腺发育的影响。通过投饲含0、30、60、120、240和400 mg.kg-1 MT的饲料30 d后,石蜡切片观察斑马鱼卵巢的发育情况。结果表明,随着MT剂量的增加,斑马鱼性腺的发育受到抑制,卵巢结构变化明显。30 mg.kg-1剂量组的成熟系数有明显的降低,至400 mg.kg-1剂量组,成熟系数仅为对照组的1/6;同时外源雄性激素严重影响或抑制了卵巢的进一步发育,较低剂量组性腺发育停滞在Ⅲ时相左右,较高剂量组的性腺明显的退化,至400 mg.kg-1剂量组,卵巢已呈明显的萎缩状态,卵母细胞几乎不可见;卵母细胞在甲基睾酮作用下的退化过程为:抑制卵母细胞发育—细胞核结构变化—累及细胞器—形成凋亡小体、破坏细胞膜结构—凋亡小体逸散、细胞固缩—崩解。结果显示外源雄性激素对雌性斑马鱼的性腺发育影响明显,较高剂量时可导致外源激素的阉割作用。     

HPLC-MS/MS快速检测水产品中甲基睾丸酮的研究

用甲基叔丁基醚提取水产样品中的甲基睾丸酮,提取物经C18粉和正己烷净化后,通过电喷雾离子源电离多反应监测(MRM)对甲基睾丸酮作定性和定量分析。结果表明,甲基睾丸酮在1.0～500.0μg/L浓度范围内线性良好(r>0.999);在4.0～200.0μg/kg添加水平下,回收率为80.4～95.2%,相对标准偏差为4.1%～10.5%,检测限为1.0μg/kg(S/N=5)。     

Nucleotide sequence and mRNA expression analysis of <Emphasis Type="Italic">β</Emphasis>-actin gene in the orange-spotted grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

The full-length cDNA, encoding the orange-spotted grouper β-actin and spanning 1920 bp including a poly (A) tail, was cloned from its brain cDNA library. The open reading frame encodes a protein of 375 amino acids. Sequence analysis indicated that it contained the typical structural features of cytoplasmic actins, and showed higher homology with other vertebrate β-actin than any other members of the actin family. The partial genomic sequence indicated that the organization of the β-actin gene in the orange-spotted grouper might also be conserved. Northern blot analysis indicated that it was expressed at high levels in the brain, spleen, adipose tissue, ovary, and liver, but at low levels in the gill filament and heart, and at a very low level in the kidney. The expression of β-actin gene in the skeletal muscle was barely detectable. These results indicated that the expression of the orange-spotted grouper β-actin gene showed significant variation in different tissues. Therefore, caution should be taken when using β-actin gene as an internal control in the normalization of gene expression among tissues. Whereas, semi-quantitative RT-PCR analysis indicated that treatment with 17α–methyltestosterone (MT) had little effect on the mRNA expression of β-actin gene in the in vitro incubated hypothalamus, pituitary, and ovary fragments of the orange-spotted grouper, suggesting β-actin can be used as an internal control for RT-PCR analysis of MT effects on gene expression in these tissues.     

Effects of aromatizable and nonaromatizable androgens on the sex inversion of red-spotted grouper (Epinephelus akaara)

The effects of aromatizable 17α-methyltestosterone (MT) and non-aromatizable 17α-methyldihydrotestosterone (MDHT) on sex inversion in red-spotted grouper, Epinephelus akaara, were investigated. Fish were implanted with MT, MDHT and MT+AI (aromatase inhibitor, AI) respectively for one month. The results showed that the three treated groups turned into transitional stage with intersex gonads, which contained atretic oocytes and spermatogenic germ cells at all stages of spermatogenesis. The controls did not change sex. The gonads of more than half MT-implanted fish were in early transitional stages of sex inversion, whereas those of more than half MDHT and MT+AI-implanted fish were in late transitional stages of sex inversion. No difference in serum estradiol-17β (E₂) levels between the controls and the treated groups were observed, whereas 11-ketotestosterone (11-KT) and testosterone (T) levels increased in all treated groups. Significantly lower gonadosomatic index (GSI) and gonadal aromatase activity were observed in the treated groups, which were in accordance with the lower mRNA expression of P450aromA. However, P450aromB mRNA expression increased in the MT group, while it did not change in the MDHT group. These results suggest that the sex inversion of red-spotted grouper by MT and MDHT implantation might be due to the suppression of P450aromA gene expression, and resulting in both the decrease of the ovarian estrogen –secretion, as well as the increase in the 11-KT levels. Furthermore, the main reason for MT being less effective than MDHT might be due to partial aromatization of MT to estrogen.     

Effects of oestradiol-17β or 17α-methyltestosterone administration on gonadal differentiation of largemouth bass Micropterus salmoides (Lacepède)

Monosex populations can be a valuable management tool in culture of larger size largemouth bass (>400 g). In this study, we investigated the effective mode and duration of oestrogen and androgen administrations to produce monosex largemouth bass populations. The experiment consisted of nine treatments. In oral administration groups, we fed 40-day-old fry either 200 mg of an oestradiol-17β (E₂) kg⁻¹ diet or 60 mg of a 17α-methyltestosterone (MT) kg⁻¹ diet for 30, 45 or 60 days. In bath treatments, we immersed fry in a 1 mg MT L⁻¹ solution for 5 h a day on three or six occasions. For control treatment, we fed fry an ethanol-treated diet for 45 days. The frequency of females in the control group was 53.1%. Oral administration of E₂ at all durations resulted in slight increases in the frequency of females (59.8–70.5%). Both modes of androgen administration at all durations were ineffective in altering phenotypic sex. The experimental results of our study indicated that male differentiation passed the point of being completely and functionally influenced by exogenous oestrogens, while female differentiation had already taken place and was no longer responsive to exogenous androgens in 40-day-old (33.5 mm) largemouth bass fry.     

甲基睾酮免疫胶体金试纸条的研制及在水产品中的应用

通过研究胶体金免疫层析技术,建立了一种快速检测水产品中甲基睾酮含量的方法。将待测样品中的甲基睾酮和试纸条检测线上包被的人工抗原(MT-BSA)竞争性结合金标结合垫上包被的胶体金-甲基睾酮单抗聚合物,根据检测线呈现的红色线条深浅来判定样品的阴阳性。结果显示:试纸条上MT-BSA抗原工作浓度为1.0mg/m L,羊抗鼠二抗工作浓度为1.5 mg/m L,试纸条的最低检测限达到10μg/kg,与甲基睾酮类似物的交叉反应率低;使用甲醇提取,4倍PBST稀释,整个检测所需时间仅10~15 min,适用于大规模样品筛选。     

Effect of aromatable androgen (17‐methyltestosterone) on induced maturation of silver European eels (Anguilla Anguilla): Oocyte performance and synchronization

The reproductive performances of silver European eel in term of gonad development and egg production, employing slow‐release implants with the androgen 17‐MT (1 mg) in combination with traditional weekly injection of carp pituitary extract (CPE) was evaluated. Wild female European eels (Anguilla anguilla) underwent a standard induction protocol with CPE and were randomly divided into three groups (N‐group, no implant; Y‐group, with implant; and control, C‐group, no treatment). The results showed that 17‐MT‐treated females (Y‐group) reproduced spontaneously about 6 weeks earlier than the N‐group females with a saving of almost 40% in CPE and time of induction. Concerning artificial induction of maturation in female silver eels, our study demonstrated that they positively respond to androgen exposure also in terms of eggs productivity. Indeed, Y‐group was more productive than N‐group: in Y‐group, 11 eels ensured an eggs production that exceeded 50% of initial body weight (BW), whereas in N‐group only three eels have exceeded this value. The results suggest that 17‐MT should be considered in future protocols for the improvement of the artificial reproduction of female silver European eels.     

The effect of 3,3′,5-triiodo-L-thyronine and 17-α-methyltestosterone on growth and body composition of the glass stage of the eel (Anguilla anguilla L)

This study was undertaken to determine the influence of orally administered 3,3,5-triiodo-L-thyronine (T₃) or 17--methyltestosterone (MT) on growth and body composition of the eel (Anguilla anguilla L.) during the glass stage.Elvers receiving 20 or 40 ppm of T₃ in the food weighed significantly more (P < 0.05) than controls. However, after 61 days of treatment a dosage of 60 ppm was not efficacious.Terminal percentages of body protein were significantly lower (P < 0.05) in elvers that received 20 ppm or 40 ppm T₃ in their diets, than in the control. Crude body fat content was higher (P < 0.05) in elvers receiving 20 ppm (29% fat) or 40 ppm (28% fat), than in the controls (26% fat). Compared to the control, diets containing T₃ at 20 or 40 ppm increased the body glucose concentration.Glass eels fed a diet containing 1 or 10 ppm MT grew significantly more slowly (P < 0.05) than the controls.Total body protein content was higher (P < 0.05), but total body crude fat content was lower (P < 0.05) in elvers fed a diet with 1 or 10 ppm MT compared to the controls. Total body glucose concentration of elvers administered 10 ppm MT was significantly lower (P < 0.05) than that of the control groups.     

甲基睾丸酮在罗非鱼体内的药代动力学

以20 mg.kg-1和2 mg.kg-1剂量对经检测不含甲基睾丸酮残留的罗非鱼单次口灌给药,用高效液相色谱法测定血浆及组织中甲基睾丸酮(MET)的浓度。用乙酸乙酯提取样品,Florisil小柱净化。以3p97软件分析药时数据,MET在两个浓度组均符合一级吸收二室开放模型,吸收半衰期分别为0.29和0.28 h;分布半衰期分别为0.65和0.57 h,达峰时间分别为是0.78和0.60 h,消除半衰期分别为18.40和5.98 h。20 mg.kg-1组血液和肌肉中MET残留至144 h仍有检出,至216 h血液和肌肉样品MET均未检出;肝脏中的药物浓度高于血液和肌肉中的浓度,MET残留至24 h有检出,48 h的样品未检出。结果表明,口灌给药,MET在罗非鱼体内吸收、分布快,肝脏为主要吸收和代谢器官,与血液和肌肉相比消除速率要快,血液和肌肉中的MET消除速率相对较缓慢,因此开展MET监督检验除了常规的肌肉样品的检测外,还应增加血液样品的检测。     

高效液相色谱法测定罗非鱼鱼苗和不同来源罗非鱼中甲基睾丸酮残留

用高效液相色谱法(HPLC)法对投喂添加不同浓度甲基睾丸酮(MET)饲料的罗非鱼鱼苗、随机采取的罗非鱼成鱼及其跟踪饲料进行检测。刚孵化的罗非鱼鱼苗投喂添加不同浓度MET饲料30 d后,HPLC检测其体内残留量。继续投喂未添加药物的饲料,30 d后再次检测MET残留量。分别从不同的水产品批发市场、农贸市场和罗非鱼主要养殖产地,随机抽取罗非鱼成鱼样品,并从养殖产地随机抽取追踪饲料样品,高效液相色谱法检测MET残留。结果表明,投喂30 d后的罗非鱼苗种,对照组无MET残留,其余5个添加MET试验组的MET残留量随着添加浓度的增加而增加;继续投喂未添加MET的正常饲料30 d后,所有试验组均无MET残留;从各批发市场、农贸市场以及各个养殖基地随机抽检的罗非鱼样品及其跟踪饲料中MET阳性率为零。