反相高效液相色谱法分离中华绒螯蟹性腺抑制激素的初步研究

采用反相高效液相色谱层析法(RP-HPLC)分离中华绒螯蟹(Eriocheir sinensis)眼柄视上神经节中的性腺抑制激素,收集在高血糖激素家族区域内出峰的洗脱液组分,得到7组单一的样品组分。通过活体注射的方法,观察和统计每一组分对去眼柄中华绒螯蟹卵母细胞直径大小的影响,以检验每个分离组分的性腺抑制活性。结果发现,其中的第7组分对中华绒螯蟹的卵巢发育具有最强的抑制作用,推测这一组分为中华绒螯蟹的性腺抑制激素。     

中华绒螯蟹大颚器分泌物的HPLC分析

利用三相提取法和高效液相色谱(HPLC)对中华绒螯蟹血淋巴及大颚器(MO)提取物进行了定性、定量的分析。结果发现在血淋巴及MO提取物中基本都可检测到三种物质:保幼激素Ⅲ(JHⅢ)、A和B。A物质的分泌受到眼柄因子的抑制调控,可能就是中华绒螯蟹MO的分泌物—法呢酸甲酯(MF)。     

中华绒螯蟹卵细胞透明液的配方

为了快速准确判断中华绒螯蟹卵细胞是否发育成熟,实验根据斑马鱼卵细胞透明液的配方进行一系列的探索,最终发现用无水乙醇、冰乙酸和甲醛按60∶1∶30的体积比混合成的透明液处理中华绒螯蟹卵细胞,在细胞内可以观察到一白色斑点。对卵巢组织进行切片观察,证实该斑点即为中华绒螯蟹卵细胞的细胞核。在发育成熟的卵细胞中则不能观察到白色斑点。研究表明,无水乙醇、冰乙酸和甲醛按60∶1∶30的体积混合成的透明液能够有效透明中华绒螯蟹卵细胞,为准确判断其卵母细胞是否发育成熟,及时对卵母细胞进行人工诱导具有重要的实用价值。     

红螯螯虾XO-SG神经细胞的原代培养

本研究采用酶解的方法获得红螯螯虾眼柄XO-SG复合体的单个神经细胞,并依据显微观察对XO-SG神经细胞进行分类,同时利用Leibovitz's L-15等作为基础培养基离体培养解离的神经细胞。目的是建立虾类XO-SG神经细胞的分类标准并确定合适的培养条件,便于在体内外开展神经内分泌系统的调控研究。结果显示,根据神经细胞形态特点,红螯螯虾XO-SG神经内分泌细胞分为6种类型,不同类型的细胞在细胞大小以及显微结构上存在明显差异;建立了红螯螯虾眼柄XO-SG神经元的体外原代培养方法,细胞在改良的L-15培养基中存活状态良好,原代培养的神经细胞可以在体外存活14 d。离体培养过程中,不同类型神经细胞的再生速率存在差异,部分细胞在第2天就出现再生的轴突或树突。再生过程基本可以持续7 d,随后细胞开始萎缩凋亡。本研究为红螯螯虾眼柄神经细胞的分类以及利用神经细胞在体外开展虾类的内分泌代谢和调节机制研究提供了基础依据和研究平台。     

中华绒螯蟹离体卵巢发育的调控作用

通过卵巢离体实验研究性激素、大豆黄酮、生物胺及神经组织对中华绒螯蟹(Eriocheir sinensis)卵巢发育的影响。结果表明，雌二醇可显著地增大卵黄发生前期、初级卵黄发生期和次级卵黄发生期卵巢卵母细胞的直径，黄体酮可显著增大卵黄发生前期卵巢卵母细胞直径，对初级卵黄发生期和次级卵黄发生期的卵母细胞直径有极显著地促进作用；大豆黄酮具有增大卵母细胞直径的作用，但作用不显著；脑和胸神经团对卵黄发生前期、初级卵黄发生期的卵母细胞直径有显著增大作用，对于次级卵黄发生期卵母细胞直径有极显著增大作用；视神经节作用不明显；5-羟色胺通过脑可显著促进卵黄发生前期、初级卵黄发生期卵巢卵母细胞直径的增大，极显著促进次级卵黄发生期卵巢卵母细胞直径的增大，还通过胸神经节极显著增大各期卵巢的卵母细胞直径；多巴胺作用不明显。     

克氏原螯虾卵巢不同发育期大颚器法尼酸甲基转移酶活力分析

法尼酸甲基转移酶(farnesoic acid O-methyl transferase,FA-O-MeT)具有催化法尼酸(farnesoic acid,FA)转变成甲基法尼酯(methyl farnesoate,MF)的功能。运用放射化学法对克氏原螯虾不同发育期大颚器(mandibular organ,MO)FA-O-MeT的酶活进行了分析,并探讨了眼柄因子对FA-O-MeT酶活的调控。结果表明,克氏原螯虾MO中的FA-O-MeT酶活左、右侧对称,差异不显著(P>0.05);FA-O-MeT酶活随卵巢发育呈周期性变化,次级卵黄发生期的MO酶活最高,达到61.75 pmol/(p·h);镊烫法破坏眼柄内的X器官窦腺复合体后,FA-O-MeT酶活逐渐上升,第5天酶活达到最高;利用眼柄内的窦腺提取物体外培养处理MO,检测到其对FA-O-MeT酶活具有强烈的抑制作用(P<0.01)。研究表明,克氏原螯虾FA-O-MeT酶活与卵巢发育密切相关,且其生理活性受到眼柄X器官窦腺复合体的负调控。     

不同pH下硫化物胁迫对中华绒螯蟹亲体卵巢发育的影响

中华绒螯蟹(Eriocheir sinensis)亲体初始体质量为(80.228±6.270)g。实验采用2×3因子设计,设置pH 6.5条件下的3个硫化物浓度组LS1(0.1 mg/L)、MS1(0.33 mg/L)和HS1(1.0 mg/L),以及pH 8.5下的3个硫化物浓度组LS2(0.1 mg/L)、MS2(0.33 mg/L)和HS2(1.0 mg/L),共6个处理组;另设一个不添加硫化物的对照组CTR(自然pH值为7.5),设2个平行处理,分别在实验后第10天、20天和30天取样,以性腺指数(GSI)、卵巢卵黄磷蛋白(Lv)含量、卵巢Lv总含量和卵巢蛋白质含量的变化为评判指标,研究了硫化物胁迫对中华绒螯蟹亲体卵巢发育的影响。结果表明,不同pH条件下各处理组的GSI在30 d时均低于对照组,其中HS组显著低于对照组(P<0.05)。在各取样时间点,所有处理组蟹卵巢的Lv含量均低于对照组,且MS和HS组在前20 d显著低于对照组(P<0.05),但这种差异随时间的推移而逐渐减小;至暴露结束时仅HS1组蟹的卵巢Lv含量显著低于对照组(P<0.05)。各处理组蟹的卵巢Lv总含量始终低于对照组,且这种差异随时间的推移而逐渐增大;第10天时仅HS1组的Lv总量显著降低,而时间延长至30 d后,各处理组Lv总量均显著低于对照组(P<0.05)。结论认为,慢性硫化物胁迫会抑制中华绒螯蟹雌体的卵巢发育,相比之下,弱酸条件下水体中硫化物的毒性作用有所增强。     

高效氯氰菊酯对中华绒螯60 d慢性毒理及病理研究

为研究高效氯氰菊酯对中华绒螯扣蟹生长发育的影响,该实验采取急性毒性试验方式,以60d为记录周期,定时测定其体质量与存活率。试验结果表明,中华绒螯蟹(Eriocheir sinensis)扣蟹在高效氯氰菊酯(0.10、1.00mg/L处理组)作用下体质量明显降低。尤其是1.00mg/L浓度组最明显。0.01mg/L处理组和对照组扣蟹无明显异常现象。且在1.00mg/L浓度组下存活率较其他浓度也下降得很快。说明相比较0.10mg/L和0.01mg/L,1.00mg/L浓度的高效氯氰菊酯对中华绒螯扣蟹毒性最强。石蜡切片结果显示:一定浓度的氯氰菊酯会对河蟹的肝胰腺、鳃和肌肉组织产生明显的病理变化。     

莠去津对中华绒螯蟹蜕皮抑制激素表达的影响

用RT-PCR方法检测了中华绒螯蟹暴露于不同质量浓度[0(DMSO对照)、0.001、0.01、0.1、1、10 mg/L]莠去津后蜕皮抑制激素在眼柄视神经节中的表达。结果表明,低质量浓度的莠去津可诱导蜕皮抑制激素的合成分泌,其中0.1 mg/L处理组作用明显。莠去津对中华绒螯蟹的蜕皮活动有一定影响。     

中华绒螯蟹、日本绒螯蟹及其杂交种性腺发育和生化组成的比较

研究了中华绒螯蟹(Eriocheir sinensis)、日本绒螯蟹(E.japonica)及其杂交种成蟹性腺指数、肝胰腺指数、出肉率、总可食率及其常规生化成分,结果显示:(1)中华绒螯蟹的精巢指数显著高于其它两种群蟹,但其肝胰腺指数、出肉率和总可食率均无显著差异;中华绒螯蟹的卵巢指数也显著高于日本绒螯蟹和杂交蟹,而杂交蟹的肝胰腺指数显著高于其它两种群蟹。(2)中华绒螯蟹、日本绒螯蟹和杂交种精巢中水分、蛋白和脂肪含量分别最低,日本绒螯蟹精巢中碳水化合物含量最高;杂交种肝胰腺蛋白、脂肪和碳水化合物含量均最高,日本绒螯蟹肌肉中脂肪含量显著低于其它两种群。(3)日本绒螯蟹卵巢水分含量显著高于其它两种群蟹,蛋白、脂肪和碳水化合物含量显著低于其它两种群;日本绒螯蟹肝胰腺的水分和蛋白含量最高,但脂肪含量最低,杂交种肝胰腺中脂肪含量最高、水分含量最低;日本绒螯蟹肌肉中的水分含量最高,其它生化成分含量最低;中华绒螯蟹肌肉的水分含量最低,其它生化成分含量最高。整体上,中华绒螯蟹具有早熟特性,不同种群绒螯蟹性腺中的主要生化成分含量可能与其发育阶段有关,杂交蟹肝胰腺中脂肪含量较高,日本绒螯蟹肌肉中脂肪含量较低。     

眼柄摘除对红螯光壳螯虾生长、性腺发育及体色的影响

为探究眼柄摘除对红螯光壳螯虾(Cherax quadriarinatus)生长、性腺发育及体色的影响,对6月龄红螯光壳螯虾[体质量(47.47±3.48)g]进行了摘除眼柄处理实验。结果显示,4周后摘除双侧眼柄组红螯光壳螯虾体质量为(84.40±13.41)g,显著高于不摘除眼柄组[(49.63±6.47)g]和摘除单侧眼柄组[(51.47±4.08)g](P<0.05);摘除双侧眼柄组红螯光壳螯虾存活率为60.0%±4.8%,显著低于不摘除眼柄组和摘除单侧眼柄组(分别为94.4%±2.3%和97.2%±2.1%);摘除双侧眼柄组红螯光壳螯虾性腺指数为1.23±0.50,显著高于不摘除眼柄组(0.20±0.06)和摘除单侧眼柄组(0.35±0.08)。利用ImageJ软件对红螯光壳螯虾体色RGB值分析表明,摘除双眼柄组红螯光壳螯虾体色G值和B值(分别为99.26±5.23和98.40±3.58)显著高于摘除单侧眼柄组(59.02±3.85和44.07±4.57)(P<0.05)。结果表明,眼柄摘除能促进红螯光壳螯虾生长和性腺发育,影响体色变化。     

Histological examination of final oocyte maturation and atresia in wild and domesticated Penaeus monodon (Fabricius) broodstock

Oocyte maturation and gonadosomatic index (GSI) of eyestalk ablated Penaeus monodon females collected from the wild and from two first‐generation domesticated lines were assessed. Frequency and diameter of the different oocytes, and the intensity of oocyte atresia, were compared among groups through histological assessments of the sections of the middle ovarian lobe. Digitized images from ovary sections were used to record the frequency and diameter of different oocyte types. Spawning performance of the three groups were expressed in terms of the percentage of females that spawned at least once (productive females), time from eyestalk ablation to first spawning (latency period) and the number of spawnings per female stocked. Final ovarian maturation was attained in all groups, as indicated by the presence of mature oocytes with cortical rods (cortical oocytes), dark‐green ovarian colour and high GSI values (5.83–6.86%). However, domesticated groups presented significant larger immature oocyte types (previtellogenic and yolky oocytes) and smaller cortical oocytes compared with wild females, indicating a reduced vitellogenic activity during final oocyte maturation. Additionally, the frequency of atresia was comparatively higher for both domesticated groups, which could be related to their inferior spawning performance. The implications of these results on the reproductive potential and development of domesticated P. monodon are discussed.     

切除单侧眼柄对不同体质量养殖斑节对虾卵巢发育及产卵的影响

在盐度31、水温28~29℃下,进行了4次卵巢诱导试验,研究了切除单侧眼柄对体质量56.4~105.4g的养殖斑节对虾卵巢发育和产卵的影响及卵巢发育、产卵与体质量之间的关系。结果显示,切除单侧眼柄诱导养殖斑节对虾卵巢发育成熟,成功率(y)与雌虾体质量(x)显著相关(P0.05),雌虾体质量越小,诱导成功率越低,回归方程为:y=133.92lnx-535.63(r2=0.9297)。养殖斑节对虾眼柄手术后,最快10d产卵,部分雌虾能重复产卵。产卵量(y)与雌虾体质量(x)显著正相关(P0.05),雌虾体质量越小,产卵量越小,回归方程为:y=122.22lnx-472.2(r2=0.9738)。雌虾累积死亡率、卵子直径和卵子孵化率与雌虾体质量无显著相关性(P0.05)。建议用体质量80g以上的养殖斑节对虾雌虾作为亲虾。     

绵羊卵丘细胞对卵母细胞减分恢复核成熟的影响

实验研究了卵丘细胞与卵母细胞通过间隙连接通道对绵羊卵母细胞核成熟的影响。采用抽吸法从2～6mm的卵巢上收集的卵丘-卵母细胞复合体COCs。将部分COCs脱去全部卵丘细胞,成为机械裸卵(DOs)后,分4种方式培养,即卵丘细胞包裹的卵母细胞(CEOs)的单独培养、DOs与COCs共培养(DOs(COCs))、DOs与卵丘颗粒细胞共培养的(DOs(CCs)),以及DOs单独培养,培养26h,用0.2%地衣红对绵羊卵母细胞进行染色检查。各组卵母细胞到达减数分裂2期的比率分别是79.6%、5.6%、9.9%和46.4%,4组间都有显著差异统计核成熟率(P<0.05)。表明卵丘细胞及间隙连接对卵母细胞减数分裂恢复核成熟有非常重要的作用。     

Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone

The present study analyses some biochemical variables in haemolymph proposed as predictive indicators of the maturation capability following eyestalk ablation. Haemolymph of captive females was obtained before and 8 days after eyestalk ablation, and levels of haemocyanin, total proteins, glucose, lactate, cholesterol, triacylglycerides and vitellogenin were determined. Biochemical variables were also analysed in the hepatopancreas at the end of the experiment. Females were grouped as immature (previtellogenic stage) and mature (vitellogenic and cortical stages) based on histological analysis done 8 days after eyestalk ablation. To analyse haemolymph variables before eyestalk ablation in relation to maturation capability, immature females were classified as those with a low maturation capability and mature females as those with a high maturation capability. Females of high maturation capability had significantly higher vitellogenin levels before eyestalk ablation than females of low maturation capability. No significant differences were found for the other biochemical variables. Vitellogenin was also higher in mature than in immature females at the end of the experiment. These results indicate that vitellogenin levels in haemolymph could be used as possible predictive criteria of maturation capability, possibly because they reflect the degree of ovarian development at the time of eyestalk ablation.     

Ovarian re‐maturation following the first spawning in the Chinese mitten crab,Eriocheir sinensis (H. Milne‐Edwards)

The present study investigated the external and histological changes in the ovary and measured the gonadosomatic index (GSI) and hepatosomatic index (HSI) during the re‐maturation of the Chinese mitten crab Eriocheir sinensis. The results show that the ovarian re‐maturation cycle of this crab species can be divided into four stages, i.e. Stage I: the ovary had an X shape and appeared milky white or with a mottled buff colour, and the dominant gametocyte at the stage were oogonia (OG, 28.3%) and previtellogenic oocytes (PR, 31.6%); Stage II: the ovary appeared light yellow or tan in colour, dominated by endogenous vitellogeic oocytes (EN, 69.3%). The yolk globules of unreleased mature oocytes (MO) from the previous spawn were absorbed, leaving many vacuoles in the retrogressing MO; Stage III: the ovary appeared crimson red or deep purple, and although the main type of gametocyte was exogenous vitellogenic oocytes (EX), near‐mature oocytes (NO) could also be found in the late phase of the ovarian development stage; Stage IV: the ovary was ripe and filled with MO that contained large yolk globules. During the period of the second ovarian maturation cycle, the GSI increased significantly from the time just after the first spawning (P<0.01). However, although HSI appeared to decrease within days after spawning (DAS), no significant correlation was found between GSI and HSI or between HSI and DAS (P>0.05). Furthermore, the final GSI and the volume of MO of E. sinensis during the second maturation cycle were drastically lower than those of the first maturation cycle.     

Preliminary Observations on the Reproductive Physiology of Female Orangemouth Corvina in Captivity

Seasonal changes In oocyte growth and plasma estradiol, testosterone, thyroid hormones and vitellogenin levels were monitored in three captive adult female orangemouth corvina Cynoscion xanthulus subjected to a condensed (8 mo) seasonal cycle of photoperiod and water temperature. Plasma concentrations of estradiol and testosterone began to rise when temperature increased to 28 C and photoperiod to 15 h light (midsummer conditions). This was accompanied by elevated circulating levels of vitellogenin and the appearance of vitellogenic oocytes (diameter > 100 μm). Estradiol concentrations and mean oocyte size increased concurrently during late summer conditions and were maximum during fall conditions, approximately 8 wk after the beginning of ovarian recrudescence. In contrast, plasma levels of thyroid hormones did not show any distinct seasonal changes. Gonadally recrudesced females contained several size classes of vitellogenic oocytes. Injection of these fish with a luteinizing hormone-releasing hormone analog (LHRHa) caused maturation and spawning of the largest oocytes (mean diameter of follicles × 2500 μm). Another group of vitellogenic oocytes had been recruited into this size class by 2 wk after the end of spawning, which suggests that this species is capable of repeated spawning during the reproductive season. Injection of LHRHa resulted in increased plasma levels of gonadotropin and repeated spawning over several days. LHRHa-induced final oocyte maturation, ovulation and spawning were preceded by increases in plasma levels of two teleostean maturation-inducing steroids, 17α,20β, 21-trihydroxy-4-pregnen-3-one (20β-S) and 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P₄). The results provide preliminary evidence that oocyte growth is stimulated in orangemouth corvina subjected to an abbreviated seasonal cycle in captivity under midsummer photoperiod and temperature conditions and is associated with seasonal increases in plasma estradiol concentrations and vitellogenin production. However, the relative importance of 20β-S versus 17α, 20β-P₄ in the control of final oocyte maturation in this species could not be determined from the results of the present study.