卵黄蛋白原研究及其进展

甲壳动物和昆虫由于进化上亲缘关系近,被统称泛甲壳动物,大多数泛甲壳动物卵黄蛋白原为雌性特异性蛋白,是卵母细胞中储存的卵黄蛋白的前体,可为胚胎发育提供营养和能量,是决定繁殖性能的关键因素,在生殖发育中起着十分重要的作用。已有研究证明,调控泛甲壳动物卵黄蛋白原的主要激素有保幼激素、蜕皮激素、神经肽和胰岛素样肽等。昆虫保幼激素和蜕皮激素发挥促进作用,而神经肽和胰岛素样肽对卵黄蛋白原合成的调控作用因种类不同而存在不同。除了以上激素,甲壳动物特有的眼柄高血糖激素家族以及促雄腺激素(IAG)对卵黄蛋白原的合成起负调控作用。本文总结了近年来泛甲壳动物卵黄蛋白原合成调控机制的相关研究,概括并比较了调控昆虫与甲壳动物的卵黄蛋白原合成的主要激素及调控机制,为养殖虾蟹甲壳动物生殖调控相关研究提供参考。

     

泛甲壳动物激素调控卵黄蛋白原合成的分子机制研究进展

昆虫和甲壳动物由于进化上亲缘关系近统称泛甲壳动物，大多数泛甲壳动物卵黄蛋白原为雌性特异性蛋白，是卵母细胞中储存的卵黄蛋白的前体，可为胚胎发育提供营养和能量，是决定繁殖性能的关键因素，在生殖发育起着十分重要作用。业已证明，调控泛甲壳动物卵黄蛋白原的主要激素有保幼激素、蜕皮激素、神经肽和胰岛素样肽等。昆虫保幼激素和蜕皮激素发挥促进作用，而神经肽和胰岛素样肽对卵黄蛋白原合成的调控作用因种类不同存在不同。除了以上激素，甲壳动物特有的眼柄高血糖激素家族以及促雄腺激素对卵黄蛋白原的合成起负调控作用。本文总结了近年来泛甲壳动物卵黄蛋白原合成的调控机制的相关研究，概括并比较了调控昆虫与甲壳动物的卵黄蛋白原合成的主要激素及调控机制，为养殖虾蟹甲壳动物生殖调控相关研究提供参考。     

甲壳动物眼柄神经内分泌系统研究概述

眼柄X器官-窦腺复合体(XO-SG)被称为甲壳动物的内分泌调控中心,如同开关一样控制着虾蟹等甲壳动物的生长代谢和生殖发育等重要的生理活动。文中综述了眼柄神经内分泌系统的结构和功能、神经肽类激素的种类和作用机制,重点阐述了甲壳动物眼柄神经肽类激素分泌和调控研究进展,以期为研究和实现在生产上控制虾蟹的繁殖和生长提供参考。     

虾蟹类眼柄激素的生理生化及基因克隆研究进展

甲壳动物内分泌学研究主要集中在虾蟹类、虾蟹类内分泌学研究对于揭示甲南动物的生长与繁殖以及指导经济虾蟹类养殖生产实践具有重要意义，尤其是眼柄内分泌激素在调控甲壳运物的蜕皮、变态发育、生殖、血糖平衡及体内的渗透压等方面起着重要的作用，一直是神经内分泌学的研究热点^[1,2]。本主要对虾蟹类的眼柄神经内分泌系统的结构、激素和种类及其生理生化特性，特别是基因结构及克隆等方面作一综述，为进一步开展虾蟹类内分泌学研究提供参考资料。     

我国鲆鲽类生殖内分泌研究进展

鱼类的生殖和苗种繁育是鱼类养殖业持续健康发展的必要前提和关键技术基础之一,是海洋生物技术的重要研究领域.近年来,我国北方沿海鱼类繁殖生理主要围绕鲆鲽类“下丘脑-垂体-性腺轴”的组织结构特征、性腺发育规律、性类固醇激素、生殖相关功能基因内分泌调控机制等方面开展了较系统的研究.本文重点介绍主要养殖鲆鲽类“下丘脑-垂体-性腺轴”中表达的重要功能基因的研究进展,并综述了鲆鲽类生殖相关组织的结构及其内分泌系统特征、性腺发育的生理特性及其与环境因子和激素诱导的关系、性类固醇激素的表达变化规律及其与水温及光周期调控关系等.旨在为鲆鲽类生殖活动的精准调控和建立苗种繁育新技术提供参考.     

甲壳动物雄性腺研究的进展

雄性腺（ａｎｄｒｏｇｅｎｉｃｇｌａｎｄ）又名促雄性腺,是甲壳动物的一种内分泌器官,只存在于雄性体内,它所分泌的物质称为雄性腺激素（ａｎｄｒｏｇｅｎｉｃｈｏｒｍｏｎｅ）。四十多年来,对于甲壳动物雄性腺的位置和形态、发生和发育、结构和功能以及雄性腺激素的...     

中华绒螯蟹类浮舰蛋白基因Esflol的克隆和组织表达分析

甲壳动物高血糖激素家族目前仅被发现存在于节肢动物中,在血糖水平调节、蜕皮、应激反应等多种生理代谢活动中发挥着重要的作用。为了解这个家族中的重要一员--甲壳动物高血糖激素(crustacean hyperglycemic hormone, CHH)如何调节河蟹体内的血糖水平,我们前期进行了转录组和表达谱分析,最终选取Esflol(Eriocheir sinensis flotillin-1 like,Esflol)作为我们的研究对象。根据转录组数据提示,结合RACE方法首次从中华绒螯蟹肝胰腺组织中克隆得到Esflol基因并进行了序列及组织表达分析。结果显示Esflol基因开放阅读框(ORF)1281bp,编码426个氨基酸,具有典型的SPFH超家族和PHB结构域,不含信号肽序列。多重序列比对分析表明,Esflol氨基酸序列与中华绒螯蟹flotillin-1相似度最高,达到57%。荧光定量PCR结果显示成熟中华绒螯蟹Esflol基因在肝胰腺组织中的表达量最高,其次为肌肉,另外在心,鳃,肠,胸神经节和胃中均有一定量表达,在脑中表达量较低。上述实验结果为下一步继续研究中华绒螯蟹糖代谢的生理机制奠定了基础,也为其他养殖类甲壳动物糖类代谢的研究提供了重要参考。     

中华绒螯蟹眼柄神经多肽激素的分离纯化及活性鉴定

采用反相高效液相色谱技术(RP-HPLC),首次分离、纯化了中华绒螯蟹(Eriocheir sinensis)眼柄神经多肽激素,获得了高血糖激素(crustacean hyperglycemic hormone,CHH)纯品。初步的生理活性鉴定结果表明,该激素有显著的升血糖作用,能使中华绒螯蟹血糖水平在30min内升到最高值,为(56.14±10.58)mg.100mL-1(n=4),比正常水平增加了(2.87±0.73)倍,被确定为甲壳动物高血糖激素(CHH)。CHH在窦腺(sinus gland,SG)中的含量最丰富,占SG粗提物蛋白总量的(12.26±2.57)%,占SG激素总量的(52.90±11.09)%。生化特征研究结果表明,河蟹眼柄CHH的分子量为5.1kD,显著低于已知甲壳动物CHH的分子量。研究结果也为进一步研究河蟹神经多肽激素的合成和分泌及调控机制等奠定了基础。     

GnIH多肽对半滑舌鳎(Cynoglossus semilaevis)下丘脑生殖相关基因表达的影响

本研究首次通过下丘脑离体孵育的方法研究促性腺激素抑制激素(Gonadotropin-inhibitory hormone,GnIH)多肽对半滑舌鳎(Cynoglossus semilaevis)下丘脑中生殖相关基因的表达调控.研究结果显示,tsGnIH-1促进了gnrh2和gnih的表达,对gnrh3和kiss2的表达无影响;tsGnIH-2抑制了gnrh3的表达,对gnrh2、kiss2和gnih的表达无影响.GnIH多肽对生殖相关基因的不同调控表明同一前体蛋白编码的不同GnIH多肽在生殖调控中的作用不尽相同.本研究结果增加了对GnIH参与鱼类生殖调控机制的认识,为深入研究奠定了基础.     

对虾繁殖内分泌调控研究进展

对虾繁殖受到内分泌系统释放的激素调控，从激素对性腺发育引起的作用，可将其分为两大类，一类为促使对虾性腺发育的诱导因子，另一类为限制性腺发育的抑制因子。诱导因子有甲基法尼醇－一种由大颚腺分泌的结构类似保幼激素的物质、性腺诱导激素、甲壳类红色素集合激素、脱皮抑制激素、类固醇激素和外激素等。     

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。     

Identification of SNPs in the 5′‐flanking region and 3′‐UTR of the MIH gene and their association with precocity of the Chinese mitten crab Eriocheir sinensis

Moult‐inhibiting hormone (MIH), an important regulator of steroidogenesis, inhibits the synthesis of ecdysteroid in Y‐organ (YO) and plays a significant role in the regulation of moulting and post‐embryonic development of crustacean. Because unsuccessful moulting have been widely observed in precocious crabs, we investigated whether genetic variants in the 5′‐flanking region and 3′‐untranslated region (3′‐UTR) of the MIH gene are associated with precocity of the Chinese mitten crab. Thirty individual DNA samples were sequenced to search for SNPs in the 5′‐flanking region and 3′‐UTR of the MIH gene. Five SNPs (g.196 T>A, g.230 C>T, g.305 T>C, g.323 C>A and g.372 C>T) in the 5′‐flanking region and 6 SNPs (g.2677 C>T, g.2759 T>A, g.2807 T>C, g.3042 A>G, g.3088 T>G and g.3295 T>G) in the 3′‐UTR of the MIH gene were selected for the individual genotyping in a two‐stage association study. We found that a SNP g.3088 T>G in the 3′‐UTR of MIH gene was consistently associated with precocity of the Chinese mitten crab in stage 1 and stage 2, with a per‐allele OR (Odds Ratio) of 1.469 (95% CI: 1.169–1.844) after two stages combined (P = 0.001). However, no significant associations were observed between the other 10 SNPs and precocity of the Chinese mitten crab. To our best knowledge, this is the first association study between various SNP genotypes and phenotype attributes in Chinese mitten crab. Our findings suggest that the SNP g.3088 T>G may be a candidate marker for effective marker‐assisted selection to decrease the precocity of the Chinese mitten crab in future studies.     

Induction of spawning in Pacific white shrimp Litopenaeus vannamei (Boone, 1931) by injection of its molt inhibiting hormone isoform II produced in E. coli

The optimization of reproductive parameters in shrimp farming continues to be a challenge for most producing countries. Although the crustacean neuropeptides have been studied extensively in the last two decades, the functions of most of these neuropeptides remained putative. Among them, molt‐inhibiting hormone isoform II (MIH II) has shown an important role in vitellogenesis. In this study, the cDNA encoding mature MIH II peptide was isolated by RT‐PCR from the L. vannamei eyestalk. The cDNA was cloned into pET28a bacterial expression vector. Recombinant MIH II was obtained in the form of insoluble inclusion bodies and purified to ~88% purity. Two doses of rMIH II and a negative control group were assayed in vivo. The stages of ovarian maturation and spawning were recorded during 72 hr post‐injection. The results showed that ovarian maturation occurred approximately in 9% and 33% of females injected with rMIH II at the doses of 300 and 600 ng/gbw respectively. Neither maturation nor spawning was detected in the negative control group. Females injected with 600 ng/gbw, which showed vitellogenic stages III and IV, spawned. These preliminary results argue that the hormone rMIH II could be a promising candidate to induce spawning in L. vannamei shrimp.     

36种药物对青苔的抑杀作用

为了筛选抑杀河蟹养殖池塘中青苔的有效药物,通过受试青苔外观生长情况及显微观察对36种药物抑杀青苔的作用效果进行了定性研究,并对筛选出的8种对青苔有较好作用效果且有一定性价比的药物,采用青苔生物量法及青苔叶绿素a含量检测法进行了定量研究,比较每种药物对青苔的EC50值,结果表明:对青苔抑杀作用最强的是50%氟乐灵,其次是40%扑草净、98%SYXA、98%五水硫酸铜、99%2,2-二溴-3-次氮基丙酰胺(DBNPA)、99%十二烷基二甲基苄基溴化铵(苯扎溴铵)、14%异噻唑啉酮、博落回等。     

海洋低温溶菌酶抗肿瘤活性的初步研究

通过观察海洋低温溶菌酶对体外培养细胞株增殖的影响、对角膜诱生血管的影响、对小鼠肉瘤S180（实体型）及对小鼠肝癌HAC（实体型）的抑制作用，探讨海洋低温溶菌酶抑制肿瘤生长活性。结果表明，该酶与蛋清溶菌酶在抑瘤作用机制上有很大区别，海洋低温溶菌酶具有血管生成抑制因子的活性，故对肿瘤生长具有明显的抑制作用。     

Enhanced survival in striped bass fingerlings after maternal triiodothyronine treatment

Elevation of the triiodothyronine (T₃) content of striped bass (Morone saxatilis) eggs by maternal T₃ injection confirms the uptake of T₃ by oocytes. The resulting offspring were influenced favorably by the T₃, as seen in quantitative indices of development. As reported previously, larvae from T₃-supplemented eggs raised under laboratory conditions exhibited increased body area, length, dry weight, and rates of swimbladder inflation and survival, compared to controls. Also, the T₃ content of unfertilized oocytes correlated positively and highly significantly with survival to two weeks of age within individual cohorts (Brownet al., 1988). In the present study, the survival of experimental and control striped bass was monitored through the fingerling stage, under hatchery production conditions. The rate of recovery of maternally T₃-treated cohorts from pond-culture was approximately fourfold that of controls. The striking effects of T₃ enrichment of eggs on offspring indicate the potential contribution of maternal hormones in striped bass development, and suggest possible applications in aquaculture.     

鳖组织浆对鼠免疫功能的影响

从体内抑制试验和体外培养巨噬细胞的杀瘤试验对甲鱼组织浆的抑瘤作用进行了研究。体内抑瘤试验采用组织浆灌胃方式,计算抑瘤率和生命延长率。体外试验将巨噬细胞与肿瘤细胞共培养,测定ＮＯ浓度与细胞毒效应。培养上清液中一氧化氮（ＮＯ）浓度采用ＮＯ试剂盒检测,细胞毒效应通过ＭＴＴ比色法检测。用黄嘌呤氧化酶法测定用鳖组织浆大鼠脑、肝、血中的超氧化物歧化酶（ＳＯＤ）含量。结果表明,不同来源鳖的效果有区别,野生鳖和仿生养殖鳖２龄与４龄对Ｓ１８０小鼠肿瘤生长有明显的抑制作用（Ｐ〈０．０１）,其抑瘤率分别达７０．６９％和６３．１７％,荷瘤小鼠的生长延长率均显著高于空白对照组（Ｐ〈０．０１）,分别为４０．５９％和３９．９５％;１龄和２龄温室鳖的抑瘤效果也显著（Ｐ〈０．０５）。不同来源和不同年龄的鳖组织抑瘤效果有差异,野生鳖、仿生鳖和温室     

Effects of 11-ketotestosterone and gonadotropin-releasing hormone on follicle-stimulating hormone and luteinizing hormone gene expression in castrated and sham-operated male red seabream <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:   In order to clarify the roles of androgen and gonadotropin-releasing hormone (GnRH) on gonadotropin (GTH; luteinizing hormone [LH] and follicle stimulating hormone [FSH]) synthesis, effects of castration and implantation of GnRH analog (GnRHa) or 11-ketotestosterone (11-KT) on expression of GTH subunit, α-glycoprotein subunit (αGSU), FSHβ, and LHβ genes, during the early spermatogenic stage in male red seabream Pagrus major were examined. Male red seabream underwent castration or sham-operation and were subsequently implanted with cholesterol pellets containing GnRHa, silicone capsules filled with 11-KT, or blank capsules (control). FSHβ mRNA levels increased due to castration, and it was reversed by treatment with 11-KT. 11-ketotestosterone treatment also decreased FSHβ mRNA levels in sham-operated fish. These results suggest that 11-KT acts on the pituitary to suppress FSH synthesis in male red seabream. On the other hand, neither castration nor replacement of 11-KT in castrated fish had effects on LHβ mRNA levels, whereas 11-KT treatment had slightly but significantly decreased LHβ mRNA in sham-operated fish. αGSU mRNA levels were not changed by castration or 11-KT treatment in both sham-operated and castrated fish. Meanwhile, treatment with GnRHa significantly decreased FSHβ mRNA levels in sham-operated fish, but not in castrated fish. This suggests that GnRHa may down-regulate expression of FSHβ mRNA through the production of 11-KT in testis. LHβ and αGSU mRNA levels in sham-operated fish, but not in castrated fish, were significantly elevated by treatment with GnRHa.     

三苯基锡对褐菖鮋卵巢发育的影响

尽管三苯基锡(triphenyltin,TPT)与三丁基锡(tributyltin,TBT)在海洋环境中经常同时存在,但相对于TBT,对TPT的毒性研究还较少。本实验以海洋经济鱼类褐菖鲉(Sebastiscus marmoratus)为对象,研究环境水平TPT对卵巢发育的影响。结果发现,TPT抑制了卵巢发育,表现为性腺指数的降低。TPT还诱导卵巢总脂质水平的增加,而卵巢脂质蓄积可能影响卵巢的正常生殖功能。另外,TPT暴露后,卵巢游离睾酮水平增加,卵巢游离17β-雌二醇水平降低;同时,血清三碘甲腺原氨酸水平降低。因此,TPT对性激素和甲状腺激素水平的影响可能是其抑制卵巢发育的重要机制。实验结果为全面了解TPT产生鱼类生殖毒性效应、途径和机制提供了新的重要的资料。