抑卵激素对家蝇卵巢周期性发育的调控

抑卵激素是调控家蝇Musca dorncstica vicina卵巢周期性发育的关键因子之一。在家蝇中,当第一个周期的卵母细胞处于卵黄发生期或卵黄发生后期时,其第二个周期的卵母细胞的发育不进入卵黄发生期。本文建立了家蝇抑卵激素的生物测定方法,即用一对卵巢提取物注射1头羽化后12h家蝇,并在羽化后60h观察卵母细胞的发育及卵黄蛋白的沉积情况。抑卵激素的作用首先是延缓了卵母细胞在卵黄发生前期的发育;其次,抑卵激素抑制脂肪体中卵黄蛋白的合成,导致血淋巴中卵黄蛋白含量的下降,从而抑制了卵母细胞的发育。抑卵激素并不抑制卵母细胞对卵黄原蛋白的摄取。卵发育神经激素可以颉抗抑卵激素的抑制作用。抑卵激素无种属特异性。     

壬基酚和雌二醇干扰罗氏沼虾卵黄蛋白原VTG基因表达的效应

对刚孵化后的罗氏沼虾(Macrobrachium rosenbergii)亲虾用高低两个浓度的壬基酚(nonylphenol,NP,100μg/L和0·01μg/L)和雌二醇(estradiol,E2,1μg/L和0·01μg/L)进行浸泡处理,分别于3d和5d对罗氏沼虾肝胰腺和卵巢中卵黄蛋白原(vitellogenin,VTG)基因表达变化进行半定量分析。结果显示,NP和E2能够提高罗氏沼虾肝胰腺和卵巢中卵黄蛋白原VTG基因的表达。100μg/LNP对罗氏沼虾表现出雌激素效应,0·01μg/LNP作用效果不明显;而E2则在1μg/L和0·01μg/L两个浓度下均对罗氏沼虾有较强的雌激素效应。在100μg/LNP作用下,卵巢VTG表达量3d和5d均保持较高水平,无明显下降,其他实验组均是3dVTG基因表达量升高,5d后表达量较3d表达量有所降低。结果表明,与其他动物一样,NP对罗氏沼虾具有内分泌干扰作用。     

小体鲟卵黄蛋白生化特性及合成途径的研究

使用葡聚糖凝胶（Sephadex G-200）从小体鲟鱼卵粗提液中,提纯卵黄脂磷蛋白(Lipovitellin, Lv )和卵黄高磷蛋白(Phosvitin, Pv)。卵黄脂磷蛋白（含糖、磷和脂,等电点7.50）具有雌性特异性, 分子量为144 kD,由97.4 kD和30 kD的大小2个亚基组成。卵黄高磷蛋白（含糖、磷,等电点8.30）其分子量为66 kD,其具有两个亚基,分子量分别为47.6 kD、16.8 kD。制备卵黄脂磷蛋白兔抗血清,采用免疫组化方法对不同年龄小体鲟的肝脏、肠、卵（Ⅱ-Ⅴ期卵巢）及血涂片,进行免疫组织化学定位研究。小体鲟卵巢发育到 Ⅳ期前,卵黄蛋白主要靠卵母细胞自身合成,这个时期内源性合成卵黄蛋白;当卵巢发育到 Ⅳ期,卵母细胞自身不合成卵黄蛋白,主要是通过肝脏合成卵黄蛋白原,通过血液循环运送到卵巢,被卵母细胞吸收后,裂解为卵黄蛋白,这个时期外源性合成卵黄蛋白。     

天蚕卵黄原蛋白的合成、运转与沉积

系统测定了天蚕Antheraea yamamai吐丝结茧至成虫期脂肪体、血淋巴和卵巢中卵黄蛋白和可溶性蛋白总含量的动态变化。结果表明,脂肪体是卵黄原蛋白(Vg)合成场所,Vg合成始于吐丝结茧后第4天;脂肪体、血淋巴中Vg滴度在吐丝结茧后第4天开始上升,化蛹后第6天或第8天达高峰,成虫羽化第1天则明显下降。卵巢对Vg摄取始于化蛹第1天,此后随蛹日龄逐渐上升,并渐趋平稳。同一卵巢管中卵黄蛋白(Vt)含量自顶端至基端随卵室增大而逐渐升高,不同日龄蛹中相应序号卵室的Vt含量以日龄大者为高;卵室中Vt含量与卵室体积大小呈正线性关系。电镜观察表明,Vg被卵母细胞摄入后以卵黄体形式存在,不同发育阶段卵巢中卵母细胞内卵黄体大小不同,以早期者为小;同一卵巢管中不同卵母细胞内卵黄体以顶端为小,基端明显增大,且卵黄体呈网状。     

三疣梭子蟹的第二次卵巢发育规律

2005年3-5月,在室内养殖条件下对第二次卵巢发育的三疣梭子蟹进行连续采样,系统研究了其第二次卵巢发育期间的卵巢指数(GSI)、肝胰腺指数(HSI)、卵巢外部特征、卵巢发育分期及其组织学变化.结果表明:①三疣梭子蟹第二次卵巢发育过程可以分为4期,Ⅰ期卵巢为乳白色或者淡黄色细带状,卵巢中主要为卵黄合成期的卵母细胞和内源性卵黄合成前卵母细胞;Ⅱ期为淡黄色或橘黄色,主要为外源性卵黄合成期的卵母细胞;Ⅲ期卵巢为橘红色,主要为外源性卵黄合成期的卵母细胞和近成熟期的卵母细胞;Ⅳ期卵巢发育基本成熟,肉眼可见卵粒,主要为成熟期的卵母细胞;②三疣梭子蟹第二次卵巢发育期间,GSI显著增加,HSI变化不显著,GSI和发育天数呈显著正相关性,HSI和GSI的变化没有显著的相关性.     

粗糙沼虾卵巢发育的组织学

利用组织切片技术,对粗糙沼虾的卵子发生和卵巢发育周期进行了组织学研究。根据细胞的大小、细胞核和核仁的大小形态及卵黄积累等情况,将卵子发生划分为4个时期,卵原细胞、卵黄合成期的卵母细胞、成熟前期和成熟期。卵黄合成期的卵母细胞又可细划分为3个时期。粗糙沼虾卵巢发育具有一定的规律性。根据卵巢的大小和颜色及每种雌性生殖细胞在卵巢中所占的比例,将卵巢发育划分为7个时期。并通过卵巢发育规律的探讨,对粗糙沼虾的人工养殖提出了合理的建议。     

三疣梭子蟹胚胎发育过程中肝胰腺的发生与卵黄物质利用的关系

通过对三疣梭子蟹胚胎进行连续采样和组织切片,系统研究了三疣梭子蟹胚胎发育过程中卵黄囊和肝胰腺的发生与卵黄物质利用的关系。结果表明:(1)三疣梭子蟹胚胎的卵黄岛和卵黄囊结构分别出现在原肠期和无节幼体期,胚胎从原肠期至卵内第一期溞状幼体期,始终存在卵黄岛结构,且卵黄岛中的卵黄物质不断被分解和利用. (2)卵内第二期溞状幼体后,卵黄囊分为两个区域,卵黄囊壁中出现肝胰腺细胞(柱状上皮细胞),此时肝胰腺前体已开始形成,卵黄岛开始融合. (3)卵内第三期溞状幼体阶段,卵黄囊发育成一双肝胰腺,由于肝胰腺中的卵黄物质互相融合,卵黄岛结构消失。此阶段胚胎对卵黄物质的利用加快, 卵黄物质中存在许多空泡状结构;(4)胚胎发育进入孵化前期后,肝胰腺腔内的卵黄物质极少,而初孵溞状幼体肝胰腺腔内卵黄物质已完全消失,肝胰腺为一对囊状结构。这些结果表明在三疣梭子蟹胚胎发育从原肠期到孵化前的过程中,卵黄岛和肝胰腺细胞对于卵黄物质分解和利用起着十分重要的作用。     

家蝇的卵黄发生及其激素调节

用5—15%SDS-PAGE分析表明,家蝇Musce domestica viaina卵黄蛋白由三个亚基组成,其亚基分子量分别为58KD、50KD、48KD.火箭免疫电泳的结果表明,脂肪体、血淋巴和卵巢内卵黄原蛋白的变化具有密切的相关性,卵黄原蛋白在体内最早出现在羽化后30小时左右,然后迅速增加,在羽化后48小时,脂肪体和血淋巴中卵黄原蛋白含量达到最大值,卵巢开始沉积卵黄蛋白在羽化后30小时,到产卵前达到最大值,脂肪体在离体培养条件下,通过测定³H-亮氨酸掺入卵黄原蛋白的量,对不同发育时期家蝇脂肪体合成卵黄原蛋白的能力及激素的调节作用进行了研究,结果表明,羽化12小时后,合成能力迅速上升,48小时时形成高峰,60小时后迅速下跌直至产卵,其合成能力一直维持在低水平,产卵后合成能力又迅速回升,激素处理结果表明,保幼激素可以促进卵黄发生前期和后期家蝇脂肪体的卵黄原蛋白合成,20-羟基蜕皮酮可以大幅度促进卵黄发生期家蝇脂肪体的卵黄原蛋白合成.当二种激素共同处理时,对卵黄发生前期和卵黄发生期的家蝇脂肪体有协同促进作用,而对卵黄发生后期的脂肪体没有这种作用.本文还对家蝇卵黄发生过程中脂肪体、血淋巴和卵巢三者之间的关系及家蝇卵黄发生的激素调节进行了讨论.     

龟纹瓢虫卵黄蛋白的分子特性及发生动态

研究了龟纹瓢虫Propylea japonica (Thunberg) 卵黄蛋白的基本特性以及卵黄发生过程中卵黄蛋白的动态变化。PAGE和SDS-PAGE实验表明,龟纹瓢虫卵黄蛋白分子量为294.81±40.70 kD,并由分子量分别为144.68±0.03 kD和51.23±0.27 kD的两种亚基组成。对卵黄蛋白的氨基酸组成和含量分析发现,其必需氨基酸总量占57.48％,略高于非必需氨基酸,其中谷氨酸（Glu）含量最高,为15.26％;色氨酸（Trp）和蛋氨酸（Met）含量较低,分别为0.50％和0.11％。采用间接竞争ELISA法,系统测定了龟纹瓢虫成虫期脂肪体、血淋巴和卵巢中卵黄蛋白的动态变化,结果表明：脂肪体是卵黄原蛋白合成的场所,卵黄原蛋白的合成始于羽化后第2天;脂肪体、血淋巴和卵巢中卵黄原蛋白的滴度在羽化后第4天开始迅速上升,至成虫期的第8天左右达到高峰期。     

日本囊对虾Cathepsin B基因在不同卵巢发育时期的表达

采用实时定量PCR技术,以18SrRNA为内标基因,检测日本囊对虾Cathepsin B基因在mRNA水平不同卵巢发育时期的表达。结果显示,Cathepsin B在卵黄合成前期表达最高,内源性卵黄合成期和外源性卵黄合成期表达水平基本一致,而在成熟期表达水平急剧下降。其中卵黄合成前期与其它3期相比具有显著性差异(P0.05),内源性卵黄合成期和外源性卵黄合成期Cathepsin B不具有显著性差异(P0.05),而成熟期与前边三期相比具有显著性差异(P0.05)。推测Cathepsin B可能在日本囊对虾卵巢发育中发挥了作用。     

Molecular Characterization of a cDNA Encoding Vitellogenin and Its Expression in the Hepatopancreas and Ovary during Vitellogenesis in the Kuruma Prawn, Penaeus japonicus

In Crustacea, reproductive function and mechanisms regulating vitellogenesis have not been fully elucidated. This is due in great part to a lack of information concerning the biochemical nature of the vitellogenin molecule, the hemolymph precursor of yolk protein, vitellin, as well as the functional expression of the vitellogenin-encoding gene. We have therefore cloned a cDNA encoding vitellogenin in the kuruma prawn, Penaeus japonicus based on the N-terminal amino acid sequence of the 91 kDa subunit of vitellin. The open reading frame of this cDNA encoded 2,587 amino acid residues. This is the first investigation reporting a full-length cDNA and its corresponding amino acid sequence for vitellogenin in any crustacean species.Northern blot analysis and in situ hybridization have revealed that mRNA encoding vitellogenin was expressed in both the follicle cells in the ovary and the parenchymal cells in the hepatopancreas. In nonvitellogenic females, vitellogenin mRNA levels were negligible in both the ovary and hepatopancreas, but in vitellogenic females, levels were dramatically increased in both tissues. In the ovary, highest levels were observed during the early exogenous vitellogenic stage, and thereafter rapidly decreased, whereas in the hepatopancreas, high levels were maintained until the onset of the late vitellogenic stage. Differing profiles of vitellogenin mRNA levels in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenin synthesis harbor separate and complementary roles during vitellogenesis.     

Hepatopancreas is the likely organ of vitellogenin synthesis in the freshwater prawn, Macrobrachium rosenbergii

The objective of the present study was to investigate the source of vitellogenin in the freshwater prawn, Macrobrachium rosenbergii. Ovarian development of M. rosenbergii was classified into five stages (stage I-V). Vitellin/vitellogenin was detected in the ovary and the hepatopancreas in different stages by native-PAGE and Western blotting. Two and three subunits of vitellin were observed in the ovary at the early- (I-II), mid- and late- (III-V) stages, respectively. The subunit of vitellogenin was not detected in the hepatopancreas at different stages of prawns. Hepatopancreas had positive immunocytological staining (against vitellin antibody) in different ovarian stages of prawn. Only vitellogenic oocyte but not previtellogenic oocytes and follicle cells had a positive immunocytological staining. Hepatopancreas could synthesize radiolabeled immunoreactive proteins after incubation with radiolabeled glycine on the basis of immunoprecipitation (against vitellin antiserum). Therefore, it is concluded that hepatopancreas is the most likely organ to synthesize vitellogenin in the freshwater prawn, M. rosenbergii.     

Vitellogenin gene expression and hemolymph vitellogenin during vitellogenesis, final maturation, and oviposition in female kuruma prawn, Marsupenaeus japonicus

In penaeid shrimps, vitellogenin (VTG), the precursor of vitellin, is synthesized in the ovary and hepatopancreas and accumulated in oocytes during ovarian development. In the present study, VTG gene expression levels and hemolymph VTG levels were determined throughout ovarian development in female kuruma prawn, Marsupenaeus japonicus. Hemolymph VTG levels and VTG mRNA levels in the ovary and hepatopancreas were high during vitellogenesis, remained high until final maturation, and then decreased after oviposition. This profile suggests that VTG synthesis activity increases during vitellogenesis and decreases after oviposition. Absence of a significant increase in ovary size in final maturation suggests cessation of yolk accumulation and low activity of VTG synthesis in spite of high VTG mRNA levels. VTG mRNA levels in ovary and hepatopancreas were both highly correlated during vitellogenesis. Thus, their contribution to yolk accumulation seems to be similar. In contrast, VTG mRNA levels in the hepatopancreas increased more slowly at the start of vitellogenesis and declined more sharply after oviposition than in the ovary. This suggests a difference in the regulation of VTG synthesis between the ovary and the hepatopancreas.     

The stage of ovarian development affects organ expression of vitellogenin as well as the morphometry and ultrastructure of germ cells in the freshwater prawn Macrobrachium amazonicum (Heller, 1862)

The objective was to characterize vitellogenin expression in the ovary and hepatopancreas, and to describe the morphometry and ultrastructure of oocytes, in the freshwater prawn Macrobrachium amazonicum at various stages of ovarian development. Five ovarian stages were defined: (I) immature, (II) maturing, (III) mature, (IV) spawned, and (V) reorganized. Ovaries and hepatopancreas were analyzed by immunohistochemistry for vitellogenin expression. Vitellogenin expression in both ovary and hepatopancreas was predominantly widespread, beginning at Stage I, peaking at Stage III, and decreasing in Stages IV and V. Characterization of the ovary included measurement of the following germ cell types: oogonia (OG), and previtellogenic (PV), early vitellogenesis (EV), advanced vitellogenesis (AV), and mature (M) oocytes. Mean ± SD diameter of OG and EV oocytes in Stages I (14.2 ± 5.5 and 119.8 ± 15.7 μm) and II (17.9 ± 4.8 and 114.3 ± 34.6 μm), respectively, were significantly different from that in Stages IV (16.6 ± 4.7 and 107.0 ± 24.6 μm) and V (14.4 ± 4.1 and 101.0 ± 25.2 μm). Both scanning and transmission electron microscopy enabled identification of EV, AV and M oocytes based on the presence of a nucleus, and the organization and distribution of yolk in the cytoplasm. In summary, vitellogenesis occurred both in the hepatopancreas and ovary, with the ovary expressing vitellogenin starting as early as Stage I. This process promoted accumulation of yolk and growth of oocytes, thus favoring sexual maturation of females. This knowledge may be applied to improve production of farmed M. amazonicum.     

Hepatopancreas and ovary are sites of vitellogenin synthesis as determined from partial cDNA encoding of vitellogenin in the marine shrimp,Penaeus vannamei

Summary

The site of yolk protein synthesis in crustaceans has long been a subject of controversy. A portion of the vitellogenin gene structure was reported recently in a freshwater giant prawn (Macrobrachium rosenbergii) and black tiger shrimp (Penaeus monodori), in which the hepatopancreas was confirmed to be the extraovarian site of vitellogenin synthesis. The ovary is also frequently reported to be the site of yolk protein synthesis in penaeid shrimp. The same PCR product was obtained using cDNA from the hepatopancreas or the ovary as a template. The deduced amino acid sequence of Vg in P. vannamei showed high identities of 57% and 78% with those from M. rosenbergii and P. monodon, respectively. The same location of the intron in the sequenced region of genomic DNA was also found between these three species. We therefore concluded that the hepatopancreas and ovary are sites of vitellogenin synthesis in P. vannamei. The partial structure of the vitellogenin gene is further presented.     

Molecular cloning and characterization of cortical rod protein in the giant freshwater prawn Macrobrachium rosenbergii, a species not forming cortical rod structures in the oocytes

The formation of cortical rod structures is a characteristic of fully mature oocytes in penaeid prawns, but such structures are absent from oocytes of giant freshwater prawn Macrobrachium rosenbergii. In the present study, we first demonstrated the presence of a 30-kDa protein, which was immunologically related to kuruma prawn cortical rod protein (CRP), in the ovary of giant freshwater prawn, and subsequently purified this protein. Furthermore, a cDNA encoding the CRP-like protein was isolated. Based on the high homology (98%) in the amino acid sequence with kuruma prawn CRP, the 30-kDa protein has been identified as a CRP homologue of giant freshwater prawn, designated mrCRP. The RT-PCR analysis revealed that mrCRP mRNA was present in the ovary from a prawn with a gonadosomatic index (GSI) of 0.2. Western blot analysis revealed the presence of a CRP-immunoreactive band of 30kDa in the ovary with GSI of 1.6. By immunocytochemistry, CRP-immunopositive signals were detected in the ovary with GSI of 0.9, that had started to accumulate considerable amounts of vitellins and lipids in the peripheral cytoplasm. With progress of vitellogenesis, mrCRP was apparently accumulated in the mature oocytes, although it was not detectable, presumably because a relatively small amount of mrCRP was masked with large amounts of vitellin and lipids. In giant freshwater prawn without forming cortical rod structures, our findings indicate that the oocytes produce mrCRP, a homologue of CRP found in penaeid prawns.     

Dynamics of vitellogenin synthesis in juvenile giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin (Vg) mRNA expression and patterns of Vg and vitellin distribution in the hepatopancreas and ovary of juvenile Macrobrachium rosenbergii were examined using real-time RT-PCR and immunohistochemical methods. Eyestalk ablation was seen to induce rapid development of the gonads and Vg synthesis in females. In the female hepatopancreas, Vg mRNA expression was observed several days following ablation, after which levels increased gradually with increasing gonadosomatic index (GSI). Vitellin accumulation in the oocytes also increased with increasing Vg mRNA synthesis; expression was however negligible in the ovary. Hemolymph Vg levels in females ranged from 0.04 to 2.2 mg/ml. SDS PAGE/Western blotting analysis of hemolymph samples revealed that juvenile Vg was composed of 199 and 90 kDa subunits; the 102 kDa subunit present in adult female Vg (Okuno et al., 2002. J Exp Zool 292:417-429) could not be detected at any stage of vitellogenesis in juveniles. Vg was not detectable in non-ablated juveniles. The results of this study confirmed that the mode of involvement of eyestalk factors in regulating vitellogenesis is intrinsic to both juveniles and adults, and that a basic pattern of Vg synthesis and processing is conserved. However, the fact that juveniles are not able to produce the same Vg levels observed in adult females, and do not reach high GSI levels culminating in spawning suggests that other factors and physiological conditions specific to adult females are necessary to demonstrate full reproductive ability.     

Dynamics of vitellogenin mRNA expression and changes in hemolymph vitellogenin levels during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin mRNA expression during ovarian maturation in Macrobrachium rosenbergii were examined by measuring hemolymph vitellogenin (Vg) levels and Vg mRNA expression in the hepatopancreas and ovary at differing reproductive stages in both intact and eyestalk ablated animals. Vg mRNA was quantified using real-time RT-PCR and hemolymph Vg was measured by enzyme immunoassay. In intact animals, Vg mRNA levels in the hepatopancreas and hemolymph Vg levels showed a gradual increase during the molt cycle concomitant with increasing gonadosomatic index (GSI), with Vg levels decreasing prior to ecdysis although GSI continued to increase. Eyestalk ablation was seen to accelerate Vg synthesis as well as ovarian maturation, although it did not alter the overall pattern of Vg expression. Vg mRNA expression was negligible in the ovary of both intact and eyestalk ablated animals, confirming that the hepatopancreas is the principal site of Vg synthesis in M. rosenbergii with the ovary being only a minor contributor. This study has shown that Vg synthesis is correlated to ovarian maturation and the molt cycle in M. rosenbergii.     

Localization of vitellogenin mRNA expression and vitellogenin uptake during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii

In situ hybridization and immunohistochemical techniques were used to investigate the dynamics of vitellogenin (Vg) mRNA expression and Vg uptake during ovarian maturation in the hepatopancreas and ovary at differing stages of ovarian maturation in both intact and eyestalk ablated female Macrobrachium rosenbergii. In the hepatopancreas of intact animals, Vg mRNA expression was detected faintly two days after ecdysis, and signals showed a gradual increase as the molt cycle advanced to the premolt stages, but decreased at the late premolt stage. Vg mRNA was detected in the R-cells of the hepatopancreas, indicating that these cells are responsible for synthesizing Vg. No Vg mRNA expression was observed in the ovary. Immunohistochemistry results for the hepatopancreas showed a pattern of staining intensity similar to that of in situ hybridization. Increases in the accumulation of yolk protein in the oocytes occurred concomitantly with increasing Vg mRNA expression. In eyestalk ablated animals, Vg mRNA expression and Vg uptake showed similar but accelerated patterns to those of intact animals. This study has confirmed on the cellular level previous results that Vg synthesis is intrinsically correlated to ovarian maturation and the molt cycle in M. rosenbergii.