Dynamics of vitellogenin mRNA expression during vitellogenesis in the banana shrimp Penaeus (Fenneropenaeus) merguiensis using real-time PCR

An open reading frame (ORF) of vitellogenin (Vg) cDNA was amplified from the ovaries of the banana shrimp, Penaeus merguiensis. An examination of Vg-deduced amino acid sequence revealed the presence of cleavage sites at a consensus motif for subtilisin-like endoproteases prior to the N-terminal sequences of purified vitellin (Vt) subunits. A comparison of the primary structures of Vg molecules in decapod crustacean species revealed the existence of a common characteristic structure, and phylogenetic analysis reflected the current taxonomic classifications of crustaceans. A PCR product of 1.1 kb encoding the 3'-end of Vg cDNA was cloned from the hepatopancreas. Although its sequence was almost identical to that of the same region of the ovarian Vg, with only 18 nucleotide differences, analysis suggests that they have been subjected to natural selection, indicating that there may be two different, tissue-specific Vg genes in P. merguiensis. This is consistent with the different expression patterns of Vg mRNA, as determined by real-time PCR. Vg mRNA levels were maintained at low levels during the previtellogenic stage and they increased as vitellogenesis progressed to reach a peak at the early vitellogenic stage in the ovary or at the vitellogenic stage in the hepatopancreas, and thereafter, levels decreased. Expression of Vg mRNA was much higher in the ovary compared to the hepatopancreas at all stages of ovarian development, implying that the ovary is mainly responsible for Vt synthesis. These indicate that penaeids constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.     

七星瓢虫的卵黄发生:脂肪体与卵巢中卵黄原蛋白的合成与分泌

本文利用[³H]亮氨酸参入及特异性抗体沉淀等方法,研究了七星瓢虫体外培养的脂肪体中卵黄原蛋白合成与分泌的动力学,以及不同发育期脂肪体与卵巢中卵黄原蛋白合成的定量变化。脂肪体中卵黄原蛋白的合成与分泌在培养1—4小时内直线上升,到6小时稍下降。保留在脂肪体内的卵黄原蛋白缓慢积累,但一直水平很低。卵黄原蛋白合成的最初30分钟,分泌速率较慢,60%以上的卵黄原蛋白保留在脂肪体内。1小时后分泌速率加快,70%以上的卵黄原蛋白被分泌,保留的卵黄原蛋白在4小时中逐渐被释放。在4小时,被分泌的卵黄原蛋白超过80%,最高可达92%。 在雌虫发育过程中,脂肪体中卵黄原蛋白合成的高峰在羽化后11—15天,所合成的卵黄原蛋白占整个发育期合成总量的80%。在合成高峰期分泌的卵黄原蛋白高达90%以上,但在发育的早期和晚期分泌的卵黄原蛋白仅占30%或稍多。 卵黄发生前的卵巢就开始合成卵黄原蛋白,但卵巢中卵黄原蛋白的合成高峰期与脂肪体中大致相同。与脂肪体相反,卵巢合成的卵黄原蛋白大部分保留在卵巢内。在卵黄发生盛期,卵巢合成的卵黄原蛋白为脂肪体合成的卵黄原蛋白的20%。     

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.     

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。     

Immunolocalization of estrogen receptor α in Neomysis japonica oocytes and follicle cells during ovarian development

Estrogen induces oocytes development and vitellogenesis in crustacean by interacting with estrogen receptor (ER) subtypes. In the present study, we detect for the first time the ERα in oocytes and follicle cells and hepatopancreas cells of mysis by immunohistochemistry using a specific ERα antibody. ERα was mainly localized in the nuclei of oocytes and follicle cells, while mainly detected in nuclei of oogonia (OG), previtellogenic oocyte (PR) and endogenous vitellogenic oocyte (EN) at previtellogenic and early vitellogenic stage (I-early III). Follicle cells in all stages of ovary (all vitellogenic stages) showed strong ERα positive reaction, and they were able to gradually move to oocytes during the development of oocytes. In addition, ERα was also localized in the nuclei and cytoplasm of four hepatopancreas cells (including E-, R-, F- and B-cell) in all ovary stages. These findings suggest, for the first time to our knowledge, that there could be a close link between oogenesis, follicle cells, hepatopancreas cells and endocrine regulation, and estrogens might be involved in the regulation of oocytes at early ovarian stage in mysis.     

The fate of follicles after a blood meal is dependent on previtellogenic nutrition and juvenile hormone in Aedes aegypti

Juvenile hormone (JH) mediates the relationship between fecundity and nutrition during the gonotrophic cycle of the mosquito in three ways: (1) by regulating initial previtellogenic development, (2) by mediating previtellogenic resorption of follicles and (3) by altering intrinsic previtellogenic follicle “quality”, physiology, and competitiveness thereby predetermining the fate of follicles after a blood meal. To support a role for JH in mediating the response of ovarian follicles after a blood meal, we explored three main questions: (1) Do changes in nutrition during the previtellogenic resting stage lead to relevant biochemical and molecular changes in the previtellogenic ovary? (2) Do hormonal manipulations during the previtellogenic resting stage lead to the same biochemical and molecular changes? (3) Does nutrition and hormones during the previtellogenic resting stage affect vitellogenic resorption and reproductive output? We examined the accumulation of neutral lipids in the previtellogenic ovary as well as the previtellogenic expression of genes integral to endocytosis and oocyte development such as the: vitellogenin receptor (AaVgR), lipophorin receptor (AaLpRov), heavy-chain clathrin (AaCHC), and ribosomal protein L32 (rpL32) under various previtellogenic nutritional and hormonal conditions. mRNA abundance and neutral lipid content increased within the previtellogenic ovary as previtellogenic mosquitoes were offered increasing sucrose concentrations. Methoprene application mimicked the effect of offering the highest sucrose concentrations on mRNA abundance and lipid accumulation in the previtellogenic ovary. These same nutritional and hormonal manipulations altered the extent of vitellogenic resorption. Mosquitoes offered 20% sucrose during the previtellogenic resting stage had nearly 3 times less vitellogenic resorption than mosquitoes offered 3% sucrose despite taking smaller blood meals and developed ～10% more eggs during the first gonotrophic cycle. Mosquitoes treated with JH III during the previtellogenic resting stage and then offered a blood meal had a ～40% reduction in the amount of vitellogenic resorption and developed ～12% more eggs. Taken together, these results suggest that previtellogenic nutrition alters the extent and pattern of resorption after a blood meal through the effect of JH on mRNA abundance and lipid accumulation in previtellogenic follicles.     

Previtellogenic development and vitellogenin synthesis in the fat body of a mosquito: an ultrastructural and immunocytochemical study

We describe two phases, previtellogenic and vitellogenic, in the activity of the trophocytes in the fat body of the mosquito Aedes aegypti. The previtellogenic phase, leading to trophocyte competence to synthesize vitellogenin (Vg), occurred during the first 3 days after eclosion. This phase was characterized by enlargement and activation of the nucleoli, proliferation of ribosomes and rough endoplasmic reticulum (RER), development of Golgi complexes, and extensive invaginations of the plasma membrane. During the vitellogenic phase, initiated by a blood meal, Vg was first detected, by immunofluorescence, 1 hr after feeding. The intensity of the immunoreaction increased for the next 24 hr, was declining at 30 hr, and had disappeared by 48 hr. Vg synthesis was characterized ultrastructurally by the enlargement of the RER and the formation of dense secretion granules in Golgi complexes. These secretion granules were two to three times larger at the peak of Vg synthesis than at the beginning. The granules discharged their contents by exocytosis. Two electron microscopical immunocytochemical methods, immunoferritin and peroxidase-antiperoxidase, confirmed this pathway of Vg processing. For the first 12 hr after feeding. Vg synthetic organelles proliferated and the active nucleoli were multilobed; thereafter, while Vg synthesis continued, the nucleoli began to regress into compact bodies. Termination of Vg synthesis was marked by autophagical degradation of Vg synthetic and processing organelles.     

Annual changes in serum levels of two choriogenins and vitellogenin in masu salmon, Oncorhynchus masou

Annual changes in serum levels of two chorion precursors, choriogenin H (Chg H) and choriogenin L (Chg L), vitellogenin (Vg) and estradiol-17beta (E2) were quantified in masu salmon, Oncorhynchus masou, using specific immunoassays. Serum Chg levels were higher than Vg during the previtellogenic growth phase when circulating E2 levels were low ( approximately 0.1 ng/mL), suggesting higher sensitivity of Chg to E2. When oocyte growth shifted to the vitellogenic phase, Vg levels increased and became the most abundant in serum coincident with elevations of E2 and GSI. Chg H, Chg L and Vg peaked 1 month prior to ovulation at 0.61+/-0.08, 0.98+/-0.18 and 10.93+/-3.24 mg/mL, respectively. These results suggest that chorion formation by Chgs occurs prior to vitellogenesis and that the sensitivity of Chgs to low circulating E2 is closely related to the sequential events of oocyte growth.     

九孔鲍卵子发生及卵巢发育的组织学观察

采用组织学方法研究了九孔鲍(Haliotis diversicolor supertexta)的卵子发生、卵巢结构及其发育.根据卵细胞的大小、形状,核仁的形态,卵黄颗粒的积累情况,滤泡的结构等.将九孔鲍卵子的发生分为卵原细胞、卵黄发生前的卵母细胞和卵黄发生期的卵母细胞3个时期;卵巢壁由外膜及内生殖上皮构成,生殖上皮分化产生卵原细胞和滤泡细胞;卵巢的结构单位是滤泡.根据卵巢的外部形态和内部组织结构,将九孔鲍的卵巢发育分为休止期、增殖期、生长期、成熟期和排放期共5期.     

Molecular analysis of nutritional and hormonal regulation of female reproduction in the red flour beetle, Tribolium castaneum

Female reproduction includes maturation of oocytes and the synthesis of yolk proteins (vitellogenin, Vg) in the fat body and their deposition into the oocytes. Our recent studies showed that juvenile hormone (JH) regulates Vg synthesis and 20-hydroxyecdysone (20E) regulates oocyte maturation in the red flour beetle (Tribolium castaneum). Here, we report on the role of nutritional signaling on vitellogenesis and oogenesis. Comparison of gene expression between fed and starved beetles by microarray analysis showed the up-regulation of genes involved in energy homeostasis and down-regulation of genes involved in egg production in the starved beetles. The RNA interference (RNAi) aided knock-down in the expression of genes involved in insulin and TOR signaling pathways showed that both these signaling pathways play key roles in Vg synthesis and oocyte maturation. Starvation of female beetles resulted in a block in Vg synthesis but not in the progression of primary oocyte development to the resting stage. Feeding after starvation induced Vg synthesis and the progression of primary oocytes from the resting stage to the mature stage. However, in the beetles where JH or 20E synthesis or action was blocked by RNAi, both Vg synthesis and oocyte maturation were affected suggesting that both these hormones (JH and 20E) and nutritional signaling and their cross-talk regulate vitellogenesis and oogenesis.     

Vitellogenin expression in queen ovaries and in larvae of both sexes of Apis mellifera

In the honeybee, Apis mellifera, vitellogenin (Vg) expression has been detected in the ovary of queens, but not in that of workers. In addition, larvae of both sexes produce Vg in significant amounts, which suggest that Vg serves for functions additional to oocyte growth and energy supply to the embryo. In vivo hormone treatment experiments suggest that the decrease of 20-hydroxyecdysone concentration occurring in previtellogenic phases allows Vg production. Southern analysis indicates that the Vg gene is present as a single copy in the honeybee genome.     

Regulation of vitellogenesis in Nereis virens (Annelida: Polychaeta): effect of estradiol-17beta on eleocytes

In the marine polychaete Nereis virens, the yolk protein precursor vitellogenin (Vg) is synthesized in specialized coelomic cells (eleocytes) during oogenesis. This process was visualized by immunohistochemistry using antibodies raised against the yolk protein. Transversal sections from male and female worms confirmed that eleocytes from females but not from males produce Vg. In order to investigate the hormonal regulation of Vg synthesis, eleocytes were incubated in vitro with estradiol-17beta (E(2)) at a concentration of 1 microg/l for up to three days. A strong increase in Vg secretion was detected by ELISA in culture media of treated eleocytes from vitellogenic females. In contrast, no response to the hormonal treatment was detectable in immature worms. Our results showed that Vg synthesis is under a complex regulation, which involves endocrine factors like estrogens. The role of E(2) in vitellogenesis of N. virens rather resembles the situation found in vertebrate than the one in insects.     

Ovarian steroidogenesis and the role of sex steroid hormones on ovarian growth and maturation of the Japanese eel

Three sex steroid hormones, estradiol-17β (E2), 11-ketotestosterone (11-KT), and 17α,20β-dihydroxy-4-pregnen-3-one (DHP), are well established as primary estrogen, androgen, and progestin, respectively, in teleost fish. Japanese eel, Anguilla japonica, would be a suitable candidate to study ovarian steroid physiology of fish because the ovarian growth and steroidogenesis is dormant under laboratory condition but can be induced by administration of exogenous gonadotropic reagents. In this review, we summarized our work on the function and production of sex steroid hormones in the ovary of the Japanese eel during ovarian growth and oocyte maturation artificially induced by treatment with extract of salmon pituitary. In vitro and in vivo assays suggest that 11-KT and E2 play primary roles in previtellogenic and vitellogenic growth of oocytes, respectively, whereas DHP is essential for induction of final oocyte maturation. We also reviewed the correlation between ovarian steroidogenesis to produce these sex steroid hormones, serum titers and gene expression.     

Ovarian function in the giant tiger prawn (Penaeus monodon) as determined by in vitro bioassay.

Ovary tissue fragments of the giant tiger prawn Penaeus monodon were incubated in vitro with L-methionine[35S] plus L-cysteine[35S] as a metabolic labeling reagent. The labeled cytoplasmic and secreted proteins synthesized in vitro during incubations under various conditions were subjected to SDS polyacrylamide electrophoresis and visualized by autoradiography. Vitellogenin (Vg) was immunologically identified and shown to be actively synthesized and released into the incubation medium. The synthesis and release of Vg into the incubation medium was optimized and shown to be linear over a 16-h period. Comparisons between different ovarian regions and different stages of development revealed that the level of Vg synthesis and accumulation in the incubation media was variable depending on stage of development and region within the ovary. Coincubation of ovarian fragments with sinus gland extracts showed a dose-related inhibition of total protein and Vg synthesis. The in vitro ovarian bioassay is suitable for examining the effect of hormonal inputs of P. monodon.     

Ovarian maturation and oogenesis in the blue swimmer crab,Portunus pelagicus (Decapoda: Portunidae)

The study was aimed at understanding the process of reproduction and the changes happening in the ovary of Portunus pelagicus during maturation, which would be useful for its broodstock development for hatchery purposes. For that, tissue samples from different regions of the ovary at various stages of maturation were subjected to light and electron microscopy, and based on the changes revealed and the differences in ovarian morphology, the ovary was divided into five stages such as immature (previtellogenic oocytes), early maturing (early vitellogenic oocytes), late maturing (late vitellogenic oocytes), mature (vitellogenic oocytes), and spent (resorbing oocytes). The ovarian wall comprised of an outermost thin pavement epithelium, a middle layer of connective tissue, and an innermost layer of germinal epithelium. The oocytes matured as they moved from the centrally placed germinal zone toward the ovarian wall. The peripheral arrangement of nucleolar materials and the high incidence of cell organelles during the initial stages indicated vitellogenesis I. Movement of follicle cells toward oocytes in the early maturing stage and low incidence of mitochondria and endoplasmic reticulum in the ooplasm during late vitellogenic stage marked the commencement and end of vitellogenesis II, respectively. Yolk granules at various stages of development were seen in the ooplasm from late vitellogenic stage onwards. The spent ovary had an area with resorbing oocytes and empty follicle cells denoting the end of one reproductive cycle and another area with oogonial cells and previtellogenic oocytes indicating the beginning of the next.     

An immunoelectron study of karyosphere and nuclear bodies in oocytes of mealworm beetle, Tenebrio molitor (Coleoptera: Polyphaga)

The karyosphere and nuclear bodies (NBs) were studied in Tenebrio molitor oocytes using immunoelectron cytochemistry. During early diplotene (previtellogenic stage), oocyte chromosomes begin to unite in a small nuclear volume forming the karyosphere. In vitellogenic oocyte nuclei, the chromatin undergoes condensation, and the karyosphere acquires a ring-shaped structure. The karyosphere is the only structure containing DNA in the oocyte nucleus. Pre-mRNA splicing factors [small nuclear ribonucleoproteins (snRNPs) and SC35] are not found in the karyosphere itself. In previtellogenic oocyte nuclei, these factors are present in NBs and in a fibrogranular substance surrounding the chromosomes in the early stages of karyosphere formation. At this stage, larger fibrillar NBs contain the non-snRNP splicing factor SC35. Smaller roundish NBs were shown to contain snRNPs. Some NBs with the same morphology contain neither snRNPs nor SC35. In the vitellogenic oocyte, there are fibrogranular NBs containing both snRNPs and SC35 splicing factors, fibrillar NBs containing snRNPs only, and complex NBs containing both. Complex NBs are often connected with the ring-shaped karyosphere. Based on the obtained immunoelectron data, we suggest that T. molitor oocyte NBs containing both snRNPs and the non-snRNP splicing factor SC35 are homologs of the well-characterized B-snurposomes in amphibian germinal vesicles and clusters of interchromatin granules in mammalian oocyte nuclei. Other NBs containing only snRNPs are suggested to represent a special class of insect oocyte snurposomes. The nuclear organelles mentioned seem to play a role as storage domains for pre-mRNA splicing factors during T. molitor oogenesis.