Determination and distribution of a female-specific protein in the brown planthopper, Nilaparvata lugens Stal (Homoptera: Delphacidae)

Electrophoretic analyses of hemolymph and body or ovary homogenates from reproducing females, males, and 5th instar nymphs of the brown planthopper, Nilaparvata lugens Stal, revealed a protein band of 175 kDa in females. An immunoblot test using antibody against this protein showed a positive reaction with a 175 kDa protein from female body or ovary homogenates. It is likely that this protein in hemolymph is vitellogenin (Vg). Distribution of Vg was determined by immunofluorescence and immunogold labeling techniques. The results showed that the positive immunofluorescence reactions were present in yolk particles, the intercellular space of follicle cells, hemolymph, and the epithelial plug of ovarioles. In addition, the yeast-like symbiotes (YLS) in mycetocytes of adults and various nymphal instars as well as those free in hemolymph or entering oocytes also exhibited a positive reaction. Electron micrographs showed that immunogold particles were found most in yolk mass and YLS over other tissues. Especially the YLS in various developmental stages all contained immunogold particles, implying that the symbiote is somewhat related with production of the female-specific protein.     

Structural diversity of symbionts and related cellular mechanisms underlying vertical symbiont transmission in cicadas

Many insects depend on symbiont(s) for survival. This is particularly the case for sap-feeding hemipteran insects. In this study, we revealed that symbionts harboured in cicadas are diverse and complex, and the yeast-like fungal symbionts (YLS) are present in most cicada species, but Hodgkinia is absent. During vertical transmission, Sulcia became swollen with the outer membrane drastically changed, while Hodgkinia became shrunken and changed from irregular to roughly spherical. Sulcia and/or Hodgkinia were exocytosed from the bacteriocytes to the intercellular space of bacteriomes, where they gathered together and were extruded to haemolymph. YLS and associated facultative symbiont(s) in the fat bodies were released to the haemolymph based on bacteriocyte disintegration. The obligate symbiont(s) were endocytosed and exocytosed successively by the epithelial plug cells of the terminal oocyte, while associated facultative symbiont(s), and possibly also YLS, may take a ‘free ride’ on the transmission of obligate symbiont(s) to gain entry into the oocyte. Then, the intermixed symbionts formed a characteristic ‘symbiont ball’ in the oocyte. Our results suggest that YLS in cicadas represent a new example of a relatively early stage of symbiogenesis in insects and contribute to a better understanding of the diversity and transmission mechanisms of symbionts in insects.     

The Differentiation of the Zona Pellucida (Vitelline Envelope) in the Lizard Tarentola mauritanica

The organization of the zona pellucida in the lizard Tarentola mauritanica was studied at the transmission electron microscope. Evidence is provided in support of the hypothesis that follicle cells and the oocyte work together to synthesize and release components that give rise to the zona .
The components of the zona consist of fibrils and amorphous electron-dense material, which are first observed in young previtellogenic oocytes. These components seem to be released by coated vesicles that are formed by the Golgi complex in both the oocyte and the follicle cells. The material relased by the coated vesicles forms patches around the microvilli that project from the oocyte and the folds of follicle cells. During the following previtellogenic stages, the patches merge together to form a continuous coat around the oocyte. The coat persists until the end of vitellogenesis.     

Abnormal vitelline envelope induced by unphysiological doses of ecdysterone in Aedes aegypti

ABSTRACT. The oocytes of 3-day-old unfed Aedes aegypti mosquitoes are in a state of oogenic arrest, but microgram doses of ecdysterone stimulate their accumulation of a variable amount of yolk. We now find that these doses also induce the deposition of plaques of vitelline envelope by the follicle cells, and with transmission electron microscopy we have compared their formation with that in normal blood-fed females. Plaques in the experimental animals were abnormally large and irregular in shape and distribution. In part, these abnormalities were attributable to the fact that the follicle cells remain in close contact with the oocyte, whereas the space between follicle cells and oocyte increase significantly in the blood-fed female. Deposition of the plaques occurred earliest after the injection of 5 μg ecdysterone, but even at this high dose the amount of plaque material deposited was less than in the blood-fed controls. Induction of the deposition of abnormal vitelline envelope in unfed females was most clearly demonstrated after two injections, 1 μg ecdysterone each, 14h apart; 24h after the second injection, the plaques had prematurely fused into a thin disorganized envelope. When females were injected with ecdysterone immediately after a blood-meal, vitelline envelope plaques formed prematurely, and their structure became increasingly abnormal with time. This early onset of activity was characteristic of follicle cells adjacent both to the oocyte and to nurse cells. Thus, the factors that normally control the formation and organization of the vitelline envelope are absent in the unfed female stimulated with high doses of ecdysterone, while in the blood-fed females, excessive ecdysterone apparently interferes with the timing and orderly sequence of envelope formation.     

Control of follicular epithelium development and vitelline envelope formation in the mosquito; role of juvenile hormone and 20-hydroxyecdysone

Using microsurgical manipulations, hormone applications, and transmission electron microscopy we have investigated the regulation of differentiation of the follicular epithelium and formation of the vitelline envelope (VE) in primary follicles in the ovary of the mosquito, Aedes aegypti. During the first 3 days after eclosion, the primary follicle grows, and cells of the follicular epithelium differentiate, their content of mitochondria, rough endoplasmic reticulum, and Golgi complexes increases significantly. Growth and differentiation of the follicular epithelium appear to be under the control of juvenile hormone (JH), because they are blocked by removal of corpora allata in newly closed adult females and can be restored by either implantation of corpora allata or application of JH III. In insects, including mosquitoes, VE is the first layer of the eggshell to be deposited. It is formed from the secretory products of the follicle cells and its deposition coincides with yolk accumulation by developing oocytes. Only follicle cells adjacent to the oocyte deposit VE. In decapitated females, given a blood meal by enema and injected with picogram doses of 20-hydroxyecdysone (20-HE), follicle cells synthesize the VE precursors and deposit morphologically normal VE, in contrast to saline injected controls which deposit no VE. We conclude that 20-HE, as well as factors originating from the blood meal and the oocyte, are required for the normal formation of VE in the mosquito follicles.     

东北梅花鹿初级卵母细胞的超微结构

研究东北梅花鹿初级卵母细胞发育的超微结构变化,目的是为探索东北梅花鹿初级卵母细胞的发育规律提供组织学和形态学依据。本研究于2003年和2004年的6月初到8月末取3只、9月中旬到10月初取4只,共计7只健康经产2~3胎的成年东北梅花鹿卵巢;卵巢经2·5%戊二醛固定液固定后,切取约1mm3的卵巢皮质和直径0·5~1·5mm及1·5~3mm的卵泡作为电镜观察用材料;该材料经0·1MpH7·2的PBS漂洗、1%锇酸固定、不同浓度乙醇脱水后,再经Epon812和丙酮等量混合液浸透,最后用Epon812包埋制块,并用半薄切片机切成0·5~2μm半薄切片;再经亚甲基兰-天青Ⅱ染色后,在光镜下进行卵泡分类和卵母细胞定位;将经定位的材料用超薄切片机切成厚度为700~800的切片,经醋酸铀和柠檬酸铅双重染色后,用透射电镜观察、记录并照相。观察时将卵泡依其直径大小、透明带的形成、卵泡腔的出现等分为原始卵泡、初级卵泡、次级卵泡和三级卵泡4类。研究结果表明,在原始卵泡阶段,卵母细胞为较规则的圆形,质膜与卵泡细胞膜紧密相贴,有时形成桥粒,细胞器多分布于近核区,高尔基体不典型,线粒体多为圆形,嵴较少;在次级卵泡阶段,2~4层的卵泡细胞局部开始形成透明带,4层以上时形成薄的透明带,微绒毛斜伸入透明带内,方向不规律;在直径为0·5~1·5mm的三级卵泡阶段,卵母细胞的透明带增厚,各种细胞器在皮质区内数量较多,皮质区内高尔基体的数目增多,粗面内质网明显减少;在直径为1·5~3mm的三级卵泡阶段,卵母细胞的透明带继续加厚,微绒毛缩短变弯,开始从透明带退出,许多皮质颗粒开始排列在卵母细胞膜下。     

Nuclear structures in the ovarioles of the butterfly Laspeyresia pomonella. Sex chromatin in trophocytes

The nuclear structures in the ovarioles have been studied in Laspeyresia pomonella by means of light and electron microscopy, autoradiography (RNA and DNA synthesis) and molecular hybridization in situ. The karyosphere was shown to form in oocyte nuclei at the beginning of oocyte growth. Numerous protein granules appeared in close contact with the karyosphere chromosomes; the true nucleolus was absent and the whole nucleus was inactive in RNA synthesis. A special attention was paid to studying nuclear structures in trophocytes. Numerous complex nucleoli actively synthesizing RNA formed in highly endopolyploid nuclei of trophocytes. Besides, each trophocyte had a spheroid vacuolized body of DNA which developed from one of meiotic bivalents soon after trophocyte differentiation and increased in diameter up to 10-15 mu. The DNA body in trophocytes and follicle cells was in close contact with the nucleolar material. Ribosomal DNA was present in these bodies as was shown by molecular hybridization in situ. A suggestion is put forward to the effect that the DNA bodies take part in the formation of complex nucleolar apparatus of trophocytes. On the basis of both the author's and literary data, a conclusion is drawn that DNA spheres in trophocytes and follicle cells are sex chromatin bodies formed, however, by both the X- and Y-chromosomes, rather than by one Y-chromosome.     

Morphology and formation of the eggshell in the tarnished plant bug, Lygus lineloaris (Palisot de Beauvois) (Hemiptera: Miridae)

Scanning and transmission electron microscopy were used to study the morphology and formation of the eggshell in the tarnished plant bug, Lygus lineolaris. Eggs are bean-shaped, with an operculum at the anterior end surrounded by a row of 36-40 respiratory horns. Three micropylar openings are on the operculum, and are sealed in oviposited eggs. The chorion consists of the chorion proper and the innermost chorionic layer. An air layer composed of colonnades is present in the chorion. The innermost chorionic layer is homogeneous and electron lucent. The follicle cells secrete electron dense materials that later coalesced into the reticulated vitelline membrane. This is followed by the deposition of the innermost chorionic layer by the follicle cells. After the primordial innermost chorionic layer is formed, follicle cells at the anterior pole of the oocyte secrete the scaffold for the colonnades in the air layer. Later, the primordial scaffold matrix is redistributed and localized at the lateral and posterior end of the oocyte where it becomes secondarily modified. At the end of choriogenesis, follicle cells at the anterior pole secrete the operculum and respiratory horns.     

不同发育时期哲罗鱼卵黄的超微结构

应用透射电镜观察了不同发育时期哲罗鱼(Hucho taimen)卵黄的超微结构.根据哲罗鱼卵黄物质在卵母细胞中的加工合成、积累以及卵母细胞中参与卵黄颗粒形成的细胞器的变化,可将该鱼卵黄发生分为4个特征时期,即卵黄发生前期、卵黄泡期、卵黄积累期和卵黄积累完成期.卵黄发生前期是指卵母细胞发育过程中的卵黄物质开始积累前的时期,此时期核仁不断分裂,出现线粒体云和早期的滤泡细胞层、基层和鞘细胞层;卵黄泡期特点主要是细胞器不断变化产生卵黄泡和皮层泡;卵黄积累期的滤泡膜由内向外依次为放射带、颗粒细胞层、基层和鞘细胞层,此时外源性卵黄前体物质不断经过血液汇集于鞘细胞层,后经微胞饮作用穿过胶原纤维组成的基层,经过多泡体作用转运至颗粒细胞内,在细胞内经过加工和修饰形成小的卵黄蛋白颗粒,卵黄蛋白颗粒经微胞饮穿过放射带进入卵母细胞边缘形成的空泡中,不断积累形成卵黄球;进入卵黄积累完成期,卵黄球体积变大,向细胞中心聚集,填满大部分卵母细胞,卵黄积累完毕.     

Localization and transport of lipids in the avian ovarian follicular layers and the structural relationship of theca and granulosa to the basement membrane

We describe the localization of lipids in the wall and superficial ooplasm of the largest avian ovarian follicles by the use of different fixatives and light and electron microscopy. We demonstrate that each yolk globule is always accompanied by one or more highly osmiophilic and sudanophilic alcohol insoluble yolk masses, which we have called satellite yolk. Together with the protein containing yolk globule it forms an integral morphological part of a compartmentalized, bipartite yolk system. Cytochemical, histoautoradiographic, biochemical, and light and electron microscopical aspects of satellite yolk were studied. At the start of satellite yolk formation in the 3–4 mm diameter follicle (when the oocyte begins to yellow) the distribution of the microcirculation of the follicle wall becomes printed on the underlying superficial ooplasm of the oocyte. The oocyte then presents so-called yolk mountains (containing satellite yolk), only localized below the thecal capillary sinus and not below the efferent and radially perforating thecal veins (black hole regions). We also describe the structural continuity between the thecal intercellular spaces and the microvilli-associated extracellular spaces of the granulosa cells via the basement membrane. The thecal cells present centripetal extensions into the basement membrane and the basement membrane material extends centripetally into the granulosa microvillar channels. Therefore, at least two cellular barriers are crossed when fat or fat precursors are transported from the thecal capillary sinus to the ooplasm.     

泥螺卵子发生的超微结构研究

利用透射电镜观察了泥螺卵子发生过程。结果表明 ,泥螺的卵子发生可划分为卵原细胞、卵黄发生早期、卵黄发生中期及卵黄发生后期卵母细胞 4个时期。卵原细胞核大而圆 ,胞质内分布有少量的线粒体和高尔基囊泡 ,细胞表面具微绒毛。卵黄发生早期的卵母细胞 ,胞质中各类细胞器发达 ,并出现数量较多的类朦胧子。卵黄发生中期的卵母细胞胞体迅速增大 ,核伸出伪足状突起 ,卵质中各种细胞器活动活跃 ,并参与形成卵黄粒和脂滴。此期还可观察到卵母细胞与滤泡细胞间的物质交换现象。卵黄发生后期的卵母细胞体积增至最大 ,细胞器数量减少。本文就卵黄发生前后卵母细胞内部构造的变化、意义及滤泡细胞与卵母细胞蛋白来源间的关系作了探讨     

刘氏蝎蛉卵巢管结构和卵子发生（英文）

卵巢管结构及卵子发生过程在探讨昆虫系统发育关系中有重要意义, 深入研究长翅目昆虫卵巢管结构及卵子发生可为确定其在全变态类昆虫中的系统发育地位提供依据。本文利用光学显微镜和扫描、透射电子显微镜技术研究了刘氏蝎蛉Panorpa liui Hua卵巢管超微结构及卵子发生过程。结果表明：蝎蛉卵巢由12根多滋式卵巢小管组成, 每个卵巢小管分为端丝、生殖区和生长区。根据滋养细胞、卵母细胞及滤泡细胞的变化, 卵子发生过程可分为5个阶段：卵黄发生前早期、卵黄发生前中期、卵黄发生前后期、卵黄发生期及卵壳形成期。在卵黄发生期, 滋养细胞为卵母细胞提供养分后逐渐消亡, 而此时的卵母细胞可通过滤泡之间的细胞间隙从血淋巴中获取营养。在卵壳形成期间, 3种不同类型的滤泡细胞参与形成不同区域的卵壳, 从而形成不同花饰的卵壳表面。据此推测, 与其他目的滋养细胞数目相比, 每个卵室中2次有丝分裂形成3个滋养细胞可能是比较原始的特征, 表明长翅目昆虫可能是全变态类群中近基部的分支。     

中华蚱蜢卵子发生中花生凝集素受体的初步鉴定和发育表达

The distributions of PNA binding glycoconjugates in the plasma membrane of Acrida cinerea Thunberg germ cells were detected using biotin labeled PNA, for better understanding of the formation and changes of glycoconjugates during oogenesis. The ultrastructure of vitellogenesis also was observed by electron microscopy for detection of the origin and track of vitelline material. In the ovary, PNA receptors appeared in the oocyte cytoplasm of the second phases of oogenesis; positive granules gradually increased from the third phase to the fourth, and they exhibited a maximum expression before the vitellogennic stage in the cytoplasm of the oocyte. From the vitellogennic to chorionation stage, positive granules gradually declined. Binding sites on follicle cells were changed with their morphological variation in every stage of oogenesis. The vitelline of A. cinerea formed within the oocyte by degrees. The results suggest that PNA receptors and yolk materials are synthesized by the oocytc at an early period. With the development of the oocyte, some exogeous materials from two sources act as PNA receptors and others take part in vitelline synthesis. One is blood lymph that offers some useful materials to the oocyte directly through follicle cell gaps; the other are follicle cells that produce and transmit some materials to oocyte to support vitellogenesis. In addition, PNA receptors secreted by follicle cells participate in the formation of yolk membrane [ Acta Zoologica Sinica 5 l （5） ： 932 - 939, 2005 ].     

Ultrastructural study of the follicle cells in the freshwater gastropod Viviparus viviparus L.

Summary The morphology and the role of the follicle cells of Viviparus viviparus were examined by means of light and electron microscopy. The follicle cells appear to contain glycogen and fat, and often lysosomes or heterogeneous inclusions. Therefore, they seem to be active in phagocytosis and storage. They are probably involved in the nutrition of the oocyte. Their role in the formation of a selectively permeable barrier is discussed.The authors thank Drs. H.H. Boer, M. de Jong-Brink and J. Wijdenes of the Free University of Amsterdam for their assistance in the translation of this paper     

Physiological roles of a yeast-like symbiote in reproduction and embryonic development of the brown planthopper,Nilaparvata lugens Stål

The physiological roles of a yeast-like symbiote in oviposition and embryonic development of its host, the brown planthopper, Nilaparvata lugens Stål, were studied using heat treatment, lysozyme injection and ligation of eggs. The eggs laid by the heat-treated females harboured only a few of the symbiotes, and their symbiote ball through embryonic development was free of symbiotes. The embryos of subsymbiotic eggs could not undergo blastokinesis and dorsal closure, and failed to hatch due to lack of differentiation of the abdominal segments. Electrophoretic profile of the eggs laid by the heat-treated females indicated the absence of several minor proteins which are usually found in the fat body of normal females. A protein (Y) of 131 kD was barely detectable in the heat-treated insects, and could not be found in the ligated eggs in which the symbiote ball was completely separated from the developing germ band. It is suggested that the symbiote supplies its host with proteins for normal embryonic and postembryonic development. The number of yeast-like symbiotes in female adults was reduced after injection with lysozyme solution, and some of the eggs were unable to hatch due to failure in blastokinesis; this was similar to the heat-treated insects. The embryos of ligated eggs could complete segmentation and differentiation normally before 110 h, but the abdominal segments failed to differentiate after dorsal closure, and regressed leaving only the head. Partially ligated eggs harboured some symbiotes and could produce normal larvae. It is concluded that the yeast-like symbiote is significant in abdominal segmentation and differentiation of the planthopper embryo.     

Development of the germinal epithelium and early folliculogenesis during ovarian morphogenesis in the cichlid fish Cichlasoma dimerus (Teleostei,Perciformes)

Formation of the germinal epithelium and folliculogenesis during ovarian development in Cichlasoma dimerus were described at the light‐ and electron‐microscopic levels. Prior to gonadal differentiation, germ cells and enveloping support cells reside within an inpocketing of the coelomic epithelium. Separation of the germinal and interstitial compartments of the gonad by a basement membrane is apparent from early gonadal development. Upon ovarian differentiation, oogonia undergo cyst‐forming divisions leading to the formation of clusters of interconnected cystocytes that synchronously enter meiosis, becoming oocytes. At the pachytene step, each oocyte becomes individualized by cytoplasmic extensions of prefollicle cells, thereby developing as an ovarian follicle. Subsequent somatic reorganization leads to the formation of the ovarian lumen in a cephalo‐caudal gradient. As a result, the germinal epithelium becomes internalized and lines the ovarian lumen. As defined by its origin from the germinal epithelium, the ovarian follicle is composed of an oocyte and the surrounding follicle cells. Thecal cells derived from the stroma encompass the basement membrane outside the follicle, thus forming a follicle complex. A common basement membrane is shared by the germinal epithelium and the follicle complex along a small portion of its surface. This point of attachment represents the site at which the oocyte would be released to the ovarian lumen during ovulation.     

Gap junctions between microvilli of an oocyte and follicle cells in the teleost (Plecoglossus altivelis)

In the teleost, Plecoglossus altivelis, intercellular junctions between microvilli of an oocyte and follicle cells were studied by electron microscopy. Microvilli, which were radiated from an oocyte and arrived at the surface of follicle cells, established contact with follicle cells. These contact areas appeared to be a seven-layered membrane with an overall thickness of about 18 microns by standard fixation. In freeze-fracture replicas, many small aggregates of intramembraneous particles were revealed on the cleavage faces of cytoplasmic membranes of follicle cells. These morphological evidences suggest that in the teleost gap junctions exist between the oocyte and follicle cells, especially on the surface of follicle cells.     

Immunocytochemical localization of a follicle specific protein of the hawkmoth Manduca sexta

A follicle specific protein (FSP-I) from the hawkmoth Manduca sexia, has been localized in developing follicles by immuno-fluorcscence and immuno-gold labeling techniques. At the light microscopical level, the protein was demonstrated to be present in both the basolateral and apical parts of foilicular epithelial cells, as well as in clearly defined, spherical compartments in the cortex of the developing oocyte. Immuno-gold labeling at the electron microscopical level revealed the localization of FSP-I in cndoplasmic compartments of the foilicular epithelial cells, in the extracellular matrix of the follicle and in endocytic compartments of the oocyte. Our results indicate that M. sexta FSP-I is synthctizcd and secreted by the foilicular epithelial cells, after which it is taken up by the developing oocyte through endocytic routes.     

Cloning and expression of the yolk protein of the tsetse fly Glossina morsitans morsitans

Two major families of nutritional proteins exist in insects, namely the vitellogenins and the yolk proteins. While in other insects only vitellogenins are found, cyclorraphan flies only contain yolk proteins. Possible sites of yolk protein synthesis are the fat body and the follicle cells surrounding the oocyte. We report the cloning of the yolk protein of the tsetse fly Glossina morsitans morsitans, a species with adenotrophic viviparity. The tsetse fly yolk protein could be aligned with other dipteran yolk proteins and with some vertebrate lipases. In contrast to the situation in most fly species, only a single yolk protein gene was found in the tsetse fly. Northern blot analysis showed that only the ovarian follicle cells, and not the fat body represents the site of yolk protein synthesis.