Yolk Droplets Research Articles

A crustacean vitellogenin-derived peptide as an oocyte-specific delivery vehicle for gene silencing

Gene silencing by dsRNA is well documented in crustaceans, but RNA interference (RNAi) in developing oocytes is yet to be achieved. The main obstacle to RNAi in the oocytes of oviparous animals derives from their protective layers, including the cytosolic membrane, the vitelline envelope, and a layer of follicular cells. These layers form a barrier preventing the entry of large nonspecific molecules, such as double-stranded RNA (dsRNA). This article describes a sophisticated tool – designated OSDel [oocyte-specific delivery] – for the delivery of dsRNA for gene silencing in the developing oocyte. The methodology exploits the process of receptor-mediated endocytosis (RME) taking place in the crustacean female for internalizing vitellogenin (Vg) (the precursor of the major yolk protein, vitellin) into oocytes. In this process, the extracellular domain of the Vg receptor (VgR) interacts with a distinct amino acid sequence of Vg and internalizes it to form yolk droplets. Here, we tested the premise that a distinct 24 amino-acid peptide derived from Macrobrachium rosenbergii Vg (designated VgP) would interact with VgR to form particles of a size suitable for piggy-backing dsRNA into oocytes via RME. We found that fluorescently labeled VgP had a micromolar affinity for the ligand-binding domain (LBD) of the VgR and could indeed be delivered to and internalized in oocytes. As model system to illustrate the applicability of the OSDel, we injected vitellogenic females with dsRNA encoding the eye development gene PAX6 piggybacked on VgP. Proof that the dsRNA had been successfully internalized into the developing oocytes and had silenced the PAX6 gene was derived from impaired eye development in 87% of the embryos. The ability to manipulate embryos by simple injection into vitellogenic crustacean females may prove to be a powerful high throughput tool for functional genomics investigation in crustacean embryos and for silencing genes relevant to crustacean aquaculture and biotechnology.

Histopathology unveiling the structural damage in gonads of Catla catla due to freshwater contamination

Potentially harmful materials, e.g., heavy metals, pesticides, dyes, and hydrocarbons, are often released untreated into freshwater bodies. When such pollutants are released in large quantities, there may be large-scale, sudden mortalities of aquatic organisms. This study summarizes experimental evidence about the disruption of gonadal development due to freshwater contamination. Adult fish specimens of Catla catla were collected from the River Chenab downstream of the Chakbandi Main Drain (CMD) entrance into the Chenab River. CMD collects domestic and industrial sewage wastes from Faisalabad city and disposes them into River Chenab, polluting this water body. Water samples were analyzed from each site of fish harvest for water quality parameters. Collected fish specimens were subjected to histopathological evaluation to detect the impact of pollution on the gonads of this fish species. The male Catla catla gonads showed marked histopathological alterations such as testes degeneration, generalized tissue degeneration produced by fragmentation and detachment of basement membrane, necrosis, and fibrosis in the testis. A loss of tubular arrangement, rupturing of the blood vessels, delimiting seminiferous lobules, vascular hypertrophy of the interstitial compartment, and appearance of a clustered organization were observed. The follicular epithelium in vitellogenic oocytes, rupturing and detachment of follicular basement membrane tissue with cytoplasmic vacuolization, immature oocytes with scattered yolk droplets, hypertrophied follicular epithelium, interstitial proteinaceous fluid deposition, and structural deterioration of follicles with early oocytes were observed in the ovary.

Effects of programmed cell death protein 10 on the Schistosoma japonicum female reproductive system

This study aimed to investigate the effects of programmed cell death protein 10 (PCDP10) on the female reproductive system of Schistosoma japonicum, one of the major infectious agents of schistosomiasis. We found that PCDP10 was widely distributed in the integument, the worm parenchymal area, and the vitellarium of the female worm, but was localized to a lesser extent in the ovary and testicles. RNAi experiments successfully achieved gene knockdown, and the ultrastructural morphology of the adult reproductive organs was observed. The results demonstrated that, compared with those of the negative control group, the number of cortical granules around oocytes decreased and the number of immature oocyte cells increased. Fusion of yolk globules occurred, and the number and the diameter of yolk droplets decreased significantly. Real-time PCR showed that the expression of yolk glands reached its peak before ovulation and then decreased. The TUNEL assay results showed that apoptosis in the RNAi group was significantly higher than that in the negative control group. These results suggested that SjPCDP10 plays an important role in the female reproductive system. In conclusion, PCD10 is involved in oocyte growth and development, especially in eggshell formation, which may provide a reference for further elucidating the molecular mechanism of PCDP10 involved in egg formation and embryo development in Schistosoma japonicum.

Ultrastructural analysis of oocytes of Rhipicephalus (Boophilus) annulatus during postengorgement period as a tool to evaluate the cytotoxic effects of amitraz and deltamethrin on the germinative cells

The present study utilizes the ultrastructural analysis of the fully engorged female Rhipicephalus (Boophilus) annulatus ticks, as a tool to evaluate the cytotoxic potential of deltamethrin and amitraz on the germinative cells. The ultrastructural analysis of the ovary of the normal (untreated) R (B.) annulatus revealed, oocytes in different stages of development, attached to the ovary wall by pedicel cells. The attachment site of oocyte to the pedicel cell was characterized by indentations of the plasma membrane. The oocyte was bound by three cell membranes viz., plasma membrane, chorion and basal lamina. The stages of oocytes were differentiated ultrastructurally based on the features of their outer membrane and the number and size of lipid and yolk droplets. Detailed day wise analysis of ultrastructural changes in the ovary during the post-engorgement period revealed the occurrence of the degenerative changes from day five onwards. These appeared first in the oocytes followed by the germinal epithelium. The ovary of ticks treated with methanol (control), revealed similar topographies as that of a normal ovary except for the presence of very few oocytes with ring shaped nucleoli. Ultrastructurally, treatment with deltamethrin produced more prominent and extensive morphological alterations when compared to amitraz. In the case of ticks treated with amitraz, the oocytes of stage IV and V showed wavy and disrupted outer boundaries along with the loss of integrity of the yolk droplets. Uneven nuclear membranes of stage II oocytes and cristolysis of mitochondria of mature oocytes were the other changes noticed. Ticks treated with deltamethrin revealed prominent modifications such as, detachment of the basal lamina, wrinkled boundary, inconsistent nuclear membrane, ring shaped nucleoli and chromatin clumping in the case of the early stage oocytes (I and II), whereas swelling and cristolysis of mitochondria were seen in mature oocytes. The study further indicated that, in addition to the previous proven neurotoxic effects, these compounds act directly on the ovary of tick.

Placentation in watersnakes II: Placental ultrastructure in Nerodia erythrogaster (Colubridae: Natricinae)

Ultrastructure of the placental tissues from redbelly watersnakes (Nerodia erythrogaster) was analyzed during late pregnancy to provide insight into placental development and function. Examination of the chorioallantoic placenta with transmission electron microscopy reveals that chorionic and uterine epithelia are extremely attenuated but intact and that the eggshell membrane is vestigial and lacks a calcareous layer. These features minimize the interhemal diffusion distance across the placenta. Scanning electron microscopy reveals that fetal and maternal components of the placentas are richly vascularized by dense networks of capillaries. Although the yolk sac omphalopleure has largely been replaced by chorioallantois by late gestation, it retains patches of yolk droplets and regions of absorptive cells with microvilli and abundant mitochondria. Transmission electron microscopy reveals that yolk material is taken up for digestion by endodermal cells. As yolk is removed, allantoic capillaries invade to occupy positions just beneath the epithelium, forming regions of chorioallantoic placentation. Ultrastructural features indicate that the chorioallantoic placenta is specialized for gas exchange, while the omphalallantoic ("yolk sac") placenta shows evidence of functions in yolk digestion and maternal-fetal nutrient transfer. Placental features of this species are consistent with those of other thamnophines, and are evolutionarily convergent on snakes of other viviparous clades.

Follicle Development in Immature Spiny Dogfish (Squalus acanthias): Histomorphometric Analysis

Squalus acanthias is a commercially exploited small coastal shark, highly sensitive to overexploitation due to a low reproductive potential and a low intrinsic rate of population increase. We collected specimens caught by commercial bottom trawls from April 2005 to January 2007 in the Adriatic Sea and investigated reproductive traits of the species in ovaries of 68 immature females (total body length range: 26.1-70.1 cm). Histomorphometric analyses included measurements of the growing follicle diameters, the width of the follicular layer and the width of the zona pellucida. We analysed 2700 ovarian follicles and grouped them into four categories: small previtellogenic, medium previtellogenic, large previtellogenic and small vitellogenic follicles. The growth of follicles was positively correlated with the body size of females (r=0.726; p<0.001). We documented significant changes in the thickness of the follicular layer and zona pellucida as follicles increased in size. Small vitellogenic follicles started to appear in small proportion (around 1% of follicles) in subadult females with the minimum size of 60 cm total body length. Yolk droplets first emerged in follicles larger than 600 μm in diameter while vitellogenesis was evident in follicles ranging 1.2-2.0 mm in diameter when both follicular layer and zona pellucida were the widest (41.1 μm and 49.1 μm, respectively).

Yolk protein uptake in the oocyte of the Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae)

The formation and uptake of the yolk protein in the oocyte of the Asian Tiger, Aedes albopictus mosquito was investigated. Light and electron microscopy of the ovaries at early resting stage as well as the structural changes associated with yolk formation were described 16 h after blood meal. The deposition of the yolk protein in the oocyte was correlated with a 15-fold increase in 138-μm pit-like depressions on the oocyte surface. These pits result by invagination of the oocyte cell membrane. They have a 20-μm bristle coat on their convex cytoplasmic side and a layer of protein on their concave extraoocyte space. The pits, by pinching off from the cell membrane become bristle coat vesicles which carry the adsorbed protein into the oocyte. These vesicles lose the coat and then fuse to form small crystalline yolk droplets, which subsequently coalesce to form the large protein yolk bodies of the mature oocyte. Preliminary radioautographs and certain morphological features of the fat body, ovary, and midgut, suggest that the midgut is the principal site of the yolk protein synthesis in A. albopictus.

Scanning electron microscopy of the placental interface in the viviparous lizard Sceloporus jarrovi (Squamata: Phrynosomatidae)

Placental membranes mediate maternal-fetal exchange in all viviparous reptilian sauropsids. We used scanning electron microscopy to examine the placental interface in the mountain spiny lizard, Sceloporus jarrovi (Phrynosomatidae). From the late limb bud stage until birth, the conceptus is surrounded by placental membranes formed from the chorioallantois and yolk sac omphalopleure. The chorioallantois lies directly apposed to the uterine lining with no intervening shell membrane. Both fetal and maternal sides of the chorioallantoic placenta are lined by continuous layers of flattened epithelial cells that overlie dense capillary networks. The chorioallantoic placenta shows specializations that enhance respiratory exchange, as well as ultrastructural evidence of maternal secretion and fetal absorption. The yolk sac placenta contains enlarged fetal and maternal epithelia with specializations for histotrophic nutrient transfer. This placenta lacks intrinsic vascularity, although the vascular allantois lies against its inner face, contributing to an omphallantoic placenta. In a specialized region at the abembryonic pole, uterine and fetal tissues are separated by a compact mass of shed shell membrane, yolk droplets, and cellular debris. The omphalopleure in this region develops elongate folds that may contribute to sequestration and absorption of this material. Fetal membrane morphogenesis and composition in S. jarrovi are consistent with those of typical squamates. However, this species exhibits unusual placental specializations characteristic of highly placentotrophic lizards.

Biological observations on the common guitarfish Rhinobatos rhinobatos from İskenderun Bay (Turkey, Eastern Mediterranean)

Abstract Histological examination of the gonads showed that spermatogenesis occurs in every male R. rhinobatos collected in autumn and winter months of the year. The testis was composed of lobes having numerous spermatocysts in a dorsoventral zonated arrangement. Present results suggest that since R. rhinobatos have a cystic mode of spermatogenesis it can be used as a model for biomedical and toxicological studies. Eight stages of maturation for females and seven stages of maturation for males were identified. Moreover, vitellogenic oocytes were consistently found in every female collected during the summer months. In vitellogenic oocytes, yolk is present as pleomorphic droplets and very often; small yolk droplets have a crystalline configuration. These data suggest that both sexes are reproductively active at different times of the year. Both, the right ovary/testis and the left ovary/testis were functional. Ripe oocytes in the ovaries were usually symmetrically distributed. The population consisted of aplacental viviparous individuals with lecithotrophic embryo development. Although, sex ratios of R. rhinobatos were not significantly different from the expected 1:1 ratio (p&gt;0.05), slightly female biased sex ration were recorded.

Ultrastructure of cyst shell and underlying membranes of three strains of the brine shrimp Artemia (Branchiopoda: Anostraca) from South India

The cyst of Artemia has shell and membranous coverings over the embryo. The membranous coverings have special adaptive features to allow the physical changes accompanying repeated hydration and dehydration cycles that might occur and adversely influence postembryonic development. Whole and slices of cryptobiotic cysts were processed for electron microscopy to study the internal details and to compare the morphological architecture of three Artemia strains of South India. Surface topography of scanning electron microscopic (SEM) studies revealed distinct button shaped structures on the cyst of Puthalam strain. Transmission electron microscopic (TEM) studies of the cysts displayed the conventional pattern of anostracan crustaceans with outer cortex and alveolar layer, cuticular membranes, and the cytoplasmic inclusions namely nucleus, yolk droplets, lipoid bodies, and mitochondria. The prominent wavy outer cortex layer of Puthalam cysts corroborates the results of SEM studies.

Morphology of canine cumulus-oocyte complexes in pre-pubertal bitches.

The morphology of canine cumulus-oocyte complexes (COCs) before puberty is still unknown. Therefore, the aim of our study was to elucidate the morphological characteristics of pre-pubertal oocytes and cumulus cells by light microscopy, scanning electron microscopy and transmission electron microscopy. The pre-pubertal oocyte was characterized by accumulation of lipid yolk droplets in the cytoplasm as well as high energy metabolism, low protein synthesis and high transcriptional activity of the cumulus cells. The cumulus cells, which revealed a prominent nucleus and few cytoplasm, communicated with each other by few short processes and exhibited merely a small amount of processes reaching the oocyte. Our studies imply that both the oocyte and the cumulus cells of canine COCs before puberty reveal characteristic morphological features which are correlated with changes in oocyte metabolism and cumulus cell communication.

Histology of the extraembryonic membranes of an oviparous snake: towards a reconstruction of basal squamate patterns.

An understanding of the evolutionary morphology of extraembryonic membranes in reptiles requires information about oviparous as well as viviparous species. We are studying histology and ultrastructure of the extraembryonic membranes of snakes to clarify the evolutionary history of reptilian fetal membranes, including determination of basal (ancestral) ophidian and squamate patterns. Microscopic anatomy of the membranes of oviparous corn snakes (Elaphe guttata) was examined using light and electron microscopy. At mid-development the inner surface of the eggshell is lined by two extraembryonic membranes, the chorioallantois and the omphalallantoic membrane. The chorioallantois consists of a bilayered cuboidal epithelium that overlies the allantoic blood vessels. During development, allantoic capillaries become more abundant, and the chorionic epithelium thins, decreasing the diffusion distance for respiratory gas exchange. The abembryonic pole of the egg is delimited by a bilaminar omphalopleure and isolated yolk mass, the latter of which is lined on its inner face by the allantois. The isolated yolk mass regresses developmentally, and patches of yolk droplets become isolated and surrounded by allantoic blood vessels. By late development, the abembryonic hemisphere has been fully vascularized by allantoic vessels, forming a "secondary chorioallantois." With regard to its extraembryonic membranes, Elaphe gutatta is similar to viviparous snakes. However, this species exhibits features that have not previously been reported among squamates, perhaps reflecting its oviparous reproductive habits. Morphological evidence for the uptake of eggshell material by epithelia of the chorion and omphalopleure suggests that the potential for absorption by extraembryonic membranes predates the origin of viviparity.

Placentation in garter snakes. III. Transmission EM of the omphalallantoic placenta of Thamnophis radix and T. sirtalis.

The omphalallantoic placenta is a complex organ that is unique to viviparous squamates. Using transmission EM and light microscopy, we examined this placenta in garter snakes in order to understand its structural organization and functional capabilities. The omphalallantoic placenta is formed from the uterine lining and the bilaminar omphalopleure, the latter of which is associated with the isolated yolk mass and allantois. A thin shell membrane separates the fetal and maternal tissues throughout gestation. The uterine epithelium contains cuboidal cells with large droplets or granules and appears to be secretory. Epithelium of the omphalopleure is specialized for absorption and contains cells with prominent microvilli and others with large cytoplasmic droplets or granules. The brush-border cells are rich in mitochondria and Golgi bodies and interdigitate extensively with adjacent cells, forming elaborate intercellular canaliculi. Their morphology is consistent with their proposed role in sodium-coupled water movement. During development, the isolated yolk mass becomes depleted as yolk droplets are digested by cells of the omphalopleure and allantois. However, the allantois does not fuse to or vascularize the inner face of the omphalopleure. Consequently, the distance between fetal and maternal circulatory systems remains large (about 250-300 microm), precluding efficient gas exchange and hemotrophic transfer. The morphology of the omphalallantoic placenta strongly suggests that it functions in nutrient transfer through uterine secretion and fetal absorption.

Histology of abortive egg sites in the uterus of a viviparous, placentotrophic lizard, the skink Chalcides chalcides.

Although abortive uterine eggs are often assumed to be resorbed by females of the viviparous skink Chalcides chalcides, little microscopic evidence of resorption of such eggs is available. Oviducts from pregnant female C. chalcides in which egg resorption was inferred were examined histologically to seek a morphological basis for resorption. Uterine histology at the site of abortive eggs was very similar to that of lizards in early pregnancy. The uterine epithelium consisted of a monolayer of pseudostratifed columnar cells that showed no evidence of yolk phagocytosis. The uterine lamina propria exhibited shell glands and modest vascularity, typical of early gestation, and contained neither yolk droplets nor accumulating leukocytes. Unattenuated regions of the lamina propria contained occasional macrophages and mast cells, some of the latter of which were undergoing degranulation. The abortive eggs often were collapsed with ruptured shell membranes, and some were undergoing extrusion from the incubation chambers down the oviduct. In eggs that had begun developing, extraembryonic ectoderm and endoderm were atypical in location, and had failed to enclose yolk leaking from the eggs. Oviducts sampled from later in the reproductive season were reproductively inactive, and showed no trace of abortive eggs or egg components. We postulate that abortive eggs are extruded from the oviduct by pregnant females under conditions of physiological stress, as a means of enhancing future reproductive effort. J. Morphol. 235:97-108, 1998. © 1998 Wiley-Liss, Inc.

Oöcytes degeneration in the queen honey bee after infection by Nosema apis

Terminal oöcytes containing yolk in both healthy and nosema infected queen honey bees were studied. In the healthy queens the terminal oöcytes exhibited a layer of follicular cells which were covered by a smooth-surfaced ovariole sheath. In the oöplasm were numerous electrondense yolk granules and lipid yolk droplets. The elecron-dense yolk granules exhibited a crystalline structure. Stacks of endoplasmic reticulum were observed in the yolk granules throughout the oöplasm. Numerous mitochondria possessing well defined cristae were also observed. Oöcytes in the ovary of queen honey bees appeared degenerated after 7 days of infection by Nosema apis. The ovariole sheath was wrinkled. In the oöplasm, yolk granules were broken down into small spheres and granular substances. Numerous ribosomes without stacks of endoplasmic reticulate were observed. Lysosomes were abundant and numerous electron-dense materials surrounded by a membrane were detected. The oöcytes appeared to be extensively autolysed. The significance of these observations is discussed.

Reproductive behaviour and early development of the honey blue-eye, Pseudomugil mellis Allen and Ivantsoff 1982 (Pisces : Pseudomugilidae), from the North-east Coast Divison, south-eastern Queensland, Australia

Specimens of Pseudomugil mellis larger than 15 mm standard length (SL) showed sexual dichromatism as well as dimorphism in fin and body shape. Pairing occurred diurnally, with multiple releases of eggs amongst submerged vegetation between late morning and early afternoon when increasing water temperature reached 28� C. Eggs were demersal, adhesive and spherical (1.29-1.64 mm diameter) and had adhesive fibres (0.5-2.0 mm long) distributed (0.2-0.3 mm apart) over the chorion. Oil droplets (0.01-0.35 mm diameter) persisted throughout egg and larval development. Eggs hatched 112-190 h after spawning when they were incubated at 25-27�C. Yolk-sac larvae were 3.60-5.00 mm SL at hatching and began feeding at the surface after absorption of the yolk and oil droplets (3-80 h after hatching). All fin rays were developed in 9.0 mm SL fry, which feed at any depth. Aquarium-reared fish first spawned at 20 mm SL, when they were 95 days old. Features of Pseudomugil reproduction as they relate to a specific survival strategy and to taxonomy are discussed briefly.

Aspects of the reproductive biology of the bass, Dicentrarchus labrax L. I. An histological and histochemical study of oocyte development

Histological and histochemical studies of oocyte development in the bass, Dicentrarchus labrax L., showed that three types of inclusions are formed during vitellogenesis. Lipid yolk accumulates first as lipid droplets, followed by protein yolk in the form of discrete protein yolk granules. The third type of inclusion are the small cortical alveoli (intravesicular yolk/yolk vesicles, i.e.‘carbohydrate yolk’) which form in the peripheral cytoplasm after both the lipid and protein yolk have started to accumulate. While the protein yolk granules maintain their structural integrity through to maturation, forming a densely packed zone in the mid‐outer cortex, the lipid yolk droplets continually coalesce and migrate centripetally, forming a prominent zone of large lipid droplets in the inner‐mid cortex. From the histological study of oocyte development, a number of distinct developmental stages are delineated, while gross examination of the paired ovary revealed that, depending on its stage of development, it can be placed into one of seven maturity stages.

Effects of colchicine- and cytochalasin B-treatment on the intracellular distribution of yolk droplets, lipid bodies, and Golgi apparatus of the chick neuroepithelial cells.

Treatment with colchicine (antimicrotubular agent) and cytochalasin B (antimicrofilamentous agent) has been used to investigate the possible role played by the cytoskeleton in the maintenance of intracellular distribution of yolk droplets, lipid bodies, and Golgi apparatus of the chick neuroepithelial cells. On the one hand, embryos treated with colchicine showed modifications in their distribution patterns of yolk droplets and lipid bodies, which suggests the involvement of the microtubular integrity of neuroepithelial cells in the maintenance of normal distribution patterns. On the other hand, the close relationships between vitelline and lipid inclusions and Golgi apparatus observed in untreated embryos seems to be kept in the embryos treated with colchicine and cytochalasin B. Moreover, from the effects of colchicine on Golgi apparatus position a possible functional role for the microtubular system in the maintenance of Golgi apparatus polarity in the chick neuroepithelial cells can be proposed. The results provided here constitute new information about the cellular mechanisms involved in chick neurulation.

Subcellular organization of the yolk syncytial-endoderm complex in the preimplantation yolk sac of the shark, Rhizoprionodon terraenovae.

The structure of the yolk syncytial-endoderm complex of the preimplantation yolk sac of the shark is examined by light- and transmission electron microscopy. The yolk syncytium is bounded by a membrane that is anchored to the plasmalemma of adjacent endoderm cells by desmosomes. Enlarged nuclei, rough endoplasmic reticulum, Golgi complexes, mitochondria, and other cellular organelles populate the syncytium. Microtubules and filamentous elements are also observed free in the syncytium. Yolk is present as pleomorphic droplets, the profiles of which are generally spherical but may be vesicular, especially at the periphery of large yolk droplets. Occasionally, large yolk droplets have a paracrystalline configuration. Small yolk droplets are modulated through the Golgi complex of the yolk syncytium, and it is suggested that acid hydrolases are added there. Small yolk droplets released from the maturing face of the Golgi complex are sequestered in membrane-limited packets. The membrane of the packets fuses with the membrane enveloping the yolk syncytium and the yolk droplets are released into the yolk syncytial-endoderm interspace. Subsequently, the yolk droplets are endocytosed by the endoderm. Yolk droplets disperse and fuse to form the large irregular yolk inclusions of the endoderm. Yolk metabolites are transported out of the endoderm through the yolk sac endothelium. The yolk sac endoderm thus mediates the transfer of metabolites from the yolk mass to the extraembryonic circulation.

Histological changes during ovarian maturation in the tarnished plant bug, Lygus lineolaris (Palisot de beauvois) (Hemiptera : Miridae)

Histological changes during the first gonotropic cycle in the telotrophic ovarioles of Lygus lineolaris (Hemiptera : Miridae) were studied by light and transmission electron microscopy. Each oocyte goes through a gonotrophic cycle that lasts for ca 7 days during which time 3 distinct stages are observed: previtellogenic, vitellogenic and choriogenic. In the previtellogenic stage, oocytes descend into the vitellarium and increase in size, while maintaining contact with the trophic core by means of nutritive cords. During vitellogenesis, ovarioles are characterized by the development of intercellular spaces in the follicular epithelium and numerous microvilli on the oocyte surface. Yolk granules are incorporated by pinocytosis and the granules coalesce, resulting in large yolk droplets. The trophic core supplies ribosomes, mitochondria and lipid to the oocytes, and its morphology remains unchanged throughout the gonotropic cycle. Vitellogenesis ends with the formation of vitelline membrane on the oocyte surface. During choriogenesis, an egg shell consisting of an exo- and endochorion is formed on the surface of the vitelline membrane. With the completion of choriogenesis, the mature oocyte is ready to be ovulated. During the gonotropic cycle, the oocyte increases in size 10–12-fold, while the germarium remains unchanged in size.