Yolk Bodies Research Articles

Ultrastructural evaluation of the oocytes and spermatozoa of the scleractinian coral Mussismilia harttii

Global coverage of living coral has declined by half since 1950s. Reef-building species have been severely impacted in this climate crisis scenario, compromising the future of coral reefs. Despite their importance, there is a lack of knowledge regarding the reproductive biology of scleractinian corals. In the present study, we evaluated through electron microscopy approaches, the gametes of the endemic Southwestern Atlantic coral Mussismilia harttii. We observed spherical oocytes with microvilli throughout the outer membrane. Fine granular material dispersed in cytoplasm, lipid granules, numerous yolk bodies, and mitochondria were identified in the oocytes. In addition, small Symbiodinium-like cells were observed, suggesting a vertical transmission from parental coral to oocytes. The spherical-head sperm presents a 9.3 ± 2.1 μm flagellum. The nucleus is located centrally in the head, and the centrioles are positioned between the nuclear base and the flagellar insertion, which is connected to the axoneme. This axoneme has a microtubular arrangement (9+2). Vesicles, underlining the inner plasma membrane, presented the same electron-dense pattern as the Golgi complex, and mitochondria positioned surrounding the axoneme. The vesicles present in the sperm may have a role as an acrosome since the oocytes do not develop any cell specialization for fertilization.

Di (2-ethylhexyl) phthalate effects on the growth, development, and reproduction of Moina macrocopa (Crustacea: Cladocera)

Di (2-ethylhexyl) phthalate (DEHP) is used as a plasticizer in plastics. The effects of DEHP on terrestrial vertebrates have been extensively reported but the effects of DEHP contamination on aquatic ecosystems have not been thoroughly studied. Since water bodies are one of the main mediums through which DEHP is released worldwide, the impacts of DEHP contamination should be manifested in water fleas. Therefore, maternal Moina macrocopa were exposed to 1, 10, 100, and 1000 μg/L concentrations of DEHP. Changes in growth and reproduction were evaluated. The findings demonstrated that DEHP exposure did not have a negative impact on growth or the ability to reproduce. An analysis of the ovary yolk body (YB) demonstrated that the average size and number of yolk bodies (YBs) produced by M. macrocopa exposed to 1000 μg/L DEHP were not significantly different to the average size and number of YBs produced in blank control and solvent control conditions. These outcomes support the cellular pathology data gathered by other researchers. Nevertheless, when M. macrocopa was exposed to 1000 μg/L DEHP for five days, a significant increase in YB numbers was observed with changes in YB morphology. The critical cellular pathology of YB showed morphological abnormalities, including rod-shaped YBs, and YB density was higher than in the blank and solvent controls. Even though these results suggest that antioxidative stress can be induced by DEHP exposure, growth, and reproduction were not significantly different among exposed water fleas compared to fleas in the blank and solvent controls. The result was attributed to the antioxidant response of the water flea. In conclusion, the present study enhances our understanding of previous findings from risk assessments of DEHP contamination in aquatic ecosystems.

Gonadal proliferation and reproductive cycle of the exotic sea squirt Cnemidocarpa amphora () (Pleurogona, Styelidae) sampled for the first time from the northern coast of Arabian Gulf in Saudi Arabia.

Specimens of Cnemidocarpa amphora were collected monthly from the Arabian Gulf from September 2017 to August 2018. Parts of their gonads were prepared for histological testing. The gonads' diameters varied by month. Each gonad contained many ovarian follicles with different morphologies and was surrounded by several testicular follicles. The ovarian and testicular follicles were separate, although the latter were always present near the former. Repeated measures ANOVA tests were conducted to investigate monthly changes in oocyte stages. In squirts measuring 12-13 cm in length, the gonads measured 30-50 mm from July to August; 20-25 mm from September to October; 15-20 mm from November to February; and 25-40 mm from March to June. Oogonia budded from the germinal epithelium with diameters of 20-30 µm. Previtellogenic oocytes measuring 70-120 µm were characterized by the deposition of small granules of protein around the nucleus, a continuous layer of follicular cuboidal epithelium, and scattered vacuoles in the ooplasm. The measurement of gonads and oocyte diameters were performed by image analysis (Image scope 2.3, Image Line, Inc.) and stage micrometer. The vitellogenic oocytes measured 130-220 µm and the follicular epithelium consisted of flattened and cuboidal layers. Beneath the vitelline membrane, scattered test cells appeared in the ooplasm and different granules of protein and MPS were deposited in the ooplasm. In the later phase, lipid droplets began to appear in the ooplasm. Yolk bodies formed after the impregnation of various granules together and the oocyte was ready to be shed. Before spawning, a yolk membrane appeared above the ooplasm. Post-vitellogenic oocytes, in which the homogeneity of ooplasm was restored, underwent gradual lysis and entered the atretic phase. Different stages of sperm development were present year-round in different follicles of the same squirt; hence, the testes were always mature.

The "Mexican dancer" in Ecuador: molecular confirmation, embryology and planktotrophy in the sea slug Elysia diomedea.

Elysia diomedea, otherwise known as the "Mexican dancer", aries in adult size and color across its geographical distribution in Ecuador. Because of morphological variation and the absence of genetic information for this species in Ecuador, we analyzed mtDNA sequences in three populations (Ballenita, La Cabuya, and Mompiche) and confirmed that individuals from the three locations belonged to E. diomedea and that there was no population structure that could explain their morphological differences. Next, we analyzed general aspects about the reproductive biology and embryology of this species. Live slugs from the Ballenita population were maintained and reproduced ex situ. Egg ribbons and embryos were fixed and observed by brightfield and confocal microscopy. We observed a single embryo per capsule, 98 embryos per mm2 of egg ribbon, and compared the cleavage pattern of this species to that of other heterobranchs and spiralians. E. diomedea early development was characterized by a slight unequal first cleavage, occurrence of a 3-cell stage in the second cleavage, and the formation of an enlarged second quartet of micromeres. We observed clear yolk bodies in the egg capsules of some eggs ribbons at early stages of development. Both reproductive and embryological characteristics, such as presence of stomodeum in the larva, and ingestion of particles after hatching confirmed the planktotrophic veliger larvae of this species, consistent with the majority of sacoglossans from the Eastern and Northeast Pacific Oceans.

Biochemical and ultrastructural changes in the ovaries of red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae) following acute imidacloprid poisoning

Abstract This study aims to inspect the effects of Imidacloprid (IMI) acute toxicity in the ovaries of the red palm weevil (Rhynchophorus ferrugineus). The LC50 of IMI was determined to be 15.7 ppm. We assessed the biochemical changes resulting from treatment with four IMI concentrations (10, 15, 20 and 30 ppm). IMI reduced acetylcholine esterase activity in a dose-dependent manner. Glutathione (GSH) content decreased with increasing concentration of IMI; however, GSH was not affected and accompanied by an increase in catalase activity with the highest concentration of IMI. Lipid peroxidation increased steadily as IMI concentration increased. Based on the ultrastructural analyses of the follicle cells and the ooplasm of developing oocytes, acute IMI toxicity produced damaged yolk bodies, pale nuclei, and shrinking microvilli. Fractionation of rough endoplasmic reticulum, deformed mitochondria, and autophagic bodies were also observed. In conclusion, these ovarian pathobiochemical changes could be used as biomarkers of acute IMI exposure.

Note on the vitellogenesis and the yolk granules in a Veronicellidae slug, Laevicaulis alte (Férussac, 1822)

The Laevicaulis alte is a terrestrial, pulmonate slug. The ovotestis of the slugs comprises only one vitellogenic oocyte in each acinus. Ultrastructural features of the vitellogenesis and various yolk granules along with their different developing stages are described for the first time in the pulmonate slugs by a transmission electron microscope. Vitellogenesis accompanied by the autosynthetic and the heterosynthetic processes. Ooplasmic Golgi complex and rough endoplasmic reticulum are involved in autosynthesis, whereas endocytosis of microvillar process of the oolemma supports heterosynthesis of yolk bodies. The follicular cleft contains various secretory materials and glycogen granules, some of which are incorporated into the distal tips of the microvilli of the oolemma. In ooplasm, three types of yolk granules are discriminated morphologically. The Type-1 yolk granules are ovoid shaped, characterized by the presence of a well-developed axial band. The Type-2 yolk granules are oval-shaped filled with a uniform electron-dense matrix. The Type-3 yolk granules are circular and vacuolated, surrounded by few cisternae of the rough endoplasmic reticulum. Morphometric characteristics of the vitellogenic oocyte, follicle cells, and different yolk granules are noted. The number of yolk granules in the ooplasm of L. alte is compared to those of other pulmonate species in different habitats.

Can snakes use yolk reserves to maximize body size at hatching?

We experimentally miniaturized freshly laid eggs of the Chinese cobra Naja atra (Elapidae) by removing ∼10% and ∼20% of original yolk. We tested if yolk-reduced eggs would produce 1) normal-sized hatchlings with invariant yolk-free body mass (and thus invariant linear size) but dramatically reduced or even completely depleted residual yolk, 2) smaller hatchlings with normal-sized residual yolk but reduced yolk-free body mass, or 3) smaller hatchlings of which both yolk-free body mass and residual yolk are proportionally reduced. Yolk quantity affected hatchling linear size (both snout-vent length and tail length) and body mass. However, changes in yolk quantity did not affect incubation length or any hatchling trait examined after accounting for egg mass at laying (for control and sham-manipulated eggs) or after yolk removal (for manipulated eggs). Specifically, yolk-reduced eggs produced hatchlings of which all major components (carcass, residual yolk, and fat bodies) were scaled down proportionally. We show that snakes cannot use yolk reserves to maximize their body size at hatching. Furthermore, our data also suggest that the partitioning of yolk in embryonic snakes is species-specific.

DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae): Purification, bioinformatic analyses and the significance of its interaction with lipophorin in the internalization by developing oocytes

DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima, is synthesized by the fat body and the ovary and functions as yolk protein precursor. Functionally, DmCatD is involved in vitellin proteolysis. In this work, we purified and sequenced DmCatD, performed bioinformatic analyses and investigated the events involved in its targeting and storage in developing oocytes. By ion exchange and gel filtration chromatography, DmCatD was purified from egg homogenates and its identity was confirmed by mass spectrometry. Approximately 73% of the full-length transcript was sequenced. The phylogeny indicated that DmCatD has features which suggest its distancing from “classical” cathepsins D. Bioinformatic analyses using a chimeric construct were employed to predict post-translational modifications. Structural modeling showed that DmCatD exhibited the expected folding for this type of enzyme, and an active site with conserved architecture. The interaction between DmCatD and lipophorin in the hemolymph was demonstrated by co-immunoprecipitation. Colocalization of both proteins in developing oocyte membranes and yolk bodies was detected by immunofluorescence. Docking assays favoring the interaction DmCatD-lipophorin were carried out after modeling lipophorin of a related triatomine species. Our results suggest that lipophorin acts as a carrier for DmCatD to facilitate its further internalization by the oocytes. The mechanisms involved in the uptake of peptidases within the oocytes of insects have not been reported. This is the first experimental work supporting the interaction between cathepsin D and lipophorin in an insect species, enabling us to propose a pathway for its targeting and storage in developing oocytes.

X-ray metal assessment and ovarian ultrastructure alterations of the beetle, Blaps polycresta (Coleoptera, Tenebrionidae), inhabiting polluted soil.

X-ray analysis was applied to estimate the percentages of heavy metals in ovarian tissues of the tenebrionid beetle, Blaps polycresta. Calcium, phosphorus, sulfur, cadmium, copper, and zinc were the most common detected metals in ovaries of insects collected from reference and polluted sites. Only cadmium showed significantly higher percentages in the polluted ovaries compared with the reference ones. Ultrastructure investigation revealed severe alterations in polluted ovaries both in the tropharium and in the vitellarium. Contraction of nuclear membrane of trophocytes was observed; therefore, cavities and spaces appeared in the cytoplasm followed by nuclear pyknosis. In the vitellarium, fragmentation of chromatin materials in nuclei of the follicular cells was detected. The cytoplasm was poor in the rough endoplasmic reticulum and mitochondria. Damage of yolk bodies occurred in addition to break off in the layer of microvilli. Accumulation of electron-dense vesicles and multivesicular bodies were observed in both reference and polluted ovaries. These alterations in ovarian ultrastructure of B. polycresta show the severe impact of cadmium pollution on cell organelles of insects and could be used as an interesting tool for monitoring heavy metals inside the body organs due to soil pollution.

Histological and statistical studies on the gonadal cycle of the mussel Lithophaga truncata (Gray, 1843) along immature and mature phases (Pelecypoda - Mytilidae)

The main purpose of this study was to investigate the gonadal cycle of the musselLithophaga truncata (Gray, 1843). Specimens were sampled regularly twice every season at depth 1- 2 meters in the year (2015 - 2016) along the northern estuarine harbor of the Arabian Gulf - Saudi Arabia. Parts of gonads (ovaries and testes) were prepared for histological and statistical studies. The histological observations of the gonad indicated that there was only one gonadal cycle in the year. The ovaries produced oocytes which undergone vitellogenesis to change these oocytes into comparatively ripe ones condensed with yolk. With slide micrometer a fixed length and width of the ovary was determined in serial histological sections (30000 µm). The different stages of oocyte development were counted and statistically analyzed. During November - February the gonadal follicles appeared small in size and in a quiescent state. Follicles were few in number and scattered throughout the vesicular connective tissue. In males the follicles were small and scattered throughout the connective tissue in the same way as females. Follicles contained undifferentiated residual cells, spermatogonia and negligible number of primary spermatocytes. The oogonia were the most dominant type. Few number of previtellogenic oocytes were observed. The ooplasm of these stages, oogonia and previtellogenic oocytes, was homogeneous and with comparatively large nucleus. In previtellogenic oocytes small granules of the same nature were scattered randomly in the ooplasm. These granules consisted of proteins (peptide linkage). Carbohydrates and lipids were entirely absent.During April - July previtellogenic oocytes began to undergo growth phase and extending towards the center of the follicles. In males spermatogenesis became more apparent and proceeded at a faster rate. In some mussels spermiogenesis was formed in great number. The gonadal follicles ramified rapidly, and the development of gametes of both sexes was very fast. In females the vitellogenic oocytes showed prominent development. Atretic oocytes and their debris were negligible. Very few number of oogonia were observed. Yolk bodies (glycoproteins) increased rapidly and replaced the vacuoles in the ooplasm. Other large granules gained a dark blue color when Aqueuos bromophenol blue was applied. These granules are basic proteins. Other large-sized granules began to appear at the periphery of the ooplasm. These granules gained positive reaction with Alcian blue, PAS and Alcian blue- PAS reactions. The vitelline envelope became conspicuous under the follicle cells. Mucosubstances were positively intense at the periphery of the oocyte while proteins surrounded the nucleus. Gradually fat deposition increased. The size range of these oocytes was 130 - 180µm with the ratio of the nucleus to the ooplasm 0.5:2. During August - October both sexes had ripe gametes. In males, the central portion of the follicles was empty because of the recent discharge of large quantities of ripe gametes. At the end of this step resorption of the gonads proceeded very rapidly and emptied follicles began to acquire an elongated form. At this step the oocyte attains its maximal size 180 - 200µm. The mean of the total number of oocyte developmental stages in all seasons showed that the ovary was active in all seasons of the year but different labors were present. It concluded that the oogonia , the postvitellogenic oocyte development and atretic oocytes were conspicuous during the first step of oogenesis ; the vitellogenic oocytes were dominant during the second step of oogenesis whereas the postvitellogenic and atretic oocytes were dominant during the third step of oogenesis. All data obtained were subjected to analysis of variance (ANOVA) means significant difference P>0.05 or P 0.05.

Ultrastructural observation of oocytes in six types of stony corals

In this study, the ultrastructure of the oocytes of 6 types of scleractinian corals was observed by transmission electron microscopy (TEM). Moreover, histological and ultrastructural analyses were performed to improve our understanding of the organelles involved in coral oocyte formation. In all 6 stony coral species, the microvilli were tubular and directly grew from the surface of the oocyte membrane; yolk bodies, lipid granules, and cortical alveoli accounted for most of the volume inside the oocytes, suggesting that they are associated with energy storage and buoyancy. Clear differences were observed in the size of yolk bodies and lipid granules in the oocytes of the 6 stony coral species, which occupied approximately 55%-80% of the inner space of the oocytes. Galaxea fascicularis exhibited the largest lipid granule volume, but the oocytes contained only an average number of 12.45 lipid granules per unit area. Only Montipora incrassata oocytes contained symbiotic algae. The smallest size and proportion of lipid granules in M. incrassata oocytes may be attributed to the presence of symbiotic algae and large yolk bodies, which may help oocytes produce energy and function as a nutritional source. This study is crucial for improving the understanding of the basic biology of coral reproduction, and the ensuing datasets is critical for conservation-oriented studies seeking to cryopreserve corals during these times of dramatic global climate change.

Dynamics of expression of two vitellogenin genes in the Chagas’ disease vector Triatoma infestans: Analysis throughout pre-vitellogenesis and vitellogenesis

The reproductive success of all oviparous species depends on vitellogenin (Vg) biosynthesis and its accumulation in the developing oocytes. The expression levels of two Vg genes (Vg1 and Vg2) were analyzed by qPCR and western blot in fat body and ovaries of adult females, at different times after ecdysis (pre-vitellogenic phase) and after blood feeding of females (vitellogenic phase). Vg genes were also evaluated in fat bodies of adult males as well as in female fifth instar nymphs. No trace of Vg mRNA was detected in adult males or in nymphs. Vg1 and Vg2 were expressed in the fat bodies and ovaries of adult females. The Vg genes start to be expressed slightly in both tissues of adult females during pre-vitellogenesis. After blood feeding, Vg1 and Vg2 were up regulated and significant levels of Vg transcripts as well as protein expression were observed in fat bodies sampled throughout vitellogenesis. During this period however, the distribution patterns of Vg1 and Vg2 transcripts showed two peaks around early and advanced vitellogenesis (days 4 and 12 post-feeding, respectively). In the ovaries, levels of mRNAs increased from the day 10 post-blood feeding onwards. In addition, the immunofluorescence assays showed a strong signal for vitellin in the yolk bodies of terminal follicles of vitellogenic females. The involvement of fat body and ovary in the synthesis of Vg suggests different roles of Vgs in supporting the growth of oocytes.

The Role of DmCatD, a Cathepsin D-Like Peptidase, and Acid Phosphatase in the Process of Follicular Atresia in Dipetalogaster maxima (Hemiptera: Reduviidae), a Vector of Chagas' Disease.

In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas’ disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia.

Ultrastructure and histopathological alteration in the ovaries of Callosobruchus maculatus (F.) (Coleoptera, Chrysomelidae) induced by the solar radiation

The ultrastructure and histopathological changes in the ovarioles were tested in normal adult females and in those developed from solar energy treated pupae (53°C for 15min) of Callosobruchus maculatus. The telotrophic ovarioles of C. maculatus contain germarium region followed by vitellarium region which constitutes the vitellarium.The germarium houses trophocytes and the vitellarium consists of ovarian follicles.At the germarium region of the ovariole, the trophocyte or nurse cell nucleus contains uniformly spaced clumps of heterochromatin. The cytoplasm contains numerous free ribosomes and mitochondria.At the vitellarium region, follicular epithelial cells form a layer around the oocyte, each cell contains a large oval nucleus with abundant heterochromatin, and the cytoplasm contains mitochondria, free ribosomes and dark spherical globules. Also the vitellarium includes the previtellogenic oocyte which is the most anterior one and two last vitellogenic oocytes at the posterior end as the yolk was deposited.Oocyte microvilli are interdigitated with those of the follicle cells. The ooplasm consists primarily of electron-dense yolk bodies and lipid droplets.These phases could be identified in the ovarioles of normal females and to a less extent in those of females developed from the treated pupae. In the ovarian follicles of the treated generation, degeneration of the cell components of trophocytes, follicular epithelium and oocytes were the most obvious signs of damage. Also, lacking of yolk bodies and vacuolation in the border of the ooplasm were observed.The damage was more pronounced in the ovarioles of (F.) progeny of the treated generation.

Ovarian nutritional resources during the reproductive cycle of the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae): focus on lipid metabolism.

In this study, we have analyzed the changes of the ovarian nutritional resources in Dipetalogaster maxima at representative days of the reproductive cycle: previtellogenesis, vitellogenesis, as well as fasting-induced early and late atresia. As expected, the amounts of ovarian lipids, proteins, and glycogen increased significantly from previtellogenesis to vitellogenesis and then, diminished during atresia. However, lipids and protein stores found at the atretic stages were higher in comparison to those registered at previtellogenesis. Specific lipid staining of ovarian tissue sections evidenced remarkable changes in the shape, size, and distribution of lipid droplets throughout the reproductive cycle. The role of lipophorin (Lp) as a yolk protein precursor was analyzed by co-injecting Lp-OG (where OG is Oregon Green) and Lp-DiI (where DiI is 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine) to follow the entire particle, demonstrating that both probes colocalized mainly in the yolk bodies of vitellogenic oocytes. Immunofluorescence assays also showed that Lp was associated to yolk bodies, supporting its endocytic pathway during vitellogenesis. The involvement of Lp in lipid delivery to oocytes was investigated in vivo by co-injecting fluorescent probes to follow the fate of the entire particle (Lp-DiI) and its lipid cargo (Lp-Bodipy-FA). Lp-DiI was readily incorporated by vitellogenic oocytes and no lipoprotein uptake was observed in terminal follicles of ovaries at atretic stages. Bodipy-FA was promptly transferred to vitellogenic oocytes and, to a much lesser extent, to previtellogenic follicles and to oocytes of ovarian tissue at atretic stages. Colocalization of Lp-DiI and Lp-Bodipy-FA inside yolk bodies indicated the relevance of Lp in the buildup of lipid and protein oocyte stores during vitellogenesis.

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.

The storage of nutritional resources during vitellogenesis of Panstrongylus megistus (Hemiptera: Reduviidae): The pathways of lipophorin in lipid delivery to developing oocytes

In this work, we have analyzed the pathways by which lipophorin (Lp) delivers its lipid cargo to developing oocytes of Panstrongylus megistus, a hematophagous vector of Chagas’ disease. Lp, vitellin, total lipids and proteins were measured in ovarian tissues at different stages of the reproductive cycle. Localization of Lp in developing oocytes, mainly at their cortical area, was demonstrated by immunofluorescence assays using an anti-Lp antibody labeled with FITC. In vivo approaches injecting fluorescently labeled Lp to follow the course of the entire particle (Lp-DiI or Lp-Oregon Green) or its lipid cargo (Lp-Bodipy-FA) were monitored by laser scanning confocal microscopy. Significant increases in the amounts of lipids, proteins and vitellin were observed in ovarian tissue with the progress of vitellogenesis. Unexpectedly, an increase in the amount of Lp was also observed. The experiments in vivo demonstrated that the uptake of fluorescent Lp labeled on its protein or lipid moiety by developing oocytes occurred very fast, being impaired at low temperatures. The co-injection of fluorescent Lp and vitellogenin (Vg) showed that both particles co-localized inside yolk bodies, confirming the endocytic pathway for Lp. When the fate of lipids transferred to oocytes was evaluated in vitellogenic females by co-injecting Lp-Bodipy-FA and Lp-DiI, the signal for Bodipy-FA was found in both lipid droplets and yolk bodies. In contrast, in injected females kept at 4°C the fluorescence was reduced, being observed exclusively in lipid droplets, implying that lipid transfer to the oocyte was diminished but not abolished. Taken together, the results demonstrate that in the hematophagous P. megistus, the storage of lipid resources by developing oocytes occurs by the convergence of different pathways by which Lp maximizes the delivery of its lipid cargo. In addition, it was also shown that, to some extent, lipids stored in the oocyte lipid droplets can also originate from endocytosed Vg. The relevance of these events in the context of the physiology of reproduction in P. megistus is discussed.

Structural changes of gonads during artificially induced gametogenesis and spawning in the giant jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae)

We conducted a histological investigation of the ovaries and testes during the gametogenesis and spawning in the giant jellyfish Nemopilema nomurai, which has bloomed in East Asian marginal seas almost annually since 2002. Oocytes arising from the ovarian epithelium make intimate contact with special epithelial cells, called trophocytes, which have microvilli on the subgenital sinus side, Golgi complexes and vesicles in the cytoplasm. In the early vitellogenic stage, yolk bodies occur in the ooplasm adjacent to the trophocytes, suggesting that the trophocytes transfer nutrients from the subgenital sinus to the oocyte. Later, yolk bodies are formed by the Golgi complexes in the entire ooplasm and accumulate until the oocyte matures. In the late vitellogenic stage, the oocyte separates from the trophocytes and forms microvilli on its surface, indicating nutrient uptake from the surrounding mesoglea. Nutrient support from the trophocytes in the early vitellogenic stage may make the oocytes mature rapidly after medusae are physically damaged. Microvilli-rich epithelial cells also associate with sperm follicles where spermatocytes arise from the follicle cells and accumulate, but their function in nutrient uptake is possibly less than that of trophocytes according to their morphology. During ovulation, which takes 1.5 hours after light exposure, trophocytes separate from each other and make an ovulation pit where the oocyte passes out to the subgenital sinus with the surrounding basal lamina. Spermiation occurs 5–20 minutes after light exposure, and spermatozoa are liberated through the spermiation pit that was formed by which the microvilli-rich cells dissociate. The trophocytes in ovaries and microvilli-rich cells in testes have important roles not only in the gametogenesis but also in the spawning of N. nomurai.

OOCYTES OF HOLOTHURIA LEUCOSPILOTA (ECHINODERMATA: HOLOTHUROIDEA): AN ULTRA STRUCTURAL STUDY

Ovaries of Holothuria leucospilota consist of simple tubules, within which oocytes grew and reached maturity. The oocytes development started at the tubule lining and as the development proceeded, the oocytes increased in size and moved to the tubule lumen. An ultra-section-method was applied to describe ultrastructural changes in the oocytes during their development, leading to the knowledge on gametogenesis of sea encumber H.leucospiota. Primary previtellogenic oocytes embedded in tubule lining was the earliest stage, which were characterized by the presence of nuage. Prior to yolk formation, clusters of mitochondria appeared in the ooplasm. This may indicate the need for energy to produce yolk substance. When yolk bodies were common in vitellogenic oocytes, nuage became invisible and mitochondria were no longer in groups. As development progressed, abundant microvilli appeared on the surface of vitellogenic oocytes, but less common on postvitellogenic oocytes when maximum growth had been attained. This structure was suggested to facilitate size increase and functional efficiency of oocyte surface.

Ultrastructure of oogenesis of two oviparous demosponges: Axinella damicornis and Raspaciona aculeata (Porifera)

We investigated the cytology of the oogenic cycle in two oviparous demosponges, Axinella damicornis and Raspaciona aculeata, during 2 consecutive years both by light and electron microscopy. Oocytes of both species were similar in their basic morphological features but differences were noticed in time required to complete oocyte maturation and mechanisms of acquisition of nutritional reserves. The oogenic cycle of A. damicornis extended for 7–8 months in autumn-spring, while that of R. aculeata did it for 3–5 months in summer-autumn. Yolk of A. damicornis was predominantly formed by autosynthesis. Oocytes endocytosed bacteria individually and stored them in groups in large vesicles. Bacteria were digested and lipidic material was added to the vesicles to produce a peculiar granular yolk hitherto unknown in sponges. Scarce cells carrying heterogeneous inclusions were observed in the perioocytic space, and were interpreted as putative nurse cells. Such cells were presumably releasing lipid granules to the perioocytic space. In contrast, large numbers of nurse cells were found surrounding the oocytes of R. aculeata. They transported both lipid granules and heterogeneous yolk bodies to the oocytes. R. aculeata also produced some of their yolk by autosynthesis. The involvement of nurse cells in the vitellogenesis of R. aculeata shortened the oocyte maturation, whereas a largely autosynthetic vitellogenesis in A. damicornis prolonged the duration of oogenesis.