Proteinase activity in the early developmental stages of the carp, Cyprinus carpio

Embryonic and early postembryonic development of the carp is associated with an increase in the content of soluble proteins and nitrogen of non-protein nitrogenous components. Simultaneously, the activity of trypsin-like and chemotrypsin-like peptide hydrolases increases together with the increase in the degree of proteolysis. These data suggest that the development of carp embryos and early larva is accompanied by intensification of two opposite processes--synthesis and catabolism of proteins. The observed changes are more evident in early postembryonic period as compared with the early stages of embryogenesis.     

Loss of yolk platelets and yolk glycoproteins during larval development of the sea urchin embryo

The fate of the yolk platelets and their constituent yolk glycoproteins was studied in Strongylocentrotus purpuratus eggs and embryos cultured through the larval stage. Previous studies have shown that the yolk glycoproteins undergo limited proteolysis during early embryonic development. We present evidence that the yolk glycoproteins stored in the yolk platelets exist as large, disulfide-linked complexes that are maintained even after limited proteolysis have occurred. We provide additional evidence that acidification of the yolk platelet may activate a latent thiol protease in the yolk platelet that is capable of correctly processing the major yolk glycoprotein into the smaller yolk glycoproteins. Because we previously showed that these yolk glycoproteins are not catabolized during early embryonic development, it was of interest to study their fate during larval development. Using a specific polyclonal antibody to a yolk glycoprotein, we found that both yolk glycoproteins and the yolk platelets disappeared in feeding, Day 7, larval stage embryos, but that starvation did not significantly affect the levels of the yolk glycoproteins. We also found that the yolk glycoproteins reappeared in 30-day-old premetamorphosis larvae.     

Effects of maternal conditions on early life history traits of black porgy Acanthopagrus schlegeli

This work examined the effects of maternal conditions on early life history traits of black porgy Acanthopagrus schlegeli . Age-II females produced significantly larger eggs as compared to the same size of Age-III females. Also, within each of the age groups, there was a positive relationship between egg size and female size. The larger eggs generally had larger volumes of oil globules, required longer incubation periods (hatching age), and produced larger larvae that endured longer to starvation. Hatching age was covaried negatively with yolk volume at hatching, indicating that embryonic development consumed primarily yolk as its energy resource. The condition factor, gonadosomatic index, hepatosomatic index, and lipid content of the females were not related to the early life history traits of their offspring.     

Cryopreservation of Mexican fruit flies by vitrification: stage selection and avoidance of thermal stress

This report presents details of a vitrification methodology for the cryopreservation of embryos of the Mexican fruit fly, Anastrepha ludens. The overall summary of the data indicates that selecting the correct developmental stage for cryopreservation is the most important criterion. The key aspect in selection of the correct stage is to balance depletion of the gut yolk content against development of the embryonic cuticle. Embryogenesis was divided into four stages between 90 and 120 h after incubation at 21.7 degrees C. The classification was based on the intestinal yolk content and the initial development of mandibular-maxillary complex. Stages having low mid-gut yolk content and the appearance of mouth hooks were found to be the most suitable for cryopreservation. Embryos developing at 30 degrees C had premature cuticle formation relative to gut development and significantly lower hatching after cryopreservation. Vitrification of embryos by direct quenching in liquid nitrogen was less effective than quenching after annealing the samples in liquid nitrogen vapor. Quenched samples of vitrification solutions containing 1,2-ethanediol as the major component exhibited fractures. Fracturing occurred less frequently when the solutions were annealed and when containing polyethylene glycol. Hatching of vitrified embryos stored in liquid nitrogen for over 12 months was not statistically different from those held for only 15 min. Our protocol yielded normalized hatching rates that ranged as high as 61%. Selecting the exact stage for cryopreservation from a population of embryos obtained by collection from ovipositing females during a span of just 30 min resulted in nearly 80% of the embryos hatching into larvae.     

Relationships among salinity, egg size, embryonic development, and larval biomass in the estuarine crab Chasmagnathus granulata Dana, 1851

We studied interrelationships between initial egg size and biomass, duration of embryogenesis at different salinities, and initial larval biomass in an estuarine crab, Chasmagnathus granulata. Ovigerous females were maintained at three different salinities (15‰, 20‰ and 32‰); initial egg size (mean diameter), biomass (dry weight, carbon and nitrogen) as well as changes in egg size, embryonic development duration, and initial larval biomass were measured.

Initial egg size varied significantly among broods from different females maintained under identical environmental conditions. Eggs from females maintained at 15‰ had on average higher biomass and larger diameter. We hypothesise that this is a plastic response to salinity, which may have an adaptive value, i.e. it may increase the survivorship during postembryonic development. The degree of change in egg diameter during the embryonic development depended on salinity: eggs in a late developmental stage were at 15‰ significantly larger and had smaller increment than those incubated at higher salinities. Development duration was longer at 15‰, but this was significant only for the intermediate embryonic stages. Initial larval biomass depended on initial egg size and on biomass loss during embryogenesis. Larvae with high initial biomass originated either from those eggs that had, already from egg laying, a high initial biomass (reflecting individual variability under identical conditions), or from those developing at a high salinity (32‰), where embryonic biomass losses were generally minimum. Our results show that both individual variability in the provisioning of eggs with yolk and the salinity prevailing during the embryonic development are important factors causing variability in the initial larval biomass of C. granulata, and thus, in early larval survival and growth.     

The origin of postembryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster

Embryonic and postembryonic neuroblasts in the thoracic ventral nerve cord of Drosophila melanogaster have the same origin. We have traced the development of threefold-labelled single precursor cells from the early gastrula stage to late larval stages. The technique allows in the same individual monitoring of progeny cells at embryonic stages (in vivo) and differentially staining embryonic and postembryonic progeny within the resulting neural clone at late postembryonic stages. The analysis reveals that postembryonic cells always appear together with embryonic cells in one clone. Furthermore, BrdU labelling suggests that the embryonic neuroblast itself rather than one of its progeny resumes proliferation as a postembryonic neuroblast. A second type of clone consists of embryonic progeny only.     

Rhesus glycoprotein and urea transporter genes are expressed in early stages of development of rainbow trout (Oncorhynchus mykiss)

The objective of this study was to determine if the genes for the putative ammonia transporters, Rhesus glycoproteins (Rh) and the facilitated urea transporter (UT) were expressed during early development of rainbow trout, Oncorhynchus mykiss Walbaum. We predicted that the Rh isoforms Rhbg, Rhcg1 and Rhcg2 would be expressed shortly after fertilization but UT expression would be delayed based on the ontogenic pattern of nitrogen excretion. Embryos were collected 3, 14 and 21 days postfertilization (dpf), whereas yolk sac larvae were sampled at 31 dpf and juveniles at 60 dpf (complete yolk absorption). mRNA levels were quantified using quantitative polymerase chain reaction and expressed relative to the control gene, elongation factor 1alpha. All four genes (Rhbg, Rhcg1, Rhcg2, UT) were detected before hatching (25-30 dpf). As predicted, the mRNA levels of the Rh genes, especially Rhcg2, were relatively high early in embryonic development (14 and 21 dpf), but UT mRNA levels remained low until after hatching (31 and 60 dpf). These findings are consistent with the pattern of nitrogen excretion in early stages of trout development. We propose that early expression of Rh genes is critical for the elimination of potentially toxic ammonia from the encapsulated embryo, whereas retention of the comparatively benign urea molecule until after hatch is less problematic for developing tissues and organ systems.     

Experimental yolk sac dysfunction as a model for studying nutritional disturbances in the embryo during early organogenesis

Our investigations concerning the importance of cell surface macromolecules during embryonic development led us to the discovery in 1961 that heterologous anti-rat kidney serum produced teratogenesis, growth retardation and embryonic death when injected into the pregnant rat during early organogenesis. It was established that IgG was the teratogenic agent, primarily directed against the visceral yolk sac (VYS) but not the embryo. Heterologous anti-rat VYS serum was prepared which was teratogenic localized in the VYS and served as a model for producing VYS dysfunction and embryonic malnutrition. The role of the yolk sac placenta in histiotrophic nutrition is now recognized to be critical for normal embryonic development during early organogenesis in the rodent. VYS antiserum affects embryonic development primarily by inhibiting endocytosis of proteins by the VYS endoderm, resulting in a reduction in the amino acids supplied to the embryo. Our laboratory has recently developed teratogenic monoclonal yolk sac antibodies (MCA) which can be utilized; to study VYS plasma membrane synthesis and recycling, to compare yolk sac function among different species, and to identify components of the plasma membrane involved in pinocytosis. MCA prepared against certain VYS antigens provide an opportunity to study embryonic nutrition with minimal interference with the nutritional state of the mother. Recent developments in the study of the human yolk sac along with our laboratory's ability to isolate a spectrum of yolk sac antigens, prepare monoclonal antibodies, and perform functional studies, should provide information that will increase our understanding of yolk sac function and dysfunction in the human and determine the relative importance of various amino acids to normal development during mammalian organogenesis.     

Changes in the antioxidant metabolism in the embryonic development of the common South American toad Bufo arenarum: differential responses to pesticide in early embryos and autonomous-feeding larvae

Amphibians may be critically challenged by aquatic contaminants during their embryonic development. Many classes of compounds, including organophosphorus pesticides, are able to cause oxidative stress that affects the delicate cellular redox balance regulating tissue modeling. We determined the progression of antioxidant defenses during the embryonic development of the South American common toad, Bufo arenarum. Superoxide dismutase (SOD) and catalase (CAT) activities were high in the unfertilized eggs, and remained constant during the first stages of development. SOD showed a significant increase when the gills were completely active and opercular folds began to form. Reductase (GR) activity was low in the oocytes and increased significantly when gills and mouth were entirely developed and the embryos presented a higher exposure to pro-oxidant conditions suggesting an environmental control. Reduced glutathione (GSH) content was also initially low, and rose continuously pointing out an increasing participation of GSH-related enzymes in the control of oxidative stress. GSH peroxidases and GSH-S-transferases showed relatively high and constant activities, probably related to lipid peroxide control. B. arenarum embryos have plenty of yolk platelets containing lipids, which provide the energy and are actively transferred to the newly synthesized membranes during the early embryonic development. Exposure to the pro-oxidant pesticide malathion during 48 h did not significantly affect the activity of antioxidant enzymes in early embryos, but decreased the activities of CAT, GR, and the pool of GSH in larvae. Previous work indicated that lipid peroxide levels were kept low in malathion-exposed larvae, thus we conclude that oxidative stress is overcome by the antioxidant defenses. The increase in the antioxidant metabolism observed in the posthatching phase of development of B. arenarum embryo, thus constitutes a defense against natural and human-generated pro-oxidants present in the aquatic environment.     

The follicular placenta of the viviparous fish,Heterandria formosa. I. Ultrastructure and development of the embryonic absorptive surface

Embryos of the poeciliid Heterandria formosa develop to term in the ovarian follicle in which they establish a placental association with the follicle wall (follicular placenta) and undergo a 3,900% increase in embryonic dry weight. This study does not confirm the belief that the embryonic component of the follicular placenta is formed only by the surfaces of the pericardial and yolk sacs; early in development the entire embryonic surface functions in absorption. The pericardial sac expands to form a hood-like structure that covers the head of the embryo and together with the yolk sac is extensively vascularized by a portal plexus derived from the vitelline circulation. The hood-like pericardial sac is considered to be a pericardial amnion-serosa. Scanning and transmission electron microscopy reveal that during the early and middle phases of development (Tavolga's stages 10–18 for Xiphophorus maculatus) the entire embryo is covered by a bilaminar epithelium whose apical surface is characterized by numerous, elongate microvilli and coated pits and vesicles. Electron-lucent vesicles in the apical cytoplasm appear to be endosomes while a heterogeneous group of dense-staining vesicles display many features characteristic of lysosomes. As in the larvae of other teleosts, cells resembling chloride cells are also present in the surface epithelium. Endothelial cells of the portal plexus lie directly beneath the surface epithelium of the pericardial and yolk sacs and possess numerous transcytotic vesicles. The microvillous surface epithelium becomes restricted to the pericardial and yolk sacs late in development when elsewhere on the embryo the non-absorptive epidermis differentiates. We postulate that before the definitive epidermis differentiates, the entire embryonic surface constitutes the embryonic component of the follicular placenta. The absorptive surface epithelium appears to be the principle embryonic adaptation for maternal-embryonic nutrient uptake in H. formosa, suggesting that a change in the normal differentiation of the surface epithelium was of primary importance to the acquisition of matrotrophy in this species. In other species of viviparous poeciliid fishes in which there is little or no transfer of maternal nutrients, the embryonic surface epithelium is of the non-absorptive type.     

Effects of temperature and salinity on the eggs and early larvae of Caranx mate (Cuv. & Valenc.) (pisces: carangidae) in Hawaii

Eggs and larvae of the carangid fish, Caranx mate (Cuv. & Valenc.), were incubated at various temperature (17.2 to 33.1 °C) and salinity (10 to 42 ‰) combinations in five experiments. The following rates were directly proportional to temperature: embryonic development, yolk absorption, eye and jaw development, and increase in length. Unfed C. mate larvae attained a maximum size at 25 °C and 20 ‰ Eyes and jaws of larvae were functional by the end of the yolk sac stage at all temperature and salinity levels tested.Hatching success and larval survival at the end of the yolk sac stage were generally greater than 50 % between 22° and 32°C. Hatching success and larval survival at the end of the yolk sac stage were reduced at salinity extremes, especially in low temperature-low salinity and high temperature-high salinity combinations. The frequency of morphological abnormalities was also high at extreme temperatures and salinities.The incipient upper thermal TL_m for unfed C. mate larvae acclimated to 23.8°C increased from 31.5°C for newly hatched larvae, to 34.2°C for 72 h larvae, but decreased to 32.0°C for starving larvae after the exhaustion of the yolk supply.     

New Insights into the Functions of Cytokinins in Plant Development

Recent breakthroughs in cytokinin research have shed new light on the role of cytokinin in plant development. Loss-of-function mutants of a cytokinin receptor reveal a role for the hormone in establishment of the vasculature during embryonic development. Cytokinin controls the number of early cell divisions via a two-component signaling system. Genetically engineered plants that have a reduced cytokinin content demonstrate the regulatory role of the hormone in control of meristem activity and organ growth during postembryonic development, with opposite roles in roots and shoots. There is increasing evidence from work with transgenic plants and mutant analysis that cytokinins do not perform the previously proposed function as a root-derived signal for the regulation of shoot branching. Root-borne cytokinins might serve as a long-range signal controling other processes at distant sites, such as responding to nutritional status, particularly nitrogen availability.     

Changes in sex steroid levels in yolks of the leghorn chicken,Gallus domesticus,during embryonic development

Yolk steroid hormones have been documented to have growth and behavior effects on hatchlings in several avian species. The purpose of these investigations was to determine initial levels of androstenedione (A), dihydrotestosterone (DHT), estradiol (E(2)), and testosterone (T), and document any changes in those hormones during the course of embryonic development in the Leghorn chicken, Gallus domesticus. Eggs were collected, labeled for hen of origin and egg sequence, incubated at 37.8 degrees C, and sacrificed at predetermined times during development. The embryos were staged, the yolk material collected, homogenized and hormones extracted. A, DHT, E(2), and T were separated via column chromatography and hormone levels determined using radioimmunoassays (RIAs). Results indicate a significant decrease in A and T during embryonic development, similar to that reported by our laboratory for the alligator, with A levels being significantly greater initially than levels of all other hormones. Changes in DHT mirrored changes in T levels. Chicken E(2) yolk content dynamics differ from those we have measured in both the turtle and the alligator. After an initial decline, E(2) in the yolks of chicken eggs undergoes a significant increase at the end of development, between embryonic stages 40 and 45 (days 14 and 20 of development). As the increase is much larger than could be accounted for by hormones present in the yolk material, this may represent early embryonic production of steroid hormones by the developing gonads.     

Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster.

The spatio-temporal proliferation pattern of postembryonic neuroblasts in the central brain region of the supra-esophageal ganglion of Drosophila melanogaster was studied by labeling DNA replicating cells with 5-bromo-2'-deoxyuridine (BrdU). There are five proliferating neuroblasts per hemisphere in larvae just after hatching: one in the ventro-lateral, and the other four in the postero-dorsal region of the brain. Dividing neuroblasts increase during the late first-late second instar larval stages, reaching a plateau of about 85 neuroblasts per hemisphere. Most neuroblasts cease dividing 20-30 hr after puparium formation (APF), while only four in the postero-dorsal region continue making progenies until 85-90 hr APF. The four distinct neuroblasts proliferating in the early larval and late pupal stages are identical; they lie in the cortex above the calyces of the mushroom bodies (corpora pedunculata), proliferating over a period twice as long as that for the other neuroblasts. Their daughter neurons project into the mushroom body neuropile, and hence are likely to be the Kenyon cells. The cell-cycle period of the four neuroblasts (named mushroom body neuroblasts: MBNbs) is rather constant (1.1-1.5 hr) during the mid larval-early pupal stages and is longer before and after that. The total number of the MBNb progenies made throughout the embryonic and postembryonic development was estimated to be 800-1200 per hemisphere.     

Developmental fate of the yolk protein lipovitellin in embryos and larvae of winter flounder, Pleuronectes americanus.

The developmental fate of the vitellogenin-derived yolk protein, lipovitellin (Lv), was investigated in winter flounder embryos and yolk-sac larvae. Since Lv is present as only one major polypeptide in ovulated winter flounder eggs, unlike the multiple yolk polypeptides found in the mature eggs of most teleosts, this system is presented as a simpler model of yolk protein structure and utilization during teleostean development. Winter flounder Lv is cleaved during embryogenesis from a 94 kD polypeptide at fertilization to 67 kD and 26 kD polypeptides at hatching. The rate of this proteolytic processing is slow during early embryonic development, but enters a more rapid phase between days 8 and 12 post-fertilization in embryos reared at 4-5 degrees C, and approaches 50% completion at day 10. Lv processing is essentially complete 3 days before hatching; nevertheless, major degradation of the Lv peptide by the developing winter flounder does not occur until after hatching. The Stokes radius of Lv changes only moderately following processing, from 4.50 nm in unfertilized eggs to 4.19 nm in late embryos and newly hatched larvae, whereas the processed Lv retains its heat stability relative to other yolk polypeptides. Nearly 50% of its lipid content, however, is released from the Lv particle during embryogenesis, concomitant with cleavage of the Lv 94 kD polypeptide. Lv processing may thus render a portion of the yolk protein-associated lipid more accessible to the developing embryo, whereas other yolk components are retained for later use by the winter flounder larva. Alternately, removal of lipid may lead to proteolytic vulnerability of the Lv polypeptide. In either case, only a portion of the lipid moiety of the Lv particle appears to play a significant nutritive role for the embryo, whereas its protein component is reserved for larval use. J. Exp. Zool. 284:686-695, 1999.     

Release of ecdysteroid-phosphates from egg yolk granules and their dephosphorylation during early embryonic development in silkworm, Bombyx mori

Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.     

Dynamics of chemical composition of the Black Sea gray mullet <Emphasis Type="Italic">Mugil cephalus</Emphasis> in the period of early ontogenesis and its dependence on temperature and salinity

Results of experiments on the study of the effect of temperature and salinity on dynamics of the chemical composition of gray mullet Mugil cephalus in the embryonic-larval period of development are presented. During the period of endogenous feeding (from the beginning of egg cleavage to transition of larvae to active feeding), lipids are spent with a greater rate than protein. After transition of larvae to exogenous feeding, an increase in the absolute concentration of proteins compared to lipids begins earlier and proceeds more intensively. The stage of irreversible starvation in larvae of gray mullet starts at a loss of 39–44% of protein and of the same amount of lipids of their initial content at the moment of hatching. Minimal protein losses over the period of embryonic development of gray mullet, as well as of protein and lipids towards the moment of termination of resorption of yolk sac, are observed in the range of temperatures of 21–25°C. Under conditions of low salinity (13–15‰) in the process of resorption of the yolk sac, lipids are spent more economically than at a higher salinity (17–19‰) at the expense of an increase in protein losses. Thus, a decrease in specific weight promoting the retention of buoyancy of larvae in water of low density is achieved.     

Roles of the af and tl genes in pea leaf morphogenesis: characterization of the double mutant (afaftltl)

The pleiofila phenotype (afaftltl double mutant) of Pisum sativum arises from two single-gene, recessive mutations known to affect the identity of leaf pinnae, afila (af), and acacia (tl). The wild-type leaf consists of proximal leaflets and distal tendrils, whereas the pleiofila leaf consists of branched pinnae terminating in small leaflets. Using morphological measurements, histology, and SEM, we characterized the variation in leaf form along the plant axis, in leaflet anatomy, and in leaf development in embryonic, early postembryonic, and late postembryonic leaves of aftl and wild-type plants. Leaves on aftl plants increase in complexity more rapidly during shoot ontogeny than those on wild-type plants. Leaflets of aftl plants have identical histology to wild-type leaflets although they have smaller and fewer cells. Pinna initiation is acropetal in early postembryonic leaves of aftl plants and in all leaves of wild-type plants, whereas in late postembryonic leaves of aftl plants pinna initiation is bidirectional. Most phenotypic differences between these genotypes can be attributed to differential timing (heterochrony) of major developmental events.     

裸项栉鰕虎鱼的全人工繁殖及其胚胎发育

目的研究裸项栉鰕虎鱼全人工繁殖及胚胎发育,为该鱼的实验动物化研究奠定基础。方法对胚胎和仔稚幼鱼各个发育期的连续取样,系统地观察了裸项栉鰕虎鱼各发育期的形态变化及生长特征。结果水温25．8℃～26．5℃,裸项栉鰕虎鱼受精后约82h仔鱼开始出膜,初孵仔鱼全长1．42～1．73mm,1d开口摄食,3d卵黄囊消失,23d第一背鳍形成,鳞片出现;33d全身被鳞;初孵仔鱼经97d发育成熟,繁殖周期约为36d;雌鱼性腺成熟系数可达40％,个体相对生殖力达每克8193．5粒。结论裸项栉鰕虎鱼具有个体小、性成熟早、生殖季节长、繁殖周期短、繁殖力强等特点,可进行室内规模化人工繁殖和培育。