Catecholaminergic and cholinergic regulation of swimming motility development in free embryos of Cichlasoma Nigrofasciatum

The divergence of progeny from the same spawners of Cichlasoma nigrofasciatum into two groups by duration of embryogenesis and the level of motor activity was studied close to the end of the embryonic period. Free embryos were also studied. During the study, eggs were treated with agents, modifying the activity of catecholaminergic and cholinergic systems. 3-Hydroxytyramine and L-Tyrosine were found to exert a weak influence on embryonic motility. After hatching, these substances modify swimming performance of free embryos, approximating movements of fish at later stages. 6-Hydroxydopamine and, still more, alpha-Bungarotoxin, decrease embryonic motility and postpone the hatching. The influence of these substances on the development of embryo motility increases during early ontogenesis, as indicated by decreased concentration of the substance, necessary for adequate reaction. Neither L-Tyrosine nor 6-Hydroxydopamine influenced the divergence of the progeny into two groups. Injection of the perivitelline fluid with high concentration of hatching enzyme from pre-hatching embryos into the perivitelline space of earlier embryos was found to induce the appearance of rotation movements, typical for more advanced embryos. Changes of correlation between the miogenic and neurogenic motor activity during early development of fish are discussed.     

Sodium balance in eggs and dechorionated embryos of the Atlantic salmon Salmo salar L. exposed to zinc, aluminium and acid waters

Ionic regulation of eyed eggs of Atlantic salmon was studied using intact eggs and embryos from which the chorion and perivitelline fluid had been removed, a preparation which remained in good condition for at least 2 weeks. Both intact eggs and embryos in freshwater showed similar Na+ influx rates (0.0064 and 0.01 microM g-1 hr-1, respectively) but Na+ efflux rates (0.0024 and 0.0064 microM g-1 hr-1, respectively) were greater in embryos, suggesting that the perivitelline fluid which is known to bind cations has an important function in preventing Na+ loss from the yolk and embryo. Over 90% of the egg Na+ is located in the yolk and embryo but only about 10% is exchangeable while the chorion contains about 8% Na+ which is non-exchangeable. Both eggs and embryos in acid water at pH 4, in Zn2+ 5 mM l-1 and in aluminium 1 mM l-1 showed greatly reduced Na+ uptake but eggs in 10 microM l-1 aluminium or 100 microM l-1 Zn2+ showed normal Na+ balance while embryos were normal in 10 microM l-1 aluminium but showed reduced Na+ uptake in 100 microM l-1 Zn2+. It is concluded that the chorion and perivitelline fluid have a capacity to protect the embryo from metal ions and acid water.     

Changes in Osmotic Pressure and Swelling in Horseshoe Crab Embryos During Development

Water influx accompanying the swelling of embryos during normal development of horseshoe crabs, Limulus polyphemus and Tachypleus tridentatus , following a rupture of the chorion, was analyzed. The increase in volume of perivitelline fluid during deveopment was about 90 percent of the increase in total embryo weight. Considerable water discharge was observed on drying the embryos in air and a reversible water influx occurred with a second immersion in sea water, even though the embryos died as a result of this treatment. Since the osmotic pressure of the perivitelline fluids decreased markedly during development until the end of swelling, a close correlation between swelling and osmotic pressure was recognized. These results indicate that certain osmoactive substances may be produced in the perivitelline fluid at the initial stage of swelling.     

Multiple extracellular activities in Drosophila egg perivitelline fluid are required for establishment of embryonic dorsal-ventral polarity.

Twelve maternal effect genes (the dorsal group and cactus) are required for the establishment of the embryonic dorsal-ventral axis in the Drosophila embryo. Embryonic dorsal-ventral polarity is defined within the perivitelline compartment surrounding the embryo by the ventral formation of a ligand for the Toll receptor. Here, by transplantation of perivitelline fluid we demonstrate the presence of three separate activities present in the perivitelline fluid that can restore dorsal-ventral polarity to mutant easter, snake, and sp?tzle embryos, respectively. These activities are not capable of defining the polarity of the dorsal-ventral axis; instead they restore structures according to the intrinsic dorsal-ventral polarity of the mutant embryos. They appear to be involved in the ventral formation of a ligand for the Toll protein. This process requires serine proteolytic activity; the injection of serine protease inhibitors into the perivitelline space of wild-type embryos results in the formation of dorsalized embryos.     

Yolk utilization in stick insects entails the release of vitellin polypeptides into the perivitelline fluid

This study investigates the developmental fate of vitellin (Vt) polypeptides generated by limited proteolysis in an insect embryo. To this end, a number of polyclonal (pAb) and monoclonal antibodies (mAb) were raised against the yolk sac and the perivitelline fluid of late embryos of the stick insect Carausius morosus. Two dimensional immuno gel electrophoresis and Western blotting demonstrate that polypeptides resulting from Vt processing are present both in the yolk sac and the perivitelline fluid. At the confocal microscope, different labelling patterns were detected in the ooplasm depending on the stage of development attained by the embryo. At early developmental stages, label is associated with large unsegmented portions of the fluid ooplasm. During embryonic development, the fluid ooplasm is gradually transformed into yolk granules by intervention of vitellophages. Prior to dorsal closure, the yolk sac is separated from the perivitelline fluid by interposition of serosa cells (the so called serosa membrane). Several mAbs raised against the perivitelline fluid react specifically with this membrane suggesting that the release of Vt polypeptides from the yolk sac occurs by intracellular transit through the serosa cells. By immunocytochemistry, gold label appears associated with the cell surface and a number of vacuoles of the serosa membrane. These data are interpreted as suggesting that Vt polypeptides resulting from limited proteolysis in stick insect embryos are not exhaustively degraded within the yolk sac, but are instead transferred transcytotically to the perivitelline fluid through the serosa membrane.     

慢病毒载体介导的绿色荧光蛋白转基因标记法研究猪嵌合体制作

目的:通过建立慢病毒载体感染猪胚胎体系实现胚胎标记,进而研究不同发育阶段猪孤雌胚胎之间的嵌合能力,为进一步研究猪早期胚胎发育以及细胞分化奠定基础.方法:首先,通过显微注射的方法把2×109I.U./ml、2×108I.U./ml和2×107I.U./ml三个梯度的表达绿色荧光的慢病毒载体分别注射到猪1-细胞胚胎和2-细胞胚胎的透明带下,进行胚胎的GFP转基因标记,在荧光显微镜下观察比较卵裂率、阳性胚胎率、囊胚率、阳性囊胚率和囊胚细胞数.然后,采用凹窝聚合法对同步发育胚胎在不同阶段(2-细胞,4-细胞,8-细胞)进行嵌合,2-细胞胚胎与不同发育阶段(2-细胞、4-细胞、8-细胞)胚胎进行嵌合以及2-细胞胚胎卵裂球互换制作嵌合体胚胎,发育到囊胚时在荧光显微镜下检测胚胎的嵌合状态.结果:2×109I.U./ml的慢病毒感染猪2-细胞胚胎组中,体外受精和孤雌胚胎感染阳性率( 80.00％、76.36％)和阳性囊胚率(90.74％、89.56％)都显著高于其它滴度组(P＜0.05),另外,慢病毒感染的两种胚胎与对照组对卵裂率、囊胚率和囊胚细胞数三个指标没有显著影响(P＞0.05).2-细胞胚胎之间嵌合囊胚率和2-细胞卵裂球互换嵌合囊胚率( 53.85％、62.50％)显著高于2-细胞胚胎与4-细胞胚胎的嵌合率(18.60％,P＜0.05),在同步发育胚胎中8-细胞胚胎之间的嵌合率(75.00％)高于4-细胞胚胎之间和2-细胞胚胎之间的嵌合率( 65.00％、53.80％).结论:2×109I.U./ml的慢病毒感染2-细胞期胚胎效率最高,另外,慢病毒感染对猪胚胎发育没有明显影响.8-细胞间的嵌合率比较高;发育同步胚胎间的嵌合率高于发育非同步胚胎间的嵌合率.     

Selective sequestration of an oviductal fluid glycoprotein in the perivitelline space of mouse oocytes and embryos

Previously, we identified a 215 kd glycoprotein, GP215, which is associated with postovulatory oocytes and embryos, but not with preovulatory oocytes (Kapur and Johnson, '85). In this paper a polyclonal antibody that specifically recognizes GP215 has been used to study the distribution of the molecule in association with ova and preimplantation embryos and in the female reproductive tract. GP215 is present in epithelial cells lining the cranial portions of the oviduct and in oviductal fluid, ovarian bursal fluid, and medium conditioned by oviductal tissue in vitro. Immunofluorescence assays of the ovum and early embryo show that GP215 is sequestered in the perivitelline space. Since preovulatory oocytes exposed to bursal fluid in vitro acquire GP215, we hypothesize that GP215 is synthesized and secreted by the oviductal epithelium and secondarily associates with the ovulated oocyte. Sequestration of GP215 within the perivitelline space is relatively specific since mouse serum albumin, a major constituent of oviductal fluid, and other high molecular weight proteins are not similarly retained. These observations indicate that the composition of the perivitelline space may be significantly different from the greater environment external to the zona pellucida such that fertilization and early development of mammalian ova potentially take place in a distinct perivitelline microenvironment.     

The importance of perivitelline fluid convection to oxygen uptake of Pseudophryne bibronii eggs

The ciliated epithelium of amphibian embryos produces a current within the perivitelline fluid of the egg that is important in the convective transfer of oxygen to the embryo's surface. The effects of convection on oxygen uptake and the immediate oxygen environment of the embryo were investigated in Pseudophryne bibronii. Gelatin was injected into the eggs, setting the perivitelline fluid and preventing convective flow. Oxygen consumption rate (M(.)o?) and the oxygen partial pressure (Po?) of the perivitelline fluid were measured in eggs with and without this treatment. M(.)o? decreased in eggs without convection at Gosner stages 17-19 under normoxia. The lack of convection also shifted embryos from regulators to conformers as environmental Po? decreased. A strong Po? gradient formed within the eggs when convection was absent, demonstrating that the loss of convection is equivalent to decreasing the inner radius of the capsule, an important factor in gas exchange, by 25%. M(.)o? also declined in stage 26-27 embryos without cilia-driven convection, although not to the extent of younger stages, because of muscular movements and a greater skin surface area in direct contact with the inner capsule wall. This study demonstrates the importance of convective flow within the perivitelline fluid to gas exchange. Convection is especially important in the middle of embryonic development, when the perivitelline space has formed, creating a barrier to gas exchange, but the embryos have yet to develop muscular movements or have a large surface area exposed directly to the jelly capsule.     

Kinetic characteristics of the sodium uptake mechanism during the development of embryos and fry of Atlantic salmon, Salmo salar L., in an improved water quality

The kinetics of active sodium uptake in dechorionated embryos, yolk-sac fry and start-feed fry of Atlantic salmon were compared in two groups reared either in low conductivity, untreated, river water (conductivity ∼ 46 μS cm⁻¹, pH 5.75), or in 'improved' river water buffered with sea water (conductivity ∼2200 μS cm ¹, pH 6.56), the latter treatment often being used in commercial hatcheries to avoid problems associated with periodic acidification.
Maximal transport rate ( V _max) increased during development in both groups but was always significantly higher in embryos and fry maintained in untreated river water. Values for K _m were not seen to vary during development up to 12 weeks after hatching and were not significantly different between groups, or from values reported for adult Atlantic salmon in fresh water.
The results are discussed with respect to the influence of Na⁺ concentrations in the perivitelline fluid of developing eggs and in the external medium surrounding fry on V _max and K _m. The ability of fry reared entirely in buffered river water to maintain sodium balance following transfer to untreated river water is also considered.     

Gastrulation defective, a complement factor C2/B-like protease, interprets a ventral prepattern in Drosophila

gastrulation defective (gd) encodes a serine protease required for specification of dorsal-ventral cell fates during Drosophila embryogenesis. Using RNA microinjection, I show that wild-type gd RNA can restore ventrolateral pattern elements with correct polarity with respect to egg shape in embryos lacking gd function. While low RNA concentrations restore ventrolateral pattern elements, higher concentrations ventralize the embryo. Gastrulation defective concentration has a rate-limiting effect on the domain of high Dorsal concentration but little effect upon the slope of the gradient. In embryos from pipe-null females, much higher RNA concentrations generate an ectopic axis oriented with respect to the site of injection. The data suggest that the Dorsal gradient is not directly determined by asymmetric cues in the eggshell but arises de novo within the perivitelline space as a consequence of self-regulatory properties of the protease cascade. A homology to the mammalian complement factors C2 and B is also described.     

Ion-exchange phenomena regulate the environment of embryos in the eggs of freshwater fish

• 1.1. Calcium-selective microelectrodes have been used to measure the activity of calcium in the perivitelline fluid of brown trout eggs, exposed to water with a range of Ca²⁺ concentrations. Trans-chorion potential differences were also measured.
• 2.2. These values are compared with those predicted by a model based on ion-exchange theory and the Donnan equilibrium. The model allows for partial-dissociation of acid-groups in both the perivitelline fluid and the chorion, and the effects of external pH and ion concentration on this dissociation. The ion-exchange properties of perivitelline fluid and chorion were determined by acid-base titration.
• 3.3. The model has the potential to predict the effects of external pH and ion concentration on the distribution of many ions in fish eggs; especially in nutrient-poor waters. It is particularly relevant, therefore, to studies of acidification and metal pollution.

     

Relationship between steroid concentrations in ovarian follicular fluid and oocyte morphology in patients undergoing intracytoplasmic sperm injection (ICSI) treatment

The objective of this study was to investigate the relationship between oocyte morphology and follicular fluid steroid concentrations in patients being treated with intracytoplasmic sperm injection. A total of 82 IVF cycles were evaluated in patients aged 24-40 years. Oocytes at metaphase II were graded into four groups according to the status of the first polar body and the size of the perivitelline space. The proportion of oocytes at the germinal vesicle and germinal vesicle breakdown stages, and the proportion of degenerated oocytes and oocytes with a large polar body were compared with different concentrations of oestradiol, progesterone and testosterone in the follicular fluid. The association between these oocyte characteristics and the ratio of oestradiol:testosterone and oestradiol:progesterone was also analysed. The results showed that oocyte morphology, as assessed by the status of the first polar body and the size of the perivitelline space, is associated with the ratio of oestradiol:testosterone and oestradiol:progesterone but not with the absolute concentrations of oestradiol, progesterone and testosterone in the follicular fluid. A ratio of oestradiol:testosterone > 200 is the best indicator for a small proportion of grade 1 and 2 oocytes (poor quality), a large proportion of grade 3 and 4 oocytes (good quality), and a small proportion of oocytes with cytoplasmic inclusions. These results will be of clinical use in evaluating oocyte quality.     

Structure, localization and potential role of a novel molluscan trypsin inhibitor in Lymnaea.

Eggs and egg masses of the freshwater gastropod mollusc Lymnaea provide a microenvironment for developing embryos. Secretions of the exocrine albumen gland of Lymnaea are packaged in the eggs of an egg mass before the eggs are laid externally. The perivitelline fluid that directly surrounds individual oocytes is the main source of nutrition for developing embryos. During early stages of development, the perivitelline fluid is initially internalized by pinocytosis and degraded by lysosomes; in later stages, the embryo ingests the fluid. We previously found that the albumen gland produces large amounts of Lymnaea epidermal growth factor. The albumen gland also appears to produce significant amounts of a novel Lymnaea trypsin inhibitor (LTI), a second peptide that was purified and characterized from Lymnaea albumen gland extracts. The primary structure was determined by microsequence analysis, mass spectrometry, and C-terminal sequence analysis, and showed that LTI is a 57-residue glycosylated peptide. Comparison of the LTI sequence with other known serine protease inhibitors indicates that LTI is a member of the bovine pancreatic trypsin inhibitor family. Reverse phase-high performance liquid chromatography, microsequence analysis, mass spectrometry, and immunocytochemistry demonstrated that abundant amounts of intact LTI are packaged in egg masses. The presence of a trypsin inhibitor in the perivitelline fluid compartment of the egg mass may minimize digestion of peptides and proteins in the perivitelline fluid that are important for the development of the embryo, for example, Lymnaea epidermal growth factor.     

Transgenic embryos and mice produced from low titre lentiviral vectors

Lentiviral vectors are now recognised as an efficient transgene delivery system which can result in greater than 90% of founder animals carrying the transgene. Vector injection into the perivitelline space has emerged as the standard delivery method but is limited by the need for high-titre lentivirus vector preparations. Based on a modified perivitelline injection method we demonstrate that transgenic animals can be generated from low-titre virus vector preparations further simplifying lentiviral transgenesis. Repeat injection of 10⁷ TU/ml vector preparation resulted in 23% of embryos carrying the transgene compare to 1% from a single injection. Embryos exposed to repeat injection of vector developed to blastocyst with the same efficiency as non-injected embryos and produced transgenic mice capable of transmitting the transgene through the germline     

In vitro and in vivo association of an oviduct estrus-associated protein with bovine zona pellucida

The interaction between the bovine egg zona pellucida and a 97 kDa estrus-associated protein produced by the oviduct was examined in vitro and in vivo. In vitro matured bovine eggs were incubated with oviduct fluid recovered throughout the estrous cycle from separate indwelling cannulae placed in the ampulla and isthmus of the same oviduct. Immunofluorescence techniques and a polyclonal antiserum against the 97 kDa protein were used to localize this protein on washed eggs previously incubated with oviduct fluid. Intensity and distribution of immunofluorescence varied with stage of cycle and to a lesser degree with region of oviduct from which the oviduct fluid was obtained. The most intense fluorescence was observed on the zonae pellucidae of eggs incubated with oviduct fluid pooled from days near estrus and ovulation compared to fluid pooled from luteal stage days. The immunofluorescence of isthmus-derived oviduct fluid was more intense than was ampulla-derived oviduct fluid collected near estrus. The zonae pellucidae of 7-day-old embryos flushed from the uterus displayed immunofluorescence comparable to that observed on the zonae pellucidae of eggs incubated in vitro with peri-estrus oviduct fluid. No immunofluorescence was observed associated with the perivitelline space, egg cytoplasm, or blastomeres. The apparent uptake of a 97 kDa estrus-associated protein by the zonae pellucidae of eggs in vitro and embryos in vivo may indicate that this protein functions in fertilization and/or early embryo development.     

Reproductive biology of annual killifishes and its relationship with embryonic survival during diapause: Millerichthys robustus (Cyprinodontiformes: Cynolebiidae) as an integrative model

Diapause is a metabolic arrest expressed by annual killifish embryos as an extreme adaptation to persist in environments that show alternate periods of favourable‐hostile conditions along the annual cycle. Survival under deteriorated condition can be considered as the final consequence of a previous set of complementary morphophysiological and behavioral processes in adult fishes. Millerichthys robustus is the only annual fish that has developed an annual life history in North America wherewith allow us to consider it as an emerging model for annual killifishes to review, analyze and integrate the knowledge about its reproductive biology involved in allowing the embryos in diapause to survive face the hostile environmental condition. First, we review developmental ecology of Millerichthys embryos throughout different periods (flood, drought and humid) of the annual life cycle showing the possible developmental trajectories in situ. We then analyze: (i) the way in which embryos achieve survive drought from protective cortical structures (perivitelline space and egg envelope) that present dynamic changes according to the conditions to which they are exposed buffering the harmful effects of high temperatures and water loss. (ii) The nature and origin of these protective structures during the ovogenesis (cortical alveoli and zona pellucida give rise to the perivitelline space and egg envelope respectively and oil droplets represent an emergency nutritional reserve). (iii) Sexual synchronization through secretion of pheromones and the reproductive behavior that allows them to spawn under the substrate. Embryonic survival is achieved by success of simplest interactions between reproductive biology elements prior to embryo development.     

Survival and development of horseshoe crab (Limulus polyphemus) embryos and larvae in hypersaline conditions

The horseshoe crab Limulus polyphemus spawns in the mid- to upper intertidal zone where females deposit eggs in nests below the sediment surface. Although adult crabs generally inhabit subtidal regions of estuaries with salinities from 5 to 34 ppt, developing embryos and larvae within nests are often exposed to more extreme conditions of salinity and temperature during summer spawning periods. To test whether these conditions have a negative impact on early development and survival, we determined development time, survival, and molt cycle duration for L. polyphemus embryos and larvae raised at 20 combinations of salinity (range: 30-60 ppt) and temperature (range: 25-40 degrees C). Additionally, the effect of hyperosmotic and hypoosmotic shock on the osmolarity of the perivitelline fluid of embryos was determined at salinities between 5 and 90 ppt. The embryos completed their development and molted at salinities below 60 ppt, yet failed to develop at temperatures of 35 degrees C or higher. Larval survival was high at salinities of 10-70 ppt but declined significantly at more extreme salinities (i.e., 5, 80, and 90 ppt). Perivitelline fluid remained nearly isoosmotic over the range of salinities tested. Results indicate that temperature and salinity influence the rate of crab development, but only the extremes of these conditions have an effect on survival.     

The influence of the zona radiata on the toxicities of zinc, lead, mercury, copper and silver ions to embryos of steelhead trout Salmo gairdneri

Eyed embryos of the steelhead trout (Salmo gairdneri) were significantly more resistant to zinc and lead but significantly less resistant to mercury, copper and silver if the zona radiata (egg capsule) was removed than if it was intact. The zona radiata appears to act as a cation exchanger and inhibits metals with high binding constants (Hg2+, Cu2+, Ag+) from entering the perivitelline fluid. Metals with low binding constants (Zn2+, Pb2+, Cd2+) rapidly penetrate the zona radiata and accumulate in the perivitelline fluid according to the Donnan equilibrium.     

A new method to efficiently produce transgenic embryos and mice from low-titer lentiviral vectors

Vector injection into the perivitelline space has emerged as the standard delivery method to transduce lentivirus to mammalian oocytes or one-cell embryos, but its application is limited by the need for high titers of lentivirus. Herein we developed a new method by using a Piezo impact micro-manipulator for injecting low titer of lentivirus into the subzonal space of two-cell embryos or the perivitelline space of one-cell embryos that were shrunk with a highly concentrated sucrose solution. The survival rate of embryos was greater than 98% using this micromanipulation strategy, which was increased compared to the normal one-cell embryo injection method. More than 90% of injected embryos were GFP positive after subzonal injection of a lentivirus vector carrying the GFP gene with titers of 2 × 10⁸ I.U./ml. Even when a low titer of lentivirus (2 × 10⁶ I.U./ml) was used, 53.26% and 40.85% transgenic embryos were obtained after two-cell embryonic injection and one-cell sucrose treated embryonic injection, respectively. The GFP-positive rates were also greater than in the conventional method of injecting one-cell embryos (25.39%). In addition, blastocysts from the two-cell embryo injection group displayed stronger GFP fluorescence than the one-cell embryo injection groups treated with or without the sucrose solution. Increased expression of GFP suggests that the embryos obtained from this injection method have higher exogenous gene expression levels compared to previous methods. Therefore, in contrast with the traditional injection method, we have demonstrated a simplified and efficient lentivirus-mediated gene transfer method based on a low-titer virus preparation.     

Electron Microscopic Study of Development in a Sea Cucumber,Stichopus tremulus (Holothuroidea), from Unfertilized Egg through Hatched Blastula

In this, the first fine structural study of sea cucumber embryology, eggs and embryos of Stichopus tremulus developing at 7.5°C are described from spawning through hatched blastulae. Spawned eggs are at about first meiotic metaphase and are surrounded by a jelly layer that remains around the embryos until hatching. No vitelline coat can be demonstrated, but whether it is truly absent or removed by electron microscopic processing is not known. Insemination initiates a rapid cortical reaction, completed within 2 min., which involves a wave of cortical granule exocytosis and fertilization envelope formation. The compactly fibrous fertilization envelope is about 50 nm thick and appears to consist entirely of ejected cortical granule material (if one assumes that there is no vitelline coat). As the fertilization envelope elevates, no hyaline layer appears in the perivitelline space. The first and second polar bodies are emitted, respectively, at about 9 and 15 min. after insemination. The first seven or so cleavages are equal, radial, and occur approximately every 4 hr. The blastocoel opens up at the four-cell stage and, during the earlier cleavages, remains connected with the perivitelline space via numerous gaps between the roughly spherical blastomeres. At the 64-cell stage, these gaps begin to close as the blastomeres start to become cuboidal; in addition, an embryonic cuticle is produced on the apical surface of each blastomere. In embryos of several hundred cells, the blastomeres become associated apicolaterally by junctional complexes, each consisting of a zonula adherens and a septate junction. Several hours before hatching, a single cilium is produced at the apical surface of most blastomeres. At hatching (about 50 hr after insemination), the ciliated blastula leaves behind the fertilization envelope and jelly layer. Swimming blastulae soon begin to elongate in the animal-vegetal axis, and a basal lamina develops on blastomere surfaces facing the blastocoel. The discussion includes a fine structural comparison of egg coats among the five classes of the phylum Echinodermata.