Dithiothreitol Affects the Fertilization Response in Immature and Maturing Starfish Oocytes.

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca2+ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca2+ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca2+ response at fertilization and the subsequent embryonic development.

POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos.

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

The Starfish Asterina pectinifera: Collection and Maintenance of Adults and Rearing and Metamorphosis of Larvae.

Here we describe methods for (a) collecting starfish during their breeding period; (b) maintaining adults with fully grown gonads in laboratory aquaria; (c) rearing fertilized eggs to brachiolaria larvae, and (d) inducing larvae to metamorphose into juveniles under laboratory conditions. Such protocols should facilitate various analyses of starfish development throughout the entire life cycle of these model organisms.

Oocyte Maturation in Starfish.

Oocyte maturation is a process that occurs in the ovaries, where an immature oocyte resumes meiosis to attain competence for normal fertilization after ovulation/spawning. In starfish, the hormone 1-methyladenine binds to an unidentified receptor on the plasma membrane of oocytes, inducing a conformational change in the heterotrimeric GTP-binding protein α-subunit (Gα), so that the α-subunit binds GTP in exchange of GDP on the plasma membrane. The GTP-binding protein ßγ-subunit (Gßγ) is released from Gα, and the released Gßγ activates phosphatidylinositol-3 kinase (PI3K), followed by the target of rapamycin kinase complex2 (TORC2) and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-dependent phosphorylation of serum- and glucocorticoid-regulated kinase (SGK) of ovarian oocytes. Thereafter, SGK activates Na+/H+ exchanger (NHE) to increase the intracellular pH (pHi) from ~6.7 to ~6.9. Moreover, SGK phosphorylates Cdc25 and Myt1, thereby inducing the de-phosphorylation and activation of cyclin B-Cdk1, causing germinal vesicle breakdown (GVBD). Both pHi increase and GVBD are required for spindle assembly at metaphase I, followed by MI arrest at pHi 6.9 until spawning. Due to MI arrest or SGK-dependent pHi control, spawned oocytes can be fertilized normally.

Fluorescence Measurement and Calibration of Intracellular pH in Starfish Oocytes.

Oocyte maturation is a process wherein an oocyte arrested at prophase of meiosis I resumes meiosis to become a fertilizable egg. In starfish ovaries, a hormone released from follicle cells activates the oocytes, resulting in an increase in their intracellular pH (pHi), which is required for spindle assembly. Herein, we describe a protocol for pHi measurement in living oocytes microinjected with the pH-sensitive dye BCECF. For in vivo BCECF calibration, we treated oocytes with artificial seawater containing CH3COONH4 to clamp pHi, injected pH-standard solutions, and converted the BCECF fluorescence intensity ratios to pHi values. Of note, if the actual pHi is higher or lower than the known pH of injected standard solutions, the BCECF fluorescence intensity ratio will decrease or increase, respectively. On the other hand, the pH of the injected solution displaying no change in fluorescence intensity should be considered the actual pHi. These methods for pHi calibration and clamping are simple and reproducible.

SGK phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation at the meiotic G2/M transition.

The kinase cyclin B-Cdk1 complex is a master regulator of M-phase in both mitosis and meiosis. At the G2/M transition, cyclin B-Cdk1 activation is initiated by a trigger that reverses the balance of activities between Cdc25 and Wee1/Myt1 and is further accelerated by autoregulatory loops. In somatic cell mitosis, this trigger was recently proposed to be the cyclin A-Cdk1/Plk1 axis. However, in the oocyte meiotic G2/M transition, in which hormonal stimuli induce cyclin B-Cdk1 activation, cyclin A-Cdk1 is nonessential and hence the trigger remains elusive. Here, we show that SGK directly phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation in starfish oocytes. Upon hormonal stimulation of the meiotic G2/M transition, SGK is activated by cooperation between the Gßγ-PI3K pathway and an unidentified pathway downstream of Gßγ, called the atypical Gßγ pathway. These findings identify the trigger in oocyte meiosis and provide insights into the role and activation of SGK.

SGK regulates pH increase and cyclin B-Cdk1 activation to resume meiosis in starfish ovarian oocytes.

Tight regulation of intracellular pH (pHi) is essential for biological processes. Fully grown oocytes, having a large nucleus called the germinal vesicle, arrest at meiotic prophase I. Upon hormonal stimulus, oocytes resume meiosis to become fertilizable. At this time, the pHi increases via Na+/H+ exchanger activity, although the regulation and function of this change remain obscure. Here, we show that in starfish oocytes, serum- and glucocorticoid-regulated kinase (SGK) is activated via PI3K/TORC2/PDK1 signaling after hormonal stimulus and that SGK is required for this pHi increase and cyclin B-Cdk1 activation. When we clamped the pHi at 6.7, corresponding to the pHi of unstimulated ovarian oocytes, hormonal stimulation induced cyclin B-Cdk1 activation; thereafter, oocytes failed in actin-dependent chromosome transport and spindle assembly after germinal vesicle breakdown. Thus, this SGK-dependent pHi increase is likely a prerequisite for these events in ovarian oocytes. We propose a model that SGK drives meiotic resumption via concomitant regulation of the pHi and cell cycle machinery.

Starfish Apaf-1 activates effector caspase-3/9 upon apoptosis of aged eggs.

Caspase-3-related DEVDase activity is initiated upon apoptosis in unfertilized starfish eggs. In this study, we cloned a starfish procaspase-3 corresponding to mammalian effector caspase containing a CARD that is similar to the amino terminal CARD of mammalian capsase-9, and we named it procaspase-3/9. Recombinant procaspase-3/9 expressed at 15 °C was cleaved to form active caspase-3/9 which has DEVDase activity. Microinjection of the active caspase-3/9 into starfish oocytes/eggs induced apoptosis. An antibody against the recombinant protein recognized endogenous procaspase-3/9 in starfish oocytes, which was cleaved upon apoptosis in aged unfertilized eggs. These results indicate that caspase-3/9 is an effector caspase in starfish. To verify the mechanism of caspase-3/9 activation, we cloned starfish Apaf-1 containing a CARD, a NOD, and 11 WD40 repeat regions, and we named it sfApaf-1. Recombinant sfApaf-1 CARD interacts with recombinant caspase-3/9 CARD and with endogenous procaspase-3/9 in cell-free preparations made from starfish oocytes, causing the formation of active caspase-3/9. When the cell-free preparation without mitochondria was incubated with inactive recombinant procaspase-3/9 expressed at 37 °C, DEVDase activity increased and apoptosome-like complexes were formed in the high molecular weight fractions containing both sfApaf-1 and cleaved caspase-3/9. These results suggest that sfApaf-1 activation is not dependent on cytochrome c.

Hormonal stimulation of starfish oocytes induces partial degradation of the 3' termini of cyclin B mRNAs with oligo(U) tails, followed by poly(A) elongation.

In yeast, plant, and mammalian somatic cells, short poly(A) tails on mRNAs are subject to uridylation, which mediates mRNA decay. Although mRNA uridylation has never been reported in animal oocytes, maternal mRNAs with short poly(A) tails are believed to be translationally repressed. In this study, we found that 96% of cyclin B mRNAs with short poly(A) tails were uridylated in starfish oocytes. Hormonal stimulation induced poly(A) elongation of cyclin B mRNA, and 62% of long adenine repeats did not contain uridine residues. To determine whether uridylated short poly(A) tails destabilize cyclin B mRNA, we developed a method for producing RNAs with the strict 3' terminal sequences of cyclin B, with or without oligo(U) tails. When we injected these synthetic RNAs into starfish oocytes prior to hormonal stimulation, we found that uridylated RNAs were as stable as nonuridylated RNAs. Following hormonal stimulation, the 3' termini of short poly(A) tails of synthesized RNAs containing oligo(U) tails were trimmed, and their poly(A) tails were subsequently elongated. These results indicate that uridylation of short poly(A) tails in cyclin B mRNA of starfish oocytes does not mediate mRNA decay; instead, hormonal stimulation induces partial degradation of uridylated short poly(A) tails in the 3'-5' direction, followed by poly(A) elongation. Oligo(U) tails may be involved in translational inactivation of mRNAs.

Block of CDK1-dependent polyadenosine elongation of Cyclin B mRNA in metaphase-i-arrested starfish oocytes is released by intracellular pH elevation upon spawning.

Meiotic progression requires the translation of maternal mRNAs in a strict temporal order. In isolated animal oocytes, translation of maternal mRNAs containing a cytoplasmic polyadenylation element (CPE), such as cyclin B, is activated by in vitro stimulation of meiotic resumption which induces phosphorylation of CPEB (CPE-binding protein) and elongation of their polyadenosine (poly(A)) tails; whether or not this model can be applied in vivo to oocytes arrested at metaphase of meiosis I in ovaries is unknown. In this study, we found that active CDK1 (cyclin-dependent kinase 1) phosphorylated CPEB in ovarian oocytes arrested at metphase I in the starfish body cavity, but phosphorylation of CPEB was not sufficient for elongation of cyclin B poly(A) tails. Immediately after spawning, however, mRNA was polyadenylated, suggesting that an increase in intracellular pH (pHi ) upon spawning triggers the elongation of poly(A) tails. Using a cell-free system made from maturing oocytes at metaphase I, we demonstrated that polyadenylation was indeed suppressed at pH below 7.0. These results suggest that a pH-sensitive process, functioning after CPEB phosphorylation, is blocked under physiologically low pHi (<7.0) in metaphase-I-arrested oocytes. The increase in pHi (>7.0) that occurs after spawning triggers polyadenylation of cyclin B mRNA and progression into meiosis II.

Arrest at metaphase of meiosis I in starfish oocytes in the ovary is maintained by high CO2 and low O2 concentrations in extracellular fluid.

During the spawning process in starfish, oocytes are arrested at metaphase of meiosis I (MI) within the ovary, and reinitiate meiosis only after they have been released into the seawater. However, this arrest does not occur if the ovary is removed from the animal. As the pH of the coelomic fluid is buffered by CO2/H(+)/HCO3(-), we investigated the involvement of gas concentrations in MI arrest. In vivo, the CO2 level in the coelomic fluid was high (∼1.5% vs. 0.04% in air) and the O2 level was low (0.1-1.0% vs. ∼20% in air). When these gas conditions were reproduced in isolated coelomic fluid or seawater, ovarian oocytes arrested at MI, just as in vivo. Isolated oocytes from the ovary required the similar high CO2 and low O2 level to remain arrested in MI and had an intracellular pH of ∼6.9. Intracellular pH increased to ∼7.3 when oocytes were transferred to seawater equilibrated with air, a condition that mimics that of spawning. We used ammonium acetate to clamp intracellular pH at different levels and found that MI arrest occurred when intracellular pH was ∼6.9. Our results support the idea that high CO2 and low O2 in the ovarian environment lead to low intracellular pH and MI arrest, while spawning into the seawater with low CO2 and high O2 results in high intracellular pH and release from MI arrest. The biological significance of MI arrest is that oocytes are spawned into seawater at the optimal physiological state of MI when the least polyspermy occurs.

Sperm from sneaker male squids exhibit chemotactic swarming to CO2.

Behavioral traits of sperm are adapted to the reproductive strategy that each species employs. In polyandrous species, spermatozoa often form motile clusters, which might be advantageous for competing with sperm from other males. Despite this presumed advantage for reproductive success, little is known about how sperm form such functional assemblies. Previously, we reported that males of the coastal squid Loligo bleekeri produce two morphologically different euspermatozoa that are linked to distinctly different mating behaviors. Consort and sneaker males use two distinct insemination sites, one inside and one outside the female's body, respectively. Here, we show that sperm release a self-attracting molecule that causes only sneaker sperm to swarm. We identified CO2 as the sperm chemoattractant and membrane-bound flagellar carbonic anhydrase as its sensor. Downstream signaling results from the generation of extracellular H(+), intracellular acidosis, and recovery from acidosis. These signaling events elicit Ca(2+)-dependent turning behavior, resulting in chemotactic swarming. These results illuminate the bifurcating evolution of sperm underlying the distinct fertilization strategies of this species.

Symmetrical division of mouse oocytes during meiotic maturation can lead to the development of twin embryos that amalgamate to form a chimeric hermaphrodite.

BACKGROUND: Gentle compression of mouse oocytes during meiosis-1 prevented the usual extrusion of a small polar body and resulted in the symmetrical division of the ooplasm into two cells of similar size within the zona pellucida. The purpose of our study was to determine whether such cells, equivalent to two small oocytes, were capable of embryonic development and would result in birth following transfer to the uterus. METHODS: IVF of the 2-celled oocytes was performed and the twin intra-zonal embryos were observed. In each case, the two embryos that originated from fertilized cells with two pronuclei were observed to amalgamate and form a single morula and subsequent blastocyst that was transferred to the uterus of a recipient of a different mouse strain. FISH analysis was performed on sectioned paraffin-embedded tissue of the offspring. RESULTS: In symmetrically divided oocytes each cell contained a metaphase II spindle. Both cells were fertilizable and cleaved to form twin embryos within the same zona pellucida. Most twin embryos amalgamated to form a single compacted morula, which progressed to hatched blastocysts that contained a single inner cell mass. In total, 104 of these blastocysts were transferred to 19 mice, two of which became pregnant, resulting in the birth of three offspring. FISH analysis showed that one newborn contained both XX and XY cells. CONCLUSIONS: We found that two small oocytes fertilized within the same zona pellucida to form twin embryos that amalgamate to establish a single chimeric embryo. This may be one mechanism that leads to the formation of a chimeric hermaphrodite when an embryo containing XX cells mixes with its intra-zonal twin containing XY cells.

Evolution of the acquisition of fertilization competence and polyspermy blocks during meiotic maturation.

In many animals, fully grown oocytes are arrested at prophase of meiosis I. Before or after ovulation/spawning, a secondary arrest occurs at metaphase of meiosis I or II (MI or II, respectively). MI arrest in the ovary is released after spawning, and is followed by fertilization, whereas MI and MII arrest after ovulation are released by fertilization. Insemination of isolated oocytes from the ovaries at an inappropriate time increases the rate of polyspermy, indicating that ovaries provide the proper environment for acquisition of the polyspermy blocks and the development of competence to be fertilized normally. Due to MI arrest in the ovaries or MI/MII arrest after ovulation/spawning, the fertilizable period can be elongated. Thus, MI and MII arrest may play a role in maintaining the cell-cycle phases to enable normal fertilization. Here, the evolution of fertilization timing is discussed.

Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization.

To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm-oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm-zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocyte's plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization.

Regulation of intracellular pH by p90Rsk-dependent activation of an Na(+)/H(+) exchanger in starfish oocytes.

Starfish oocytes arrest at metaphase of the first meiotic division (MI arrest) in the ovary and resume meiosis after spawning into seawater. MI arrest is maintained by lower intracellular pH (pH(i)) and release from arrest by cellular alkalization. To elucidate pH(i) regulation in oocytes, we cloned the starfish (Asterina pectinifera) Na(+)/H(+) exchanger 3 (ApNHE3) expressed in the plasma membrane of oocytes. The cytoplasmic domain of ApNHE3 contains p90 ribosomal S6 kinase (p90Rsk) phosphorylation sites, and injection of a constitutively active p90Rsk and the upstream regulator Mos to immature oocytes, stimulated an increase in pH(i). This increase was blocked by 5-(N-ethyl-N-isopropyl)-amiloride, a NHE inhibitor, and SL0101, a specific Rsk inhibitor. The MAPK kinase (MEK) inhibitor U0126 blocked the Mos-induced, but not the p90Rsk-induced, pH(i) increase, suggesting that the Mos-MEK-MAPK-p90Rsk pathway promotes ApNHE3 activation. In a cell-free extract, the Mos-MEK-MAPK-p90Rsk pathway phosphorylates ApNHE3 at Ser-590, -606, and -673. When p90Rsk-dependent ApNHE3 phosphorylation was blocked by a dominant-negative C-terminal fragment, or neutralizing antibody, the p90Rsk-induced pH(i) increase was suppressed in immature oocytes. However, ApNHE3 is up-regulated via the upstream phosphatidylinositol 3-kinase pathway before MAPK activation and the active state is maintained until spawning, suggesting that the p90Rsk-dependent ApNHE3 phosphorylation is unlikely to be the primary regulatory mechanism involved in MI arrest exit. After meiosis is completed, unfertilized eggs maintain their elevated pH(i) ( approximately 7.4) until the onset of apoptosis. We suggest that the p90Rsk/ApNHE3-dependent elevation of pH(i) increases fertilization success by delaying apoptosis initiation.

Association of spindle midzone particles with polo-like kinase 1 during meiosis in mouse and human oocytes.

Polo-like kinase 1 (Plk1) has been reported to localize to the spindle midzone during meiosis in mouse oocytes. However, it has not been reported in human oocytes. In this study, the interaction of the meiotic structures and chromosome segregation in mouse and human oocytes were studied by time-lapse differential interference contrast microscopy. Using immunocytochemical studies, the localization of polo-like kinase 1 and its association with microtubules were examined during the extrusion of first and second polar bodies. It was found that Plk1 was localized in the spindle midzone in human oocytes at anaphase I and telophase I. Also, three-dimensional confocal laser microscopy showed that the meiotic spindle midzone contained numerous dot-like particles that were stained by anti-Plk1 antibody. These particles were aligned in the plane of the meiotic midzone in mouse and human oocytes.

Damage of embryo development caused by peroxidized mineral oil and its association with albumin in culture.

OBJECTIVES: To examine the effect of free radicals from peroxidized oil and the role of albumin on the passage of radicals. DESIGN: Prospective study. SETTING: Clinical IVF laboratory and university department. PATIENT(S): Blood samples were donated by laboratory staff. INTERVENTION(S): Examination of the effects of mineral oil samples with various peroxide value (POV) on culture of erythrocytes and on the passage of a lipophilic tracer, DiI, into the zona pellucida. MAIN OUTCOME MEASURE(S): Time required for hemolysis of red blood cells by peroxidized oil, staining of zona pellucida from human oocytes and embryos by lipophilic tracer, and POV analysis of mineral oil samples in relation to various storage conditions. RESULT(S): The time for hemolysis was related to the POV levels of oil samples covering the culture medium. Albumin in the medium facilitated hemolysis and the passage of DiI into the zona. Peroxidized oil (POV >0.02 meq/kg) blocked the entry of DiI into the zona. CONCLUSION(S): The presence of albumin in the medium was associated with the entry into the human zona of agents present in peroxidized mineral oil. This process and variable oil peroxidation could be deleterious to embryos in culture.

Involvement of mitogen-activating protein kinase and intracellular pH in the duration of the metaphase I (MI) pause of starfish oocytes after spawning.

The metaphase I (MI) arrest of starfish oocytes is released after spawning. In this study using starfish Asterina pectinifera, the duration of MI after spawning was ~20 min and approximately 30 min in fertilized and unfertilized oocytes, respectively. This prolongation of MI in unfertilized oocytes, referred to as the MI pause, was maintained by mitogen-activating protein kinase (MAPK) as well as low intracellular pH (approximately 7.0). Contrary to previous reports, MI arrest was not maintained by MAPK, since it was inactive in the oocytes arrested at MI in the ovary and activated immediately after spawning. Also, cyclin B was not degraded at pH 6.7 in the cell-free preparation without MAPK activity, whereas it was degraded at pH 7.0, suggesting that MI arrest was solely maintained by lower pH (< 7.0). Normal development occurred when the spawned oocytes were fertilized before the first polar body formation, whereas fertilization after the first polar body formation increased the rate of abnormal development. Thus, due to MI pause and MI arrest, the probability for fertilization before the polar body formation might be increased, leading to normal development.

Hormone-induced cortical maturation ensures the slow block to polyspermy and does not couple with meiotic maturation in starfish.

Meiotic progression in starfish oocytes is reinitiated by a maturation-inducing hormone called 1-methyladenine (1-MeAde). In addition to meiotic maturation, 1-MeAde induces cortical maturation in which cortical granules become competent to discharge in response to fusion of a single sperm, which results in the formation of the fertilization envelope. We found that subthreshold concentrations of 1-MeAde induce cortical maturation without germinal vesicle breakdown (GVBD). During cortical maturation, the IP3 sensitivity of calcium stores was increased as well as during meiotic maturation. When oocytes were exposed with 1-MeAde only on a hemisphere of oocytes, the IP3 sensitivity of the cortical region was increased only in the exposed hemisphere, suggesting that signals and components involved in cortical maturation do not readily spread in the cytoplasm. Although a specific inhibitor of phosphatidylinositol-3 kinase, LY294002 blocked both GVBD and cortical maturation, a Cdc2 kinase inhibitor, roscovitine did not block cortical maturation. Inhibition of Akt activation by injecting the competitors for Akt phosphorylation and membrane recruitment also blocked cortical maturation. These results suggest that the signaling pathway leading to Akt activation is common in cortical maturation and meiotic maturation, and Cdc2 activation was not required for cortical maturation.