Relaxin increases the release of plasminogen activator, collagenase, and proteoglycanase from rat granulosa cells in vitro.

Relaxin (Rlx) is shown in vitro to increase the release of plasminogen activator (PA) activity from granulosa cells obtained from 28-day-old rats after priming 48 h before with PMSG. Priming with PMSG was essential for the subsequent marked increase in PA by the addition of Rlx to these cells in vitro. Under the same conditions Rlx also increased the release of both total collagenase and total proteoglycanase activities but not of beta-glucuronidase activity. The total collagenase and proteoglycanase activities of control cells are made up of essentially equal amounts of their respective active and latent enzymes. Rlx stimulation increases the amounts of the respective active enzymes while the latent collagenase and proteoglycanase activities are unchanged or decreased, respectively. The enzyme beta-glucuronidase was not stimulated by Rlx and appears not to be involved in follicular proteoglycan degradation. Granulosa cells harvested from preantral follicles responded most to FSH by PA production whereas cells from antral follicles responded more to LH, reflecting the known changes in concentration of FSH and LH receptors on these cells. The release of PA is maximal by all four hormones studied (FSH, LH, prostaglandin E1, and Rlx) on granulosa cells harvested from rats 48 h after PMSG treatment and this suggests that the follicles at this time are a mixture of both preantral and antral stages. The PA response to FSH is lost by 60 h after PMSG at the same time that the response to prostaglandin E1 is maintained at the same level, whereas that to Rlx and LH, although still significantly higher than controls, were decreased. By 70 h after PMSG, postovulatory, the responses to all hormones studied were lost. Thus, the involvement of PA in ovarian connective tissue alterations appears to be greatest in the period of follicular antrum formation rather than just before ovulation. Rlx is one of a number of hormones involved in the sequence of events culminating in follicle connective tissue remodeling as shown by its action on the release of three intrafollicular enzymes.

Pulmonary insulin responsivitiy: in vivo effects of insulin on the diabetic rat lung and specific insulin binding to lung receptors in normal rats.

Adult rats were rendered diabetic by a single iv injection of streptozotocin (70 or 75 mg/kg). In these rats, serum insulin fell to minimal levels during the 48 h following drug treatment, and this was roughly paralleled by a progressive decrease in the ability of the lung to oxidize glucose. The addition of insulin to diabetic rat lung slices in vitro had no restorative effect on the depressed glucose oxidative rate during a 2 h incubation period; however, two daily treatments of the rats with 1 unit of protamine, zinc insulin completely restored lung glucose oxidation rate to normal, without significantly reducing the hyperglycemic state of the rats. An examination of the temporal changes in glucose utilization by the rat lung after acute insulin treatment revealed that the diabetic lung responded directly to serum levels of insulin, whereas the normal lung appeared to be unaffected by serum insulin levels as hihg as 87 ng/ml. The reduced rate of glucose oxidation in the diabetic lung was apparent after perfusion of the lung with glucose-free medium, and was characterized by a significant reduction in Vmax without an alteration in Km. This was attended by a depressed ability of the lung to incorporate [3H]leucine into protein and an increased ability to produce lactate, but hexose monophosphate shunt activity was normal. Specific receptors for insulin have been identified and partially characterized in crude membrane preparations of normal rat lung. The interaction of insulin with these receptors was rapid, reversible, saturable, and was dependent upon time and temperature. The binding of labeled insulin was inhibited by low concentrations of unlabeled insulin and by high concentrations of proinsulin, whereas it was unaffected by the presence of glucagon, gastrin, prolactin, ACTH, or growth hormone in microgram amounts. These observations suggest that insulin regulates the transport and utilization of glucose in the rat lung, and that this tissue contains specific receptors for insulin.

Endometrial relaxin: effects of mastectomy in the cyclic and pregnant guinea pig.

Mastectomy in the guinea pig increases the incidence of still births and neonatal deaths compared with intact animals. The guinea pig has the endometrial gland cells (EGC) as the major source of relaxin, and this hormone has possible roles in uterine quiescence, cervical dilatation, and lengthening of the interpubic ligament in pregnancy. An effect of mastectomy on uterine relaxin has been sought by immunocytochemical localization during the estrous cycle, mid and late pregnancy and on endometrial relaxin gene expression in the late pregnant mastectomized animal by Northern analysis. Endometrium from midpregnant (day 35) and late pregnant (day 63) and from lactating (days 5, 21, and 28) guinea pigs immunostained with antiserum to porcine relaxin by the avidinbiotin technique. By increasing the sensitivity of the latter, relaxin immunostaining was also detected for the first time in EGC from cyclic animals (days 9 and 14). A pattern and intensity of relaxin immunostaining could be readily assigned to each of the stages examined: estrous cycle, midpregnancy, late pregnancy, lactation, and post weaning. The dark uniform staining of the EGC in late pregnancy was followed by sporadic staining of the EGC in lactation, returning to the cyclic picture after weaning. Endometrium from mastectomized cyclic and late pregnant guinea pigs showed a reduction in the amount of immunostaining compared with the relevant control animals. The reduction was most pronounced in the mastectomized late pregnant guinea pig. This result was reflected in an apparently lower level of relaxin mRNA in the endometrium of these animals compared to intact controls. These data indicate a novel linkage between the mammary gland, either directly or indirectly, to the nonpregnant or pregnant uterus. The loss of this signal appears to be associated with subsequent problems at parturition which may be linked to the reduction in endometrial gland relaxin production. The nature of this signal from the mammary gland, normally considered to be an exocrine rather than an endocrine gland, is unknown.

Relaxin detected by immunocytochemistry and northern analysis in the mammary gland of the guinea pig.

Relaxin is a product of the endometrial gland cells in the guinea pig. Mammary tissue was collected from intact cyclic animals, on days 35 and 63 of pregnancy, days 5 and 21 of lactation, and day 28 postpartum. Tissue was collected from six cyclic hysterectomized animals. Sections of mammary gland were immunostained with the avidin-biotin immunoperoxidase method, and antisera to porcine relaxin which were raised to different preparations in different laboratories. Light uniform staining was evident in the cytoplasm of all of the cuboidal epithelial cells forming the mammary duct system in cyclic animals; mammary gland from hysterectomized animals showed similar staining. At midpregnancy light staining was seen in some epithelial cells, and by late pregnancy there was only faint staining. On day 5 of lactation there was intense and uniform staining throughout the epithelial cells of the alveoli. By day 21 of lactation the cells still immunostained for relaxin, but on day 28 postpartum there was a return to the cyclic staining pattern. Endometrium from animals at 55-60 days of pregnancy and mammary gland from day 6 of lactation were used for poly(A)+ RNA isolation and Northern analysis. Three 48-mer oligonucleotide probes were used. Poly(A)+ RNA from endometrium of late pregnant guinea pigs and from the mammary gland in lactation hybridized with the same two probes and failed to hybridize with a third under moderate stringency conditions. The results suggest that the major source of relaxin in the guinea pig is sequential, the pregnant uterus and the lactating mammary gland. The local and/or systemic significance is not known.

The complementary deoxyribonucleic acid sequence of guinea pig endometrial prorelaxin.

The nucleotide sequence of the relaxin gene transcript in the endometrium of the late pregnant guinea pig has been determined. The strategy used was a combination of polymerase chain reaction (PCR) with primers designed from the mRNA sequence of porcine preprorelaxin, rapid amplification of cDNA ends-PCR, and blunt end cloning in M13 mp18. With heterologous primers, a 226-basepair (bp) segment of the guinea pig relaxin gene sequence was obtained and was used to design a guinea pig-specific primer for use with the rapid amplification of cDNA ends-PCR method. The latter allowed completion of the sequence of 336 bp, with a 96-bp overlap. The sequence obtained shows greater homology at both the nucleotide and amino acid levels with porcine and human relaxins H1 and H2 than with rat relaxin, supporting the thesis that the guinea pig is not a rodent. The transcription of the guinea pig endometrial relaxin gene during pregnancy was confirmed by Northern analysis of guinea pig endometrial tissues with a species-specific cDNA probe. The endometrial relaxin gene is transcribed during pregnancy, but not in lactation, consistent with the observed immunostaining for relaxin.

Expression of the human relaxin H1 gene in the decidua, trophoblast, and prostate.

Relaxin is a peptide hormone whose A- and B-chains are derived by posttranslational cleavage from a single 185-amino acid preprorelaxin. Two genes in the human genome (H1 and H2) code for two polypeptides significantly different in amino acid sequence. The full spectrum of biological activities of these two polypeptides has not been examined, but transcription appears to be limited to the H2 relaxin gene in the human corpus luteum. Relaxin is also synthesized by the human decidua, placental trophoblast, and prostate gland; therefore, the expression of the human relaxin genes in these tissues has been examined using the reverse transcription polymerase chain reaction. The mRNA from decidua, placental trophoblast, and prostate was reverse transcribed and then amplified by polymerase chain reaction, using a series of oligonucleotide primers that were specific for but would not distinguish between human H1 and H2 relaxins. Using mRNA from these tissues, two amplified cDNA species were detected, whose identities were confirmed by Southern blots, HpaI and HpaII restriction enzyme analysis, and dideoxy sequencing. We have confirmed that the corpus luteum does not contain detectable H1 relaxin mRNA. However, we demonstrated for the first time relaxin H1 gene expression in the decidua, placental trophoblast, and prostate, and we have also shown that there are marked tissue differences in the relative amounts of expression of the H1 and H2 relaxin mRNA forms. The functional significance is unknown, but if both mRNAs are translated, differential expression of the two genes may result in tissue-specific differences in the production of these relaxins as well as in their binding and actions.

The human fetal membranes: a target tissue for relaxin.

The purpose of this study was to adduce further evidence for a paracrine role for human decidual relaxin (Rlx) in the remodelling of collagen in the fetal membranes in the peripartal period. The binding of [125I]porcine Rlx to membrane-enriched fractions from fetal membranes as well as from dispersed cells from the fetal membranes was used to demonstrate the presence of specific Rlx receptors. Rlx added in vitro to cultured amnion/chorion cells increased the release of plasminogen activator and collagenase into the medium. Rlx had no effect on the release of beta-glucuronidase. An in vivo correlate of these in vitro results was obtained, the detection of plasminogen activator and collagenase in amniotic fluids. The active fraction of collagenase was increased in amniotic fluids collected after spontaneous rupture of the membranes. PRL, hCG, estrogen, and progesterone added in equimolar amounts to cultured amnion/chorion cells from elective cesarean sections and normal term deliveries also effected the release of plasminogen activator and collagenase. The greatest effects were found in cells from cesarean section tissue, in terms of the stimulation of plasminogen activator release by Rlx and PRL and of collagenase release by prostaglandin F2 alpha and, to a lesser extent, by Rlx, PRL, and hCG. We conclude that human fetal membranes are targets for a number of hormones, including the decidual paracrine hormones Rlx, PRL, and prostaglandin F2 alpha as well as estrogen, progesterone, and hCG. These hormones act to release or inhibit the enzymes involved in collagen breakdown before rupture of the fetal membranes.

Human relaxin in the amnion, chorion, decidua parietalis, basal plate, and placental trophoblast by immunocytochemistry and northern analysis.

Immunocytochemistry and Northern analysis were used to show that relaxin is a product of intrauterine tissues of pregnancy. In addition, tissues from a patient without ovaries had similar results on both immunocytochemistry and Northern analysis as tissues from intact patients. The parietal decidua was clearly the major source of relaxin within the uterus and the relaxin mRNA (1.2 kilobases) from this tissue was detected with a 48-mer oligonucleotide probe designed to hybridize with both H1 and H2 relaxin gene transcripts. The mRNA isolated from the placental trophoblast was slightly smaller (1.1 kilobases), and the placental basal plate which has both maternal and fetal cells contained relaxin mRNAs of both sizes. Two monoclonal antibodies (Mabs) raised to synthetic human relaxin (H2) gave different patterns of localization in the fetal membranes, decidua and placenta. One Mab (RLX8) stained the chorionic cytotrophoblast in the fetal membranes and all of the cells in the placental basal plate. The other Mab (RLX6) stained the chorionic cytotrophoblast in some instances and selectively stained the decidua-like cells of the placental syncytiotrophoblast, whereas Mab RLX8 failed to detect this relaxin. Tissues obtained after spontaneous labor and delivery contained significantly less relaxin mRNA than tissues obtained at elective cesarean section without labor, but their hormone contents, as judged by immunocytochemistry, were not different. We conclude that the relaxin gene (H2) is expressed in intrauterine tissues, but that expression and hormone synthesis are not ubiquitous. Whether the relaxin gene H1 is expressed has not been determined.

The human Leydig insulin-like (hLEY I-L) gene is expressed in the corpus luteum and trophoblast.

A novel member of the insulin superfamily has previously been shown to be expressed only in porcine pre and postnatal Leydig cells and its human analogue demonstrated in the human testes but not in other organs and hence has been tentatively termed Leydig insulin-like peptide (Ley I-L). However, we have detected hLey I-L gene expression in the cyclic human corpus luteum and trophoblast by the reverse transcriptase-polymerase chain reaction (RT-PCR), with primers selected from the published human Ley I-L sequence. Normal and neoplastic breast tissue and fetal membranes with adhering decidua did not express the gene. The overall sequence of the trophoblast gene was in agreement with that reported with minor changes only in the putative connecting peptide, confirmed by restricted enzyme digestion. A 290 bp RT-PCR product was cloned and used as a cDNA probe in Northern analyses; hybridization was readily shown with cyclic corpora lutea but not with other tissues. The broader spectrum of the expression of this gene will warrant a new nomenclature when its biological activities are known. The different intensity of expression in the corpus luteum and trophoblast suggest endocrine and autocrine/paracrine roles respectively in these tissues in which H2 relaxin and H1/H2 relaxins coexist respectively and at similar levels of expression. Operationally the amino acid sequence homologies between the processed H1 and H2 relaxins and hLey I-L may qualify the specificity claimed for immunostaining the human relaxins in the corpus luteum and trophoblast.

Hormonal evaluation of the intrauterine progesterone contraceptive system.

Nine women were studied for one menstrual cycle prior to the insertion of an intrauterine progesterone contraceptive system (IPCS) delivering 65 microng progesterone/day into the uterus and again at 1 month after its insertion. Eight of these women were again studied between 6-8 months after the insertion of the IPCS. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol-17beta, progesterone, prolactin and relaxin were measured in each plasma sample. The data from each study were combined according to the day of the LH peak. Ovulation occurred in all the cycles studied in spite of an elevation in plasma estradiol-17beta and a depression of prolactin and relaxin immunoactivities at the 6-8 month follow up. Menstruations noted at the 6-8 month of use occurred while levels of estradiol-17beta and progesterone were elevated.

PIP: Plasma levels of luteinizing hormone, follicle stimulating hormone, estradiol-17beta, progesterone, prolactin, and relaxin were studied in 9 women before and after insertion of an Alza, progesterone-releasing, IUD. The IUD releases approximately 65 mcg/day of progesterone into the uterus. The lengths of the preovulatory and postovulatory phases of the menstrual cycle were not affected by the device. Ovulation occurred in all the cycles, even though plasma levels of estradiol-17beta were significantly (p less than .05) increased and plasma levels of prolactin and relaxin were significantly (p less than .05) decreased as late as 6-8 months after insertion. During menstruation at 6-8 months after insertion, plasma levels of estradiol-17beta and progesterone were high. The results are discussed.

Relaxin purification from human placental basal plates.

A crude relaxin preparation has been obtained from the basal plate region of electric cesarean section human placentae, as well as normal term placentae. The relaxin isolated has different charge properties by ion exchange chromatography from porcine relaxin, although it is of approximately the same molecular size. The most potent fraction obtained has approximately 0.7% of the immunoactivity of purified porcine relaxin when compared with porcine relaxin in a RIA based on porcine material, suggesting significant amino acid sequence differences between the putative human relaxin and its porcine counterpart.

Relaxin stimulates collagenase and plasminogen activator secretion by dispersed human amnion and chorion cells in vitro.

Dispersed cells from human amnion and chorion were cultured with and without relaxin. The addition of this hormone caused an increased secretion of both collagenase and plasminogen activator into the culture medium over a 32 h period, but had no effect on proteoglycanase or beta-glucuronidase secretion. The increase in plasminogen activator was dose-related to the amount of relaxin added in vitro. The results show that the fetal membranes are a novel target tissue for relaxin in the human, and suggest that relaxin in vivo may cause a similar release of collagenolytic enzymes, leading to the weakening and eventual rupture of the fetal membranes.

Specificity of radioimmunoassays for relaxin.

The specificities of two radioimmunoassays (RIA) for relaxin, based upon crude porcine relaxin (NIH-R-P1; RIA I) and a highly purified porcine relaxin (RIA II) have been studied concurrently using purified hormones and plasma samples. A labelled fraction, selected from radio-iodinated NIH-R-P1 and used in that RIA, was also bound to antiserum raised to the highly purified relaxin. Hence a third RIA was possible in which both the crude and the purified relaxins inhibited in the ng/ml range. Porcine insulin and the connecting peptide of porcine proinsulin did not inhibit any of the assay systems whereas porcine proinsulin did inhibit in each assay at the microgram/ml range. Concurrent measurements by assays I and II have been made in sheep plasma obtained during both delivery of the lamb and suckling. The peak values obtained by assays I and II are 3 and 6 min out of phase during suckling and delivery respectively; the NIH-R-P1 relaxin immunoactivity appearing first. The plasma inhibition curves of both appear to be the sum of individual contributions from relaxin and relaxin-like peptides, such as prorelaxin and its fragments, as seen by different antisera. Both assays, however, give qualitatively similar indices of relaxin immunoactivity. The RIA developed for the more purified peptide would be expected to yield a better quantitative estimate of relaxin secretion but this, like specificity, cannot be shown absolutely.

Identification of mRNA for relaxin in the endometrium of the pregnant guinea-pig.

The endometrium of late pregnant guinea-pigs was found to contain mRNA for relaxin. Poly(A)+RNA hybridized to 32P-labelled porcine relaxin-specific oligonucleotide probes. These probes corresponded to the C-peptide region of the prohormone. There was no hybridization to a 32P-labelled probe to the B-chain of porcine relaxin even under conditions of low stringency. The size of the relaxin mRNA was approximately 1.0 kb and similar to that found for relaxin mRNA from the pregnant sow ovary. This is the first study on relaxin mRNA from a uterine source.

Characteristics of the binding of 32P-labelled human relaxins to the human fetal membranes.

The two human relaxin genes termed H1 and H2 are expressed in the choriodecidua and placenta and have been proposed to act via specific receptors as local modulators of collagenolysis in the fetal membranes. Such receptors have been inferred, but not demonstrated, from studies of the effect of adding exogenous relaxin to these tissues. Thus conditions were optimized for the binding of 32P-labelled human relaxin H2 to membrane-enriched particulate fractions of human fetal membranes, amnion and chorion, with adhering decidua. The membrane protein concentration was optimal at 250 micrograms, when incubated at 27 degrees C for 60 min, at pH 7.5 with Mn2+ and Mg2+ ion concentrations of 2.0 mM. Incubation of membrane particulate fractions with increasing amounts of labelled relaxin H2 suggested the presence of a single class of binding sites with an affinity constant (Ka) of 2.15 nM. The binding was primarily to the chorion and decidua with very little to the amnion layer. The competition for binding of the 32P-labelled human relaxin H2 with unlabelled relaxin H2 gave an IC50 of 28 pM, while an IC50 of 60 pM and 280 pM was obtained for relaxin H1 and porcine relaxin respectively. In contrast, unlabelled guinea-pig relaxin inhibited this binding by only 10% even at a 1000-fold greater concentration than H2, and human recombinant insulin failed to inhibit even at a million-fold concentration of unlabelled relaxin H2. Relaxins H2 and H1 can readily displace the binding of either 32P-labelled human relaxins H1 or H2 and gave very similar displacement curves.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical identification of a relaxin-like protein in gastrointestinal epithelium and carcinoma: a preliminary report.

Immunostaining with antibodies to human relaxin (H2) suggests the presence of a relaxin-like peptide in the gastrointestinal tract and its tumours. The peptide is present in the cytoplasm of non-neoplastic cells at sites of cell exfoliation: the surface cells of the stomach, the small intestinal villi and the surface cells of the colon. It also occurs in the cytoplasm of gastric parietal cells and carcinomas of both the stomach and the colon. Relaxin has been shown to stimulate collagenase gene expression and to down-modulate collagen synthesis and secretion in human skin fibroblasts. The distribution of relaxin shown here suggests that it could play a role in the detachment of non-neoplastic cells from their basement membranes at the end of the cell cycle and in the penetration of carcinoma cells through the basement membrane.

Initiation of parturition and lactation in the sow: effects of delaying parturition with medroxyprogesterone acetate.

The synthetic progestagen, medroxyprogesterone acetate (MPA), was administered to sows in late pregnancy with the objective of slightly delaying the time of farrowing and thereby providing more marked associations between hormonal changes and the termination of pregnancy, and the initiation of farrowing and lactation in this species. MPA was administered orally (140 mg, twice daily) to eight sows in late pregnancy on days 112, 113 and 114 of gestation. Parturition was then induced to occur on day 116 by injecting 200 micrograms cloprostenol i.m. on day 115 of gestation. The peripartum changes in the plasma concentrations of progesterone, cortisol, oestradiol-17 beta, relaxin, prolactin, lactose and 13,14-dihydro-15-keto prostaglandin F2 alpha (PGFM) were measured in these sows together with a group of untreated sows. The gestational length for the MPA-treated sows (116.3 +/- 0.3 days, mean +/- S.E.M.) was significantly (P less than 0.01) greater compared with the untreated sows (114.9 +/- 0.3 days). Plasma progesterone declined earlier (P less than 0.05) with respect to the time of parturition in the treated sows compared with the untreated group. With respect to the timing of parturition, the time at which maximal concentrations of relaxin were attained and the timing of the subsequent decline were earlier in the MPA-treated sows. In both groups of sows, the concentration of relaxin increased before the decline in plasma progesterone. In the untreated sows, the concentration of PGFM increased either slightly before or at the same time as the decline in plasma progesterone, whereas in sows treated with MPA, progesterone concentrations began to decline before any significant increase in the plasma concentration of PGFM. The profiles of cortisol, oestradiol-17 beta and PGFM were similar in both groups of sows. In both groups of sows, the timing of the initial increase in the concentration of plasma prolactin coincided with a similar rise in plasma lactose (P less than 0.01). Plasma progesterone either declined earlier or at the same time as the rise in plasma lactose (P less than 0.01) in the treated group of sows only. We conclude that since the prepartum changes in the concentration of progesterone and relaxin occurred before significant changes in the concentration of PGFM in the MPA-treated sows, the nature of the luteolytic factor and the mechanism by which it exerts its action remains obscure. The higher concentration of lactose in the mammary secretion at birth in the MPA-treated sows compared with the untreated group suggested that lactogenesis was initiated earlier with respect to parturition following MPA treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Assessment of hypothalamic pituitary function in endocrine disease.

The insulin test carried out with adequate safeguards under standardized conditions yields valuable information regarding hypothalamic and pituitary function when plasma levels of sugar, cortisol, and growth hormone are determined. The use of a test based on the plasma cortisol response to the infusion of lysine-vasopressin, a polypeptide with a corticotrophin-releasing action, is also of value as a test of pituitary function. Used in conjunction with the insulin test it enables pituitary disorders to be differentiated from those involving the hypothalamus.

Cervical ripening with combinations of vaginal prostaglandin F2-alpha estradiol, and relaxin.

The first of a 2-part trial consisted of a double-blind randomized pilot study in which 4 groups of 10 patients near term received 1 of the following hormonal combinations in a vaginal gel 15 hours before surgical induction of labor: 1) prostaglandin F2 alpha (PGF2 alpha) and relaxin; 2) relaxin and estradiol; 3) estradiol and PGF2 alpha; and 4) relaxin, estradiol, and PGF2 alpha. In each group the mean cervical score improved after treatment; the relaxin/PGF2 alpha combination was associated with the greatest improvement in cervical score (4.8). The highest incidence of subsequent labor was also seen in the relaxin/PGF2 alpha group (40%). However, with the exception of the latter group, the clinical effects of these hormonal combinations were neither greater nor smaller than the previously published effects of these hormones used individually in similar circumstances. The second part of the study further explored the possibility of an additive effect of relaxin and PGF2 alpha in combination as suggested by the pilot study, and an additional 40 patients were given this combination. Analysis of these larger numbers showed no additive effect when these hormones were used in combination compared with when they were used individually. Thus, in the circumstances described, there is no clinical advantage to the concurrent administration of any combination of relaxin, PGF2 alpha and estradiol with regard to cervical ripening and/or the initiation of parturition.