Heterosynapsis and axial equalization of the sex chromosomes of the northern bobwhite quail.

The pairing behavior of the Z and W chromosomes in the female northern bobwhite quail (Colinus virginianus) was analyzed by electron microscopy of silver-stained synaptonemal complexes (SCs). After autosomal pairing was completed, synapsis of the sex chromosomes initiated at the short-arm end of the W chromosome and one end of the Z chromosome. Synapsis then progressed unidirectionally, producing a sex bivalent in which the entire length of the W axis was paired with an equivalent length of the Z axis. Progressive contraction and asymmetrical twisting of the Z axis ultimately resulted in a fully paired configuration with aligned axial ends. Further contraction of the Z axis reduced the extent of asymmetrical twisting such that only the nonaligned centromeric regions distinguished the SC of the ZW bivalent from SCs of similar-sized autosomes in late-pachytene nuclei. Quantitative analyses indicated that the length of the Z axis shortened significantly during the adjustment process, whereas no significant difference occurred in the length of the W axis. The nonalignment of the centromeric regions during transitional stages of ZW synapsis indicates that direct heterosynapsis of nonhomologous segments, followed by axial equalization of the length inequality, is responsible for the length adjustment during synapsis in the sex chromosomes of the bobwhite quail.     

Axial shortening during pachynema unrelated to nonhomologous synapsis

The pachytene behavior of chromosomes participating in quadrivalent formation in male mice heterozygous for T(X;4)7Rl or T(X;4)8Rl was analyzed in electron micrographs of microspread spermatocytes. In each population of nuclei from the translocation heterozygotes, the longest 4X axes were approximately the proportional length expected from the respective contributions of the 4 and the X estimated from breakpoint positions in mitotic chromosomes. However, the 4X axis of these translocation quadrivalents undergoes extensive shortening. In both R7 and R8 the shortest 4X axis observed in the population of nuclei was approximately the length of the normal 4 axis. This equalization of axial lengths suggests that there may be an interchromosomal interaction between synapsed chromosomes. In R8, axial shortening of the 4X occurs as pachynema progresses. In both translocations, shortening is accompanied by twisting of the 4X around the 4. Both axial shortening and twists are characteristics exhibited by chromosomal axes of unequal length as part of the meiotic phenomenon described as "synaptic adjustment" (Moses, 1977). Synaptic adjustment involves, in addition, nonhomologous synapsis, which is delayed until the latter part of pachynema. However, axial shortening in R7 and R8 is not accompanied by nonhomologous synapsis. In R7, nonhomologous synapsis does not occur; in R8, it is confined to quadrivalents in which the 4X axis is near its maximum length (i.e., early). This behavior suggests that axial shortening and nonhomologous synapsis during the progression of pachynema (previously considered collectively under the term "synaptic adjustment") are not necessarily coupled events.     

Synaptonemal complex analysis of mouse chromosomal rearrangements

Electron microscopy of surface-spread spermatocytes from mice heterozygous for a tandem duplication shows the heteromorphic synaptonemal complex (SC) to comprise two lateral elements of unequal length, the longer of which is buckled out in a characteristic loop, representing the unsynapsed portion of the duplication. The loop is a regular feature of late zygotene-early pachytene nuclei; it is longest at these early stages, but, through equalization of the two axes as a consequence of synaptic adjustment, it is replaced by a normal appearing SC at late pachytene. Because equalization, as indicated by a decrease in the percent difference between axes, may begin shortly after completion of synapsis, estimates of duplication segment length are restricted to a sample selected for least adjustment. — Although the mean position of the loop is constant at various pachytene substages, individual positions vary widely from cell to cell, consistent with the behavior expected of a duplication, but not of a deletion or an inversion. The length of the segment that is duplicated is estimated to be 22% of the normal chromosome, the midpoint of the segment is mapped at 0.61 of the chromosome distal to the kinetochore, and the ends of the segment are mapped at 0.50 to 0.72. Measurements of G-banded mitotic chromosomes give comparable values: duplication length, 24%; midpoint, 0.60, and segment ends, 0.48 and 0.71. This agreement constitutes further validation of the SC/spreading method for detecting and analyzing chromosomal rearrangements at pachytene and substantiates the fidelity with which the axes and SCs represent the behavior of chromosomes in synapsis.     

家鸡联会复合体的亚显微结构分析

本文以表面铺展——硝酸银染色技术,对家鸡的联会复合体(Syneptonemal Complex,SC)作亚显微结构分析。根据对10个精母细胞和10个卵母细胞SC的测量结果,绘制组型图。发现雌雄家鸡的常染色体的SC组型相同。在精母细胞中,性染色体(ZZ)的行为与常染色体相似。在卵母细胞中,性染色体ZW的长度不同,长轴为Z,短轴为W,两者之间只有部分配对,形成SC。从早粗线期到晚粗线期,由同源配对调整为非同源配对。另外,在一只雌鸡中,第一次观察到,有些细胞的常染色体能正常配对,而性染色体完全不配对的现象。     

Quadrivalent formation in a tetraploid chicken oocyte

Synaptonemal complex analysis of an exceptional tetraploid oocyte from a diploid chicken heterozygous for the MN t (Z;1) rearrangement was performed by electron microscopy of a spread preparation. Ten separate quadrivalents (26% of the chromosomal axes) were analyzed, as well as 50 autosomal bivalents. All the axes less than 2.5 microns in length formed bivalents (38) only, while axes in the 2.5-4.2 micron range formed 5 quadrivalents and 12 bivalents. The longer, separate axes formed quadrivalents only. Partner switches in excess of one were documented. The two identical W chromosomes paired only at the ends of their short arms. Quadrivalent formation may require a threshold length (2.5 microns), at least in this species. The tip of the short arm of the W chromosome may be a pairing initiation point, and it corresponds to the region associated with a localized recombination nodule previously described in diploid oocytes.     

Ultrastructure and behavior of the achiasmatic,telosynaptic XY pair of the sand rat (Psammomys obesus)

The behavior of the X and Y chromosomes in somatic and testicular cells of the sand rat (P. obesus) has been investigated with light and electron-microscope procedures. The Y chromosome has been identified as the fourth longest of the complement, both by C-banding and by its meiotic behavior. The X chromosome is the longest of the complement and carries two major C-heterochromatic blocks, one in the distal part of the long arm and the other forming most of the short arm. During presynaptic stages in spermatocytes, separate C-heterochromatic blocks, representing the sex chromosomes, are observed in the nuclei. An XY body is regularly formed at pachytene. During first meiotic metaphase the X and Y chromosomes show variable associations, none of them chiasmatic. Second meiotic metaphases contain, as in other mammals, a single sex chromosome, suggesting normal segregation between the X and the Y. — Electron microscopic observations of the autosomal synaptonemal complexes (SCs) and the single axes of the X and Y chromosomes during pachytene permit accurate, statistically significant identification of each of the largest chromosomes of the complement and determination of the mean arm ratios of the X and Y axes. The X and Y axes always lie close to each other but do not form a SC. The ends of the X and Y axes are attached to the nuclear envelope and associate with each other in variable ways, both autologously (X with X or Y with Y) and heterologously (X with Y), with a tendency to form a maximum number (four) of associated ends. Analysis of 36 XY pairs showed no significant preference for any single specific attachment between arm ends. The eighth longest autosomal bivalent is frequently partially asynaptic during early pachytene, and only at that time is often near or touching one end of the X axis. — It is concluded that while axis formation and migration of the axes along the plane of the nuclear envelope proceed normally in the X and Y chromosomes, true synapsis (with SC formation) does not occur because the pairing region of the X chromosome has probably been relocated far from the chromosome termini by the insertion of distal C-heterochromatic blocks.     

The relationship between chromosomes and axes in the chiasmatic XY pair of the Armenian hamster (Cricetulus migratorius)

The XY pair of the Armenian hamster has been studied in spreads and in three-dimensional reconstructions during the main stages of first meiotic prophase and metaphase I. The general pattern of the axes is similar to that of other mammals. There is a differential and a common region. In the latter a synaptonemal complex (SC) is formed by the pairing of the axes. This SC is longer than in other mammals. Heteropycnosis in the differential region is mirrored by differential chromatin packing at the ultrastructural level. The differential regions of the X and Y chromosomes can be identified both at the light and at the electron microscope level. The location of the axes at the interchromatid space in the differential region has been established. The visualization of the axes with the light microscope is facilitated by their bulgings at the beginning of mid-pachytene. These intermittent deformities change into a coiled and thinner axis during mid-pachytene. A chiasma originates in the common region of the XY body and it is seen near the ends of the sex chromosomes at diakinesis and metaphase I. The ultrastructure of this chiasmatic region is similar to that of autosomal chiasmata in the mouse. The axes separate from each other and leave a remaining piece of SC in which the central space is replaced by dense fibrillar material. During metaphase I the ultrastructure of this chiasmatic region cannot be identified because of the partial loss of the marker axes.     

The synaptic behaviour of the X and Y chromosomes in the marsupial Monodelphis dimidiata

The pairing behaviour of the X and Y chromosomes of Monodelphis dimidiata was studied with light and electron microscopy. Pairing of the sex chromosomes is delayed with respect to autosome synapsis. Both the X and the minute Y chromosome show an axis attached by its two ends to the nuclear envelope. Synapsis of the sex chromosomes occurs by the joining of the chromatin sheaths that surround the axes and by a small, three-layered structure close to the nuclear envelope. The X and Y chromosomes remain joined to each other during the diffuse stage and diplotene-diakinesis but they do not show a synaptonemal complex. During the diffuse stage a dense plate is formed at the boundary between the X-Y body and the nuclear envelope. During early metaphase a folded sheet is attached to the periphery of the X-Y body. This sheet is formed by a piece of the nuclear envelope carrying the dense plate and it shows transverse fibrils and a central element similar to synaptonemal-complex remains. No evidence of a non-chiasmate segregation mechanism was observed. Polarization of the axial ends of the sex chromosomes is observed after X-Y synapsis. These important departures from the X-Y pairing pattern of eutherian mammals are discussed and assumed to present a special mechanism for holding the minute Y joined to the X chromosome in this marsupial.     

High density of REC8 constrains sister chromatid axes and prevents illegitimate synaptonemal complex formation

During meiosis, cohesin complexes mediate sister chromatid cohesion (SCC), synaptonemal complex (SC) assembly and synapsis. Here, using super‐resolution microscopy, we imaged sister chromatid axes in mouse meiocytes that have normal or reduced levels of cohesin complexes, assessing the relationship between localization of cohesin complexes, SCC and SC formation. We show that REC8 foci are separated from each other by a distance smaller than 15% of the total chromosome axis length in wild‐type meiocytes. Reduced levels of cohesin complexes result in a local separation of sister chromatid axial elements (LSAEs), as well as illegitimate SC formation at these sites. REC8 but not RAD21 or RAD21L cohesin complexes flank sites of LSAEs, whereas RAD21 and RAD21L appear predominantly along the separated sister‐chromatid axes. Based on these observations and a quantitative distribution analysis of REC8 along sister chromatid axes, we propose that the high density of randomly distributed REC8 cohesin complexes promotes SCC and prevents illegitimate SC formation.     

Synaptonemal complex karyotyping in spermatocytes of the Chinese hamster (Cricetulus griseus)

Using the Counce-Meyer spreading technique, in over 70 spermatocytes it was possible consistently to obtain whole, flattened nuclei containing complete sets of pachytene SCs. The SCs are visible in both the phase and electron microscopes. Each SC is morphologically intact, preferentially stained, and attached to the nuclear envelope by a dense, terminal plaque. It is thus possible to trace each SC for its entire length. Also, a structure representing the kinetochore is clearly visible in each autosomal SC. Karyotypes comparable to the somatic karyotype can be constructed by arranging SCs according to length and kinetochore position. The observed regularity of SC morphology implies structural stability sufficient to withstand the stresses imposed by the procedure.— A coarse network of closely packed nuclear annuli connecting SC attachment plaques often provides end-to-end associations and may tend to immobilize SCs during processing.— Three kinds of perturbation of SC structure are encountered. Twists in the SC frequently occur, but no regular pattern or correspondence with chiasma distribution is observed. SCs occasionally hook around each other without disruption, but in two instances the unpaired axis of the X apparently was interlocked within an autosomal SC. Stretching of the SC is infrequent; it is conspicuous when it occurs and is usually associated with other obvious distortions of the nucleus.— Distinctive morphologies of the X and Y chromosomes facilitate their identification in all preparations. — During zygotene, autosomal synapsis, i.e., the formation of SCs from the pairing of single axial elements, initiates at distal ends and terminates at the kinetochore region; neither initiation nor termination is synchronous among all autosomes.     

The cohesin REC8 prevents illegitimate inter‐sister synaptonemal complex assembly

During meiosis, a specialized chromosome structure is assembled to promote pairing/synapsis of homologous chromosomes and meiotic recombination, a process yielding chiasmata between homologs to ensure accurate segregation. Meiosis‐specific cohesin complexes mediating sister chromatid cohesion play pivotal roles in almost all these events, including synaptonemal complex (SC) formation. In this issue of EMBO Reports, Agostinho and colleagues have examined chromosome axes and SC structures by taking advantage of a hypomorphic Stag3 mutant in which the levels of the cohesin subunit REC8 are partly reduced 6 . Using super‐resolution microscopy, the authors illuminate previously unforeseen chromosome axis structures, showing locally separated axes in regions where REC8 is absent, regardless of RAD21L or RAD21 cohesin localization. Furthermore, they assessed the relationship between sister chromatid cohesion and inter‐sister SC formation, demonstrating that “axial opening” in the REC8‐free region is accompanied by illegitimate SC formation between sister chromatids. This study highlights the physiological importance of REC8 in sister chromatid cohesion and proper SC formation during meiosis, suggesting a new model in which a high density of REC8 deposition along the chromosome prevents illegitimate inter‐sister SC formation.     

Expression and functional analysis of <Emphasis Type="Italic">TaASY1</Emphasis> during meiosis of bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

Background  

Pairing and synapsis of homologous chromosomes is required for normal chromosome segregation and the exchange of genetic material via recombination during meiosis. Synapsis is complete at pachytene following the formation of a tri-partite proteinaceous structure known as the synaptonemal complex (SC). In yeast, HOP1 is essential for formation of the SC, and localises along chromosome axes during prophase I. Homologues in Arabidopsis (AtASY1), Brassica (BoASY1) and rice (OsPAIR2) have been isolated through analysis of mutants that display decreased fertility due to severely reduced synapsis of homologous chromosomes. Analysis of these genes has indicated that they play a similar role to HOP1 in pairing and formation of the SC through localisation to axial/lateral elements of the SC.     

Synaptic behaviour and morphological modifications of the X and Y chromosomes during pachytene in three species of Ctenomys (Rodentia, Caviomorpha, Ctenomyidae).

Synaptic behaviour and the progression of morphological differentiation of the XY chromosome pair during pachytene was studied for the first time in three species of the South American subterranean rodents of the genus Ctenomys (tuco-tucos). In general, synapsis progression in the sex pair could be subdivided into four substages: (i) initial partial synapsis of the X and Y chromosome axes and beginning of the differentiation of the unsynapsed regions; (ii) complete or almost complete synapsis of the Y axis accompanied with morphological differentiation of the unsynapsed region of the X chromosome; (iii) a novel stage exclusive to Ctenomys perrensi consisting in a retraction of the free X axis, associated with the formation of a homogeneous and dense structure along the synaptic region, which leads to the achievement of full synapsis between sex chromosomes; or (iv) an increase in morphological complexity involving extreme splitting of the XY pair. The implications of the peculiar synaptic behaviour displayed by sex chromosomes in C. perrensi, a species complex highly polymorphic for Robertsonian translocations, are discussed in relation to both the triggering of the pachytene checkpoint and the avoidance of non-homologous associations between sex chromosomes and the asynaptic pericentromeric regions of trivalents in translocation heterozygotes.     

Pairing of ZW gonosomes and the localized recombination nodule in two Z-autosome translocations in Gallus domesticus

Electron microscopic observations of synaptonemal complexes of oocytes from chickens heterozygous for two Z-autosome translocations have been used to identify and study the pairing region of the Z and W chromosomes. The two translocations, MN t(Z;1) and t(OH 10), have breakpoints in opposite arms of the Z, and the arm having the breakpoint of MN t(Z;1) is marked by the terminal C+ band. In both translocations the short arm of the W was specifically paired with the euchromatic short arm of the Z. In MN t(Z;1) only open quadrivalents (74%) and trivalents plus W univalents (26%) were observed, whereas t(OH 10) exhibited, in addition to the prevalent quadrivalents (62%), III + I (19%) and II + II (19%) configurations. The extent of W pairing was slightly decreased in MN t(Z;1) (68.4% of the W chromosomes paired) and considerably decreased in t(OH 10) (25.3% of the W chromosomes paired). Nonhomologous synapsis occurred regularly at the quadrivalent crosspoint in MN t(Z;1) and also in bivalents from t(OH 10). The recombination nodule normally located in the terminus of the pairing region in normal ZW pairs is present in both translocations without any alteration of its frequency or its strict terminal position. Based on these data and previous observations (Rahn and Solari, 1986), it is proposed that an obligatory recombination event occurs at a locus between 0.7 microns and 0.15 microns of the paired ZW telomeres, establishing a recombinational region and a pseudoautosomal region which determine partial sex-linkage and no sex-linkage, respectively. Most of the pairing region of the ZW pair is nonhomologously paired.     

Meiotic chromosome synapsis in a haploid yeast

An extensive synaptonemal complex (SC) is found at pachytene in whole mount spread preparations of a haploid yeast, Saccharomyces cerevisiae, strain. Whereas unsynapsed axial elements are present only in a few nuclei, in others non-homologous synapsis involves virtually the whole chromosome set. This suggests that homology is not an indispensable precondition for SC formation in yeast but that chromosomes engage in non-homologous synapsis if no homologous partner is available. Recent evidence that in the sporulation deficient yeast mutants rad50 and mer1 axial elements do form but remain unsynapsed in the majority of nuclei is discussed in the light of the above findings.by D. Schweizer     

Synaptonemal complex spreading in Allium

A surface-spreading technique for synaptonemal complexes was applied to triploid Allium sphaerocephalon L. (Liliaceae). In early pachytene two of the three axial elements of each set of three homologues are synapsed, the third is intimately aligned with and accompanies them throughout their whole length. The unsynapsed axis is attached to the synaptonemal complex of the other 2 at up to 50 association sites per trivalent. The distribution of these sites within the trivalents is not even; they are under-represented in the proximal regions. From nought to eight switches (pairing partner exchanges), where the accompanying axis joins in synapsis in exchange for one of the two other strands, occur per trivalent. Very often the telomeres of the aligned axes are attached to their synapsed counterparts by dense spherules, which makes this type of association different from the interstitial ones. Frequently the unsynapsed axes show a double structure along short distances. In late pachytene the intercalary associations are abolished, allowing the unsynapsed axes to engage in various types of non-homologous pairing. Since the association sites involve homologous chromosomes and are less abundant in the pericentric regions (which are usually the last to synapse), it is conceivable that similar structures are responsible for the pre-synaptic alignment of homologues and provide the initiation sites for synaptonemal complex formation in diploids.     

Synaptic adjustment,non-homologous pairing,and non-pairing of homologous segments in sex chromosome mutants of Ephestia kuehniella (Insecta,Lepidoptera)

Microspread pachytene nuclei of wild-type and W chromosome mutants of the mealmoth Ephestia kuehniella were used to study synaptonemal complex (SC) formation. In structurally heterozygous bivalents, axial elements of considerable length differences were brought to the same length by synaptic adjustment. The adjustment length was a compromise between the mutant and the wildtype homologue length in a structural heterozygote of a W chromosome-autosome translocation, T(A; W). The translocated non-homologous W segment really participated in SC formation as could be seen from the W chromosomal heterochromatin, used as a cytogenetic marker. Pachytene pairing of the wild-type W-Z bivalent extended from about two-thirds to the full length of the W chromosome, though from cytogenetic and genetic evidence W and Z are largely — if not completely — non-homologous. Nonhomologous pairing was even more conspicuous in sex chromosome bivalents containing a deleted W chromosome, Df(W). In one of the pairing configurations the halves of the Z chromosome were synapsed to either side of the Df(W). Thus, one side was pairing with the Df(W) in reversed order. The pairing behavior of the W with homologous chromosome segments was tested by introducing supernumerary W segments via the T(A; W) translocation. Pairing between the W and the translocated homologous W segment never occurred, whereas the Z frequently synapsed with it. Even in T(A; W) homozygotes, pairing between the two translocated W segments was not regularly found while the autosomal parts of the translocation chromosomes were always completely paired. Homologous chromosomes and the ability to form an SC are not sufficient for pairing initiation. Specific loci or sequences are postulated for this function. They are either absent from the W chromosome or are present in only low concentrations.     

Altered distribution of MLH1 foci is associated with changes in cohesins and chromosome axis compaction in an asynaptic mutant of tomato

In most multicellular eukaryotes, synapsis [synaptonemal complex (SC) formation] between pairs of homologous chromosomes during prophase I of meiosis is closely linked with crossing over. Asynaptic mutants in plants have reduced synapsis and increased univalent frequency, often resulting in genetically unbalanced gametes and reduced fertility. Surprisingly, some asynaptic mutants (like as1 in tomato) have wild-type or increased levels of crossing over. To investigate, we examined SC spreads from as1/as1 microsporocytes using both light and electron microscopic immunolocalization. We observed increased numbers of MLH1 foci (a crossover marker) per unit length of SC in as1 mutants compared to wild-type. These changes are associated with reduced levels of detectable cohesin proteins in the axial and lateral elements (AE/LEs) of SCs, and the AE/LEs of as1 mutants are also significantly longer than those of wild-type or another asynaptic mutant. These results indicate that chromosome axis structure, synapsis, and crossover control are all closely linked in plants.     

Unequal crossing over and heterochromatin exchange in the X-Y bivalents of the deer mouse,Peromyscus beatae

Differences in length of the heterochromatic short arms of the X and Y chromosomes in individuals ofPeromyscus beatae are hypothesized to result from unequal crossing over. To test this hypothesis, we examined patterns of synapsis, chiasma formation, and segregation for maleP. beatae which were either heterozygous or homozygous for the amount of short-arm sex heterochromatin. Synaptonemal complex analysis demonstrated that mitotic differences in heterochromatic shortarm lengths between the X and Y chromosomes were reflected in early pachynema as corresponding differences in axial element lengths within the pairing region of the sex bivalent. These length differences were subsequently eliminated by synaptic adjustment such that by late pachynema, the synaptonemal complex configurations of the XY bivalent of heterozygotes were not differentiable from those of homozygotes. Crossing over between the heterochromatic short arms of the XY bivalent was documented by the routine appearance of a single chiasma in this region during diakinesis/metaphase I. Sex heterochromatin heterozygotes were characterized by the presence of asymmetrical chiasma between the X and Y short arms at diakinesis/metaphase I and sex chromosomes with unequal chromatid lengths at metaphase II. These data corroborate our hypothesis on the role of unequal crossing over in the production and propagation of X and Y heterochromatin variation and suggest that, in some cases, crossing over can occur during the process of synaptic adjustment.     

Synaptonemal complexes in insects

The synaptonemal complex (SC) is the key nuclear element formed in meiotic prophase I to join 2 homologous chromosomes at the pachytene bivalent. It is a highly conserved structure that is universally present in eukaryotes. The SC is presented as a tripartite protein structure, which consists of 2 lateral elements and a central region. In insects, the central region is particularly distinct and highly ordered. This made it possible to describe the fine structure of the central region and propose a model of its architecture. Chromatid DNA is arranged in chromatin loops extending radially from the SC. The loops appear to consist of a basic chromatin fiber with a diameter of 20–30 nm. In many insect species, synaptonemal polycomplexes occur in postpachytene cells. They represent one of the possible ways of SC degradation. Another process, which occurs beyond pachytene, is the formation of proteinaceous chromatid axis, the silver-stained chromatid core. Based on results in insect models, the chromatid cores have been related to the structure and formation of the SC. Research on insect models significantly contributed to understanding individual steps of the SC formation and temporal sequence of chromosome pairing. These include the formation of lateral elements of the SC, pairing initiation, interlocking of chromosomes, and synapsis of homologous chromosomes. Attention is also given to non-homologous pairing, including synaptic adjustment, correction of pairing, and pairing of sex chromosomes. In the next section, chiasmatic and achiasmatic modes of meiosis are compared with respect to the SC formation. In the chiasmatic mode, the SCs display recombination nodules that are believed to mediate the process of recombination. These nodules were discovered in insects, and indirect evidence for their role comes from insects. Two different examples of achiasmatic meiosis, occurring in the heterogametic sex of several insect orders, are given: one involves the SC formation, whereas in the other, SCs are absent. Finally, the potential of SC karyotyping for analysis of the insect genome is discussed.