Localization of anionic sulfate groups in the tectorial membrane

Hearing Research, 45 (1990) 283-294 Elsevier

283

HEARES 01361

Localization of anionic sulfate groups in the tectorial membrane J o r g e J. P r i e t o , M a r i a

E. Rubio

and Jaime A. Merchan

Department of Histology, Faculty of Medicine, University of Alicante, A licante, Spain

(Received I August 1989; accepted 3 December 1989)

Colloidal iron hydroxide (CIH) staining demonstrates the existence of anionic sulfate groups of glycoconjugates associated with several constituents of the tectorial membrane (TM). In the adult animal, labelling in the main body of the TM appears as long, electron-dense patches surrounding type A fibrils which show alternating stained and unstained zones. On the other hand, labelling of the fibrils of the matrix of the TM appears as single, CIH particles with no special arrangement. Some of the structurally distinct regions of the TM are also labelled (limbal zone, Hensen's stripe and inner portions of the cover net), while others are not (marginal band and outer portions of the cover net). Staining of type A fibrils in the major TM is already present in newborn animals; while, both the outermost region of the TM closest to the cells of the organ of K~511ikerand the minor TM are not labelled. The implications of these distributions of sulfated glycoconjugates for the electrochemical properties of the TM are discussed. Organ of Corti; Tectorial membrane; Sulfated glycoconjugates; Donnan equilibrium

Introduction

The tectorial m e m b r a n e (TM) is an acellular structure c o m p o s e d of a complex matrix of fine fibrils. Kronester-Frei (1978) described two types of fibrils in the T M : type A fibrils are straight, unbranched, and measure 10 n m in diameter; type B fibrils are coiled, branched, and measure 15 to 20 nm. A recent study by Hasko and Richardson (1988) revealed that the type B fibrils of Kronester-Frei are, in fact, sheets of finer ones (7 n m in diameter), showing that the T M matrix has a high degree of structural organization. The most widely accepted role of the T M in the auditory transduction process is that of passively bending the stereocilia to which it is coupled when the basilar m e m b r a n e is displaced upwards (Davis, 1958), a process which m a y trigger an inward flow of potassium and subsequent depolarization of the hair cells (see the review of Hudspeth, 1985). In

Correspondence to: Jorge J. Prieto, Department of Histology, Faculty of Medicine, University of Alicante, 03690, Alicante, Spain.

addition, the results of recent physiological studies suggest that the decrease in the c o m p o u n d action potential of the V I I I t h nerve elicited b y the activation of the crossed olivocochlear bundle m a y be due, at least in part, to the mechanical coupling of the stereocilia of inner and outer hair cells b y means of the T M (Brown and Nuttall, 1984; Strelioff et al., 1985; Kim, 1986). Nevertheless, it is possible that besides its mechanical role, the electrochemical properties of the T M could also influence the physiology of sensory cells. In fact, although the T M has been assumed for a long time to be electrically and ionically transparent, there is evidence that it m a y sustain a D o n n a n equilibrium with the surrounding e n d o l y m p h (Steel, 1983). A D o n n a n equilibrium is characterized by the assymmetrical distribution of ions between two separated compartments, and is established when a fluid containing diffusible ions bathes a distinct phase containing fixed charges; because the fixed charges cannot diffuse, the equilibrium potential can only be established b y means of the diffusible ions. F r o m the results of this study, Steel suggested that fixed negative charges of the T M proteins would create an electrical gradient tending to draw positive ions

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284 into the TM (Steel, 1983). A first corollary of this is that these electrochemical properties of the TM may create a special ionic microenviroment near the tips of the stereocilia, a fact that could be of great importance if the transduction channels which become open when the stereocilia are deflected are located at or near their tips as suggested by Hudspeth (1982). Another consequence is that the TM could act as an ion barrier between the fluid within the endolymphatic duct above the TM and the subtectorial fluid, thus providing one explanation for the possibly different ionic composition of both compartments (Runhaar and Manley, 1987). As mentioned above, Steel (1983) suggested that the explanation for the negative potential found in the TM could be that the proteins of which it is composed are negatively charged at physiological pH. However, other authors have proposed that some glycoconjugates of the matrix of the TM may be responsible for the charge density within the TM (Santi and Anderson, 1986; Khalkhali-Ellis et al., 1987). The glycoconjugates of the TM have been studied several times by means of conventional light microscope histochemistry (Plotz and Perlman, 1955; Belanger, 1956; Iurato, 1960; Mangabeira-Albernaz, 1961; Igarashi and Alford, 1969; Ross, 1974), electron microscope histochemistry (Dohlman, 1971; Kronester-Frei, 1978; Kuttner, 1977; Santi and Anderson, 1987; Hasko and Richardson, 1988), autoradiography (Belanger, 1953, 1956; Kuijpers and Manni, 1986; Manni and Kuijpers, 1987), lectin cytochemistry (GilLoyzaga et al., 1985; Tachibana et al., 1987a,b; Rueda and Lim, 1988; Rueda et al., 1989; Prieto et al., 1989) and biochemical methods (Bairati et al., 1957; Iurato, 1962; Naftalin et al., 1964; Saito and Daly, 1970; Steel, 1980; Khalkhali-Ellis et al., 1987). Little is known, however, about the distribution of anionic glycoconjugates (i.e., those which bear a negative charge at physiological pH) within the TM. For this purpose, the use of cationic dyes for transmission electron microscopy seems to be the most suitable method, although the Alcian blue technique has failed to show any new features of TM ultrastructure (Santi and Anderson, 1987). On the other hand, ruthenium red staining shows electron-dense particles periodically distrib-

uted along the type A fibrils of the TM (Hasko and Richardson, 1988). It has been previously demonstrated that ionized groups on cells and tissue components are detectable in situ by using cationic ferric hydroxide colloids visible by light and electron microscopy (Mowry, 1958, 1963; Gasic et al., 1968; Schrevel et al., 1981). Seno et al. (1983a) succeeded in obtaining ferric hydroxide colloid particles of small size and stabilizing their positive charge at pH 1.6-7.6. By boiling a mixture of ferric chloride in a cacodylate buffer solution, they obtained a ferric hydroxide colloid of finer grain than that of the previous techniques, which proved to be a valuable cationic probe for the detection of anionic sites, as revealed by histochemical observation of various tissues (Seno et al., 1983b, 1985; Tsuji et al., 1984a,b; Prieto and Merchan, 1987). The specificity of the method depends on the pH of the colloidal iron hydroxide (CIH) solution because the anionic groups of acid polysaccharides are ionized (i.e. negatively charged) at a pH higher than their isoelectric point. Actually, carboxyl groups have a pK (the negative logarithm of the dissociation constant) about 3.5-4.0, while the sulfate groups have a pK about 1.8-2.0, which means that if a CIH solution at pH higher than 4.0 is used, all the anionic groups of acidic glycoconjugates will be labeled, but if the solution is at a pH around 1.8, only the sulfate groups will be ionized and thus bound by the cationic ferric colloid particles (Seno et al., 1985; Seno, 1987). The specificity of the method was established by testing absorption to ion-exchange resin particles at different pHs: the particles were adsorbed to sulfonate groups at pH 2.0 and 4.0, and to carboxyl groups at pH 4.0 but not at 2.0 (Seno et al., 1985; Seno, 1987). Thus, from the point of view of colloid chemistry, CIH is a suitable method to detect sulfate groups histochemically by treating tissue sections or fixed cells with the probe at pH 1.8 while the carboxyl groups of acid polysaccharides are shown together with sulfate groups at a pH of about 4. Accordingly, the goal of this study is to use the CIH method in TEM to detect anionic sulfate groups in the TM, and so further our understanding of the electrochemical properties of the living TM.

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Fig. 1. (A) A n overall view of the free portion of an adult TM stained with CIH. Small electron-dense particles appear forming discontinuous stripes. These stripes are not present in the uppermost region of the T M and are increasingly frequent towards its basal surface, where they are closely packed in a thin band (short arrows). Although the inner portions of the cover net are labelled by CIH, the outer ones lack any staining. The marginal band (long arrow) is not labelled. × 4,600. (B) W h e n the plane of section is parallel to the type A fibrils of the TM, C I H labelling appears as long, electron-dense ribbons displaying the same orientation as the T M fibrils. The C I H particles label only part of the TM fibrils, thus appearing as electron-dense patches alternating with unstained areas. × 17,700. (C) On the left, C I H particles seem to surround type A fibrils, the middle zone of each electron-dense patch being of greater thickness than the fibrils itself (13-40 nm), and tapering toward the ends. O n the right, single C I H particles appear associated with the fibrils of the matrix of the T M closest to the cover net; unlike the labeling pattern of type A fibrils, that of the fibrils of the matrix does not form stripes. × 5,580.

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Material and Methods

Cochleae were obtained from seven one-daypostpartum rat pups and five 7 - 8 weeks-old rats. After intraperitoneal anaesthesia with chloral hydrate (0.3 g / k g body wt) the animals were decapitated, the bullae removed and the cochleae exposed. Holes were made at the apex and at the basal turn of the cochleae, through which the fixative was gently perfused. The fixative consisted of a mixture of 2.5% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M cacodylate buffer, p H 7.4. The fixation always began less than 4 min after the death of the animal, and was carried out at room temperature for 1 h.

The organ of Corti (OC) was then excised under the dissecting microscope and cut into 1 mmlong pieces. Reissner's m e m b r a n e and the lateral wall were removed to facilitate diffusion of the C I H particles. Samples were then placed for 5 h at room temperature in the C I H solution prepared as follows: 1 vol of 0.1 M FeC13 • 6H20 was added to 10 vol of 0.1 M sodium cacodylate buffer solution, p H 7.4. This solution was boiled until the color turned to red-brown. After cooling at room temperature, the mixture was diluted with 2 vol of sodium cacodylate buffer solution. The p H was adjusted to 1.8 with 1 N HC1 and the solution was made isotonic with sucrose. Afterwards, the specimens were washed several times in 0.1 M cacodylate buffer, p H 7.4, and

Fig. 2. C I H labelling of different parts of the adult TM.(A) The phalangeal process of an interdental cell (arrow) between the underlying limbus spiralis' matrix and the timbal portion of the TM. At this latter location, labelling of the fibrillar layer appears as electron-dense stripes similar to that of the free portion of the TM. In addition, small single particles are also present both in the amorphous layer close to the interdental cell microvilli (arrowheads) and in the upper surface of the TM. x 9,600. (B) A portion of the cover net adjacent to the marginal band lacks any CIH staining. Type A fibrils located below the cover net show m a n y electron-dense patches of CIH particles, x 12,600. (C) At the undersurface of the free portion of the TM, Hensen's stripe is labelled with a few, single CIH particles (arrowhead). A thin, 0.8-1 t~m-thick layer of closely-packed type A fibrils (arrows) shows a density of electron-dense patches higher than the rest of the TM. x 12,600.

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Fig. 3. CIH labelling of the T M in one day-old animals. (A) Electron-dense patches are present in the fibrillar layer of the limbal portion of the TM, over an interdental cell. x 26000. (B) Inner region of the major TM. The upper half of the T M shows patches of C I H particles parallel to the spreading type A fibrils. Over the cells of the organ of K611iker the labelling of the T M appears as single particles. × 5000. (C) Neither the outer region of the T M (star), over the cells of the organ of K6Uiker (arrowheads) nor the minor T M (asterisk) over an inner hair cell are labelled. × 8000.

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postfixed in ice-cold 2% OsO 4 for 2 h, dehydrated through an acetone series and embedded in Epon-Araldite. Silver-to-gold sections cut on a Reichert Ultracut ultramicrotome were mounted on copper grids and examined without any counterstaining in a Zeiss EM-10 electron microscope, operated at 80 kv. Results

In an overall view of the adult TM, the CIH particles appear as fine electron-dense precipitates arranged in strings (Fig. la). At a low magnification the labelling seems to be absent from the marginal band and the outer portions of the cover net; it is more intense in the lower two thirds of the main body of the TM than in the upper third, and especially prominent in a band (0.8-1 ~mthick) close to the lower surface of the TM (Fig. la). When the TM is sectioned parallel to type A fibrils, the labelling of the main body appears organized in long, parallel electron-dense ribbons (Fig. lb). CIH particles seem to be attached to the type A fibrils, as the ends of each electron-dense ribbon are continuous with an unstained portion of type A fibril (Fig. lb). The unstained regions seem to be larger than the stained ones (Fig. lb). At a higher magnification the electron-dense precipitates seem to surround type A fibrils forming patches which display a greater diameter (13-40 nm-thick) than the fiber itself (Fig. lc). Precipitates also appear in the matrix of the TM, not in large strings, but in small groups of single particles without any defined arrangement (Fig. lc). This pattern, which is typical of the main body of the TM, is somewhat different in other regions Thus, the fibrillar layer of the limbal region of the TM also contains the electron dense strips, but there are in addition small, single CIH particles both in the amorphous layer (the closest to the interdental cells' surface) and in the apical surface of the TM (Fig. 2a). Labelling is absent from the marginal band (Fig. la) and from the portions of the cover net closest to it (Figs. la, 2b). The Hensen's stripe and the inner portions of the cover net, however, are labelled but in small and infrequent patches (Figs. 'la, 2c). A narrow band of type A fibrils (0.8-1 /~m-thick) at the lowest re-

gion of the TM shows a density of CIH particles higher than the rest of the body of the T M (Figs. la, 2c). In newborn animals, patches of CIH particles can also be detected in the limbal region of the TM, over the interdental cells (Fig. 3a). The major T M (the largest portion of the T M located over the organ of K~511iker) is stained in a similar manner to the main body of the adult TM, and short, parallel strips of CIH particles appear associated with type A fibrils (Figs. 3b, 3c). However, the lowest, amorphous region of the major TM close to the apical surface of K~511iker's organ cells shows a distinct pattern with regional differences: the inner region displays small patches of CIH particles (Fig. 3b), but the outer one, near the inner hair cells, lacks this labelling (Fig. 3c). In addition, the minor T M (a small portion of the developing TM, projecting from the outer edge of the major T M to the outermost Deiters' cells and covering the hair cells) is not stained (Fig. 3c). Discussion

The present study has demonstrated the existence of anionic sulfate groups associated with different components of the TM. Previous studies using histochemical methods to reveal glycoconjugates at the ultrastructural level failed to show such a particular distribution of anionic groups of glycoconjugates within the TM (Dohlman, 1971; Kronester-Frei, 1978; Kuttner, 1977; Santi and Anderson, 1987; Hasko and Richardson, 1988). This disparity may be due to the fact that, in previous studies, the staining solutions used were all at about p H 7.4 and, thus all the anionic groups of glycoconjugates would be ionized (Seno, 1987). As demonstrated by Seno et al. (1985) and Seno (1987), the CIH method is highly specific for the sulfate groups of glycoconjugates when it is used at p H 1.8, which strongly suggests that the results presented here reflect the distribution of sulfate-containing glycoconjugate/s in the TM. The labelling pattern obtained by CIH staining of the adult T M is remarkably different from that obtained using ruthenium red (Hasko and Richardson, 1988). In addition to labelling in the form of single, small particles, we have found large patches of CIH particles apposed to single

289 type A fibrils and, apparently, surrounding them. Within the main body of the free portion of the TM, the labelling is notably very intense for the type A fibers; since there are stained and unstained zones alternating in a single fiber, it is apparent that the glycoconjugate composition varies along the length of each fibril, a fact not reported before. The finer fibrils forming the matrix, as described by Hasko and Richardson (1988), are not seen surrounding the type A fibers using this staining method, but in some regions where they are especially frequent, such as below the cover net, it is possible to see that they are decorated with some scarce, single CIH particles. This shows that sulfated glycoconjugates are also present in the TM matrix. The higher specificity and sensitivity of the CIH method for sulfate glycoconjugates may be the explanation for the differences between the two techniques. In fact, both methods gave different results when they were used to demonstrate the anionic groups of the cell coat of hair cells (Slepecky and Chamberlain, 1985; Prieto and Merchan, 1987). However, the exact nature of the CIH-stained sulfated glycoconjugate is not known, as several glycoconjugates bear sulfate groups. Glycoconjugates (glycolipids, glycoproteins and proteoglycans) are the cell membrane components responsible for the net negative charge observed on the cell surface of most normal cells, because the saccharide residues of their carbohydrate chains bear carboxyl and sulfate groups, which are ionized at a physiological pH (see Cook, 1986, for a review). Besides the cell surface, where the glycoconjugates form the cell coat (or glycocalyx), they are also present in the extracellular matrix. Several results suggest that the T M contains both glycoproteins and proteoglycans: in a recent study on the polypeptide composition of the T M (Richardson et al., 1987), collagen types II, V and IX were found, and it is well known that collagen type II is a glycoprotein, containing mainly glucose and galactose (Butler, 1978). Proteoglycans (large molecules containing a core protein with one or more chains of glycosaminoglycans) usually contain larger amounts of sulfate than glycoproteins (Lindahl and H~5~Sk, 1978), but their occurrence in the TM is at present a matter of controversy. Type IX collagen has been shown to

have covalently linked chondroitin sulfate (Bruckner et al., 1985). Nevertheless, chondroitin sulfate contains uronic acid, and this latter compound has never been detected in the TM (Khalkhali-Ellis et al, 1987; Richardson et al., 1987). The only proteoglycan which does not contain uronic acid is keratan sulfate, and thus it is the only proteoglycan which could be a constituent of the TM; in fact, Khalkhali-Ellis et al. (1987) suggested that keratan sulfate may be present in the TM, linked to collagen type II, as the former is composed of a repeating disaccharide unit of N-acetyl glucosamine and galactose, and both sugars have been detected in the T M (Khalkhali-Ellis et al., 1987). In addition, in the biochemical study of Richardson et al. (1987) a high molecular weight, polydisperse material sensitive to keratanase treatment was found. Moreover, in an ultrastructural study of the different types of fibers forming the TM, Hasko and Richardson (1988) found that the highly organized matrix of the TM, which was revealed by adding tannic acid to the fixative, had properties similar to the non-collageneous polypeptidic materials found in the biochemical study of Richardson et al. (1987). The use of a cationic dye, ruthenium red, demonstrated the existence of small particles distributed periodically along the type A fibrils in young animals, a fact which led these authors to suggest that the particles were granules of an unidentified proteoglycan linked to type II collagen. Although keratan sulfate would seem, from the aforementioned evidence, to be a suitable candidate, it is nevertheless unlikely that it represents the CIH-stained sulfated glycoconjugates. This is because it does not usually bind to collagen since this depends on a high charge density on the glycosaminoglycan molecule, and that of keratan sulfate is low because of the absence of carboxyl groups (Lindahl and HiSiSk, 1978). The limbal zone is the only portion of the adult TM that keeps a close contact with underlying cells (except for the tips of the hair cells' stereocilia) and this, together with the secretion displayed by the interdental cells (Prieto et al., 1988) suggest a possible turnover mechanism for T M components in the adult cochlea for which these cells may be responsible. In this region, CIH particles are present in the fibrillar layer, arranged in a way similar to that of the stripes associated with

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type A fibrils in the main body of the TM. In the underlying amorphous layer, however, labelling appears as small particles, a fact which could indicate that sulfated glycoconjugates may be secreted as single molecules, which later would become associated with the developing type A fibrils. Kronester-Frei proposed that the cover net, the marginal band, Hensen's stripe and the limbal undersurface of the TM were composed of tightly packed, weakly hydrated, type B protofibrils (Kronester-Frei, 1978). Our results show that, the cover net, Hensen's stripe and the limbal undersurface all have a similar composition in terms of sulfated glycoconjugates, presenting discrete labelling by single CIH particles, while the marginal band and the portions of cover net closest to it lack any labelling. Although it is not known whether all these specializations of the TM have the same polypeptide composition, it seems, at least, that they may have a dissimilar glycoconjugate composition, as has been previously suggested by Rueda and Lim (1988). In the newborn animals, the labelling of the limbal zone and the major TM is similar to that of the adult. Strikingly, Hasko and Richardson (1988) found ruthenium red-stained granules associated with type A fibrils in early postnatal TMs, but not in the adult animals. They proposed that this difference was due to the disappearance of the proteoglycans from the matrix of the TM at some time during the development of the animal, thus explaining the absence of uronic acid in the adult TM. Our results, however, show that, whatever the nature of the sulfated glycoconjugate labelled by the CIH particles, it is present from birth to adulthood, at least in the limbal zone and main body of the TM. It is widely accepted that in early developmental stages most of the major TM is secreted by the cells of the underlying organ of KOlliker (Lim, 1972; Gil Loyzaga et al., 1985; Lim and Anniko, 1986; Prieto et al., 1989). However, until now it was generally assumed that all the cells in the organ of K~511iker had the same secretory properties, as their morphology is similar. Using lectin cytochemistry, we have recently shown (Rueda et al., 1989; Prieto et al., 1989) that there are functional differences between different regions of the

organ of K/Slliker: N-acetyl glucosamine and Nacetyl galactosamine are detected in the cell cytoplasm but the labelling, which at birth is distributed over the entire inner half of the organ of K/511iker, is reduced to the inner third of it by the 5th postnatal day (Prieto et al., 1989). However, this cannot be explained by the degeneration process which takes place from the region adjacent to the inner hair cells towards the spiral limbus at around the 8th postnatal day, since the direction of degeneration of the normal organ of K/511iker is opposite to that of the pattern of reduced labelling (Uziel et al., 1981, 1983). The results of the present study support those of lectin cytochemistry as CIH particles are found close to the apical surface of the cells of the inner portion of the organ of K~511iker, and are less frequent towards the outer portion, where they are distinctly separated from the microvilli of these cells. This fact may suggest that only the secretion products of the innermost cells in the organ of K611iker contain sulfated glycoconjugates. In a similar way, the finding that there are no sulfated glycoconjugates in the minor T M points to qualitative differences between the secretion processes of the supporting cells of the organ of Corti (which are thought to form the minor TM: Lim, 1987), and both the interdental cells and the inner cells of the organ of KiSlliker. The results presented here constitute morphological evidence which supports the idea that a Donnan equilibrium exists between the T M and the surrounding fluid: the high negative charge derived from the ionization at a physiological p H of the sulfate groups of glycoconjugates may account for an electrical gradient tending to attract positive ions into the TM, as suggested by Steel (1983). The results of Manley and KronesterFrei (1980) and Runhaar and Manley (1987), demonstrated that the endocochlear potential cannot be recorded in the subtectorial fluid of the inner sulcus, and that the transition between the zero potential in the inner sulcus and the endocochlear potential occurs across the lower surface of the TM. These latter authors suggested that the resistance of the lower region of the TM might then be much higher than that of the sensory epithelium, and related this property to the closely-packed band of fibers present at this location, as described by Kronester-Frei (1978), although there

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was no evidence presented at that time for functional specialization of this zone. However, our CIH staining demonstrates that sulfated glycoconjugates are much more concentrated in the lower region than the upper region of the T M (Fig. la), particulary in the 0.8-1/~m-thick lower layer (Figs. la, 2c). Such a polyanionic layer in the undersurface of the TM may act as an ion barrier, since it has been demonstrated several times that a polyanionic glycoconjugate layer can affect ion mobility in several systems, for example, as in the basement membrane (Rennke et al., 1975; Gupta and Hall, 1979; Johansson, 1983) and the cell coat (Glick and Githens, 1965; Langer et al., 1976; Robertson and Wann, 1987). Thus, it might be expected that a larger concentration of immobile cations (mainly potassium) could exist within the T M than of free cations in the surrounding endolymph. An additional observation which supports this hypothesis, is that the decrease in endocochlear potential caused by anoxia has a slower time course in the TM than in the scala media (Manley and Kronester-Frei, 1980). An unresolved question about the subtectorial fluid composition is whether it is the same in the inner sulcus and in the space over the sensory epithelium. If it is accepted that the sulfated glycoconjugates of the TM are capable of binding cations, and furthermore are responsible for an ionic border between the TM and the inner sulcus fluid, the fact that the band which bears the highest density of sulfated glycoconjugates extends along the entire undersurface of the T M suggests that the ionic composition of the fluid in both subtectorial compartments may be similar. Against this hypothesis it could be argued that the concentration of K + in the inner sulcus fluid is lower than in the endolymph (Runhaar and Manley, 1987), and an appropriate accessibility of K + to the tips of hair cells stereocilia is needed to allow the inward flow of this cation into the hair cells when the channels become open (Hudspeth, 1982). However, since the tips of the tallest stereocilia are embedded in the lower band of the T M this difficulty would be overcome because the tips would be surrounded by a K+-rich ionic microenvironment created by the highly anionic, sulfated glycoconjugates at this location. Although the permeation of ions through the T M would be re-

stricted, a flow of ions could still occur, as pointed out by Runhaar and Manley (1987); in that case not all the K + ions would be irreversibly fixed to the ionized sulfate groups, and an appropriate supply of free K ÷ ions to the tips of the stereocilia would be achieved.

Acknowledgements The authors are grateful to Mrs. Maria Dolores Segura for sectioning the specimens and Mr. Emilio Gutierrez for the illustrations. The authors wish to thank Dr. Enrique Alcaraz for the style corrections, and Dr. David N. Furness and one of the reviewers for helpful comments. This work was supported by a grant from the Spanish Government (CICYT PB 86-0277) and a personal fellowship from the FISS (J.J.Prieto).

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