735
J. Anat. (1977), 123, 3, pp. 735-743 With 4 figures Printed in Great Britain
Pre-elastic (oxytalan) fibres in the developing elastic cartilage of the external ear of the rat v
V
ZELIMIR BRADAMANTE AND ANTON SVAJGER
Institute of Histology and Embryology, Faculty of Medicine, Zagreb, Yugoslavia
(Accepted 1 June 1976) INTRODUCTION
Elastic fibres were first defined by their staining properties. However, the specificity of elastic staining is doubtful, and the staining mechanism is poorly understood. Consequently, we lack a true histochemistry of elastic fibres (Ross, 1973). In recent years electron microscopic and biochemical investigations have shed light on the nature of elastic fibres (Greenlee, Ross & Hartman, 1966; Ross & Bomstein, 1969). According to current concepts the elastic fibre is a morphologically and chemically heterogeneous structure, with two components (see for review Ross & Bornstein, 1971; Ross, 1973; Fullmer, 1973; Robert, Robert & Robert, 1974), namely: (a) an amorphous component, which has been identified on the basis of its amino acid composition as the cross-linked protein elastin; (b) a microfibrillar component, which is chemically different from both elastin and collagen. It is represented by microfibrils, 10-12 nm in diameter, which appear to be composed of structural glycoproteins. The development of elastic fibres proceeds in two steps; longitudinal aggregates of microfibrils appear first, and these are subsequently impregnated with the amorphous component. Consequently, during elastic tissue histogenesis, the structural glycoprotein shows a relative decrease while the elastin content increases (Greenlee, et al. 1966; Fahrenbach, Sandberg & Cleary, 1966; Ross & Bornstein, 1969, 1971; De Simone-Santoro & Renda, 1971; Giro, Castellani & Volpin, 1974). Bundles of 12 nm thick microfibrils have also been observed in the intercellular matrix of connective tissues generally. These 'microfibrillar fibres' (SerafiniFracassini & Smith, 1974) seem to be identical with the oxytalan fibres which were first described and histochemically defined by Fullmer in 1958 (Carmichael & Fullmer, 1966; Griffin & Harris, 1967; Fullmer, 1973). The histochemical characteristics of oxytalan fibres are as follows: (a) they fail to stain with the stains commonly used for collagen, elastin and reticulin; (b) they stain with acid orcein, aldehyde fuchsin and resorcin fuchsin after previous oxidation; (c) they fail to stain with these dyes after digestion with testicular hyaluronidase, /8-glucuronidase or lysosyme between oxidation and staining; and (d) they are resistant to digestion with elastase prior to oxidation (Fullmer, 1958; Fullmer & Lillie, 1958; Rannie, 1963). As to the significance of oxytalan fibres, two possibilities are currently being proposed: (a) they remain unchanged throughout life in those tissues which do not
736 Z. BRADAMANTE AND A. SVAJGER contain appreciable amounts of elastic fibres in maturity (true oxytalan fibres); and (b) they represent the fibrillar glycoprotein matrix for the subsequent deposition of the amorphous elastin (pre-elastic fibres, Fullmer, 1960a, 1962; Feher, Jennings & Rannie, 1971). The aim of the present study was to establish whether or not during the differentiation of elastic cartilage oxytalan fibres are the forerunners of elastic fibres, and can therefore be regarded as pre-elastic fibres. The cartilage of the external ear of the rat was chosen for this investigation because its abundant elastic fibres appear relatively late in ontogenesis (gvajger, 1970). 7A
V
MATERIAL AND METHODS
The inbred Fischer strain of albino rat was used in the investigation. The day on which sperms were found in the vaginal smear early in the morning was considered as the first day of embryonic development. On the next morning the embryos were considered to be 1 day old. The rudiments of the external ear of embryos from the sixteenth to twenty second day of development (i.e. 15 to 21 day old embryos) and the external ear of newborn animals from the first to the fifth postnatal day (0 to 4 day old rats) were examined. The embryos were isolated from uteri of pregnant females killed with ether. The newborn rats were killed by decapitation. The entire external ear (auricle+ external meatus) was excised, rinsed in isotonic saline and fixed in Lillie's buffered neutral formol (Romeis, 1968) for 12 hours. Specimens were dehydrated, embedded in paraffin wax and sectioned serially at 7 ,m. The sections were examined after the following treatments: (1) Staining with Gomori's aldehyde fuchsin (Gomori, 1950) for 15 minutes. (2) Staining with aldehyde fuchsin after previous oxidation of de-paraffinized sections with peracetic acid for 20 minutes (Fullmer, 1958). (3) Staining with aldehyde fuchsin after previous digestion of sections with 0 01 % elastase (ESFF, 1FB, Worthington) in Holmes' 0-2 M borate buffer, pH 8-8, at 37 °C for 30 minutes (Bodley & Wood, 1972). (4) Staining with aldehyde fuchsin after previous digestion of sections with elastase and oxidation with peracetic acid. (5) Staining with aldehyde fuchsin after oxidation with peracetic acid and digestion with 0-05 % bovine testicular hyaluronidase (EC 3.2.1.35, Fluka AG, Buchs SG) in Sorensen's phosphate buffer, pH 6 1, at 37 °C for 20 hours (Pearse, 1968). (6) Gomori's silver impregnation of reticular fibres (Romeis, 1968). For clear demonstration of the finest intercellular fibres counterstains were not used in the above listed staining procedures. (7) Mallory's aniline blue collagen stain (Sheehan & Hrapchak, 1973). As a control, aortae from 20 day fetal rats were subjected to the same treatments, except Gomori's silver impregnation and Mallory's staining of collagen.
Pre-elastic fibres in the rat ear cartilage
737
Table 1. Staining of elastic and pre-elastic (oxytalan) fibres in developing rat ear cartilage Oxidation +
Aldehyde fuchsin Prenatal development
Postnatal development
16th day 17th day 18th day 19thday 20th day 21st day 22nd day 1st day 2nd day 3rd day 4th day 5th day
Elastase + Elastase+ Oxidation + oxidation aldehyde + aldehyde aldehyde fuchsin fuchsin fuchsin
-
-
-
-
+/+ + + +
-
hyaluronidase+ aldehyde fuchsin -
+ + + + + + + +
+ + + + + + + +
+
+
-
+ +
+ +
+/+/-
-
-
RESULTS
In sections stained with aldehyde fuchsin (without or after previous oxidation), the elastic and pre-elastic fibres respectively appeared as thin purple threads on a colourless background. The keratinized cells of the epidermis and mast cell granules were the only tissue constituents which also stained with aldehyde fuchsin. Cartilage ground substance, which shows a marked affinity for aldehyde fuchsin, appears in rat ear cartilage at a later stage in development than any examined here (Svajger, 1970). Elastic ear cartilage The results of staining are partly demonstrated in Table 1 and are illustrated in Figures 1-4. In sections stained with aldehyde fuchsin alone, a very few elastic fibres were visible on the second postnatal day. From the second to the fifth day they increased in number, being localized predominantly in the central part of the pre-cartilaginous plate (Fig. 4). They failed to stain in sections pre-treated with elastase. In sections stained with aldehyde fuchsin after previous oxidation, single, very fine fibrils could be detected in the pre-cartilage on the seventeenth day of development. On the nineteenth day they were already abundant in the pre-cartilage, in the perichondrium and in the dermis of the auricle (Fig. 1). Beginning on the twenty second day these fibres displayed the definitive pattern of distribution of future elastic fibres in the ear cartilage as seen in the 10 days old rat (Figs. 3 and 4). This pattern remained unaltered in sections treated with elastase (30 minutes) before oxidation and staining (Fig. 2). When pre-oxidized sections were digested with testicular hyaluronidase, aldehyde fuchsin failed to stain fibrillar intercellular structures in the pre-cartilage. A very few faintly coloured fibrils could be seen at the fourth and fifth postnatal days. After Gomori's silver impregnation fine reticular fibres were visible in the chondro-
738
Z. BRADAMANTE AND A. SVAJGER _r-
.AP"*".
wl-
'i Id
I
,r
.,. :X=
A
Fig. 1. Oxytalan fibres in the pre-cartilage of the ear of a 20 day rat embryo. Oxidation+ aldehyde fuchsin. x 1000.
2
J. Anat. (1977), 123, 3, pp. 735-743 With 4 figures Printed in Great Britain
Pre-elastic (oxytalan) fibres in the developing elastic cartilage of the external ear of the rat v
V
ZELIMIR BRADAMANTE AND ANTON SVAJGER
Institute of Histology and Embryology, Faculty of Medicine, Zagreb, Yugoslavia
(Accepted 1 June 1976) INTRODUCTION
Elastic fibres were first defined by their staining properties. However, the specificity of elastic staining is doubtful, and the staining mechanism is poorly understood. Consequently, we lack a true histochemistry of elastic fibres (Ross, 1973). In recent years electron microscopic and biochemical investigations have shed light on the nature of elastic fibres (Greenlee, Ross & Hartman, 1966; Ross & Bomstein, 1969). According to current concepts the elastic fibre is a morphologically and chemically heterogeneous structure, with two components (see for review Ross & Bornstein, 1971; Ross, 1973; Fullmer, 1973; Robert, Robert & Robert, 1974), namely: (a) an amorphous component, which has been identified on the basis of its amino acid composition as the cross-linked protein elastin; (b) a microfibrillar component, which is chemically different from both elastin and collagen. It is represented by microfibrils, 10-12 nm in diameter, which appear to be composed of structural glycoproteins. The development of elastic fibres proceeds in two steps; longitudinal aggregates of microfibrils appear first, and these are subsequently impregnated with the amorphous component. Consequently, during elastic tissue histogenesis, the structural glycoprotein shows a relative decrease while the elastin content increases (Greenlee, et al. 1966; Fahrenbach, Sandberg & Cleary, 1966; Ross & Bornstein, 1969, 1971; De Simone-Santoro & Renda, 1971; Giro, Castellani & Volpin, 1974). Bundles of 12 nm thick microfibrils have also been observed in the intercellular matrix of connective tissues generally. These 'microfibrillar fibres' (SerafiniFracassini & Smith, 1974) seem to be identical with the oxytalan fibres which were first described and histochemically defined by Fullmer in 1958 (Carmichael & Fullmer, 1966; Griffin & Harris, 1967; Fullmer, 1973). The histochemical characteristics of oxytalan fibres are as follows: (a) they fail to stain with the stains commonly used for collagen, elastin and reticulin; (b) they stain with acid orcein, aldehyde fuchsin and resorcin fuchsin after previous oxidation; (c) they fail to stain with these dyes after digestion with testicular hyaluronidase, /8-glucuronidase or lysosyme between oxidation and staining; and (d) they are resistant to digestion with elastase prior to oxidation (Fullmer, 1958; Fullmer & Lillie, 1958; Rannie, 1963). As to the significance of oxytalan fibres, two possibilities are currently being proposed: (a) they remain unchanged throughout life in those tissues which do not
736 Z. BRADAMANTE AND A. SVAJGER contain appreciable amounts of elastic fibres in maturity (true oxytalan fibres); and (b) they represent the fibrillar glycoprotein matrix for the subsequent deposition of the amorphous elastin (pre-elastic fibres, Fullmer, 1960a, 1962; Feher, Jennings & Rannie, 1971). The aim of the present study was to establish whether or not during the differentiation of elastic cartilage oxytalan fibres are the forerunners of elastic fibres, and can therefore be regarded as pre-elastic fibres. The cartilage of the external ear of the rat was chosen for this investigation because its abundant elastic fibres appear relatively late in ontogenesis (gvajger, 1970). 7A
V
MATERIAL AND METHODS
The inbred Fischer strain of albino rat was used in the investigation. The day on which sperms were found in the vaginal smear early in the morning was considered as the first day of embryonic development. On the next morning the embryos were considered to be 1 day old. The rudiments of the external ear of embryos from the sixteenth to twenty second day of development (i.e. 15 to 21 day old embryos) and the external ear of newborn animals from the first to the fifth postnatal day (0 to 4 day old rats) were examined. The embryos were isolated from uteri of pregnant females killed with ether. The newborn rats were killed by decapitation. The entire external ear (auricle+ external meatus) was excised, rinsed in isotonic saline and fixed in Lillie's buffered neutral formol (Romeis, 1968) for 12 hours. Specimens were dehydrated, embedded in paraffin wax and sectioned serially at 7 ,m. The sections were examined after the following treatments: (1) Staining with Gomori's aldehyde fuchsin (Gomori, 1950) for 15 minutes. (2) Staining with aldehyde fuchsin after previous oxidation of de-paraffinized sections with peracetic acid for 20 minutes (Fullmer, 1958). (3) Staining with aldehyde fuchsin after previous digestion of sections with 0 01 % elastase (ESFF, 1FB, Worthington) in Holmes' 0-2 M borate buffer, pH 8-8, at 37 °C for 30 minutes (Bodley & Wood, 1972). (4) Staining with aldehyde fuchsin after previous digestion of sections with elastase and oxidation with peracetic acid. (5) Staining with aldehyde fuchsin after oxidation with peracetic acid and digestion with 0-05 % bovine testicular hyaluronidase (EC 3.2.1.35, Fluka AG, Buchs SG) in Sorensen's phosphate buffer, pH 6 1, at 37 °C for 20 hours (Pearse, 1968). (6) Gomori's silver impregnation of reticular fibres (Romeis, 1968). For clear demonstration of the finest intercellular fibres counterstains were not used in the above listed staining procedures. (7) Mallory's aniline blue collagen stain (Sheehan & Hrapchak, 1973). As a control, aortae from 20 day fetal rats were subjected to the same treatments, except Gomori's silver impregnation and Mallory's staining of collagen.
Pre-elastic fibres in the rat ear cartilage
737
Table 1. Staining of elastic and pre-elastic (oxytalan) fibres in developing rat ear cartilage Oxidation +
Aldehyde fuchsin Prenatal development
Postnatal development
16th day 17th day 18th day 19thday 20th day 21st day 22nd day 1st day 2nd day 3rd day 4th day 5th day
Elastase + Elastase+ Oxidation + oxidation aldehyde + aldehyde aldehyde fuchsin fuchsin fuchsin
-
-
-
-
+/+ + + +
-
hyaluronidase+ aldehyde fuchsin -
+ + + + + + + +
+ + + + + + + +
+
+
-
+ +
+ +
+/+/-
-
-
RESULTS
In sections stained with aldehyde fuchsin (without or after previous oxidation), the elastic and pre-elastic fibres respectively appeared as thin purple threads on a colourless background. The keratinized cells of the epidermis and mast cell granules were the only tissue constituents which also stained with aldehyde fuchsin. Cartilage ground substance, which shows a marked affinity for aldehyde fuchsin, appears in rat ear cartilage at a later stage in development than any examined here (Svajger, 1970). Elastic ear cartilage The results of staining are partly demonstrated in Table 1 and are illustrated in Figures 1-4. In sections stained with aldehyde fuchsin alone, a very few elastic fibres were visible on the second postnatal day. From the second to the fifth day they increased in number, being localized predominantly in the central part of the pre-cartilaginous plate (Fig. 4). They failed to stain in sections pre-treated with elastase. In sections stained with aldehyde fuchsin after previous oxidation, single, very fine fibrils could be detected in the pre-cartilage on the seventeenth day of development. On the nineteenth day they were already abundant in the pre-cartilage, in the perichondrium and in the dermis of the auricle (Fig. 1). Beginning on the twenty second day these fibres displayed the definitive pattern of distribution of future elastic fibres in the ear cartilage as seen in the 10 days old rat (Figs. 3 and 4). This pattern remained unaltered in sections treated with elastase (30 minutes) before oxidation and staining (Fig. 2). When pre-oxidized sections were digested with testicular hyaluronidase, aldehyde fuchsin failed to stain fibrillar intercellular structures in the pre-cartilage. A very few faintly coloured fibrils could be seen at the fourth and fifth postnatal days. After Gomori's silver impregnation fine reticular fibres were visible in the chondro-
738
Z. BRADAMANTE AND A. SVAJGER _r-
.AP"*".
wl-
'i Id
I
,r
.,. :X=
A
Fig. 1. Oxytalan fibres in the pre-cartilage of the ear of a 20 day rat embryo. Oxidation+ aldehyde fuchsin. x 1000.
2
