Histochemical immunohistochemical and ultrastructural observations on the iris muscles of Gallus gallus.код для вставкиСкачать
THE ANATOMICAL RECORD 221:687-699 (1988) Histochemical, Immunohistochemical, and Ultrastructural Observations on the Iris Muscles of Gallus gallus P.A. SCAPOLO, S.M. PEIRONE, G. FILOGAMO, AND A. VEGGETTI Institute of Veterinary Anatomy, University of Bologna, 40126 Bologna, Italy (P.A.S., A . V); Institute of Veterinary Anatomy (S.M.P.)and Institute of Human Anatomy (G.FI), University of Torino, 10126 Torino, Italy ABSTRACT The distribution and typology of fibers in the two muscular systems (sphincter and dilator) of the iris in Gallus gallus were determined histochemically, immunohistochemically, and ultrastructurally. The sphincter muscle in proximity to the ciliary margin was composed predominantly of slow fibers. In the intermediate tract, a large group of fast oxidative fibers were evident and the pupillary margin was exclusively composed of slow fibers. The fast fibers had histochemical and immunohistochemical patterns similar to the a fibers in the skeletal control muscle (biventer cervicis). In contrast, the slow fibers were composed of at least three slow types, which were comparable to the isoforms of the different myosins in 01 and & skeletal fibers. In the dilator muscle, the oblique system was uniquely composed of fast oxidative fibers. The radial system was predominantly composed of slow fibers with isoforms of myosins different from the slow fibers of the sphincter and control muscles. Ultrastructural features (width of Z bands, extension of the sarcoplasmicreticulum and SR-T tubule junctions, and number of mitochondria) confirm the histochemical and immunohistochemical assessments of fiber types, even if some peculiar aspects in several fibers were observed. Smooth muscle cells separated from striated fibers were evident at the pupillary margin. The hypothesis of a mesenchymal origin for all irideal striated muscles is discussed. It is generally known that the musculature of the avian iris is composed of striated muscle fibers organized into circumferential (sphincter muscle) and radial (dilator muscle) systems (Lewis, 1903; Leplat, 1912; Oehme, 1969). In particular, in Gallus gallus, the opinion has been that both systems (sphincter and dilator) of striated fibers are derived from neuroectodermal cells of the retinal epithelium in proximity to the pupillary border (Walls, 1942;Duke-Elder, 1958;Rohen, 1964).Therefore, researchers in the last 20 years have focused on the embryonic development of this striated muscle and its innervation (Hess, 1966;Zenker and Krammer, 1967; Pilar and Vaughan, 1971; Lucchi et al., 1974; Marchi et al., 1980; Narayanan and Narayanan, 1981; Mussini et al., 1982; Ferrari and Koch, 1984a,b; Mussini, 1984; Giacobini et al., 1984; Mussini et al., 1984). Specific research on the ultrastructure of striated iris muscle fibers is rare, both in adult Gullus gallus (Zenker and Krammer, 1967) and other avian species (Oliphant et al., 1983). Moreover, at present, histochemical and immunohistochemical data on these muscles is lacking. Recently, the attention of researchers has shifted to a smooth muscle component that, in the species so far studied, is poorly developed and of which little is known (Gabella and Clarke, 1983; Fischer and Dieterich, 1985). The present work investigates the nature of both components of the iris musculature in the adult chicken at Q 1988 ALAN R. LISS, INC. the ultrastructural, histochemical, and immunohistochemical levels, the typology and distribution of the iris muscle fibers in the sphincter and dilator systems, and their relationship to smooth muscle cells. A preliminary report has been given elsewhere (Peirone et al., 1986). MATERIALS AND METHODS The study was conducted on the irises of male and female chickens (Gallus gallus) between 6 months and 2 years of age. Histochemistry and lmmunohistochemistry Five subjects were decapitated while deeply anesthetized with ketalar (ketamine hydrochloride, Parke-Davis) and the irises were removed from the ocular globe after removal of the cornea. The irises were then dissected free at the root from the back of the vitreous body and the lens. Each iris was Placed on a sample of skeletal muscle (biventer cervicis) from the same subject and frozen in isopentane cooled in liquid nitrogen. The irises were radially sectioned in a cryostat and treated for the following activity: 1) Ca-dependent myosin ATPase Received August 3,1987; accepted October 28,1987. 688 P.A. SCAPOLO ET AL TYPOLOGY OF AVIAN IRIS MUSCLE FIBERS 689 Fig. 2. Chicken iris. Transverse sections. a,b: Ciliary portion of sections equidistant from the iris margins. a, m-ATPase at pH 4.6. x86. b, am-ATPase at pH 9.4. ~ 2 6 0c,d: . Sections equidistant from the iris margins. m-ATPase at pH 4.6 (c) and pH 10.2 (d). ~ 6 0e,E . Sections next to the ciliary margin. m-ATPase at pH 4.6 (el and pH 10.2 (0. ~ 1 5 8 S, . sphincter muscle; R and 0, radial and oblique systems, respectively, of the dilator muscle; M, control muscle. Fig. 1. a-c: Fiber typology of the control skeletal muscle, biventer cervicis. ~ 2 9 0 a: . Double stain with a-ALD (yellow) and a-IIA (blue) sera. b: m-ATPase at pH 4.6. c: m-ATPase at pH 10.35. d-g: Chicken iris. Transverse sections. Double stain with a-ALD (yellow) and a-IIA (blue) sera. d: Section next to the ciliary margin. ~ 7 6 e:. Detail of panel d. ~ 1 9 6f:. Section equidistant from the ciliary and pupillary margins. ~ 7 0 g:. Radial section. ~ 5 0 Inset: . Detail of the pupillary margin. ~ 1 7 0 h: . Distribution of the fast and slow fibers in an iris sector. S, sphincter muscle; R and 0, radial and oblique systems, respectively, of the dilator muscle; M, control muscle. 690 P.A. SCAPOLO ET AL. RESULTS Observations With the Light Microscope Typology of the control muscle Three fundamental types of fibers in the control muscle (biventer cervicis) were histochemically identified as slow tonic fibers, 01 and Pz, and fast CY, according to Toutant et al. (1981). The p1 and 0 2 fibers were positive to SDH and had an m-ATPase activity very resistant to the acid preincubation and a certain degree of alkaline stability. However, the 01 fiber had less activity with both acidic and alkaline preincubation than the 02 (Fig. lb,c). The CY fibers had a high m-ATPase activity, alkaline stable and acid labile (Fig. lb,c). These fibers could be further subdivided into two subgroups based on SDH oxidative reactions of varying degrees of intensity (very intense to totally absent) that resulted in different stains. The immunohistochemical tests confirmed the presence of slow and fast myosins in the 0 and CY fibers, respectively (Fig. la). The (Y fibers reacted only to sera against the fast myosin (a-IIA, a-FW) and not to those against the slow Fig. 3. Chicken iris. Transverse section next to the ciliary margin. myosin. The 0 fibers were positive to the sera against SDH. x86. S , sphincter muscle; R and 0, radial and oblique systems, the slow myosin (a-S, a-SHC, and a-ALD).Nevertheless, respectively, of the dilator muscle; M, control muscle. the fibers had a major affinity for the a-ALD serum compared to the 02 fibers, with an evident cross-reaction in the immunohistochemically double-stained preparations with serum against the myosin of the white muscle (adenosine triphosphatase) with acid and alkaline prein- of teleosts (a-FW). cubation (m-ATPase) according to Brooke and Kaiser (1970) as modified by Lewis et al. (1982), and Guth and Sphincter muscle Samaha (1969); 2) Ca-Mg-dependent actomyosin The sphincter muscle, the more developed of the two ATPase, pH 9.4 (am-ATPase)according to Mabuchi and Sreter (1980); and 3) succinic dehydrogenase (SDH), ac- muscles in the iris, occupies the anterior part of the iris cording to Nachlas et al. (1957). The sections were also stroma. Next to the pupillary margin, the sphincter immunostained with indirect immunoperoxidase and muscle is reduced to a few threads of fiber that are immunobetagalactosidase (Bondi et al., 1982). In se- bypassed by the retinal epithelium (Fig. lg). The fibers lected slides, a double immunostaining technique was progressively increase in number toward the ciliary applied (Gugliotta et al., 1982) using one or more of the margin to occupy a greater part of the stroma. The following antisera: 1)fast twitch antimyosin of mam- sphincter muscle fibers follow a circular arrangement, mals (a-IIA);2) fast antimyosin from the white muscle and are generally larger (diameter 10-18 pm) than the of teleosts (a-FW); 3) slow twitch and slow tonic anti- fibers of the dilator muscle. The types of fibers described below are located in difmyosin of mammals (a-S); 4) slow tonic antimyosin of chicken (a-ALD); and 5) antisera against the heavy ferent sectors of the muscle. Almost all the fibers in chains of the slow myosin from the red muscle of teleosts proximity to the ciliary margin were positive only to the (a-SHC).For details concerning the specificity of sera see sera against the slow myosin (a-S, a-SHC, and a-ALD) Carpene et al. (1982) and Mascarello et al. (1982) for 1 (Fig. lf,g) with a low or medium am- and m-ATPase and 3; see Rowlerson et al. (1985) for 2 and 5; and see activity. The m-ATPase activity was moderately alkaline and acid-stable. Because the degree of acid-stability Pierobon Bormioli et al. (1980)for 4. was less than the 01 and 02 fibers of the control muscle Electron Microscopy (Fig. 2a,b), the slow fibers at the ciliary margin were The irises were rapidly removed from four subjects called slowl. Only a few of the fibers in the intermediate tract were anesthetized with ketalar. Prior to revival, the subjects were sacrificed. The irises were immediately immersed slowl, most being positive only to fast antimyosin serum in a 2.5% solution of glutaraldehyde in a phosphate (a-IIA and a-FW) (Fig. lf,g). These fast fibers were simibuffer for 4 hr. After immersion in the fixative, each iris lar to the fast fibers of the control muscle, having a high was fragmented with 8 radial cuts. The fragments were am- and m-ATPase activity, and being acid-labile and postfixed in 1%OsO4 for 1 hr, dehydrated, placed in alkaline-stable (Fig. 2c-f). A small group of these fast araldite, and oriented in the direction of the various fibers in proximity to the ciliary margin merged with the slowl fibers (Figs. lf,g, 2b). The fast fibers decreased muscular systems. Semithin sections were stained with toluidine blue for in number toward the pupillary margin until only those analyzing the whole iris muscle in complete sections. fibers positive to the slow antimyosin sera (reacting According to the distribution of the fibers as determined uniformly to the a-S and a-SHC sera) remained at the by light microscope observations, ultrathin sections were pupillary margin (Fig. lg). The majority of these fibers, called slow2, were made with lead citrate and examined with a Siemens Elmiskop 1A electron microscope. strongly positive to a-ALD serum, as were the slowl TYPOLOGY OF AVIAN IRIS MUSCLE FIBERS fibers (Fig. l g inset). Those that were less reactive were called slow3. All the fibers of the pupillary margin had a low am- and a low m-ATPase activity, moderate alkaline stability, and a variable degree of acid stability, although always less than slowl fibers. All the fibers of the sphincter muscle group were positive to SDH (Fig. 3). Dilator muscle The dilator muscle, smaller than the sphincter, occupies the posterior part of the iris stroma immediately in front of the retinal epithelium. It is composed of radial fibers of very small diameter (4-10 pm) and oblique fibers of slightly larger diameter (10-12 pm). The boundary between the two fiber systems is indistinct because the fibers are intermixed. The radial system is more extensive than the oblique, extending from the ciliary margin to the pupillary limit of the sphincter. In contrast, the oblique fibers are in proximity to the ciliary margin, directly in contact with the radial system and intermixing with sphincter muscle fibers (Fig. lh). The oblique system was predominantly formed from fibers positive to anti-fast myosin sera (a-IIA, a-FW) (Fig. ld,e), having a high am- and m-ATPase activity and being alkaline-stable and acidlabile (Fig. 2c-0. These fibers were similar to the fast fibers in the sphincter muscle and to the Q! fibers in skeletal muscle. On the other hand, the radial system was formed predominantly from fibers positive only to sera against the slow myosin (a-S, a-SHC, and a-ALD) (Fig. Id-g). However, a small group ( 5 4 % ) next to the ciliary margin was positive only to sera against the fast myosin (a-IIAand a-FW).Sporadic fibers cross-reacted with sera against the fast and slow myosins as seen in the immunohistochemically double-stained preparations (Fig. le). The evaluation of the am- and m-ATPase activity in the single fibers of the radial system was problematic, since the smallness of fibers did not permit observation of the light-chromatic variations. These variations differentiated the types of fibers in other parts of the iris musculature as well as in the control muscle. The amand m-ATPase activity appeared to be medium-low, moderately alkaline-stable (Fig. 2e,0 and acid-stable at pH 4.65, but not at a pH lower (Fig. 2c,d) than the slowl and slow2 fibers of the sphincter muscle (Fig. 2c). The radial slow fibers, different from the other slow fibers in the iris in ATPase activity, were called slow4. In some radial fibers, however, the degree of acid and alkaline stability of the am- and m-ATPase reaction seemed to be higher than in the slow4 fibers. These fibers were probably the very small group of fibers that reacted to antifast sera or to both anti-fast and anti-slow sera, but it was difficult to follow the same fiber in the serial sections. Both the radial and oblique systems of the dilator muscle were positive to SDH (Fig. 3). Ultrastructural Observations General aspects Even if the iris muscle fibers ultrastructurally resemble skeletal muscle fibers, they differed in diameter size and structural organization. All of the iris fibers had an abundance of variously distributed mitochondria with a matrix of different electron densities. They were loosely dispersed or clumped together among the myofibrils or 691 accumulated along the fiber until they formed sarcolemma1 protrusions (Fig. 40. The elongated nuclei of the fibers were usually in the subsarcolemmal position. Frequently in the sarcoplasm, large lipidic vacuoles were observed that sometimes contained membrane-like profiles (Figs. 4a,C 7e,0. These vacuoles were isolated among the organelles of the fiber or arranged in stacks that filled the fiber and seeped into the myofibrils. In some cases, disorganized tracts along the fiber were noted in which the nuclei were centrally located, the myofibrils were missing, and bands of myofilaments were dispersed in all directions in a sarcoplasm rich in ribosomes, vesicles, cisterns of sarcoplasmic reticulum, and numerous small, round mitochondria (Fig. 4a,b). The fibers, independently of type and diameter, were interconnected at the ends by deep indentations. On the sarcoplasmic side, thin filaments of myofibrils were often anchored to subsarcolemmal groups. The space between two contiguous fibers contained a flaky substance (Fig. 44. In every section of the iris muscle, only en grappe myoneural junctions were observed. The synaptic buttons contained large accumulations of clear, rounded vesicles, and some scattered dense-core vesicles. The subneural apparatus was smooth and lacked infoldings (Fig. 4e). Types of fibers The following parameters that differentiate skeletal fibers were used to characterize the iris muscle fibers: density and compactness of myofibrils, width of the Z bands, and the development and distribution of sarcoplasmic reticulum and T system. Even with the extreme variability of these ultrastructural characteristics, the fundamental order that characterizes the principle types of skeletal fibers was recognized in the iris muscles. Sphincter muscle. The sphincter muscle, with the exception of the pupillary margin, had two fundamental types of fibers that represent the extremes of a wide range of variability. Fiber A (Fig. 5a,b) was characterized by registered myofibrils with a thin Z band, mostly medium and large mitochondria, and a discretely developed sarcoplasmic reticulum. At the level of the I band, the sarcoplasmic reticulum formed a dense network of tubules that enveloped each myofibril. From this network, single tubules or a loosely woven network of tubules formed part of a small tract in the A band. In cross section, the single myofibrils appeared to be partially surrounded by elements of sarcoplasmic reticulum. The T system was also well developed with triads formed from two terminal cisterns, recognizable by their granular content (dark tubules), bordering a transverse light tubule. They were frequently visible in longitudinal and transverse sections. Atypical groups of five tubules, formed from four paired dark tubules with a light tubule and diads of a light and a dark tubule were observed (Fig. 5c-el. In contrast, the B fibers (Fig. 5f) were differentiated from the A fibers by smaller and more numerous mitochondria and a less developed sarcoplasmic reticulum. In transverse section the myofibrils were not distinct. The T system was poorly developed and the triads and other complexes were infrequent. Between these two extreme types, A and B, there were fibers with intermediate characteristics. In correspondence to the pupil- Fig. 4. Chicken iris. Sphincter muscle. a,b: Atypical muscle fibers (arrows) next to normal fibers. a: Semithin section. Note the numerous vacuoles in the fiber. X560. b: Ultrathin section. The myofilaments are not organized to form myofibrillar bands. X 11,000. c , d Myomuscular contacts. F, Flaky material between the fibers; arrows indicate subsarcolemmal densities. c, X36,OOO. d, ~13,000.e: Synaptic button on muscle fiber. Note the absence of infoldings. ~23,000. f: Mass of mitochondria peripherally located in a muscular fiber. Arrows indicate some vacuoles containing membrane-like profiles. x 11,400. TYPOLOGY OF AVIAN IRIS MUSCLE FIBERS Fig. 5. Chicken iris. Sphincter muscle. a,b: Longitudinal and transverse sections (respectively) of fibers of type A. Note the well-developed sarcoplasmic reticulum (SR) and the triads (arrowheads)placed in the A band. a, x 16,800. b, ~22,000.c-e: Unusual forms of the junctions 693 between sarcotubules and T tubules. c: Diads. d, e: Pentads of different forms, c, e, x 73,600. d, ~55,200.E Type B fiber. Note the reduced dimensions of the mitochondria and the poorly developed sarcoplasmic reticulum (SR). ~23,000. 694 P.A. SCAPOLO ET AL. Fig. 6. Chicken iris. Region next to the pupillary margin. a-d: Lon- triads (arrowheads). a: Type C fiber. ~9,400.b: Type D fiber. x 13,000. gitudinal sections of striated fibers of sphincter muscle. Note the dif- c: Detail of panel a. x 17,600. d: Detail of panel b. ~27,600.e: Smooth ference in the thickness of the Z band and the different positions of the muscle cells with pigment granules (P). x 11,000. TYPOLOGY OF AVIAN IRIS MUSCLE FIBERS 695 Fig. 7. Chicken iris. Dilator muscle, radial system. a: Muscle fibers close to the retinal epithelium (RE); note the poorly defined structure. at various orientations (arrows). Note the small diameter. RE, retinal c, x 15,000. d, X27,OOO. e, f: Longitudinal and transverse sections e,.X6,OOO. f, epithelium. ~6,000.b: Transverse section of type F fiber close to the (respectively)of type G fiber. Note the large vacuoles 0’) retinal epithelium (RE). ~8,000.c , d Smallest-diameter type E fibers x 21,000. 696 P.A. SCAPOLO ET AL. TABLE 1. Summary of the histochemical and immunohistochemicalstaining and ultrastructural properties of fiber types in the iris muscle (sphincter and dilator) and their likely correlations Dilator muscle Oblique system Suhincter muscle Radial system Pupillary margin Histochemical and immunohistochemical criteria Cross- Fiber types Anti-FW Anti-IIA Anti-S Anti-SHC Anti-ALD SDH am-ATPase m-ATPase after: Alkali preincubation Acid preincu- Slow1 Slow2 slow3 - - - - - - Fast +++ +I+ t + +++ +++ ++ ++ ++ + + ++ +++ +++ ++I +1 +1 - + - ++ ++ ++ ++ - - Fast reaction ++ ++ ++ ++ ++ ++ +++ +++ - - +++ - +++ +++ +++ ++ +++ +++ +++ ++ slow4 - ++ +I+ + bation Ultrastructural characteristics Fiber types Fibrils3 B - D C - +- F A + G + E - + Very few thick organized Irregular and poorly Z-line Sarcoplas. Ret.4 Triads4 Diameters thick + - thick + (I band) + (I band) thin + + (A band) 10-18 Fm thin +++ (I band) ++ (A&A/Iband) thick + + ++ (A/Iband) ++ - (A band) 4-12 um Histochemical and immunohistochemical criteria: relative stain intensities, on an arbitrary scale, increasing from 'Positive above pH 4.5. 'Positive above pH 4.65. + = distinct (Fibrillenstruktur-like);- = indistinct (Felderstruktur-like). 4 + + +, + +, +, - = very well, well, poorly, very poorly developed, respectively. lary margin, two additional types of fibers were present. The first, fiber C (Fig. 6a), was characterized by very compact myofibrils in register, a thick Z band, and a very electron-dense matrix of mitochondria, extending primarily along the axis of the fiber. At the level of the I band, the sarcoplasmic reticulum made a collar that enveloped the myofibril with a few extensions that intruded into the A band (Fig. 6c). In contrast, fiber D (Fig. 6b) had fewer compacted myofibrils and thinner Z bands and was more uneven. The sarcoplasmic reticulum formed a collar a t the level of the I band with small extensions that intruded into the A band at times for the entire sarcomere (Fig. 6d). The tubular system in both C and D fibers was not very developed. The triads, oriented in various ways, were located in fibers a t the level of the I band in the C fibers and at the level of the A band in D fibers. A well-developed smooth muscular component was intermixed with striated fibers at the pupillary margin. These smooth cells, oriented in the same manner as the sphincter striated muscles, contained altered pigment granules (Fig. 6e). - - to ++f. Dilator muscle: Radial system. Not all the fibers seemed to have the same radial disposition when observed ultrastructurally (Fig. 7a), but they all had very small diameters with heterogeneous characteristics that were not identifiable with the sphincter types. At least three types of fibers were discernible. The first type, E fibers (Fig. 7c,d), corresponded to the smaller-diameter fibers immediately juxtaposed to the retinal epithelium, with scarce and poorly organized myofibrillar bands and sarcoplasmic reticulum. These fibers are reminiscent of the embryonic muscle elements during myofibril formation. A second type, F fibers (Fig. 7b), next to the epithelium but with a slightly larger diameter than that of E fibers, had well-organized myofibrils, a poorly developed sarcoplasmic reticulum a t the level of both the I and the A bands, few elements of the T system, and abundant mitochondria. The last type, G fibers (Fig. 7e), had the largest diameter and was characterized by abundant scattered mitochondria, developed and organized myofibrils, a welldeveloped sarcoplasmic reticulum a t the level of the I band, and triads at various levels of the A band (Fig 70. TYPOLOGY OF AVIAN IRIS MUSCLE FIBERS Dilator muscle: Oblique system. The fibers of this system were similar to those of the A fibers in the sphincter muscle. The histochemical, immunohistochemical, and ultrastructural characteristics of the iris muscle fibers are summarized in Table 1. DISCUSSION Generai Considerations In the adult chicken, the striated muscle fibers in the iris sphincter and dilator muscles are very small in diameter (4-18 pm). Yet the structural features, although extremely heterogeneous, are reducible to distinct typological categories based on histochemical, immunohistochemical, and ultrastructural parameters that are valid for striated skeletal fibers. Even so, the previously described categories are not comparable to those of the control muscle, confirming the pecularity of the striated iris musculature with respect to the skeletal type of somitic origin. Very important differences also result in the histochemical, immunohistochemical, and ultrastructural typologies of the dilator and sphincter components. These differences have not been reported in the literature because of the paucity of ultrastructural data concerning the adult, and have instead been attributed either to the iris musculature as a whole or only to the sphincter muscle. The iris musculature is fundamentally composed of fast and slow fibers of a primarily oxidative metabolism. These results are in agreement with the findings of Mussini et al. (1983), who have shown the presence of light chains characteristic of slow and fast myosin in the iris muscular complex by means of electrophoretic analyses. Histochemical and lmmunohistochemical Characteristics Fast fibers always have histochemical and immunohistochemical patterns similar to the (Y fibers of skeletal muscle. In contrast, the slow fibers are composed of distinct myosin isoforms that are different from the slow tonic 01 and 62 fibers of skeletal muscle (Toutant et al., 1981). The myosin polymorpnism in the slow fibers of the iris muscles is revealed histochemically by different degrees of acid resistance to m-ATPase activity. Their acid resistance is similar to that of slow fibers and lower than that of fast fibers. The different isoforms of the myosin of slow2 and slow3 at the pupillary margin of the sphincter muscle have been recognized immunohistochemically by a-ALD serum that differentiates the PI from the & fibers in the skeletal muscle. The histochemical and immunohistochemical differences described above preclude the certain discrimination of slow twitch from tonic myosin isoforms in the iris musculature. Given what has been demonstrated in the ALD muscle (Pierobon Bormioli et al., 1980), the group of slow iris fibers with a more acid-stable m-ATPase activity should be referred to as the slow twitch type, and those that are less acid-stable as the slow tonic type. The possibility of recognizing the slow twitch and tonic myosins could not be examined in reference to the control muscle, biventer cervicis, in which slow & and & fibers have a different degree of m-ATPase activity to the acid preincubation, and are considered by Toutant et al. (1981)to be tonic. The anti-ALD serum is obtained against the myosin of the anterior latissimus dorsi muscle, which is largely composed of slow tonic fibers (Ash- 697 more et al., 1978; Rouaud and Toutant, 1982). Positivity to anti-ALD serum discriminates the slow tonic from the twitch fibers in mammals, reptiles, and amphibians but not in birds, because the slow twitch fibers are also labeled (Pierobon Bormioli et al., 1980;Mascarello et al., 1982, 1983; McVean et al., 1987). Ultrastructural Characteristics The iris fibers, in spite of the analogy to skeletal fibers, have specific ultrastructural aspects. The SR-T complexes are situated at different levels of the A band or at the center of the I band (C fibers of the sphincter). They have variable orientations, are composed of triads and of tetrads and pentads in the fast fibers. In skeletal fibers, the SR-T complexes are situated at the level of the I band in proximity to the I-A junction, have a transverse orientation, and are composed of triads in the fast fibers and diads in the slow fibers. However, since a systematic study has not been done, one cannot exclude the possibility that a different degree of contraction of single fibers at the moment of fixation may have an effect on the position of the triads. The arrangement of the SR-T complexes in the iris fibers, which of necessity are contractile, is probably tied to the architecture and innervation of the iris muscles. According to the literature, these en grappe motor terminations are exclusively typical of slow fibers in skeletal muscle. Disorganized tracts along the fibers of the sphincter muscle could be due to growth processes-fusion of uncertainly derived myogenic elements, given the few satellite cells, or to atrophic processes, as described in the denervated skeletal fibers (Zelena and Jirmanova, 1973; Hikida and Bock, 1976; Eisenberg et al., 1984)-consequent to muscular remodeling. Other peculiar aspects of the iris muscle, or myofibers, that have been reported in the literature, have to do with the density of mitochondria and the connections among the fibers. The distribution of mitochondria in skeletal muscle is dependent on the type of contraction, whereas mitochondria appear to be randomly distributed in the iris muscle (Eisenberg and Salmons, 1981; Ovalle, 1982; Ogata and Yamasaki, 1985). The myomuscularjunctions, at the level of what AChE activity has been demonstrated (Zenker and Krammer, 1967; Mussini et al., 1984), are reminiscent of adherent fascia of the cardiac cells in respect to structure. However, they differ by a lack of closed junctions. Distribution of the Different Types of Fibers The frequency and distribution of the fast and slow fibers in the two muscular components of the iris musculature have been demonstrated with preparations of double immunostaining. In the sphincter muscle at the ciliary margin, the long fibers are slowl. In the intermediate tract, the slowl fibers are intermixed with a large group of fast fibers. At the extreme pupillary margin, the sphincter is exclusively composed of slow fibers (slow2 and slow3). The oblique system of the dilator muscle is composed of fast fibers, whereas the radial system is composed of slow fibers (slow4)with diameters among the smallest of the iris muscles. Electron microscope observations of the fiber types determined by the light microscope permit an indirect synthesis of the histo-immuno properties of these fibers with the ultrastructural data. This includes the extension of the sarcoplasmic reticulum and its relationship 698 P.A. SCAPOLO ET AL with the T system, the myofibril arrangement, and the thickness of the Z band. The sphincter muscle at the extreme pupillary margin is composed exclusively of fibers characterized by two distinct isoforms of slow myosin (slow2 and slow3). The ultrastructural examination demonstrated two types of fibers (C and D) with characteristics similar to those of the slow tonic fibers of skeletal muscle (Page, 1969).The concentration at the pupillary border of slow fibers, which are probably tonic since they maintain prolonged contractions, could mean that this section of sphincter muscle is implicated in the maintenance of myosis. The presence also of a smooth muscle component reinforces this possibility and may maintain the degree of tonicity. The same hypothesis has been recently advanced by Oliphant et al. (1983). In Bubo virginianus, a nocturnal raptorial bird, the constricting musculature of the iris at the pupillary border is completely smooth. In the other sections of the sphincter muscle, the wide variability of ultrastructural features ranges between the two extremes expressed by fibers A and B. The A fibers may be related to the fast oxidative type, whereas the B fibers may be similar to the slow1 fibers. The fact that the B fibers are not a homogeneous type histochemically (with a low or medium m-ATPase activity) could explain many of the intermediate ultrastructural features in the fibers of this sector. The fast component, which is particularly concentrated in the intermediate tract, may be responsible for the rapidity of the sphincter response to light stimuli. At the ultrastructural level of the oblique system in the dilator muscle, A fibers have been demonstrated that are referable to the fast oxidative type. However, it is more problematic to correlate the histochemical and immunohistochemical data to the radial system, in which at least three types of fibers have been demonstrated. Fiber F, at the “Felderstruktur” myofibrillar organization, could correspond to the slow4 fibers; fiber G, at the “Fibrillenstruktur” organization, is a small group of fibers positive to anti-fast sera; and fiber E, characterized by the smallness of the diameter and few organelles, could correspond to the few fibers that cross-reacted with the sera against the fast and slow myosin. The significance of E fibers remains obscure, although the histochemistry could reflect the immature elements of myofibril organization. In fact, in fibril genesis, the myosins pass through a phase in which they react indistinguishably to fast and slow antimyosin sera (Masaki and Yoshizaki, 1974; Gauthier et al., 1978). The various typological compositions, when compared in the two dilator muscle systems, can be attributed to their different functional roles. For example, the oblique system would not be implicated in the dilation of the pupil but in the mechanism of accommodation of the lens neplat, 1912). Smooth Muscle As recently demonstrated in Bubo virginianus (Oliphant et al., 1983) and Pica pica (Fischer and Dieterich, 1985), smooth muscle cells have been shown among the striated fibers of the sphincter muscle in proximity to the pupillary margin in the adult chicken, confirming preliminary observations (Peirone, 1986). All of the observed smooth muscle cells are rich in pigment _ - .=an., ules, unlike the striated fibers. The presence of distinct smooth muscle in the adult contradicts the results of some authors who have only observed smooth muscle in embryos (Mussini et al., 1976). Our results, however, are in agreement with the findings of those authors who recently proposed reconsidering the embryonic origin of the iris muscle (Filogamo, 1981; Gabella and Clarke, 1983; Nakano and Nakamura, 1985; Yamashita and Sohal, 1986). These authors suggest that the smooth muscle is of neuroectodermic origin and the striated muscle derives from the iris mesenchymal cells. These cells could be neurally induced, similarly to skeletal muscle, or induced by neural crest cells (Filogamo, 1981)that are already present at the fourth incubation day (Johnston et al., 1979). From this developmental stage, elements with myogenic potentiality in the iris stroma (in vitro) (Peirone and Vercelli, 1984), which contain desmin, ACh, and ACh receptors (in vivo) (Sisto Daneo et al., in press; Filogamo, personal communication),are observed. ACKNOWLEDGMENTS The authors wish t o thank professor S. Schiaffino, of the University of Padova, who kindly furnished the serum a-ALD. 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