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Histochemical immunohistochemical and ultrastructural observations on the iris muscles of Gallus gallus.

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THE ANATOMICAL RECORD 221:687-699 (1988)
Histochemical, Immunohistochemical, and
Ultrastructural Observations on the Iris Muscles of
Gallus gallus
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
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).
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.
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.
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.
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.
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
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
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.
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
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.
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.
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.
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
Suhincter muscle
Radial system
Pupillary margin
Histochemical and
immunohistochemical criteria
Fiber types
m-ATPase after:
Alkali preincubation
Acid preincu-
+I+ t
+I+ +
Fiber types
Very few
and poorly
(I band)
(I band)
(A band)
10-18 Fm
(I band)
(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).
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
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.
Dilator muscle: Oblique system. The fibers of this system
were similar to those of the A fibers in the sphincter
The histochemical, immunohistochemical, and ultrastructural characteristics of the iris muscle fibers are
summarized in Table 1.
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
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
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-
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
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.
The authors wish t o thank professor S. Schiaffino, of
the University of Padova, who kindly furnished the
serum a-ALD.
This work was supported in part by CNR grant No.
8500645.04 to Professor G. Filogamo and by CNR and
MPI grants to Professor S.M. Peirone and Professor A.
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ultrastructure, muscle, iris, immunohistochemical, observations, gallus, histochemical
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