Histochennical properties of some jaw muscles of the lizard Tupinambis nigropunctatus (teiidae).код для вставкиСкачать
THE ANATOMICAL RECORD 203:345-352 (1982) Histochemical Properties of Some Jaw Muscles of the Lizard Tupinambis nigropunctatus (Teiidae) GAYLORD S. THROCKMORTON ANI) CARL W. SAUBERT I V Department of Cell Biology, The University of Texas Health Science Center at Dallas, Dallas, T X 75235 ABSTRACT In many vertebrate limb and jaw muscles constituent fibers with differing contractile and metabolic properties are distributed so as to produce distinct intramuscular oxidative and glycolytic regions. The purpose of this investigation was to determine if similar compartmentalization exists in jaw muscles of the teiid lizard Tupinambis nigropunctatus. Nine jaw muscles from two adults and one juvenile were examined, and serial sections from each muscle were analyzed using histochemical techniques to indicate relative contractile, oxidative, and glycolytic capacities of the fibers and their patterns of distribution. Three distinct fiber types were observed. The histochemical profile of type 1 fibers most closely resembled that of tonic muscle fibers, while profiles of type 2 and type 3 fibers corresponded to those of fast-twitch glycolytic (FG)and fast-twitch oxidative (FO) fibers, respectively. Three muscles contained only type 2 (FG)fibers, and two muscles contained a noncompartmentalized mixture of all three fiber types. The remaining four muscles were distinctly compartmentalized, having a small, inner oxidative region containing primarily type 1 (tonic)and type 3 (FO)fibers and a larger, outer region consisting entirely of type 2 (FG)fibers. The possible relationships between fiber types, compartmentalization, and jaw function are discussed. Comparison among many mammalian skeletal muscles shows a variable distribution of fiber types, which differ in their contractile and metabolic properties (Guth and Samaha, 1969; Yellin, 1969; Baldwin et al., 1972; Peter et al., 1972; Ariano et al., 1973; Collatos et al., 1977; Gunn, 1978; Armstrong, 1980; English, 1980). In general deep muscles within muscle groups are composed mainly of high-oxidative fastand slow-twitch fibers, while more superficial muscles of the group have high proportions of low-oxidative fast-twitch fibers, and this type of stratification is more prominent in antigravity muscle groups than in their antagonists (Ariano et al., 1973; Collatos et al., 1977; Armstrong, 1980; Armstrong et al., 1982). These differing properties among muscles are reflected in different patterns of muscle utilization during locomotion (Armstrong et al., 1977; Smith et al., 1977; Sullivan and Armstrong, 1978; Walmsley et al., 1978). A similar stratified distribution of fibers also exists within mammalian muscles, giving rise to specialized regions or compartments (Guth and Samaha, 1969; Yellin, 1969; Baldwin et al., 1972; 0003-276x18212033-0345102.50 C 1982 ALAN R. LISS. INC. Gonyea and Ericson, 1977; Gunn, 1978; Armstrong, 1980;English, 1980)that may function differentially (English, 1980). Numerous nonmammalian vertebrates show similar muscle compartmentalization. The iliofibularis muscle of the toad (Liinnergren and Smith, 1966), frog (Engel and Irwin, 1967; Smith and Ovale, 19731, and desert iguana (Gleeson et al., 1980)have a deep (medial)oxidative compartment containing both twitch and tonic fibers (Liinnergren and Smith, 1966; Luff and Proske, 1979; Gleeson et al., 1980) and a superficial glycolytic region. Most other hindlimb muscles of the desert iguana exhibit similar compartmentalization (Putnam et al., 1980). There have been few comparable studies on vertebrate jaw muscles. Several investigators, using various histochemical procedures, have observed regional differences in the fiber composition of several mammalian jaw muscles (Hiiemae, 1971; Suzuki, 1977; Maxwell et al., . ~~ ~~ Received Septemher 18. 1981. accepted March 15. 1982 346 G.S. THROCKMOKTON AND C.W. SAUUERT IV 1979;Throckmorton and Saubert, unpublished observations). Using morphological, histochemical, and electrophysiological techniques, Herring et al. (1979)demonstrated that muscular activity within different histochemical portions of the pig masseter was related to specific phases of the masticatory cycle. Similar histochemical and physiological studies have not been conducted on jaw muscles of lower vertebrates. The purpose of this study was to characterize the composition and compartmentalization of nine jaw muscles of the lizard Tupinambis nigropunctatus using standard histochemical procedures. We observed three types of fibers in these muscles. Four of the jaw muscles were very distinctly compartmentalized with an inner oxidative region surrounded by a glycolytic region. The other muscles showed no compartmentalization and were composed of either pure glycolytic fibers (two muscles) or a mixture of all fiber types (three muscles). MATERIALS AND METHODS The species chosen for study was the teiid lizard Tupinambis nigropunctatus (Peters and Donoso-Barros, 1970).Tupinambis is an omnivorous lizard which will feed on a variety of foods including plant material, raw egg, and small mammals, and thus exhibits a variety of feeding behaviors and serves as a general model for lizard jaw musculature. Three specimens of Tupinambis were used in the study; two adults (1.16 and 8.5 kg body weight) and one juvenile (0.60kg body weight). The specimens were purchased from animal dealers and were housed in wooden cages with sun and heat lamps available and were fed raw egg and baby mice. One animal was purchased in September and maintained in captivity for 10 months, and the other two were purchased in July and were sacrificed within 1 month of arrival. There were no noticeable differences in the muscles among the three specimens except that the muscle fibers of the juvenile were of much smaller diameter than those of the adults. Nine muscles were examined in this study: m. depressor mandibulae, m. levator anguli oris, m. adductor externus superficialis, m. adductor externus medius, m. pterygoideus superficialis, m. pterygoideus profundus, m. pseudotemporalis superficialis, m. pseudotemporalis profundus, and m. adductor posterior (Rieppel, 1980).The muscles were removed bilaterally one at a time, weighed, and then, if it was a large muscle, it was divided into smaller portions for ease of sectioning (See Table 1).In addition, the plantaris and soleus muscles of rats were used as control tissue. Muscles were prepared as described below (see also Table 1). In all instances where sampling occurred the entire thickness of the muscle was retained in the sample for analysis. 1) Depressor mandibulae: In one adult the muscle was cut transversely across the belly to produce origin and insertion portions. In the other adult a sample was taken from the middle of the muscle belly. No specimen was collected from the juvenile. 2) Levator anguli oris: In both adults the entire muscle was used, and no specimen was collected from the juvenile. 3) Adductor externus superficialis: In both adults the muscle was divided into four portions, two at the origin end and two at the insertion end. In the juvenile the entire muscle was used. 4) Adductor externus medius: In both adults a sample was taken from the middle of the muscle belly and divided into anterior and posterior portions. In the juvenile the muscle on the right side was prepared as in the adults and the muscle on the left side was used whole. 5) Pterygoideus superficialis: After removal, the muscle was spread out flat. In the adults the muscle was divided into four parts, producing medial and lateral origin and insertion portions. In the juvenile the muscle was left intact. 6) Pterygoideus profundus: In the adults a sample was taken from the middle of the muscle belly. No specimen was collected from the juvenile. 7) Pseudotemporalis superficialis and pseudotemporalis profundus: In the adult animals, samples were taken from the middle of each muscle belly, while in the juvenile both muscles were used whole. 8) Adductor posterior: In one adult a sample was taken from the middle of the muscle belly; in the other adult the entire muscle was used, and no specimen was collected from the juvenile. Each muscle sample was embedded in tragacanth gum, quick frozen in liquid freon, and then stored at - 70°C until sectioning, when 10-mmserial sections were cut on a microtome cryostat at - 20°C. One section was incubated for myofibrillar adenosine triphosphatase (ATPase) activity following preincubation at pH 10.3 (Guth and Samaha, 1970). Intense staining under these assay conditions indicates fast-twitch contractile properties (Burke et al., 1967; Gleeson et al., 1980). Other serial sections were incubated for reduced diphosphopy- 347 HISTOCHEMICAL PHOPEKTIES OF LIZARD JAW MUSCLES TABLE 1. Locations of muscle repions examined Animal number ~~ Muscle ~~ ~ Depressor Mandihulae Levator Anguli oris ~~ ~~ Middle Whole muscle Whole muscle Anterior near origin Posterior near origin Anterior near insertion Posterior near insertion Adductor Externus Medius Anterior middle Posterior middle Pterygoideus Superficialis Medial near origin Lateral near origin Medial near insertion Lateral near insertion Middle Adductor Posterior T-11 ~ Near origin Near insertion Adductor Externus Superficialis Pterygoideus Profundus Pseudotemporalis Superficialis Pseudotemporalis Profundus ~ T-6 (Juvenile) - Whole muscle Left side Whole muscle Right side Anterior middle Posterior middle Whole muscle Anterior near origin Posterior near origin Anterior near insertion Posterior near insertion Anterior middle Posterior middle Medial near origin Lateral near origin Medial near insertion Lateral near insertion Middle Middle Whole muscle Middle Middle Whole muscle Middle Middle ridine nucleotide diaphorase (DPNH) (Novikoff et al., 1961) and alpha-glycerophosphate dehydrogenase (a-GPDH) (Wattenberg and Leong, 1960) activities to indicate the relative oxidative and glycolytic capacities of the fibers, respectively. Using light microscopy, fibers were classified as either type 1, 2, or 3, depending on the histochemical profile they presented for these three assays. RESULTS Fiber types Based on the histochemical analyses used we have identified three major muscle fiber types in the jaw muscles of Tupinambis (Figs. 1,2,3). Because the histochemical properties of these fibers differ somewhat from those of mammals (Peter et al., 1972)and also from those of lizard limb muscle fibers (Gleeson et al., 1980), we have simply referred to these fibers as types 1, 2, and 3. The possible relationship of these designations to more standard nomenclature for vertebrate muscle fibers types is indicated in Table 2 and will be discussed below. Type 1 fibers stained lightly for ATPase, DPNH, and a-GPDH activities (Table 2, Figs. ~~ Whole muscle 1,2,3)and were found only in muscles with mixed fiber populations or in the oxidative regions of compartmentalized muscles. Type 2 fibers stained darkly for ATPase and a-GPDH activities and lightly for DPNH activity (Table 2, Figs. 1,2,3)and were ubiquitous to all muscles and muscle regions studied. Type 3 fibers stained darkly for ATPase and DPNH activities and lightly for a-GPDH activity (Table 2, Figs. 1,2,3). Like type 1 fibers, type 3 fibers were found only in muscles with mixed fiber populations or in the oxidative regions of compartmentalized muscles. Muscles The jaw muscles of Tupinambis fall into three groups in terms of distribution of fiber types (Table 3);homogeneous, mixed, and compartmentalized. Three muscles, the pterygoideus profundus, adductor posterior, and pseudotemporalis superficialis, consisted entirely of type 2 fibers. Two muscles, the depressor mandibulae and levator anguli oris, contained a mixture of three fiber types, and although there was some regional variation in fiber distribution, there 348 G.S. THROCKMOKTON AND C.W. SAUBEKT 1V T A B L E 2. Chmparison of histochemical properties Alkaline ATPase IY-GPIIH DPHN Light Dark Light Dark Light Light Dark Very dark Very light Hattus noruegicus so Light Intermediate to dark Light Dark FG Very dark . .. FOG Light Dark Dark Dark ~ ~~ SDH ATPase -. (Y GPUlI ~ Dipw\uurus d o r s d i s (Gleeson et a1 , 1980) Dark Dark Light FG FOG Tonic TABLE 3. Muscle characteristics Pure type 2: Pterygoideus profundus Adductor posterior Pseudotemporalis superficialis Completely mixed Depressor mandibulae Levator anguli oris Compartmentalized: Adductor externus superficialis Adductor externus medius Pseudotemporalis profundus Pterveoideus suuerficialis' 'See results on p. 350 was no clear-cut compartmentalization. In the depressor mandibulae approximately half of the fibers were type 1 and they appeared to be evenly distributed throughout the muscle. Type 2 fibers were the least numerous. Although no direct measurements of fiber diameter were made, type 3 fibers appeared to be Fig. 1. Serial section ( x 45) through the m. levator anguli oris of Tupinambis nigropunctatus (specimen T-5) showing ATPase activity following preincubation at pH 10.3. Fiber number 1 represents a lightly stained type 1 (tonic) fiber. Fiber number 2 represents a darkly stained type 2 (FG) fiber. Fiber number 3 represents a darkly stained type 3 (FO)fiber. Fig.2. Serial section as in Figure 1 showing DPNH activity. Fibers 1 and 2 represent lightly stained type 1 (tonic) and type 2 (FG)fibers, respectively. Fiber number 3 represents a darkly stained type 3 (FO)fiber. Light Dark Intermediate to dark Dark Dark Intermediate to dark smaller in diameter than the other types and tended to be found in groups of three or more surrounded by type 1 fibers. In the levator anguli oris type 3 fibers were located primarily around the lateral surface of the muscle where they formed a layer several fibers thick. Deeper in the muscle type 1 fibers predominated, though some type 2 and a few type 3 fibers were also present. Type 2 fibers were most numerous in the deepest portions of the muscle. As in the depressor mandibulae, the type 1 and type 2 fibers tended to be larger than the type 3 fibers. The remaining four muscles were distinctly compartmentalized (Fig. 4). They contained a single, usually small, inner or medial area of mixed fiber population and a larger, outer or more superficial region containing only type 2 fibers. In all samples the boundaries between these two compartments were clear cut with little, if any, intermingling of fiber popula- Fig. 3. Serial section as in Figure 1 showing a-GPDH activity. Fibers 1 and 3 represent lightly stained type 1 (tonic) and type 3 (FO) fibers, respectively. Fiber number 2 represents a darkly stained type 2 (FG)fiber. Fig. 4. A section (16 x ) from the m. pseudotemporalis profundus of Tupinambis nigropunctatus (specimen T-5) showing ATPase activity following preincubation a t pH 10.3. 0 - t h e oxidative region. G - the glycotic region. HISTOCHEMICAL PROPERTIES OF LIZARD JAW MUSCLES 349 350 G.S. THROCKMORTON A N D C.W. SAUHER'I' I V tions. We have termed the outer compartment the glycolytic region because it is composed exclusively of type 2 fibers that exhibit highglycolytic and low-oxidative metabolic potentials. We have termed the inner compartment the oxidative region because this was the only area in these compartmentalized muscles where fibers possessing notable oxidative metabolic capacity (type 3) were found. This fiber organization and regional terminology corresponds to those of compartmentalized lizard hindlimb muscles (Putnam et al., 1980). Of the eighteen samples of the adductor externus superficialis, nine showed a distinctive oxidative region containing all three fiber types; three, all in the anterior region of the muscle from animal T-5, contained only type 2 fibers; the remaining six samples consisted of primarily type 2 fibers with some type 1 fibers found near one edge of the sample. When present, the oxidative region accounted for 25% or less of the total number of fibers in these samples and was located next to the major tendon running through the muscle. The oxidative region contained approximately 60% type 1, 20-3070 type 2, and 10-20% type 3 fibers. The type 3 fibers were located along the medial surface of the oxidative region. Of the eight samples of the adductor externus medius, four contained a distinctive oxidative region located near the main tendon of the muscle. Two oxidative regions had a few type 3 fibers along one edge and the other two consisted entirely of type 1 and type 2 fibers. The type 1 fibers accounted for 60% or more of the fibers in these regions. Again, the oxidative compartment accounted for 25% or less of the muscle fibers present. Of the six samples of the pseudotemporalis profundus, only one, the left side of animal T-11, lacked an oxidative compartment. In the other samples, the oxidative region was relatively large, accounting for 25-30% of the fibers in the sample. Type 1 fibers accounted for 30-4070 of the fibers in the oxidative region and type 3 fibers accounted for 50% or more. Of nine samples of the pterygoideus superficialis examined, only one, the medial portion on the right side of animal T-5, contained fibers other than type 2. In this one sample there were a few type 1 and type 3 fibers along one edge of the sample. Because we did not section the entire length of this large muscle, it seems likely that somewhere within the muscle there is an oxidative region, and a more complete examination of this muscle will be needed to determine the size and location of such an area. DISCUSSION At present most investigators use one of three systems for classifying skeletal muscle fiber types (Brooke and Kaiser, 1970; Burke et al., 1967; Peter et al., 1972).We have used the system proposed by Peter et al. (1972)in which classification is based on three criteria- relative contractile speed, relative oxidative capacity, and relative glycolytic capacity-all of which can be demonstrated either histochemically or biochemically. In addition, two recent studies on lizard hindlimb muscle fiber populations (Gleesonet al., 1980; Putnam et al., 1980) employed the system of Peter et al. (1972),and our use of the same system simplifies comparison of results. We have identified three major muscle fiber types in the jaw muscles of Tupinambis and labeled them as types 1, 2, and 3 because their histochemical profiles are not completely compatible with those reported for mammalian and lizard muscle fiber types. We have employed these labels for discussion purposes only and do not propose their adoption as standard nomenclature. Our type 1 fibers are the most difficult to interpret. Without direct experimental evidence we have identified them as tonic fibers for several reasons. First, their histochemical profile is the same as that for tonic muscle fibers in toad (LBnnergren and Smith, 1966) and frog (Engel and Irwin, 1967; Smith and Ovale, 1973) skeletal muscle, and no twitch fibers in vertebrate skeletal muscle show such a profile. Second, in the compartmentalized jaw muscles of Tupinambis, type 1 fibers are found only in the oxidative region, and an identical pattern of restricted tonic fiber distribution occurs in compartmentalized muscles of frogs (Engel and Irwin, 1967; Smith and Ovale, 1973),toads (Lhnergren and Smith, 1966), and lizards (Putnam et al., 1980). Third, tonic fibers are commonly found in a wide variety of nonmammalian skeletal muscles (see Putnam et al., 1980). The confusing factor is that our type 1 fibers do not have the oxidative capacity of limb tonic fibers in Dipsosaurus (Gleesonet al., 1980), but this may reflect specialization of fiber function, either between lizard limb versus lizard jaw muscles, between lizard species, or between lizard limb versus other vertebrate limb tonic fibers. In addition to their presence in oxidative regions of compartmentalized muscles, type 1 fibers were also present in significant numbers in the two mixed muscles. Our type 2 fibers clearly appear to be fast- IIIS'I'OCHEMICAI, P w P E w I E s OF LIZARD JAW MUSCIXS twitch glycolytic (FG) fibers, as their profiles are identical to those of fibers classified as FG in mammalian (Peter et al., 1972) and lizard (Gleeson et al., 1980)hindlimb muscles. These type 2 (FG) fibers were present in all muscles examined and they represent the dominant jaw muscle fiber population, as they also do in most mammalian (Ariano et al., 1973; Collatos e t al., 1977) and lizard (Putnam et al., 1980) limb muscles. Three jaw muscles and the glycolytic regions of the four compartmentalized muscles were composed entirely of type 2 (FG) fibers, conferring on these muscles and muscle regions the ability to contract rapidly, forcefully, and anaerobically, but limiting their ability to provide high tension levels for prolonged periods of time. Type 2 (FG)fibers were also present in the oxidative regions of compartmentalized muscles but were less numerous than type 1 and type 3 fibers. Our type 3 fibers have histochemical profiles and sizes most closely resembling those of mammalian fast-twitch oxidative (FO) fibers (Nemeth et al., 1979).They lack the glycolytic capacity of fast-twitch oxidative glycolytic (FOG) fibers found in lizard (Putnam et al., 1980)and mammalian (Peter et al., 1972)hindlimb muscles, suggesting that they may be more specialized than similar fibers in locomotory muscles. The pterygoideus profundus, adductor posterior, pseudotemporalis superficialis, and most of the pterygoideus superficialis of Tupinambis are composed entirely of type 2 (FG)fibers, and this may indicate a functional specialization in these jaw muscles. If type 2 fibers behave as mammalian FG fibers do, then these muscles would normally be recruited only when high tension outputs or bursts of power are required or when the more oxidative fibers become fatigued (Armstrong et al., 1974; Gillespie et al., 1974; Armstrong et al., 1977; Smith et al., 1977; Sullivan and Armstrong, 1978; Walmsley et al., 1978). The levator anguli oris and depressor mandibulae muscles have mixed fiber populations and lack definitive oxidative and glycolytic regions. The levator anguli oris retracts the skin of the corner of the mouth during jaw closure in lizards (Lakjar, 1926), and the presence of both twitch and tonic fibers would ensure that this happens quickly, as during prey capture, while the tonic fibers may function to retract the skin during chewing cycles. In other lizard species electromyographic activity from the depressor mandibulae was observed only during the fast part of jaw opening (Throckmor- 351 ton, 1978,1980).However, because tonic fibers produce a very lowfrequency electromyographic signal, the tonic fibers of the depressor mandibulae may have been active during slow opening of the jaws. The organization of four lizard jaw muscles is similar to that of lizard limb muscles (Gleeson et al., 1980).These muscles contain a relatively small oxidative region containing primarily type 1 (tonic)and type 3 (FO)fibers surrounded by a large glycolytic region composed of type 2 (FG) fibers. In these lizard jaw and limb muscles the oxidative region is usually located deep within the muscle and often is near a major tendon. Similar compartmentalization has been reported in mammalian limb (Guth and Samaha, 1969; Yellin, 1969; Baldwin et al., 1972; Gonyea and Ericson, 1977; Gunn, 1978; Armstrong, 1980; English, 1980) and jaw (Hiiemae, 1971; Suzuki, 1977; Herring et al., 1979) muscles, though in these species the boundaries between regions are not as sharp as in lizard muscle. The presence of anatomically different regions in a muscle suggests functional differentiation within the muscle. In mammals the oxidative fibers are utilized during postural support and low to moderate levels of muscular tension, but they may be recruited during all types of activity (Armstrong et al., 1974; Gillespie et al., 1974; Armstrong et al., 1977; Smith et al., 1977; Sullivan and Armstrong, 1978; Walmsley et al., 1978). I t is not unreasonable to propose that our type 3 (FO) fibers are utilized in a similar fashion. We can only speculate that the functional role of our type 1(tonic)fibers is one of joint stabilization, which has been suggested as one possible function of tonic fibers in limb muscles (Simpson, 1979; Putnam et al., 1980).Given this information, we postulate that the oxidative regions of Tupinambis jaw muscles are used for manipulative or postural activities or for stabilizing joints during movement, while the glycolytic regions are used when rapid movement and strong forces are needed, as in chewing or capturing prey. Electromyographic recordings from the different regions of individual muscles are needed to confirm this pattern of differential function, as has been done in mammalian limb (English, 1980) and jaw (Herring et al., 1979)muscles. The presence of organizational specializations in lizard jaw muscles is consistent with the variety of functions and behaviors observed in these animals. Tupinambis exhibits a range of feeding behaviors that involve differ- 352 G.S. 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