THE ULTRASTRUCTURE OF THE NEUROMUSCULAR JUNCTIONS OF MAMMALIAN RED, WHITE, AND INTERMEDIATE SKELETAL MUSCLE FIBERS

Distinct ultrastructural differences exist at the neuromuscular junctions of red, white, and intermediate fibers of a mammalian twitch skeletal muscle (albino rat diaphragm). The primary criteria for recognizing the three fiber types are differences in fiber diameter, mitochondrial content, and width of the Z line. In the red fiber the neuromuscular relationship presents the least sarcoplasmic and axoplasmic surface at each contact. Points of contact are relatively discrete and separate, and axonal terminals are small and elliptical. The junctional folds are relatively shallow, sparse, and irregular in arrangement. Axoplasmic vesicles are moderate in number, and sarcoplasmic vesicles are sparse. In the white fiber long, flat axonal terminals present considerable axoplasmic surface. Vast sarcoplasmic surface area is created by long, branching, closely spaced junctional folds that may merge with folds at adjacent contacts to occupy a more continuous and widespread area. Axoplasmic and sarcoplasmic vesicles are numerous. Both axoplasmic and sarcoplasmic mitochondria of the white fiber usually contain intramitochondrial granules. The intermediate fiber has large axonal terminals that are associated with the most widely spaced and deepest junctional folds. In all three fiber types, the junctional sarcoplasm is rich in free ribosomes, cisternae of granular endoplasmic reticulum, and randomly distributed microtubules.     

CORRELATION BETWEEN FIBER LENGTH, ULTRASTRUCTURE, AND THE LENGTH-TENSION RELATIONSHIP OF MAMMALIAN SMOOTH MUSCLE

The length-tension relationship was determined for strips of guinea pig taenia coli and correlated with the length and ultrastructural organization of the component fibers. The mean fiber length in "stretched" strips (passive ≥ active tension) was 30% greater than that for fibers in "unstretched" strips (active >> passive tension). In stretched fibers the dense bodies and 100 A diameter myofilaments were consolidated into a mass near the center of fibers in cross-sectional profile. The thick myofilaments were segregated into the periphery of the fiber profiles. In unstretched fibers the dense bodies-100 A diameter filaments and the thick myofilaments were uniformly distributed throughout cross-sectional profiles. A tentative model is proposed to account for the change in fiber length and ultrastructural organization that accompanies stretch. The basic features of the model require the dense bodies to be linked together into a network by the 100 A diameter filaments. The functional consequences of stretching the fibers are discussed in relation to the model proposed for this network.     

THE ULTRASTRUCTURE OF A MAMMALIAN CELL DURING THE MITOTIC CYCLE

With a technique of preselecting the mitotic cell in the living state for subsequent electron microscopy, it has been possible to examine the ultrastructure of the various stages of mitosis with greater precision than has been reported previously. The early dissolution of the nuclear envelope has been found to be preceded by a marked undulation of this structure within the nuclear "hof." This undulation appears to be intimately related to the spindle-forming activity of the centriole at this time. Marked pericentriolar osmiophilia and extensive arrays of vesicles are also prominent at this stage, the former continuing into anaphase. Progression of the cell through prophase is accompanied by a disappearance of these vesicles. A complex that first makes its appearance in prophase but becomes most prominent in metaphase is a partially membrane-bounded cluster of dense osmiophilic bodies. These clusters which have a circumferential distribution in the mitotic cell are shown to be derived from multivesicular bodies and are acid phosphatase-positive. The precise selection of cells during the various stages of anaphase has made it possible to follow chronologically the morphological features of the initiation of nuclear membrane reformation. The nuclear membrane appears to be derived from polar aggregates of endoplasmic reticulum, and the process begins less than 2 minutes after the onset of karyokinesis. While formation of the nuclear envelope is initiated on the polar aspects of the chromatin mass, envelope elements appear on the equatorial aspect long before the polar elements fuse. Apparently interfering with this fusion are continuous spindle tubules which traverse the chromatin mass in striking density at characteristic points. Several cortical changes, also most pronounced in anaphase, have been described, as has the kinetochore which is seen to good advantage only in this stage. The Golgi complex has been found to disappear both morphologically and histochemically during mitosis and to reappear rapidly in telophase. Evidence is presented which implicates the continuous spindle tubules in certain phases of chromosome movement.     

THE ULTRASTRUCTURE OF THE Z DISC IN SKELETAL MUSCLE

This electron microscopic study deals with the structure of the Z disc of frog's skeletal muscle, with special regard to the I filaments—whether they pass through the Z disc or terminate at it. In most longitudinal sections the I filaments terminate as rod-like projections on either side of the Z disc, one I filament on one side lying between two I filaments on the opposite side. This indicates that the I filaments are not continuous through the Z disc. The rod-like projections are often seen to consist of filaments (denoted as Z filaments) which meet at an angle. In cross-sections through the Z region the I filaments and Z filaments form tetragonal patterns. The I filaments are situated in the corners of the squares; the oblique Z filaments form the sides of squares. The tetragonal pattern formed by the Z filaments is rotated 45 degrees with respect to the tetragons formed by the I filaments on both sides of Z. This structural arrangement is interpreted to indicate that each I filament on one side of the Z disc faces the center of the space between four I filaments on the opposite side of Z and that the interconnection is formed by four Z filaments.     

THE ULTRASTRUCTURE OF STRIATED MUSCLE AT VARIOUS SARCOMERE LENGTHS

1. Rest and equilibrium length muscle sarcomeres are composed of thin filaments (actin) which traverse the sarcomeres from the Z membranes up to the H band; at this level the filaments are considerably thicker and less numerous. 2. Shortening of muscle is associated with a transformation of thin into thick filaments in the A band. 3. These observations are discussed in terms of interaction of actin and myosin to form a supercoiled structure as the basis of contraction.     

ULTRASTRUCTURE OF BARNACLE GIANT MUSCLE FIBERS

Increasing use of barnacle giant muscle fibers for physiological research has prompted this investigation of their fine structure. The fibers are invaginated by a multibranched system of clefts connecting to the exterior and filled with material similar to that of the basement material of the sarcolemmal complex. Tubules originate from the surface plasma membrane at irregular sites, and also from the clefts They run transversely, spirally, and longitudinally, making many diadic and some triadic contacts with cisternal sacs of the longitudinal sarcoplasmic reticulum. The contacts are not confined to any particular region of the sarcomere. The tubules are wider and their walls are thicker at points of contact with Z material. Some linking of the Z regions occurs across spaces within the fiber which contain large numbers of glycogen particles. A-band lengths are extremely variable, in the range 2.2 µm–20.3 µm (average 5.2 µm) Individual thick filaments have thin (110 Å) hollow regions alternating with thick (340 Å) solid ones. Bridges between thick filaments occur at random points and are not concentrated into an M band The thin:thick filament ratio is variable in different parts of a fiber, from 3:1 to 6:1. Z bands are basically perforated, but the number of perforations may increase during contraction.     

哺乳动物不活动化肌纤维的动作电位

用河豚毒素(TTX)慢性阻断大鼠坐骨神经的冲动传导,使后肢不活动,经过不同时间(最长7d)后离体观察了快肌伸趾长肌(EDL)和慢肌比目鱼肌(SOL)肌纤维终板区的诱发动作电位。我们发现在不活动期间动作电位超射和上升速率逐步下降,并从第4天起部分肌纤维能在含有1×10~(-7)g/ml TTX的溶液中被诱发产生动作电位(称抗TTX动作电位),待至第7天时全部SOL肌纤维和90％的EDL肌纤维都能被诱发出抗TTX动作电位。与去神经肌纤维相比,不仅抗TTX动作电位出现较晚,并且其超射和上升速率较低。在去掉TTX阻断使肌肉恢复活动后,动作电位超射和上升速率渐趋恢复,抗TTX动作电位逐渐消失。无论是动作电位的恢复还是抗TTX动作电位的消失,EDL肌纤维均快于SOL肌纤维。本文还讨论了不活动化使肌纤维动作电位变化以及快、慢肌差别的可能原因。     

A MORPHOMETRIC STUDY ON THE NEXUS OF RAT CARDIAC MUSCLE

A morphometric study of the nexus of rat cardiac muscle was carried out. The nexus surface of one intercalated disk of one 15 µm thick fiber is found to range between 47 µm² and 94 µm², the latter value taking into account the maximal underestimation caused by tangential sectioning. Dividing the lower, minimal value by the surface of the observed subunits (90 Å periodicity), one obtains for one intercalated disk 6.7 x 10⁵ subunits, each of them assumed to be permeated by a central pore. These pores are thought to be equivalent to intercellular channels in a recently proposed model. Taking our morphometric and recently reported physiological values, this model is examined for its consistency with a low resistance pathway between cardiac muscle cells.     

THE ULTRASTRUCTURE AND DISPOSITION OF VESICULATED NERVE PROCESSES IN SMOOTH MUSCLE

The walls of the gastrointestinal tract and urinary bladder of rats were fixed in osmium tetroxide, embedded in methacrylate, and sectioned for electron microscopy. The examination of sections of smooth muscle tissue with the electron microscope reveals the presence of bundles of unmyelinated nerve fibers within the intercellular spaces. In addition, vesiculated nerve processes, bounded on their outer surfaces by delicate plasma membranes and typically containing varying quantities of synaptic vesicles and mitochondria, make intimate contact with the surface of smooth muscle cells. These nerve processes are similar in structure and disposition to nerve endings previously described in skeletal muscle, in the central nervous system, in peripheral ganglia, in receptors, and in glands. It is concluded that the relationships existing between vesiculated nerve processes and the surface of smooth muscle cells constitute neuromuscular junctions. Profiles of protrusions of smooth muscle cells are often seen protruding into the intercellular spaces. Here they occur singly or in groups, originating from one or more cells. Because of the plane of section the protrusions may sometimes appear as individual entities between the muscle cells. In such cases care must be exercised in their identification because they have characteristics similar to sectioned nerve processes which also occur in the intercellular spaces.     

大鼠离体心肌粘性劲度定量评定的研究

本实验依据心肌工作模型和心肌顺应性的理论,从大鼠离体心肌静态、动态应力-应变指数曲线和应力松弛对数曲线中,求得反映心肌粘性劲度的指标η_1和η_2,并对心肌组织粘性成分的粘性劲度进行了定量评定。η_1和η_2值与心肌粘性劲度呈同向性变化、且重复性好。本工作提示,η_1和η_2可作为实用可靠的定量评定心肌顺应性的指标。     

肌苷对缺氧心肌跨膜电位和收缩强度的影响

本工作在正常和缺氧情况下,观察肌苷对豚鼠心室乳头肌跨膜电位和收缩强度的影响。结果表明肌苷使正常心肌细胞动作电位时间(APD_(10)、APD_(50)延长。在缺氧心肌,肌苷使细胞静息电位增大,动作电位去极化幅度增高,零期最大去极化速度加快和动作电位时间延长。肌苷增加正常心肌收缩力,使缺氧心肌收缩的衰减显著缓和,亦即使收缩功能改善,且表现剂量-依从性。肌苷对心肌细胞跨膜电位的影响提示它很可能有抗心律失常作用,特别是在缺氧心脏。肌苷对离休乳头肌收缩的影响,证明其对心肌有直接的强心作用。     

THE ULTRASTRUCTURE OF CARDIAC DESMOSOMES IN THE TOAD AND THEIR RELATIONSHIP TO THE INTERCALATED DISC

The fine structure of desmosomes and intercalated discs in the toad heart is discussed. A definite relationship between the dense components of these structures and the dense region of the Z band is demonstrated. The dense region of the Z band characteristically widens at its approach to the plasma membrane, and often terminates beneath it in a distinct discoidal plaque. Cardiac desmosomes appear to be structures which result from the intimate apposition of plaques of Z band material. These desmosomes retain the Z band function as sites of attachment for myofilaments. The suggestion is made that rotation of a desmosome through 90° and splitting of filaments from the adjacent sarcomere could result in the formation of a simple step-like intercalated disc. Intermediate stages in this process are illustrated. Complex discs present in the toad probably represent the alignment of groups of simple discs produced by contractile forces. Possible physiologic functions of the disc and desmosome are discussed. Other morphologic features of toad cardiac cells include a distinct amorphous outer coat to the sarcolemma, a prominent N band, and a granular sarcoplasm with poorly developed reticulum.     

垂体后叶素和加压素对离体心肌的直接作用

本实验采用大鼠离体右心房和右心室肌条模型,观察了垂体后叶素和加压素对右心房和右心室肌的直接作用。结果表明:垂体后叶素对右心房的自主性收缩频率和幅度及右心室肌的收缩幅度均有剂量依赖性抑制作用;加压素对右心房和右心室肌收缩幅度也有剂量依赖性抑制作用,但对右心房自主节律无影响;催产素对右心房的收缩频率和幅度则均无影响。加压素V_1、V_2受体拮抗剂d(CH_2)_5Tyr(Me)AVP和d(CH_2)_5(D-Ile~2,Ile~4,Ala(NH_2)~9)AVP对垂体后叶素的负性变力作用具有不同程度的阻断作用,但对垂体后叶素的负性变时作用无阻断作用。以上结果提示,垂体后叶素的负性变力作用主要是由加压素产生的,加压素对心肌有直接的负性变力作用;垂体后叶素的负性变时作用可能是非加压素和催产素成分的作用结果。     

ARCHITECTURE AND NERVE SUPPLY OF MAMMALIAN SMOOTH MUSCLE TISSUE

Smooth muscle tissue from mouse urinary bladder, uterus, and gall bladder has been studied by means of the electron microscope. The smooth muscle cells are distinctly and completely separated from each other by a cytolemma comparable to the sarcolemma of striated muscle. The tissue is thus cellular and not syncytial. With this evidence, supported by electron microscopy of other tissues, we question the existence of true syncytia in animal tissues. Individual cell membranes necessary for the electrophysiologic events exist in smooth muscle, and its nerve and conduction in a tissue such as uterus or bladder can occur at the cellular level as well as at the tissue area level. The smooth muscle cell contains myofilaments, nucleus, endoplasmic reticulum, mitochondria, Golgi complex, centrosome, and pinocytotic vesicles. These structures are described in some detail, and their probable interrelations and functions are discussed. The autonomic nerves innervating smooth muscle cells are composed of axons and lemnoblasts. The axon is suspended by the mesaxon formed by the infolded plasma membrane of the lemnoblast. The respective plasma membranes separate axon and lemnoblast from each other and from surrounding muscle cells. The axons of autonomic nerves never penetrate the plasma membrane of the muscle cell, but pass or intrude into muscle cell pockets, forming a contact between axonal plasma membrane and smooth muscle plasma membrane. The lemnoblast shows well developed endoplasmic reticulum with Palade granules, mitochondria, and a long, elliptical nucleus. The axon contains neurofilaments, mitochondria, and synaptic vesicles; the quantity of the latter two being significantly greater in the periphery of lemnoblasts and near axon-muscle contact regions. We regard the contact regions as the synapses between the autonomic nerves and the smooth muscle cells.     

SYNTHETIC STRANDS OF CARDIAC MUSCLE : Formation and Ultrastructure

Spontaneously active bundles of cardiac muscle (synthetic strands) were prepared from isolated cells of 11–13-day old embryonic chick hearts which were disaggregated with trypsin. Linear orientation of the cells was obtained by plating them on agar-coated culture dishes in which either grooves were cut in the agar film or a thin line of palladium was deposited over the agar. The influence of cell-to-cell and cell-to-substrate interactions was observed with time lapse cinematography and the formation of the synthetic strand was shown to involve both random and guided cell movements, enlargement of aggregates by accretion and coalescence, and the compact linear arrangement of cells along paths of preferential adhesion. Electron microscope investigations of these strands showed that a dispersed population of heart cells organized into an inner core of muscle cells and an outer sheath of fibroblast-like cells. The muscle cells contained well-developed, but widely spaced myofibrils, a developing sarcoplasmic reticulum associated in part with the myofibrils and in part with the sarcolemma, an abundance of nonmembrane bound ribosomes and glycogen, and a prominent Golgi complex. Numerous specialized contacts were observed between the muscle cells in the strand, e.g., fasciae adherentes, desmosomes, and nexuses. A distinct type of muscle cell characterized by its pale appearance was regularly observed in the strand and was noted to be similar to Purkinje cells described in the adult avian conduction system and in developing chick myocardium. The present findings were compared with other observations of the developing myocardium, in situ, and it was concluded that, by a number or criteria, the muscle cells of the strand were differentiating normally and suitably organized for electrophysiological studies.     

THE GENETICS OF THE MAMMALIAN GAMETE

1. Study of the association of the somatic and gametic genotypes with the gametic phenotype has been termed the genetics of gametes. The latter is closely associated with problems of fertility and infertility; with the general validity of the postulate of random union of the gametes; with experimental attempts to control sex ratio or other Mendelian segregations; and with the study of chromosomal anomalies. Effects of the post-segregational genotype (after meiosis) are of exceptional interest. 2. The dimensions of mammalian spermatozoa show numerous patterns of genetic behaviour, such as strain and breed differences, additive variation, heterosis, high heritability, maternal effects, and a tentative ‘paternal cytoplasmic effect’. The high degree of additive genetic determination is reflected in the smooth progress of a genetic selection programme that brought into existence strains of mice with either long or short spermatozoan midpieces. The balance sheet of variation in mammalian spermatozoan dimensions is as follows. Apart from the natural variation between individual spermatozoa of a male, the paramount factors are the genetic ones. Variation is extraordinarily independent of other biological sources of variation and of environmental ones. The high heritability of most spermatozoan dimensions suggests that they are not closely associated with reproductive fitness, whereas a measure of fitness (resistance of spermatozoa to eosin staining) shows a low heritability. 3. Post-segregational gene action has been sought but not conclusively demonstrated in attempts to recognize X- and Y-bearing spermatozoa visually, and in attempts to control sex ratio by separating them according to electrophoretic or sedimentation behaviour. 4. Variations in the DNA content of spermatozoa, and gross morphological defects, are associated with infertility and their incidence is often genetically controlled. 5. There is controversy over the balance between specific spermatozoan antigens and ‘coating antigens’ acquired from the seminal fluid. Reports that AB individuals (heterozygous for blood-group antigens) produce phenotypically A and B spermatozoa would indicate post-segregational gene action but are also controversial. Sex ratio has not yet been controlled by subjecting spermatozoa to anti-Y-chromosome antibodies. Genetic (strain) differences exist in spermatozoan antigens. 6. The incidence of non-eosinophil spermatozoa is a measure of semen fertility. Strain and breed differences exist but the heritability is low. An absence of demonstrable genetic variation in spermatozoan motility may be due to a swamping effect of non-genetic factors. 7. Diploid spermatozoa differ genetically and phenotypically from the normal haploid ones. The incidence of diploid spermatozoa is associated with infertility and is controlled genetically. The incidence of polyspermy and of supplementary spermatozoa are also controlled genetically. 8. Heterospermic (mixed) insemination from two or more sires discriminates efficiently between the sires in terms of the relative numbers of offspring produced. One experiment showed a genetic control (strain differences) in heterospermic performance. Heterospermic performance of a sire is constant over periods of time and is a predictor of the relative fertility obtained in ‘ordinary’ fertility tests. Heterospermic insemination per se has no apparent practical value. 9. Species crosses in animals tend to produce diploid oocytes and triploid embryos. ‘Foreign’ mammalian eggs or spermatozoa did not fertilize in the same proportion as native ones. 10. The most recent determination shows a I : I sex ratio in the very early embryo. 11. Mammals are scarcely likely to be exempt from certain unorthodox genetic mechanisms, but positive evidence is only indicative. Micro-organisms have been detected and sometimes have a genetic role in spermatozoa and eggs of animals. There is a theory that mitochondria (and therefore most of the spermatozoan midpiece) are modified bacteria. A tentative ‘paternal cytoplasmic effect’ on mouse spermatozoan dimensions, mediated by mitochondria1 inheritance, has been invoked. Conditions may exist for possible transfer of genetic information from spermatozoa to body cells via leucocytes. There is no confirmation of experiments for transfer of genetic information by injection of DNA of specific genotypes prepared from spermatozoa or other tissues. 12. The behaviour of ‘tailless’ alleles in mice provides a unique example of post-segregational gene action, the t-spermatozoa of heterozygous Tt males yielding more offspring than the T-spermatozoa. This work explains the aberrant transmission of t through the male parent, and also the high frequency of the allele in wild populations. It is the best example of post-segregational gene action in animals, provides an exception to the random union of gametes, and permits a novel way of controlling the transmission of genetic information between generations. There is presumptive post-segregational gene action in other segregations in mouse and man. 13. The study of heteroploidy (abnormal chromosome numbers) provides massive evidence of anomalies in the mammalian egg that are in themselves genetic (e.g. diploidy) and phenotypic (e.g. abnormal numbers of polar bodies or pronuclei); that come into being under genetic control, and that have genetic consequences (e.g. triploidy) and phenotypic ones (e.g. inviability) for the embryo. This is illustrated by suppression of the cytoplasmic second meiotic division of the egg, giving diploid parthenogenetic embryos from unfertilized eggs and triploid ones from fertilized eggs. Triploidy is the dominant form of polyploidy in vertebrates. Triploidy is lethal in the mammalian embryo and is a main cause of the ‘natural’ level of prenatal mortality in man. 14. The groups of cells produced by meiosis in both sexes of animals are virtually syncytial. This has a bearing on post-segregational heterosis and gene effects. 15. The unique and apparently indisputable instance of post-segregational gene action in ‘tailless’ mice is to be contrasted on the one hand with numerous persuasive arguments (especially effective in Drosophila) that such action cannot or does not occur in animals or else is mimicked by another phenomenon, meiotic drive; and on the other hand contrasts with an hypothesis that post-segregational gene action occurs on a gigantic scale in all organisms in which crossing-over takes place. It is concluded for the time being that post-segregational gene action does occur in mammals, but is rare. Because of the rarity, a reasonable first interpretation in all gamete genetics is that gene action is mediated by the somatic genotype, unless there are specific reasons to the contrary. There is urgent need for a technique whereby the biological potential of a given spermatozoon can be recognized by fertilizing a given egg with it. 16. As a series of working hypotheses, it is proposed that the general architecture and course of differentiation of gametes are determined by the genotype of the soma and the same pre-segregational genotype of the germ line, the initial gene action being a synthesis of messenger RNA before meiosis in the germ line or at any time in the soma. These effects of the somatic genotype bring the gamete into existence and are also responsible for most of the widespread variation in its phenotype. The gamete so produced can be varied by a very few genes or sets of genes synthesizing messenger RNA in the germ cells after segregation, or else exerting action in some other way connected with their direct physical and chemical nature or their arrangement. These post segregational effects are more likely to be mediated by whole blocs of genes, especially heterochromatic and nucleolar ones, than by ‘billiard-ball’ single genes in the narrow sense.