J. Anat. (1979), 128, 3, pp. 637-667 Printed in Great Britain
Proceedings of the Anatomical Society of Australia and New Zealand AUGUST 1978
The sixteenth annual meeting of the Anatomical Society of Australia and New Zealand was held on 28-30 August 1978 at Monash University, Clayton, Victoria. In addition to scientific communications invited papers were given to symposia on Biological Control Mechanisms and Medical Education. Workshop sessions were conducted in the following areas: photographic techniques; freeze-fracture techniques; conducting group discussions and the formulation of educational objectives. The various sessions were chaired by Professors M. J. Blunt, J. B. Carman, R. M. Clarke, E. F. Glasgow, Dr J. R. Haight, Professors D. M. de Kretser, J. Priedkalns, A. W. Rogers, F. W. D. Rost, Dr P. J. M. Tutton and Professor C. P. Wendell Smith. The following are the authors' abstracts of papers presented.
COMMUNICATIONS 1. Biological control mechanisms. By D. METCALF. Walter and Eliza Hall Institute Knowledge of the biological control systems operating to control the continuous proliferation of haemopoietic populations has advanced rapidly in the last decade with the development of semisolid culture systems permitting the clonal growth of haemopoietic progenitor cells to form colonies of up to 100000 differentiating progeny cells. Cloning systems are available for granulocyte-macrophage, eosinophil, erythroid, megakaryocyte, T- and B-lymphoid and mixed haemopoietic populations. These cloning systems have an extremely high plating efficiency for primary cultures and can detect most progenitor cells in the bone marrow, spleen or blood. In each system, cell proliferation is wholly dependent on the continuous presence of a highly specific regulatory factor (colony stimulating factor, CSF). There is no direct evidence for the necessity for additional factors promoting differentiation and the CSFs either have a double action, stimulating proliferation and differentiation by distinct intracellular processes or differentiation is preprogrammed in the genome to occur after a fixed number of cell divisions. The best-characterised regulators, controlling granulocyte-macrophage and erythroid proliferation (GM-CSF and erythropoietin) have been purified, are glycoproteins and are active at 10-11 M concentrations. All haemopoietic progenitor cells are generated by a small number of multipotential haemopoietic stem cells that are also capable of extensive self-replication. The factors controlling the commitment of stem cells to specific progenitor cells partly involve cell contact processes with neighbouring microenvironmental cells but the recent development of an in vitro cloning assay for multipotential cells now indicates that this commitment process is also influenced by humoral regulatory factors. Modulating inhibitory factors have also been detected by the various cloning techniques but their specificity and in vivo relevance are unclear at present. 2. Control of spermatogenesis. By D. M. DE KRETSER. Monash University Modulation of spermatogenesis is achieved at several levels by a number of different biological mechanisms. These control mechanisms can be grouped as (i) hormonal; (ii) receptor; and (iii) cellular. Function of the spermatogenic apparatus requires the adequate secretion of FSH and LH by the pituitary gland. The levels of these gonadotrophic hormones are modulated by the stimulatory action of a gonadotrophic releasing factor (GnRH) from the hypothalamus and inhibitory feedback signals from the gonads. FSH acts directly on the seminiferous epithelium which in turn exerts negative feedback control of FSH secretion probably via inhibin, a non-steroidal hormone. LH does not act directly on the seminiferous epithelium but influences spermatogenesis indirectly by the stimulation of testosterone secretion from the interstitial cells.
0021-8782/79/2828-6160 $00.35 C 1979 Anat. Soc. G.B. & L 41
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The action of hormones necessitates their binding to specific receptors on target tissues as the first step in the initiation of their biological effect. Thus receptors for FSH are found on Sertoli cells and spermatogonia, for LH on the interstitial cells and for testosterone on Sertoli cells. Adequate receptor numbers appear to be necessary for optimal hormone action and evidence exists that high levels of a specific hormone can decrease or down-regulate its receptor numbers and interfere with the subsequent biochemical events. Since the rate of spermatogenesis is a biological constant for each species, it is evident that cellular control mechanisms exist to regulate the process. Germ cells entering the spermatogenic process either progress at specific rates or degenerate. Though FSH has been demonstrated to influence the rate of entry of stem cells into spermatogenesis the subsequent factors which regulate the rate of the process remain unknown. The different times taken for spermatogenesis in different species indicate that substantial interspecies variations occur in the rate of a basic process such as meiosis. 3. Why are tissues recognisable histologically? By A. W. ROGERS. Flinders University of South Australia We accept, often without discussion, that tissues and organs may be identified from their histological appearance. Our ability to recognise them implies a remarkable constancy in the size, shape, orientation and spacing of cells and extracellular elements. This constancy often extends across species, so that it may be easier to identify the tissue than the animal from which it came. Such structural organisation implies local control mechanisms of great precision, so that a cell complies not only on the basis of cell type (differentiational history), but also on its position within the tissue. Our ignorance of these local control mechanisms is almost total. The principal barriers to investigating these mechanisms are technical. Observations must be related to cell type and position, and must be made on very small volumes of tissue. Morphometry and autoradiography meet these requirements, and are capable together of giving a lot of information about cell activity. The uterus of the ovariectomised rat is a useful model. It is structurally simple and composed of few cell types. The size, shape, spacing and activity of many of its cells can be dramatically altered by a single injection of either progesterone or oestradiol. This will be a progress report rather than a definitive summary. It is already clear, however, that existing techniques can generate a great deal of information about these local control mechanisms. 4. Control of epithelial cell proliferation in the small intestine - the villouslongistat. By P. J. M. TUTTON. Monash University Mechanisms for the control of cell proliferation in those tissues where continuous cell loss is balanced by continuous cell production are still poorly understood. In this review experiments which perturb the rate of either crypt cell proliferation and/or villous cell loss are analysed and it is hypothesised that cell division in the intestinal crypt is regulated by the interaction of two systems, one of which is highly localised, responding to changes in villous cell loss (the villous longistat), whilst the other is generalised throughout the small intestine, and responds to the nutritional requirements of the animal. Villous length tends to be maintained at a constant value despite circumstances which alter the life expectancy of villous cells. Thus, although villous cells live longer in the intestine of gnotobiotic animals and in by-passed segments of intestine, villi do not become abnormally long, but instead, the rate of cell production is reduced. Conversely, when villous cell loss is increased by ischaemia or by lysis, the rate of crypt cell production is increased and villous shortening is avoided. When crypt cell proliferation is temporarily suppressed by cytoxic drugs or irradiation villi do become shorter. However, when the replicating cells have recovered from the inhibiting agent cell production proceeds at a very rapid rate until, and only until, the villi have been restored to their normal length. These responses by which crypt cell production is adjusted to match villous cell loss are highly localised and can be demonstrated in one segment of gut whilst cell proliferation occurs at its unperturbed rate in an adjacent segment. By contrast, when the nutritional needs of the animal are increased, for example during lactation, there is a generalised increase in crypt cell proliferation and manifest lengthening of the villi.
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5. Influence of light on gonadotrophic function in the Zebra finch (Taeniopygia guttata castanotis). By J. PR1EDKALNS AND R. K. BENNETT. University of Adelaide The onset of breeding activity in the Zebra finch (Taeniopygia guttata castanotis) inhabiting the arid central regions of Australia is normally not induced photoperiodically but follows rainfall. It is, however, not known whether greater variation of day/night length than under field conditions may influence its gonadal growth and function. To examine the question, and also to determine if a possible effect of light may interact with pineal function, Zebra finches obtained in the Alice Springs area were maintained in the laboratory at constant temperature (27 °C), humidity (30 %), and regimen of dry food and water ad libitum. In six experimental groups, consisting of 72 birds, the variables were gradually increasing (12-16-20 hours/day) or decreasing (12-8-4 hours/day) photoperiods, with or without prior pinealectomy or sham pinealectomy. Birds were maintained for 2 weeks at the final photoperiod. In the control group (20 birds) all parameters were kept constant. It was observed that long photoperiods increased (to 33-6 ± 2-2 mg, p < 0-05) and short photoperiods did not significantly alter (19-2 ± 2 5 mg) testes weight, cf. control weight of 19-4 ± 2-4 mg. These effects were not significantly varied by prior pinealectomy, although, under both long and short photoperiods, the pinealectomised birds had somewhat higher testes weights than intact birds. Body weights did not vary significantly. It is concluded that, at greater variation of day/night length than under field conditions, the gonadotrophic system of the Zebra finch inhabiting the arid central regions of Australia may respond to increased photoperiods and pinealectomy does not significantly influence the response. 6. Retino-geniculo-cortical pathway in two new world primates: an autoradiographic study. By M. H. ROWE, M. REZAK AND L. A. BENEVENTO. University of Illinois, U.S.A. The pattern of geniculostriate projections has been examined in the owl monkey and the squirrel monkey by means of transneuronal autoradiography. One mCi each of tritiated fucose and proline were injected into the vitreous chamber of one eye of an adult squirrel monkey and an adult owl monkey. The animals survived 3 weeks and the brain sections were exposed to Kodak NTB-2 emulsion for 6-8 weeks. First order grains were visible to the naked eye in the superior colliculus, pretectum and dorsal lateral geniculate nucleus. With the aid of dark field microscopy, second order grains were readily apparent in structures such as the inferior pulvinar and striate cortex, presumably due to transsynaptic transport from the superior colliculus and lateral geniculate nucleus, respectively. In both animals, grains were seen only in cortical area 17 and not in area 18, suggesting that the lateral geniculate nucleus does not project to this area in either species. In both animals, grains were largely confined to layer IV, but there was a slight suggestion of a projection to layer VI. In striate cortex of the squirrel monkey, both ipsilateral and contralateral to the injected eye, grains were found in layer IVA and in both subdivisions of layer IVC. In this respect, the geniculocortical projection in this species is identical to that described in the macaque. In the squirrel monkey, however, there was no tendency for inputs originating in the two eyes to be segregated into horizontally arranged patches or columns. In this respect, the squirrel monkey differs from the macaque. In the owl monkey, both ipsilateral and contralateral to the injected eye, grains were entirely confined to the two subdivisions of layer IVC in striate cortex, with no evidence of a projection to layer IVA. In some regions of the ipsilateral striate cortex, grains in layer IVCa were arranged in discrete horizontally arranged patches. Such patches were not seen in striate cortex contralateral to the injected eye or in layer IVC/ in the ipsilateral side. Thus, a limited degree of ocular segregation does appear to be present in the geniculostriate projection of the owl monkey. It is suggested that laminar separation of geniculocortical terminals is associated with laminar segregation of functional cell types in the lateral geniculate nucleus, while horizontal separation represents a segregation of inputs of the basis of ocularity. 7. Effect of haemodynamic factors on the distribution of anionic groups in the glomerular capillary wall. By G. B. RYAN AND M. J. KARNOVSKY, University of Melbourne and Harvard Medical School, U.S.A. Using an ultrastructural colloidal iron technique, the distribution of anionic groups was examined in the rat glomerulus following fixation under different haemodynamic conditions. Whereas the entire glomerular capillary wall showed heavy staining for anionic groups following 4I-2
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immersion fixation, superficial glomeruli fixed in situ during good blood flow showed little labelling within or deep to the endothelial fenestrae. These findings were unexpected in view of recent data indicating that plasma albumin is normally held up at the level of the endothelial fenestrae, and that such barrier function breaks down as blood flow stops. If, as has been suggested, intrinsic negative groups within the endothelial fenestrae are primarily responsible for limiting the passage of polyanionic plasma proteins between the capillary lumen and urinary space, the opposite result would have been predicted. Thus current concepts of the distribution of anionic groups in the glomerulus and their involvement in normal and abnormal glomerular permeability need to be reassessed. The heavy labelling detected in the region of the endothelial fenestrae in immersion-fixed material probably results from the clumping of albumin and other plasma polyanions following their diffusion into the glomerular capillary wall as blood flow stops. The epithelial podocytes label most heavily with colloidal iron whatever the method of fixation, and may, by imposing a negatively charged field across the width of the basement membrane, play the most significant role in charge-related glomerular barrier function. 8. FITC-labelled antibodies to identify smooth muscle. By JULIE CHAMLEY-CAMPBELL, R. CAMPBELL AND UTE GROSCHEL-STEWART. Baker Medical Research Institute and Institut fur Zoologie, Darmstadt, German Federal Republic Antibodies have been prepared against native actin, myosin, tropomyosin and denatured 55000 dalton subunit of 10 nm filaments from chicken gizzard smooth muscle. In immunofluorescence (where pre-immune sera and antigen absorbed antisera give negative results), the antibodies against actin, tropomyosin and the 10 nm filament protein stain appropriate structures in smooth muscle cells, irrespective of phenotypic state, in culture and in fresh frozen sections; i.e. staining occurs in smooth muscle cells in the contractile functional state, where the cytoplasm is filled with myofilaments and contraction can occur given the appropriate stimulus, and in the ' modulated' phenotypic state (e.g. developing or regenerating smooth muscle, subculture), where the smooth muscle cytoplasm is filled with organelles involved in synthesis (rough endoplasmic reticulum, ribosomes) with scattered bundles of thin filaments and no thick filaments and contraction cannot be induced. The antibody against myosin stains smooth muscle cells in the contractile functional state but not in the 'modulated' state. None of the antibodies stain fibroblasts present in cultures or frozen sections, and only the antibody to the 10 nm filament protein will stain endothelial cells. These antibodies provide a quick and reliable method for distinguishing smooth muscle cells from fibroblasts and endothelial cells in culture and in fresh frozen sections.
9. An ultrastructural and histochemical study on the bulbourethral glands of the short-nosed bandicoot, Isoodon macrourms. By B. W. MACDONALD and R. L. HUGHES. Queensland Institute of Technology and University of Queensland The bulbourethral glands from sexually mature animals were fixed by immersion or by perfusion using aldehydes. The periodic acid-Schiff reaction, alcian blue pH 1 0 and 2 5 procedures, and diastase neuraminidase, and hyaluronidase digestions were employed for the carbohydrate histochemical characterisation of columnar secretory cells. The columnar secretory cells form a series of dilated branched tubules within the gland. The chromatin pattern within the nuclei of the secretory cells was either diffuse or clumped. The basal region contained mitochondria, rough endoplasmic reticulum, and in some instances, many secretory granules. A supranuclear Golgi apparatus and numerous secretory granules of varying sizes were prominent in the apical region of the cells. A few microvilli were present on the luminal surface. A strong periodic acid-Schiff reaction was observed in the cytoplasm of the secretory cells. This reaction was not abolished by diastase treatment. While most cells displayed a moderate to strong alcianophilia following alcian blue pH 2-5, a few cells gave a negative reaction. A negative reaction was observed following the alcian blue pH 1-0 procedure. The alcianophilia was reduced following neuraminidase digestion and was abolished by acid hydrolysis. The alcian blue pH 2 5 reaction was unaffected by hyaluronidase digestion. The carbohydrate histochemical characterisation indicates that the secretory cells contain sialomucin and neutral proteoglycan. The ultrastructural findings support the concept that the secretory cells are capable of synthesis and storage of carbohydrate and proteins.
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10. Anatomical segregation of W-, X- and Y-cell inputs to cat striate cortex. By A. G. LEVENTHAL. University of New South Wales Small ionotophoretic injections (01-O 5 mm in diameter) of horseradish peroxidase (HRP) were made into different layers of area 17 of the cat. The size and location of labelled relay cells in the lateral geniculate nucleus (LGNd) were then studied. Three trends were evident in the results. First, injections of HRP into the deeper portions of layer III or layer IV labelled mostly medium sized relay cells in laminae A and Al. Second, an injection into the superficial portion of layer IV labelled many large relay cells in laminae A and Al, a lesser number in laminae C and very few in the medial interlaminar nucleus
Third, although small relay cells located in laminae C1-3 were labelled infrequently, their proportions were highest following injections superficial to layer IV Considerable evidence indicates that medium sized geniculate relay cells in laminae A and Al correspond to the physiological class X-cells, large relay cells in laminae A, Al, C and MIN correspond to Y-cells and small relay cells in laminae Cl-3 correspond to W-cells. Hence, it is suggested that in area 17 of the cat there is a Y-cell projection to superficial layer IV flanked by two X-cell projections, one to the deeper portions of layer III and the other to the deeper portion of layer IV. A distinct W-cell projection to the superficial laminae may also exist. 11. Influence of exercise and age on the strength of the cruciate ligaments in rats. By A. W. PARKER and N. P. LARSON. University of Queensland, St Lucia Ligament strength is influenced by changes in an animal's body weight, hormonal state and level of physical activity. This study examined the influence of exercise on the strength properties of the anterior and posterior cruciate ligaments from the knees of rats during the period just prior to and following puberty. Forty male Wistar rats assigned to four exercise and four control groups were killed serially at 40, 54, 68, and 82 days of age following 2, 4, 6 and 8 weeks training respectively, and their ligaments tested on an Instron tensile testing machine. Ligament strength increased with age (P < 0-01), with the largest percentage increase occurring between the 40 and 54 days old animals, coinciding with the active growth phase associated with puberty in the rat. Ligaments from exercised rats were stronger than controls, with the largest difference occurring after 6 weeks of training when the animals were 68 days old. These changes are discussed in relation to hormonal influences associated with exercise and puberty and the duration of the exercise programme. The anterior cruciate ligament was much stronger than the posterior cruciate ligament in both exercise and control groups. This was related to the natural weight-bearing position of flexion of the knee in the rat and its resultant increased mechanical stress on the anterior cruciate ligament. The predominance of interligament failures in the test specimens was due to an improvement in grip design and tensile testing techniques. This technique, devised as a part of the study, permits collection of data more truly representative of ligament strength rather than as assessment of the weakest point in a bone-ligament-bone complex.
12. Resin casts of vasa vasorum. By W. H. BROOK. Monash University This study was performed in order to obtain a three dimensional picture of the arrangement of the vasa vasorum on the abdominal aorta and inferior vena cava of the rat. Adult SpragueDawley rats were heparinised and perfused through the thoracic aorta or left ventricle with heparinised saline followed by methyl methacrylate. The animals were corroded in hydrochloric acid, the casts were photographed, and then examined under the scanning electron microscope. Other rats were perfused with a silicone rubber injection compound (Microfil), dehydrated in alcohols, cleared in methyl salicylate, examined under a dissecting microscope and photographed. Methyl methacrylate casts showed longitudinally orientated vasa vasorum draining into tributaries of the inferior vena cava. In the silicone rubber casts vasa vasorum were seen on
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the abdominal aorta and the inferior vena cava running both longitudinally and transversely, and more vasa vasorum were seen in the silicone rubber casts than in the methyl methacrylate casts. The silicone rubber casts showed that a number of vasa vasorum came from the surrounding tissues, and this is the explanation of the different appearances of the corrosion and cleared casts, the vasa vasorum from the adjacent tissues being lost in the corrosion process. It is concluded that resin casts of vasa vasorum using silicone rubber produce a more accurate picture than casts of methyl methacrylate. 13. The topography of the retina in three primates: the bush baby, monkey and human. By ELIZABETH M. JOHNSTON and J. STONE. University of New South Wales The distribution and soma sizes of ganglion cells have been studied in the retinas of three primate species, the bush baby (Galago senegalensis), monkey (Macacca irus) and human (Homo sapiens). The retinas were prepared as Nissl-stained whole mounts following formalin fixation. The human material was made available by the Sydney Eye Bank. The three species differ considerably in phylogenetic history, and their retinas vary greatly in size, the bush baby retina measuring approximately 23 mm across, the monkey retina 30 mm and the human retina 40 mm. Nevertheless, their retinas show considerable similarities in topography. In all three, for example, a fovea is apparent a few disc diameters temporal to the optic disc; it is least developed in the bush baby. Maps of the distribution of ganglion cells in peripheral retina have isodensity lines which in all three species are elongated horizontally, suggesting the presence of a visual streak. This horizontal elongation was least marked in human retina. In all three species the somas of the ganglion cells ranged considerably in size; they were smallest in the bush baby (< 16,um) and largest in man (< 27,um). In all three species, most markedly in the human, cells at the small end of the soma size range were particularly common in the visual streak region, suggesting that, as in the cat, this feature of primate retina may be largely formed by a concentration of small cells. Ganglion cell soma size also tended to be larger in temporal than in nasal retina, a trend also reported in the cat.
14. The small luteal cells of the sheep. By J. D. O'SHEA, D. G. CRAN and MARY F. HAY. A. R. C. Institute of Animal Physiology, Cambridge, U.K. The ovine corpus luteum (CL) contains two distinct populations of luteal cells, large and small. The ultrastructure of the large luteal (LL) cells has been described by many workers. Here we report observations on the ultrastructure and enzyme histochemistry of the small luteal (SL) cells in cycling and pregnant sheep. The major cell types in CL at all stages studied were endothelial cells and pericytes, fibroblasts, and LL and SL cells. The SL cells had large amounts of predominantly smooth, tubular endoplasmic reticulum, and both tubular and lamellar mitochondrial cristae. Some lipid droplets and lysosome-like bodies were always present, but more numerous in late pregnancy. Both LL and SL cell cytoplasm contained A5-3fl-hydroxysteroid dehydrogenase. SL cells differed from LL cells in their smaller size, more elongated or angular shape, fewer and less uniform mitochondria, and far fewer membrane-bound cytoplasmic granules of 0 2/t,m in diameter. SL cells were often in close surface contact with LL cells, and interdigitation between cytoplasmic processes of the two cell type increased these areas of contact. In areas lacking close contact with other cells, SL cells possessed an incomplete basal lamina. These observations suggest that SL cells are likely to possess a steroidogenic capacity, and to interact functionally with LL cells. 15. The function of the accessory fibre in spermatozoan propulsion. By M. A. SWAN, R. L. LINCK, D. W. FAWCETT and S. ITO. University of Sydney The function of accessory fibres in sperm-tail movement is controversial. One theory proposes that they are stiffening elements, another holds that they are motile. In mammalian and most other sperm they cannot be observed in isolation since they form a ring around the axoneme. However, amphibian sperm have an undulating membrane containing a supporting or accessory fibre which is displaced some distance from the axoneme. Analysis of phase contrast cinemicro-
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graphs of toad (Bufo marinus) sperm filmed at about 30 frames/s shows that the supporting fibre does not move independently of the axoneme. Similarly, toad sperm demembranated with Triton X-100 reactivated well in a solution containing ATP, with no decrease in beat frequency, and the supporting fibre did not move independently. Movies of these untreated and reactivated sperm were presented. On the basis of its position in the neck region, the supporting fibre of the toad appears analogous to the accessory (or dense) fibre of mammalian sperm. SDS gel electrophoresis confirms that a protein of molecular weight similar to that of the medulla of rat dense fibres is present in toad sperm-tails. To speculate on the function of the undulating membrane of toad sperm - it does not make the sperm swim faster, but may stiffen the tail without incurring an energy deficit large enough to require a long middle-piece. The toad sperm has only a few mitochondria in the neck region, whereas other sperm with accessory fibres around the axoneme have many mitochondria.
16. Prenatal growth in the marsupial, Macropus rufogriseus fruticus. By R. RoSE and M. T. WALKER. University of Tasmania The phenomenon of embryonic diapause in the family Macropodidae enables an embryological account of accurately aged specimens. Prenatal growth was examined histologically after inducing quiescent uterine blastocysts to resume development by the removal of pouch young (RPY). Organogenesis was described from serial sagittal sections at 6 gim. The outer shell diameter of unilaminar blastocysts ranged from 260 gm (0 days RPY) to 290 ,um at 6 days RPY. At 9 days RPY (330 gum diameter) the zona pellucida and mucoid coat were apparently absent and endoderm had proliferated (early bilaminar). Mesoderm had formed in the early trilaminar blastocyst (4-8 mm diameter, 15 days RPY). By 18 days RPY the shell membrane had ruptured and was 3 0 gim in thickness (7 0 ,um at 0 days RPY). The pre-fetal embryo at 18 days RPY (4.2 mm greatest length) had acquired a forelimb ridge, mesonephros, optic stalks and 25 somites. Persistent features included the amniopore, posterior neuropore and an intact stomodeal membrane. The lung buds and allantois were rudimentary. Cervical, lumbosacral and cranial flexures were present. By 20 days RPY digital anlagen were noted in the forelimb paddle of the pre-fetal embryo (6-9 mm greatest length, 32 somites). The gonadal ridge glomeruli and optic cup had formed. The near-term fetus (25 days RPY, 15-30 mm crown-rump) had forelimbs with claws on the separate digits, hind limb paddles, pontine flexure and cardiac valves. The metanephros, pigmented and nervous retina and villous intestine were present. The nuclei of voluntary muscle were aligned in a single axial row. A neonate (26 days RPY) was 16-90 mm crown-rump. As in other marsupials, the establishment of primary embryonic germ layers occupies a disproportionately long period in gestation, while fetal differentiation and organogenesis are largely confined to the terminal trimester of pregnancy. 17. Alterations in the differentiation of developing pulmonary epithelium. By D. ALCORN, T. M. ADAMSON, J. E. MALONEY, B. C. RITCHIE and P. M. ROBINSON. University of Melbourne and Monash University Alveolar development in the fetal lamb begins as a prenatal occurrence. In rats, mice, rabbits and man, this process is thought to occur postnatally. Thus the developing lamb lung allows close investigation of the changes in types of alveolar epithelial cells over this important developmental phase without additional changes caused by the transition from liquid- to airbreathing lungs. During the last trimester in this species (147 days gestation) undifferentiated epithelial cells are replaced by the alveolar epithelial Type I (TI) and Type II (TIl) cells suggesting that an initial pool of reserve cells gives rise to either type of cell. Just prior to birth, undifferentiated cells are rarely counted, indicating that some of the cells lining the bronchioles may give rise to alveolar lining cells as further growth occurs. By altering the conditions under which the lung develops (following tracheal ligation, tracheal drainage or bilateral phrenectomy as described in previous presentations to this Society) the ratios of the cell types lining the future alveoli change, indicating that the pattern of differentiation has also altered. Following enhanced alveolar formation (tracheal ligation) the ratio of TI cells is increased. Following retarded alveolar formation (tracheal drainage and bilateral phrenectomy) the TI cell ratio is decreased.
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These experiments demontstrate that not only is the growth of the fetal lung dependent on the volume relationships within the fetal thorax but so is the differentiation of the alveolar epithelium and hence alveolar development and function.
18. Studies of the structure and function of the Sertoli cell in a seasonally breeding rodent. By Y. M. HODGSON, D. C. IRBY, J. B. KERR and D. M. DE KRETSER. Monash University Rattus fuscipes has been demonstrated to have a seasonal reproductive cycle. During winter the testes are regressed and FSH levels are low while in summer testes are active and FSH levels are higher. Since Sertoli cells are known to play an important role in spermatogenesis and have been shown to be a target tissue for FSH, this study investigated Sertoli cell function as judged by (i) electron microscopy, and (ii) androgen binding protein (ABP) levels in testicular and caput epididymal cytosols in the breeding and non-breeding season. Radioimmunoassay was used to evaluate serum LH, FSH and testosterone levels and showed that the concentrations of these hormones were lower in the regressed period (Table). The seminiferous tubule diameter was decreased and the epithelium consisted of Sertoli cells and spermatogonia. The height of the Sertoli cells was reduced and the nucleus was displaced toward the lumen. Nuclear shape was more regular, its size was decreased and the nucleoplasm was more granular in comparison to the active state. Diminished smooth endoplasmic reticulum and an accumulation of lipid droplets in the Sertoli cell cytoplasm also characterised the regressed state. ABP levels in testicular and caput epididymal cytosols were significantly lower (P < 0 5) in regressed rats (Table), indicating reduced secretory function of the Sertoli cell. The altered morphological and secretory characteristics of the Sertoli cell may be one of the prime factors leading to the cessation of spermatogenic activity.
Testis length (cm) 1-24 +0 10
(ng/ml) 235 50 ± 58 04
(ng/ml) 0-02 ±0-06
0-83 ±0-14 Mean ± SEM; ND, Not detectable.
41-90 +4 56
19. Spermiogenesis in the marsupial mouse, Antechinus stuartii. By L. M. JONES and R. L. HUGHES. University of Queensland The ultrastructural aspects of spermiogenesis remain unknown for the majority of marsupials. The degree of structural diversity between the spermatozoa of the major marsupial families, as visualised by light microscope, is widely recognised. This suggests that parallel ultrastructural studies might be expected to provide important indicators of phylogenetic relationships between marsupial species. In Antechinus stuartii the formation of the acrosomal vacuole from the Golgi apparatus marks the beginning of spermiogenesis. The expanding vacuole subsequently collapses, covering 4/Sths of the dorsal nuclear surface. The distal margins of the acrosome are delineated by spurs of the Sertoli cell reaction. Concurrently an elaborate articular structure, together with the segmented sheath made up of longitudinal columns develop in the neck region of the sperm
tail. Nuclear condensation and dorsoventral flattening occur prior to nuclear rotation from an angle of 90 to 20 ° with respect to the long axis of the tail. A second nuclear rotation occurs in the caput epididymis returning the nucleus to its original orientation. A third and final rotation occurs in the corpus epididymis so that in the mature spermatozoon the head and tail axes are parallel. The axoneme of the tail is surrounded by nine peripheral fibres. These are initially 80 nm in diameter but increase differentially along the flagellum to reach a maximum diameter in the
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distal middle piece and proximal principal piece (range: 140-260 nm). All peripheral fibres are widely separated from the axoneme by connecting laminae made up of parallel filaments 77 nm diameter. The middle piece is surrounded by two sheaths, an inner mitochondrial sheath of over 90 gyres long, and an outer helical fibre network. All the peripheral fibres of the axonemal complex except numbers 4 and 7 deeply indent the inner surface of the mitochondrial sheath. Spermiogenesis in Antechinus stuartii culminates in the formation of a spermatozoon that strikingly resembles that of other dasyurid marsupials. However the ultrastructural morphogenesis of the individual sperm organelles exhibits variable differences from the non-dasyurid marsupials. The pattern of spermiogenesis in Antechinus stuartii suggests a phylogenetic affinity with the peramelid marsupials. 20. The corpus luteum of the guinea-pig during the oestrous cycle and pregnancy. By A. K. H. POON and R. T. GEMMELL. University of Queensland There is evidence in a number of species (sheep, goat and cow) that secretion of progesterone from luteal cells occurs by exocytosis of granules (approximately 0-2 #m in diameter). Furthermore, variations in the number of granules in the luteal tissue of the pregnant sheep and the pregnant goat support the view that steroid secretion takes place through exocytosis. In the guinea-pig progesterone is produced by the corpus luteum until day 30 of pregnancy, the placenta being the major source during the latter half of pregnancy. Thus it would be of interest to know of the morphological structure of the corpus luteum of the guinea-pig during pregnancy. Guinea-pigs at various stages of the oestrous cycle and pregnancy were perfused with a paraformaldehyde-glutaraldehyde mixture and the luteal cells were examined with the electron microscope. The ultrastructure of the luteal tissues active in steroid hormone production consisted of an extensive network of agranular endoplasmic reticulum (AER), numerous mitochondria with tubular cristae, moderate quantities of lipid, large dense bodies, and active Golgi regions; granules were present in large numbers throughout the cytoplasm. After day 27 of pregnancy, the luteal cells possessed an accumulation of lipid and a decreased number of granules. The networks of AER persisted through pregnancy and there was no evidence of cellular regression until after parturition. The fluctuation in the number of 0-2 ,um granules in the luteal cells during the oestrous cycle and pregnancy correlated closely with the ability of this tissue to secrete progesterone.
21. The teaching of anatomy in Britain. By K. F. RUSSELL. University of Melbourne Anatomical teaching based on universities was very late in coming to Britain. The early teaching was in the hands of the various Companies of Barber-Surgeons, notably those of London and Edinburgh. The Universities of Oxford and Cambridge laid down attendance at anatomical dissections as early as the 16th century, but a reader was not appointed at Oxford until 1624 and a professor at Cambridge until 1707. At Edinburgh the Monro dynasty of anatomists commenced in 1720. The 18th century saw the rise of the private schools of anatomy owing to the large numbers of students seeking training in medicine, but it was not until 1832 that legislation was introduced to legally supply subjects for dissection. After that the teaching gradually switched to the universities. From the beginning all teaching was based on anatomical dissection; in the earlier period as formal demonstrations and later as individual dissection by the students. 22. Student assessment. By W. E. FABB. Royal Australian College of General Practitioners This paper focuses on the process of student assessment and outlines the principles of assessment, the assessment methods which are available and their application. Methods of summative assessment are described, and the way they can be used in formative assessment. The importance of determining what is to be tested befor eselecting testing techniqes is stressed, and the value of using a three dimensional model, which lists the behaviours, the clinical skills and the clinical conditions or problems to be tested, is demonstrated. Using the behavioural categories of recall of factual knowledge, interpretive skill, problem solving skill, affective
Proceedings of the Anatomical Society of
behaviour and psychomotor behaviour, as applied in a clinical setting, as a basis for assessment, a number of tests are described which enable assessment of the extent to which the desired behaviours and clinical skills are exhibited by the candidate. Such tests include the multi-choice, the short answer question, the traditional essay question, the case commentary, and more recently developed tests such as the Patient Management Problem, the Diagnostic Interview and the Management Interview, as well as tests of perceptual and manual skills. The use of each is described, and the details of administration are given with particular attention to the Patient Management Problem as a test of clinical problem solving skill, and the Diagnostic and Management Interviews as tests of problem solving skill and affective behaviour in a clinical setting. The paper concludes by underscoring the importance of formulating tables of specifications before constructing an examination and the need for a comprehensive range of valid and reliable tests. 23. Medical education - prognosis and priorities. By RUFUS M. CLARKE. University of Newcastle, N.S. W. Medical education is facing a crisis because it is not responding sufficiently rapidly to the changing expectations of society and the changing practice of medicine. Medical education must change because medical educators have a financial and moral obligation to society. Three major areas for changes in medical education are identified: helping doctors to cope with people; helping doctors to cope with change; and helping doctors to promote change. Helping doctors to cope with people involves giving students opportunities to come to terms with their own sexuality and mortality, opportunities to practise and perfect interpersonal and communication skills, opportunities to learn about people as well as diseases, opportunities to reflect on unfashionable areas of human suffering, like old age and alcoholism. Helping doctors to cope with change includes the acceptance of the need for self-evaluation, peer-evaluation and continuing self-education through the critical appraisal of new information. Helping doctors to promote change includes the acquisition of a sound ethical framework as a basis for assisting both individuals and society to make appropriate decisions. All of these changes require a greater emphasis on 'process' at the expense of 'content' what the doctor does rather than what he knows. The greatest obstacles to these changes lie in the entrenched position of Departments, the existence of discrete scientific disciplines, the general absence of a faculty philosophy on education and the executive machinery to implement it, and on the lack of overt career reward for education competence. 24. Studies on Leydig cell fanction in cryptorchid rats. By D. M. DE KRETSER, R. M. SHARPE and J. B. KERR. Monash University Surgically induced cryptorchidism in adult rats results in spermatogenic disruption accompanied by elevated serum FSH levels but variable effects on Leydig cell function have been reported. The present study was designed to assess the effects of cryptorchidism on Leydig cell function by surgically placing the testes of male rats (80 days of age) in the abdominal cavity. In animals bled twice weekly after surgery, serum LH levels rose significantly by 10 days (Control (C) 1-2 ±0O6 ng/ml (SD); cryptorchid (CD) 2-6 ± 04 ng/ml) and remained elevated at 4 weeks (CD 4-0± 1-7 ng/ml) whereas serum testosterone (T) decreased significantly at 7 days (C 3-2± 09 ng/ml; CD 1 9 ±5 ng/ml) and gradually returned towards normal at 4 weeks (CD 2-6 ± 0 ng/ml), the near-normal testosterone being maintained by elevated LH levels. Ultrastructural studies were performed at 4 weeks on tissue fixed by vascular perfusion using a mixture of 5 % glutaraldehyde and 4 % formaldehyde and demonstrated Leydig cell hypertrophy characterised by increased smooth endoplasmic reticulum and mitochondria and enlarged Golgi complexes, features characteristic of HCG-stimulated testes. Decapsulated testes from cryptorchid and control animals were studied in vitro by assessing their response to HCG stimulation (166 mu/ml) over 4 hours incubation at 37° C in Krebs-Ringer bicarbonate. Basal T production was not significantly different (C 171 ± 27-2 ng/ml; CD 155 ± 46 ng/ml) but HCG stimulated T production was significantly greater in the cryptorchid testes (C 410±116 ng/ml; CD 912+ 291 ng/ml, P < 0-01). Similar results were found during HCG stimulation of Leydig cells
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isolated following collagenase dispersion (C 1-44±0-2 ng/106 cells; CD 4.37+ 1-5 ng/106 cells, P < 0-01). In vitro binding of [1251-]HCG to testis homogenates revealed an increased binding in cryptorchid testes per unit weight (228 ± 37 pg -HCG; CD 390 + 50, P < 0-001) but when considered per testis, binding was decreased by 58 % (C 277 ± 45 ng/testis; CD 11-18 ± 1 8, P < 0-001). These studies demonstrated that compensated Leydig cell failure exists in vivo as evidenced by high LH and low normal T levels. However in vitro Leydig cells from cryptorchid rats show increased capacity for stimulated T production consistent with their ultrastructural characteristics. The reasons for this paradox remain to be explored but may be related to decreased sensitivity of cryptorchid testes to LH stimulation. 25. Evidence for sensory perceptors in arteriovenous anastomoses in the nose of the handicoot, Isoodon macrourus. By G. S. MOLYNEUX, University of Queensland As part of an investigation of thermoregulation in marsupials the structure of arteriovenous anastomoses (AVAs) in the nose of the bandicoot, Isoodon macrourus has been studied by light and electron microscopy, tissue being fixed by immersion or perfusion with a glutaraldehydeparaformaldehyde mixture. Arteriovenous anastomoses of epithelioid type occurred in high density near the tip of the nose just lateral to the mid-line and were similar in structure to those described in other mammalian species. In addition to the dense peripheral innervation characteristic of AVAs in the extremities, the wall of the proximal segment of the anastomoses was penetrated by unmyelinated axons located between the outer circular smooth muscle fibres. The axons showed terminal swellings which contained vesicles, glycogen particles and whorls of myelin-like membranes. The structure of these nerve endings is similar to bulboid endings described in skin as mechanoreceptors and their close relationship and arrangement in parallel to the muscle fibres suggests their function as pressure receptors stimulated on dilation of the anastomoses. The demonstration of presumed sensory endings in anastomoses is relevant to the increased blood flow through AVAs which occurs reflexly and with CNS warming (Hales et al. Pflugers Arch. 1978) and suggests the presence of a feed-back mechanism to monitor AVA blood flow. 26. Observations on the cranial endocast of the Miocene marsupial, Wynyardia bassiana. By J. R. HAIGHT and P. F. MURRAY. University of Tasmania and the Tasmanian Museum Wynyardia, a robust, possum-like diprotodontid retaining many primitive polyprotodontid characters, is among the oldest known Australian marsupials. The edentulous, but otherwise nearly intact, skull is similar in size to the tiger cat, Dasyurus maculatus (basicranial length, c. 100 mm), some 15 mm longer than that of the Tasmanian Trickosurus vulpecula which Wynyardia somewhat resembles. We hoped that an examination of the endocranial cast would reveal whether the brain, in terms of its external morphology, more closely resembles those of the more generalised polyprotodontid line or whether steps had already been taken toward the morphology displayed by the advanced diprotodontid marsupials. The volume of the latex cranial endocast is 18-8 ml. Endocasts were also made from skulls of the contemporary polyprotodontid, Dasyurus maculatus (vol. = 9-8 ml) and the diprotodontids Trichosurus vulpecula (vol. = 12-5 ml) and Potorous apicalis (vol. = 13-8 ml). This latter group of endocasts accurately reflected external brain morphology, hence our confidence that the Wynyardia endocast portrays the surface features of its brain. The few sulci present in the large, nearly lissencephalic neocortical mantle follow the pattern present in Trichosurus, not that of Potorous or of Dasyurus. Proportionately, the olfactory bulbs are not large, unlike those of both the Potoroinae and the Dasyuridae, but are like those of the Phalangeridae. Nonetheless, the rhinencephalon is relatively larger than in living phalangerids. The foliated cerebellum is large and well-developed. Grooves for the passage of prominent optic nerve bundles and substantial trigeminal nerves are present in the endocranial base. Overall, the brain of Wynyardia displays phalangerid, as against dasyurid affinities. The reduced olfactory bulbs, large cerebellum and prominent optic nerves could suggest an arboreal habitat, a suggestion in keeping with Ride's assessment based upon postcranial observations. In spite of primitive features such as the large rhinencephalon and neocortical lissencephaly, Wynyardia is considerably advanced over the present day dasyurids in terms of relative brain size. Additionally, its external brain morphology places Wynyardia firmly on the phalangerid side of the phalangerid-macropodid dichotomy.
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27. The broadbeach aboriginal burial site, Southeast Queensland: stature estimates and crural indices. By W. B. WOOD and A. M. C. MURPHY. University of Queensland The mean maximum lengths of 100 tibiae and 94 femora from the Broadbeach skeletal collection w%ere used to calculate stature estimates and crural indices for both males and females. Stature was calculated using the Negro formulae of Trotter & Gleser (1952) which have been demonstrated as suitable for stature estimates on Australian Aboriginal tibiae and femora without alteration. Estimated mean adult statures for both male (168-5 cm) and female (157-3 cm) were then compared with the actual range of mean statures reported by various field studies of living Aboriginal population groups, and found to fall well within this normal range (male, 166-2172-5 cm; female, 155-9-159-5 cm). The crural indices were also calculated. The mean male index (78 9) is significantly less than that of the female (80-9, P < 05) and both are much less than the mean for Europeans (83 3) and for Negroes (86 2) according to Krogman (1962). These results indicate that the major contribution to the characteristically long lower limb of the Australian Aborigine is a result of the relatively greater length of the femora.
28. Arterial changes in spontaneously hypertensive rats. By G. R. CAMPBELL and JuLIE CHAMLEYCAMPBELL. Baker Medical Research Institute The ultrastructure of the caudal artery of spontaneously hypertensive rats (SHR) of the Okamoto-Aoki strain has been examined and compared with that from Wistar-Kyoto normotensive controls. In the 12-16 weeks animal the caudal artery demonstrates both hyaline arteriosclerosis and hyperplastic arteriosclerosis. The intimal proliferation of smooth muscle cells is localised to regions which appear intermittently throughout the length of the vessel. These areas are initially observed about 3 weeks after the onset of hypertension in these rats (i.e. at about 7 weeks of age). In older animals the intimal proliferative areas are enlarged and often contain large numbers of small smooth muscle cells oriented parallel to the long axis of the artery (i.e. at right angles to the smooth muscle cells of the media). In most of these smooth muscle cells the major portion of cytoplasm is filled with myofilaments although some contain a large number of organelles usually associated with synthesis. Irregular-shaped cells occur, many with an appearance which previously has been termed 'moth-eaten'. Between cells are long strands of material resembling basal lamina and a large number of electron-dense granules and membranous figures. The regions of artery between proliferative areas appear relatively normal in young animals. However, in older SHR, interspersed between areas of intimal proliferation, are regions in which there is an interrupted or completely absent internal elastic lamina. Smooth muscle cells from this region are often separated by a wide extracellular space which contains reduplicated layers of basal lamina-like material and many electron-dense granules. SHR treated with 6hydroxydopamine (which destroys adrenergic nerves) from 3 weeks of age do not develop hypertension by 9 weeks, nor do they develop intimal changes in the caudal artery, demonstrating that these changes are due to increased blood pressure. j 29. The reaction of the pia mater in Wallerian degeneration of the optic nerve. By COLIN YEO and DARREL FERNANDO. University of New South Wales The behaviour of the pia mater in Wallerian degeneration of the optic nerve was studied by removing the right eye in a series of rats. After survival periods of 4, 7, 9, 14, 21 and 35 days, the optic nerves proximal to the optic chiasma were examined with the EM. The normal pia mater consists of a 3-5 laminae of fusiform pial cells and scattered pial macrophages. The pial cells are fusiform in shape, containing a central electron-dense elongate nucleus and two long slender processes extending from either end of the cell. The scant cytoplasm contains a few cell organelles. The pial macrophages are round cells, containing a round dark nucleus and a cytoplasm with numerous vacuoles, a few mitochondria, lysosomes and ER.
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By 4, 7 and 9 days post-operation (PO), there is an increase in pial fibroblasts. Some of the older pial fibroblasts exhibit numerous inclusions in their processes. These include round lipid inclusions, a few round dense bodies and an occasional myelin figure. The pial macrophages become very reactive. These cells are engorged with pleomorphic inclusions and myelin figures. At 14-21 days PO, the meningeal reaction appears to have subsided. By 5 weeks PO the nerve is replaced by a glial scar. The pial cells and pial macrophages appear normal. The results indicate that pial cells increase in number and are intensely reactive when the degenerative process is at its peak. Some pial fibroblasts exhibit inclusions suggestive of an uptake of lipid material. The presence of numerous myelin figures and lipid inclusions in the macrophages suggests that these cells may in some waycontribute to theremoval of the degenerating nerve with some contribution from pial fibroblasts. 30. Surface ultrastructure of the hypothalamic third ventricle of the guinea-pig. By I. TORK. University of New South Wales The surface ultrastructural features of the ependyma and supra-ependymal cells of the hypothalamic third ventricle were examined in the guinea-pig. Several distinct regions could be distinguished using the scanning electron microscope. The lateral walls of the hypothalamic region are lined by ciliated ependyma except for a distinct anterior area which is devoid of cilia and is covered by small supra-ependymal cells while the ependymal cells are furnished with flat microvilli. The floor of the third ventricle exhibits three regions. An anterior, ciliated region which, at the point above the rostral end of the median eminence, changes over to a non-ciliated area. This non-ciliated surface stretches into the infundibular recess and is furnished by a large number of short microvilli. The posterior part of this area is characterised by an accumulation of ependymal spherules and of phagocyte-like supra-ependymal cells. These cells have small bodies and flat processes extending over the surface of several ependymal cells. The anterior part of the non-ciliated floor is devoid of spherules and phagocyte-like supra-ependymal cells. Along the line of transition from the floor to the lateral wall, large, neuron-like supra-ependymal cells could be found. These have long, slender processes several hundred micrometres in length. Dorsal to the entrance of the infundibular recess a special ependymal area consisting of at least 40 round surfaced cells of 5-10 #sm in diameter could be observed. The structural similarity and the close relationship of these cells suggest that they are of similar secretory or absorptive function. This area could be considered as a small circumventricular organ specially related to the infundibular recess. 31. The structure of the gastrointestinal tract of the flying fox Pteropus poliocephalus. By D. B. MANLEY and L. M. WILLIAMS. University of Queensland The gastointestinal tract of the Australian flying fox Pteropus poliocephalus has been examined to study the adaptations required by a frugivorous flying mammal. Sections were cut after perfusion with paraformaldehyde and embedding in paraffin. Staining was carried out utilising H and E, PAS, phloxine/tartrazine and alcian blue at pH 1-0 and 2-5. The tract is approximately 140 cm in length (small intestine 120 cm, large intestine 15 cm) with no discernible gross variations between intestinal segments. A caecum is absent. The stomach shows a greatly expanded cardiac vestibule and fundic caecum. In the stomach, there is no true cardia; the pylorus is a relatively short organ ending 1 cm proximal to the entrance of the bile duct into the duodenum. Parietal cells show an overall moderate PAS-positive reaction, with an intense reaction being found in the intracellular canaliculus. The structure of the small intestine varies from that of the human in the most proximal segments. Initially the villi are long, thin and appear to anastomose. The crypts of Lieberkiihn are extremely short with few goblet cells. At the bases of the crypts, cells resembling Paneth cells are visible. Approximately 20 cm distally, the villi become broad and contain large lacteals. The epithelial cells contain numerous absorption vacuoles. The large intestine and rectum are not greatly variant in structure from humans.
Proceedings of the Anatomical Society of
Since the fruit bat's diet is high in carbohydrate and low in protein, large quantities of food must be digested and this could explain the apparent high absorption in the proximal small intestine. 32. The effect of hydrochloric acid and ammonia vapours on the formaldehyde-induced fluorescence of noradrenaline and dopamine. N. G. M. WREFORD and G. C. SMITH. Monash University The influence of hydrochloric acid (HCI) and ammonia vapours on the excitation and emission spectra of the formaldehyde-induced fluorophores of noradrenaline (NA) and dopamine (DA) has been investigated in a model system consisting of Sephadex beads infiltrated with known concentrations of each amine. After embedding in Spurr's medium 3 ,m sections of the model were mounted on quartz slides, exposed to formaldehyde vapour (2 hours, 80 'C, 50 % relative humidity) and subsequently to HCI or ammonia vapour for periods ranging from 0 to 500 seconds. Coverslips were applied and spectra were obtained on a fully corrected microspectrofluorimeter fitted with a quartz sub-stage illumination system. Exposure to HCI produced a small shift (5-10 nm), towards longer wave lengths, of the emission maximum of both DA and NA. After similar treatment the major excitation peak of each fluorophore was shifted to shorter wave lengths; only a short exposure to HCI ( < 15 seconds) was required. In the case of NA, a shift of the excitation peak back to longer wave lengths was apparent after a 30 seconds exposure whereas the peak of the DA fluorophore remained at the lower wave length. No other significant differences were observed between the spectra of the amines. Exposure of the models to ammonia vapour sometimes shifted the excitation spectra of both amines to longer but similar wave lengths, but this phenomenon provided no basis for reliable differentiation. It is concluded that the shift back to longer wave lengths apparent following prolonged exposure of the NA fluorophore to HCI provided reliable differentiation of the formaldehydeinduced fluorophores of DA and NA.
33. Observations on the 'waist' of the scaphoid bone. By C. RZEPCZYK and M. FAHRER. University of Queensland The scaphoid is the carpal bone most frequently fractured. Fractures through the 'waist' of the scaphoid, separating the proximal part of the bone from the tuberosity, require at least 3 months of immobilisation to heal. Pseudarthroses and necrosis are quite common, despite correct treatment of these fractures. On the radial and palmer aspects of the scaphoid, the waist is represented by a marked groove, classically described as non-articular, rough, perforated by vascular orifices. The radiocapitate band of the palmar radiocarpal ligament runs through this groove. A constant hyaline cartilage lining to the groove has been equated to an articular surface. We have investigated the groove of the scaphoid bone in a series of ten dissections. Articular cartilage was absent in three cases, present only on the radial aspect of the groove in three cases, present only on the proximal half of the groove in one case and present on the whole surface of the groove in three cases. In the last four cases, the corresponding segments of the deep aspect of the radiocapitate ligament were encrusted with a hard, smooth, shiny tissue. Histologically, this tissue was typical
fibrocartilage. 34. A light microscope demonstration of alkaline phosphatase in normal and diseased large intestine and the effects of temperature on the staining reaction. By L. E. BELL and L. M. WILLIAMS. University of Queensland There are several reports in the literature that human large intestine, both normal and diseased, has very little or no alkaline phosphatase. One of the authors has demonstrated a unique alkaline phosphatase in normal large intestine using starch gel electrophoresis. The present investigation was undertaken to demonstrate histochemically the presence of alkaline phosphatase in the large intestine. The alkaline phosphatase demonstrated by electrophoresis is heat labile so during this study the tissues were maintained below 4 'C. Surgical tissue was frozen in liquid nitrogen, freeze dried at - 40 'C and embedded in glycol methacrylate at 4 'C. Some tissue was fixed in ice cold
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formol calcium and frozen sections cut. The simultaneous coupling Azo dye method using the substrate sodium ac-naphthyl phosphate at 4 °C, 22 and 37 °C was used to demonstrate the enzyme. Controls were incubated without substrate. In normal caecum, alkaline phosphatase activity was demonstrated at 4 °C on the surface of the epithelial cells, in the middle and upper crypts, blood vessels and some lamina propria cells. The surface of the goblet cells was negative. At 22 and 37 °C, the reaction was weaker. Similar results were obtained with caecum tumour tissue. However the reaction was much stronger even after a 10 minutes incubation period. The invasive tumour tissue was negative. Positive results were also obtained using the substrate naphthol AS-BI phosphate. Alkaline phosphatase activity has been demonstrated in the large intestine by using fresh specimens obtained at operation and maintaining the tissue at 4 °C or less throughout the processing and staining procedures. 35. 6-hydroxydopamine induced specific depletion of dopamine-containing nerve terminals in the median eminence. By G. C. SMITH, N. G. M. WREFORD, D. M. DE KRETSER and M. M. WALKER. Monash University 6-hydroxydopamine (6-OHDA), when given by single intravenous injection in a dose of 150 mg/kg, produces specific destruction and subsequent regeneration of catecholaminecontaining nerve terminals in the median eminence of adult male Sprague-Dawley rats. Luteinising hormone levels are affected by such treatment. A method for obtaining specific depletion of noradrenaline by adjustment of the dose schedule has been described; in theory the specificity of the depletion obtained can be checked by microspectrofluorimetry, but although this has proved possible in model studies, fading of the fluorophores and increase in background fluorescence following the necessary treatment with hydrochloric acid has prevented successful application to median eminence tissues. An alternative method of obtaining specific depletion has been used; animals were pretreated with desipramine 25 mg/kg, given intraperitoneally 30 minutes before the injection of 6-OHDA. Desipramine has been shown to block the effects of 6-OHDA on noradrenalinecontaining fibres. After 24 hours, such animals had a markedly different distribution of depleted fibres, but this was not consistent with previously reported descriptions of the differential distribution of noradrenaline- and dopamine-containing fibres. It is concluded that chemical methods of depletion will require histological confirmation and that there is a need for further refinement of the methods available for microspectrofluorimetric differentiation of noradrenaline and dopamine fluorophores.
36. The southern elephant seal - changes in testicular histology over a 12 months period. By D. J. GRIFFITHS. University of Queensland The southern elephant seal (Microunga leonina) is a seasonal breeder. Blocks of testis, collected monthly from mature adults at Macquarie Island (55 0 S, 159 ° E) were fixed in Bouin's solution, wax embedded and sectioned at 5,um. Sections were examined to determine the structural differences between the fertile and non-fertile testis. A 42 point Weibel graticule was used as a basis for all quantitative histology. During the non-breeding season males are infertile. Testicular mass is 100-150 g tonne of seal. Sperm are not produced and the lumen of the seminiferous tubule is absent or greatly reduced. The proportions of seminiferous tubule and interstitial material is 0-54 and 0-46 respectively. With the onset of the breeding season (September-October) the testicular mass increases to 300-350 g/tonne of seal. The cellular component of the seminiferous tubule does not greatly change, but the lumen increases threefold in volume. At the height of the breeding season the lumen is again reduced, due to a proliferation of secondary spermatocytes and hypertrophy of Sertoli cells. Breeding proportions of tubule and interstitial material are 0-64 and 0-36 respectively. Sperm are seen in the testis in August but are not in the epididymis until September. Sperm production is effectively finished by November. The non-breeding seminiferous tubule comprises Sertoli cells and spermatogonia, and completely lacks spermatocytes and spermatids. In July and August prior to the breeding season, Sertoli cells develop and enlarge, thus increasing the size of the tubule, and spermatogenesis
Proceedings of the Anatomical Society of
begins. Presumably this is due to an increased plasma gonadotropin concentration, or an increasing number of available gonadotropin receptors on the tubule. Spermatogenesis begins to decline in October, before the end of the breeding season, and it is assumed that, as in other seasonally fertile animals, sperm stores in the epididymis supply the sperm for the remainder of the breeding season. The above work was part of the 1977 Australian National Antarctic Research Expedition. 37. The spherulite mode of calcification related to ossification in one day old rabbit epiphyses. By G. G. CARMICHAEL and P. P. PHAKEY. Monash University The process of provisional calcification of cartilage was studied in relation to the secondary centre of ossification in epiphyses of long bones of 1 day old rabbits. Tissues were examined histologically and electron microscopically. The arrangements of crystals corresponded in general with those seen in osteoid and in the calcifying cartilage of the epiphyseal plate, but were characterised by the presence of well developed spherulites. These forms, described in both organic and inorganic crystallising systems, consist of spherical space filling radial arrays of fibils that are subject to low angle branching. In vitro the spherulite mode of crystallisation is favoured by a high viscosity of the medium and by the presence of impurities. These conditions also govern the size of the crystals, and thus affect the properties of the calcified matrix as a material. Under the light microscope Alcian blue staining indicated the presence of droplets of acidic polysaccharide at the sites of spherulite formation, a distribution that corresponds to that of extraction-resistant proteoglycans. This is consistent with the viscosity requirements for this mode of crystallisation. Ultrastructurally the crystallites measured approximately 5 nm in diameter and up to 150 nm in length and frequently exhibited a radial pattern. The process of spherulite formation and amalgamation into confluent crystalline masses is repeated as the centre of ossification advances towards the articular surface. When ossification is complete the zone of provisional calcification survives as the calcified zone of articular cartilage.
38. Biochemical and ultrastructural studies of the synthesis of elastin, proteoglycans and collagen by neonatal rat aortic smooth muscle cells in tissue culture. By B. W. Oakes, A. C. BATTY and C. J. HANDLEY. Monash University The synthesis by smooth muscle cells of collagen, elastin and proteoglycans in the region of intimal hyperplasia as seen in arterial repair, atherosclerosis and hypertension is well recognised. However, the factors responsible for smooth muscle cell proliferation and connective tissue protein synthesis are poorly understood. This report describes the characterisation of a tissue culture system using neonatal rat aortic smooth muscle cells. High density multilayer cultures of neonatal rat aortic smooth muscle cells (1 x 106 cells/25 cm3 Falcon flask) were grown for 3-4 weeks using daily medium changes. After a 3-4 day lag period, the cells grew exponentially for 10-12 days followed by further slow increase in cell numbers. The elastin and proteoglycan content of the cultures was measured by gravimetric analysis after alkali extraction and hexuronic acid analyses respectively, continued to increase with age of the culture after an initial 4-5 day lag period. Using differential digestion of the glycosaminoglycans with the enzymes chondroitin lyase AC and chondroitin lyase ABC, 40 % of the uronic acid was associated with a fraction corresponding to chondroitin sulphate and hyaluronic acid and 60 % with a fraction corresponding to dermatan sulphate. Collagen content as determined by hydroxyproline analyses tended to parallel cell numbers in that a rapid increase occurred between days 5-10 of incubation followed by a further slow increase with age. Amino acid analyses of alkali-extracted elastin showed an elastin profile with the presence of desmosine and isodesmosine and a high lysine content similar to that described by Looker and Berry (J. Anat. 113, 1972). Transmission electron microscopy revealed large elastin aggregates coalescing between the deeper layers of 'embryonic smooth muscle cells' together with collagen fibres and proteolgycan granules when ruthenium red staining was used. Such a model system will be useful for examining factors influencing smooth muscle cell proliferation and the connective tissue components synthesised.
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39. Development of the pig parotid salivary gland. By J. PATTERSON, L. C. LLOYD and D. A. TITCHEN. Melbourne University and C.S.I.R.O. In studies of the postnatal development of the parotid salivary gland of the pig, the gross appearance, histology and secretory responses were examined in animals less than 24 hours old as well as at 28 days and 110 days after birth. Tissues fixed in Carnoy's solution were stained with H & E or PAS reagent. Injections of India ink, methyl methacrylate or polyester resins via the main duct defined the extent of the glands in neonates. Bethanechol (up to 5 mg kg-', s.c.) was used as a stimulus to induce secretion in pigs less than 24 hours and those 28 days old. The injected parotid of the neonate showed an extensive duct system arborising widely over an area comparable to that occupied by the more mature parotid. Gland tissue was sparse and present as isolated masses of tissue associated with terminal regions of larger ducts. In more mature animals the ducts were less obvious as they were buried in the extensive parenchyma of the gland. The predominant cell groups of the neonate were arranged in acini in lobules with obvious connective tissue stroma. Most of the acini consisted of cells with a centrally placed nucleus and a uniformly staining cytoplasm which was faintly PAS-positive. Obvious lumina were infrequently seen. A few acini present in the neonates were similar to those seen in the more mature animals; in these the cells had the nucleus located near to the basement membrane and a vesiculated cytoplasm which was PAS-negative. Ducts with strongly PAS-positive cells (as in more mature animals) were detected in the parotid of the newly born pigs. Groups usually of two to three cells, exhibiting a strongly PAS-positive reaction similar to goblet cells of the larger ducts of more mature pigs, were another type of cell present in the lobules of the newly born animals. Bethanechol stimulated salivary secretions, although not from parotid glands, of the neonates. Parotid salivary secretion has, however, been stimulated by it in piglets at 28 days of age. It is concluded that at birth the parotid gland of the pig is not fully differentiated and lacks secretory activity. 40. Kinematic analysis of the gait of Down's syndrome children. By A. W. PARKER, R. BRONKS and P. COOPER. University of Queensland This investigation arose from a need for objective data relative to normal variability of multiple gait components of Down's syndrome children, to serve as a base for comparison and therapeutic management. Specifically, two complete walking cycles of six children were filmed simultaneously in the frontal and sagittal planes at 64 frames per second, and the temporal components of the stance and swing phases were recorded in addition to the amplitudes of upper and lower limb joint segments. Preliminary analysis of the data revealed that the stance phase occupied 64 % of the cycle. The subjects demonstrated a wide base of support and consistent out-toeing. Reduced hip flexion in the swing phase was compensated by increased abduction of the thigh. Striking deviations from normal patterns were increased wrist and head flexion. The subjects exhibited a greater development range in walking skill than reported for normal children. Fifty per cent of subjects had an immature heel-toe mechanism in the stance phase with a tendency to use a pattern characterised by toe-touching first, or a flat footed contact instead of the heel. Physical characteristics common to some of the children included protruding abdomen, lumbar lordosis, and increased pelvic tilt, factors which resulted in a decreased amplitude of flexion and extension at the hip. The high variability in the ranges of flexion and extension at the ankle and consequent foot placement, revealed poor control of distal segments perhaps attributable to hypotonia which is characteristic of this syndrome.
Proceedings of the Anatomical Society of
41. A new stereotaxic atlas of the rabbit brain. By M. GIRGIs and S. WANG. Sydney University The problems involved in obtaining monkeys, and sometimes even cats, have prompted a search for smaller and more easily available animals for neurophysiological and behavioural experimentation. Rabbits seem to provide a good solution. Rabbits are especially appropriate for limbic system studies because such structures are unusually prominent. Rabbits are docile, are easily restrained, and their temperament reduces the probability of pulling off head-mounted chronic electrode assemblies. EEG artefacts from unanaesthetised rabbits are minimal because rabbits do not move much. An accurate and detailed stereotaxic atlas for the rabbit brain is badly needed. The need for such an atlas has become more apparent as more investigators have recognised today the special advantages of rabbits in brain research. In the case of the most commonly used rabbit atlas, the brain was embedded in celloidin. Most investigators find it inaccurate and unsatisfactory. Other rabbit atlases are based on unstained sections. The lack of nuclear differentiation precludes their use for detailed studies. The atlas presented in this communication consists of copies of untouched photographic prints of frozen sections, at 0 5 mm intervals, stained by Nissl and Weil techniques. A x 10 magnification grid was first contact printed on to photographic paper, and the similarly magnified brain sections were then overprinted on to the pre-exposed grid. 42. Cytology of the pars intermedia of the adult sheep. By R. A. PERRY and P. M. ROBINSON. University of Melbourne This investigation of the microscopic structure of the adult sheep pars intermedia is the first stage in a study of the development of the sheep adenohypophysis. Light microscopy reveals a conspicuous pars intermedia about 15-20 cells thick, situated between the pars distalis and pars nervosa. A prominent hypophysial cleft and occasional follicles containing a colloid-like substance are seen. At the electron microscope, several cell types can be distinguished. The pars intermedia glandular cells are polygonal to pear-shaped, although some interdigitation of cell processes occurs. Dense-cored, membrane-bound granules are often seen near the well-developed Golgi complex and larger, irregular vesicles with finely granular contents of varying electron density occupy much of the remaining cytoplasm. The cells lining the cleft bear irregular microvilli on their free surface. Interstitial cells are characterised by deeply indented nuclei, elaborate junctional complexes and the absence of cytoplasmic granules. The pars intermedia is much less vascular than the remainder of the hypophysis and only occasional fenestrated capillaries are seen. 43. Comparative morphology of colonic epithelium and colonic adenocarcinomata. By D. H. BAUKLA and P. J. M. TUTTON. Monash University The study was undertaken in order to compare the ultrastructural morphology of epithelial cells in normal rat colon and tumour cells in rat colonic adenocarcinomata. Tumours of the large intestine were induced using weekly s.c. injections of 1,2-dimethylhydrazine (DMH) at a dose of 21 mg/kg for a period of 21 weeks. Specimens of colonic tissues from untreated rats and from tumour-bearing rats were prepared using routine procedures and examined using light microscopy and transmission electron microscopy. The results show that the majority of tumour cells are morphologically similar to the undifferentiated cells present in normal colonic crypt epithelium and that a minority of tumour cells are morphologically similar to the mucous, vacuolated, enterochromaffin and absorptive cells present in normal colonic crypt epithelium. Whilst differences between sections of normal colon and sections of adenocarcinomas were obvious because of differences in tissue architecture and cell types present, the differences between the two tissues were less obvious when the morphology of individual cells was compared and, on occasions, tumour cells and normal cells were morphologically indistinguishable. The study was unable to identify a single morphological feature characteristic of tumour cells, and the morphological criteria for distinguishing between 'malignant' and 'non-malignant' colonic epithelial cells are yet to be established.
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It is proposed that differentiated tumour cells in colonic adenocarcinomata are the progeny of undifferentiated tumour cells, arising in a similar way to their morphological counterparts in normal colonic epithelium. The production of differentiated cells that either cease dividing or divide infrequently may be an important factor limiting the rate of growth of colonic tumours. The results suggest that further studies to investigate possible factors regulating the differentiation of tumour cells in colonic adenocarcinomata and to investigate the capacity for cell division of particular types of differentiated cell in colonic adenocarcinomata are indicated. 44. The structure and function of the pericardial nerve plexus. By JANE T. COURTNEY and A. S. WILSON. University of Western Australia The aim of this study was to investigate the structure and function of the pericardial nerve network. Silver, catecholamine fluorescence, and cholinesterase methods were applied to whole preparations of the pericardial membranes in rabbits, guinea-pigs and quokkas (Setonix brachyurus). These studies were followed by electrophysiological investigations. In eleven adult rabbits, stimulation was provided by an intrapericardial inflatable balloon connected to a mechanical pump. An averaging technique was used to measure the resultant change in synchronous electrical activity in the left vagus nerve. Structural similarities were evident in the species examined, the nerve plexus consisting of an open meshwork of fibres and possessing no demonstrable specialised nerve endings. Where fasciculi intersected, there was evidence of flattening of nerve fibres. Guinea-pigs possessed a plexus of overall uniform density while, in rabbits and quokkas, the network was dense superolaterally and sparse inferomedially. In the physiological studies two animals died before records could be made while, in a further seven, the results had to be excluded for technical reasons. In the remaining two experiments, a distinct response was recorded in the vagus nerve, 70 msec following initiation of the stimulus. The investigation has shown that, in two rabbits, visible distortion of the pericardial membranes by an intrapericardial balloon results in a neural response in the left vagus nerve. It is suggested that this method may be applicable to the investigation of other possible sources of pericardial nerve fibres.
45. Seasonal changes in the ultrastructure of the Leydig cells of the bat (Myotis adversus). By H. S. F. LOH and R. T. GEMMELL. Queensland Institute of Technology and University of Queensland In the bat, Myotis adversus, spermatogenesis does not occur during January and February but does so during the rest of the year. A comparison was made of Leydig cell ultrastructure during the quiescent and active periods. Testicular tissues which were collected throughout 14 months were fixed by intraventricular perfusion with a buffered formaldehyde and glutaraldehyde mixture, and examined with the electron microscope. The concentration of testosterone in the plasma was estimated by radioimmunoassay. During the quiescent period, plasma testosterone was not detected and the testis was characterised by seminiferous tubules with an occluded or small lumen, containing no or few sperms. The Leydig cells possessed nuclei with an irregular membrane, and the agranular endoplasmic reticulum was not organised into 'compartments'. During breeding, the plasma testosterone concentration rose to 1-3 ng/ml, the testis enlarged in size, the seminiferous tubules expanded, and active spermatogenesis became evident. The Leydig cells increased approximately two and a half times in size, their nuclei were spherical, and the areas of agranular endoplasmic reticulum became enlarged and distinct. There was an approximate fourfold decrease in lipid droplets and dense bodies and a significant increase in the number of 0-2 ,um, moderately-stained granules. The study shows an increase in plasma testosterone concentration which parallels changes in the structure of the Leydig cells of the bat testis and reflects the capability of these cells to produce testosterone and of the testis to produce sperm. 42-2
Proceedings of the Anatomical Society of
46. Histochemical and ultrastructural. characterisation of a new muscle fibre type: Type I White. By M. A. KHAN. University of Queensland At the gross anatomical level the serratus metapatagialis (SMP) muscle of the pigeon is clearly divisible into a peripheral whitish band and a red portion comprised predominantly of 'pale' and 'red' fibres respectively. This important muscle was studied histochemically and ultrastructurally since such data were not available in the literature. The pale fibres of the SMP are atypical, in that they possess low succinic dehydrogenase (SDH), low mitochondrial content, absence of subsarcolemmal mitochondrial aggregates, low fat, moderate glycogen, high phosphorylase, low to moderate regular myofibrillar adenosine triphosphatase (M-ATPase), activation of M-ATPase following acid pre-incubation and jagged Z bands. Since M-ATPase and SDH activities correlate with contractility and fatiguability, the pale fibres are possible adapted for slow, unsustained and infrequent contractile activity. Consequently, on the basis of the above characteristics, these unique muscle fibres are 'Type I White or slow-twitch glycolytic'. The red fibres, however, possess high aerobic as well as glycolytic capacity, high M-ATPase activity which is labile after pre-incubation, and thick but straight Z bands. These muscle fibres, therefore, are the typical and well-known 'Type II Red or fast-twitch oxidative-glycolytic'.
47. The major features of embryonic development in the marsupial Potorous tridactylus. By R. L. HUGHES, R. T. GEMMELL, R. ROSE and G. SHAW. University of Queensland and University of Tasmania The purpose of this study was to determine the major stages of embryonic development in the rat-kangaroo Potorous tridactylus and to relate these to the levels of progesterone in the maternal peripheral blood plasma. A total of 15 embryos of known age was obtained by inducing the development of quiescent uterine blastocysts following the removal of suckling pouch young (RPY). In seven of these animals samples of peripheral blood plasma were obtained by heart puncture and levels of progesterone were determined by a method of radioimmunoassay supplied by the Hormone Assay Development Group, C.S.I.R.O. Division of Animal Production. Peripheral blood samples were also obtained from 16 other Potorous females after RPY. The sensitivity of the assay was 0 025 ng of progesterone. Quiescent unilaminar blastocysts persisted for at least 2 days RPY with their spherical protoderm measuring 150-200 /ttm in diameter. The protoderm was successively invested by three accessory egg membranes: zona pellucida (2-6 jum); mucoid coat (15-20 /&m); and shell membrane (50 ,um). The zona pellucida was not apparent at the bilaminar blastocyst stage (5-6 days RPY) and only remnants of the mucoid coat remained. However the intact shell membrane persisted beyond the medullary plate stage (14 days RPY) until at least after the primitive streak was established (18 days RPY). The early stages of pregnancy were characterised by increasing levels of progesterone above the basal value of about 0-3-0-8 ng/ml. Maximum levels of progesterone ranging between 1-3 and 5 0 ng/ml of peripheral blood plasma (mean 3-3 ng/ml) were found in eight females during the period when stages of embryonic development ranged between the formation of the bilaminar blastocyst and until at least the pre-fetal stage at about 26 days RPY. Births took place between days 29 and 33 RPY. Fetal development and interdigitation between fetal and maternal microvilli, in a restricted region of the vascularised fetal yolk sac, occurred during the latter half of the terminal trimester of pregnancy when progesterone levels again returned to basal values. It is therefore considered unlikely that either the developing fetus or its membranes contribute significantly to the midterm maxima of progesterone secretion. 48. Effects of ovarian hormones on junctional complexes of epithelial cells lining the rat uterus. By C. MuRPHi, J. G. SwIT, A. W. ROGERS and T. M. MUKHERJEE. Flinders University of South Australia and Institute of Medical and Veterinary Science The effect of progesterone and oestrogen on tight and gap junctions of epithelial cells lining the rat uterus was examined using the freeze-etching technique. Hooded Wistar rats were bilaterally ovariectomised and 1 month later were treated with various regimes of progesterone
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and oestrogen. Tissue was then processed for freeze-etch electron microscopy by routine procedures. Ovariectomised control (hormonally untreated) animals had tight junctions consisting of a network of closely packed laterally arranged circles in most areas seen. Small gap junctions of 10-25 particles were also seen within the tight junctions. Animals treated with progesterone for 3 days had virtually no gap junctions whilst the tight junctions were elongate. The circular pattern described above for controls had become irregularly shaped polygonal structures with some loose strands extending below the tight junction proper. Few spaces appeared in the junctions but the whole complex now extended two to three times further down the lateral cell membrane. Animals treated for 3 days with oestrogen showed a different pattern again. Gap junctions were absent but the, tight junctions lacked circular or polygonal compartments and comprised instead parallel arrays of loosely arranged lines. Large spaces appeared within the junctional elements which often resembled dashed lines. Animals treated with both progesterone and oestrogen for 3 days in such a way as to render the ovariectomised uterus receptive to implantation of blastocysts had tight junctions most nearly resembling the progesterone only treated animals but with more irregular strands extending below the major junctional region. This study clearly shows that junctional complexes in these cells are very sensitive to ovarian hormones and this probably reflects the dynamic state in which uterine epithelial cells find themselves during the oestrous cycle and early pregnancy. The very irregular tight junctions seen in animals treated with both progesterone and oestrogen may also indicate that the cells are not tightly joined just prior to implantation and this may facilitate penetration of the blastocyst. 49. Aspects of adrenal development in the marsupial, Isoodon macrourus. By R. J. LESLIE. University of Queensland The adrenal glands of newborn marsupials are undeveloped in comparison with those of neonatal eutherian mammals. A histological study to trace the devlopment of the adrenal gland of the bandicoot, Isoodon macrourus, included 30 developmental stages ranging from advanced fetal stages to young at weaning which occurs after approximately 65 days of pouch life. The development may be described in four morphological stages. In the initial stage, during late fetal development, proliferatingcoelomic epithelium formed a tight nodule of undifferentiated tissue in the mesoderm lateral to the dorsal aorta. Organisation of this tissue occurred in the second phase extending from the late fetal stage to the 14 day pouch young (PY). At birth, the cortical tissue was composed of large acidophilic cells arranged in thin irregular cords separated by capillaries. Small dark cells from the primitive autonomic ganglion invaded the gland on the medial aspect. In the 14 day PY the adrenal consisted of groups of cortical cells intermingling with clusters of basophilic neural cells. In stage three, these basophilic cells continued to migrate towards the central regions so that a thin cortex incompletely surrounded a large medulla early in the fourth week. During the final stage (35-65 days PY) growth in width and differentiation of cortical zones occurred and large sympathetic neurons penetrated the gland through pores in the capsule. The method of adrenal development in Isoodon macrourus is thus similar to that in most eutherian mammals; however, unlike these species, the major aspects of development took place after birth, during the period of pouch life. 50. The kidney of the Weddell seal (Leptonychotes weddelli). By P. H. VARDY and M. M. BRYDEN. University of Queensland In an attempt to correlate the renulate architecture of the marine mammalian kidney with the unique physiological responses observed in marine mammals, the kidney of the Weddell seal (Leptonychotes weddelli) was studied by corrosion casting, India ink injections and histological methods. The kidney of Leptonychotes is renulate but represents only a variation of the typically mammalian plan: a peripyramidal muscle (sporta) is present and the venous drainage is characterised by a large extrinsic system and a small intrinsic system which, along with the sporta, have not been described in a phocid before.
Proceedings of the Anatomical Society of
Parameters relating to concentrating ability (specialised fornices, relative medullary thickness, the percentage distribution of thin loops in the medulla and glomerular differentiation) show that the renule of Leptonychotes is similar to the whole kidney of a small non-desert rodent and this is consistent with the relatively mediocre concentrating ability attributed to the kidneys of whales and seals. However the percentage ratio of the glomerular volume to cortical volume is high and is consistent with the post-prandial glomerular filtration rate seen in these animals. Therefore renulation is thought to be an adaptation of the kidney to the large size of most marine mammals and not to some factor directly related to the marine environment, there being an upper limit to the size of an unmodified renal unit (unipapillate kidney/renal lobe/renule). The extrinsic venous drainage system may have developed as a supplement to the more primitive intrinsic system whose capacity is probably unequal to the relatively high renal blood flow in the recently fed seal, the large size of the extrinsic system being consistent with the generally increased size of veins in pinnipeds. 51. Glomerular cultures from normal and diseased animal and human kidneys. By C. A. L. NEAL, E. F. GLASGOW, R. C. ATKINS and N. M. THOMSON. Monash University and Prince Henry's Hospital Glomeruli have been isolated from normal and diseased rat, rabbit, sheep and human kidneys and cultured in various media to investigate their cellular outgrowth. Using scanning and transmission electron microscopy, phase contrast, light microscopy, cinemicroscopy, and immunofluorescence, three types of cell have been distinguished. Type I and Type II cells have been seen in outgrowth from both normal and diseased tissue but Type III cells more commonly in certain disease conditions. Type I cells are large (100150,cem) flattened, vacuolated, multinucleate cells. They are relatively immobile and show the presence of FC receptors. Type II cells are smaller (15-50 ,tm), mobile, microvillous cells. They are phagocytic, filamentous and stain positively with antiactinomyosin serum. They are more sensitive to media changes than Type I cells. Type III cells are small, 10-15 gum in the human and very mobile. Seen rarely in cultures of normal glomeruli, they are numerous in cultures of kidneys in crescentic glomerulonephritis. They are ,-glucuronidase-, esterase- and acid phosphatase-positive; are capable of ingesting yeast, latex particles and cellular debris and are similar to macrophages in culture. Their life span ranges between 2-14 days. Because of their morphological and functional characteristics Type I cells are believed to be epithelial, Type II cells are mesangial and Type III cells are macrophages. Mitotic figures in all types are rarely seen in the outgrowth, and the glomerulus in culture collapses in time; thus the cellular outgrowth is thought to be mainly derived from migrating cells. Variations in media tonicity, percentage of added fetal calf serum, in antibiotic and bicarbonate concentrations affect the attachment and amount of outgrowth seen surrounding the glomerulus. Glomeruli can be isolated from renal biopsies and cultured to evaluate the role of individual cell types in various glomerulonephropathies. 52. A horseradish peroxidase study of the projection upon the cerebellum of the nuclei pontis and nucleus reticularis tegmenti pontis in the rat. By J. B. CARMEN and P. A. BROWN. University
of Auckland Numerous experimental studies in several species show that the afferent and efferent connexions of brain stem nuclei projecting to the cerebellum are complex. Retrograde axonal transport of horseradish peroxidase (HRP) was used to investigate the projection of the nuclei pontis (Np) and reticularis tegmenti pontis (Rtp) upon the cerebellum in the rat. In 16 adult male rats, using pentobarbitone/ether anaesthesia, 0-2 ,ul of 30-50 % horseradish peroxidase was injected over 15 minutes through a fine needle into the exposed cerebellum. The animals were killed 20 hours post-operatively by perfusion, under ether anaesthesia, with Karnovsky's fixative. The brains were stored in additional fixative for 24-28 hours, and sectioned at 40(um. Alternate sections were reacted with diaminobenzidine-H202 and mounted, some being counterstained with cresyl violet. Each injection involved about 10 % of the cerebellar cortex; areas with the granule cell layer significantly labelled were taken as a guide to the area HRP uptake. The positions of HRPlabelled cells in Np and Rtp were plotted using dark field microscopy.
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Injections in the vermis labelled, bilaterally, small groups of cells in pars lateralis and the medial areas of Np and a narrow lamina of cells forming the medial and dorsolateral aspects of Rtp. Injections more laterally labelled many cells in pars intermedia of the contralateral Np, moderate numbers of cells in the corresponding area on the ipsilateral side and, bilaterally, many cells throughout Rtp. Injections placed far laterally labelled the pars intermedia similarly but did not label cells in Rtp. 53. The sequence of morphological changes in the neuritic growth cone and target neuron during synaptic junction development. By ROSEMARY P. REES. University of Maryland and National Institute of Neurological and Communicative Disorders and Stroke, National Institute of Health, Maryland, U.S.A. Assembly of synaptic active zones was studied in culture, the presynaptic element being provided by neurites growing from segments of fetal rat spinal cord (SC), and the postsynaptic element by isolated rat superior cervical ganglion neurons (SCGN). Ultrastructural techniques used were (a) tannic acid staining to display plasmalemmal coat material, (b) labelling of acetylcholine receptor molecules by peroxidase-conjugated antibody, and (c) EM autoradiography to monitor the passage within the neuron of membrane precursors. Prior to contact, synapse-related organelles were absent. Upon contact, SC growth cone filopodia were extensively applied to SCGN plasmalemma. Tannic acid staining revealed patches of bilateral membrane specialisation, with underlying cytoplasmic densities and interdigitation of apposed coat material. Following the initial contact, the Golgi apparatus of target neurons hypertrophied and production of coated vesicles at the maturing face increased. EM autoradiography and tracer studies indicated that the coated vesicles then passed toward and fused with the plasmalemma. They appeared to contribute a region of postsynaptic membrane density, complete with cytoplasmic undercoating, transmitter receptor molecules and membrane coat material. Subsequent to the appearance of this area of postsynaptic membrane specialisation, filopodia were lost from the growth cone and the synaptic vesicles appeared in its cytoplasm. As the synapse matured, cleft width and content increased. Golgi-derived coated vesicles continued to join the postsynaptic membrane at all stages of maturation. Results suggest that contact between the coat material of presumptive synaptic partners triggers a precisely timed sequence of events leading to the formation of a mature synapse.
54. Experimental alteration of neocortex by removal of vibrissa follicles from pouch young brushtail possums, Trichosurus vulpecula. By W. L. WELLER. University of Tasmania In the brain of the brush-tail possum much of layer IV within SI neocortex consists of an ordered array of discrete clusters of neurons (like the 'cortical barrels' of mice and rats). Several lines of evidence show that most barrels are functionally related to vibrissae. For instance, experimental removal of selected vibrissa follicles from mice during the first week after birth (when neocortex is developing) alters the morphology of the corresponding cortical barrels. The subcortical levels of the murine somatosensory system, however, develop earlier. By contrast the embryonic state of the marsupial nervous system at birth provides a good opportunity for studying the development more nearly of the entire somatosensory system. Groups of vibrissa follicles were surgically removed from pouch young possums of different ages (1 to 74 days). The surgery was readily done without having to detach the young from the anaesthetised mothers' teats. The lesioned possums were fixed by transcardial perfusion of formalin at ages ranging from 3 to 16 months. Serial 150,um sections of each isolated, flattened, and frozen neocortex were stained with thionin. Examination of the sections revealed altered arrangements of barrels which were consistent with the peripheral lesions, according to the somatotopic vibrissa-barrel relationships previously shown. No such apparent barrel disarrangement resulted after removing follicles from the 74 days old possum. These results suggest that peripheral structures exert a potent influence upon normal neocortical development up to the tenth week of pouch life. This is about the time when individual barrels begin to appear.
Proceedings of the Anatomical Society of
55. The laminar location of corticocortical cells in the sensorimotor cortex of the cat. By H. G. MURRAY and C. J. HEATH. University of Otago The axonal connexions confined within the primary sensorimotor cortex (SmI) have been explored by intracortical microstimulation and extracellular recording of single unit activity. Adults cat were lightly anaesthetised with 'Althesin' (Glaxo). Within SMI stimulus pulses of 0-1 msec duration, 1 Hz frequency and less than 50 #tA amplitude (electrode tip negative) were applied at a depth of 1 mm and responses were recorded by micropipettes with impedances of 2-6 MQ. Typical responses (a wave accompanied by spike discharges) were found in an area which extended from the stimulus point rostrally into area 4 and 1-2 mm medially and laterally. There was no evidence that the major topographic or cytoarchitectonic boundaries limited the local spread of connexions within SmI. Recording tracks were labelled by injection of Alcian blue and stimulus tracks by the Prussian blue method. On histological reconstruction the stimulus point was usually found to be centred in lamina IV. About twice the number of responsive units occurred in laminae II and III as occurred in lamina V and VI. Few units were found in lamina IV and none in lamina I. Conduction velocity estimates suggest that the responses were mediated by small myelinated and unmyelinated fibres. The corticocortical connexions described here may mediate the longer latency, lower voltage fringe zones observed around the foci of cortical potentials evoked by stimulation of the periphery. They are also seen as a possible link in a long afferent-efferent loop involving the sensorimotor cortex.
56. Comparative studies of myelinated nerves in the sympathetic system. By J. W. HEATH. University of Newcastle, N.S. W. The presence of doubly myelinated axons in sympathetic nerves of untreated rats has been previously reported. This report describes the preliminary results of comparative studies on the sympathetic nerves of two rodent species, the mouse and guinea-pig. Superior cervical ganglia were prepared for electron microscopy by routine methods. Some tissue was fixed by perfusion and the remainder by immersion techniques. Thick (0 5 ,um approx.) sections and a group of thin sections were cut alternately, and stained for light and electron microscopy respectively. This method allows three dimensional reconstruction of particular nerve fibre segments by both light and electron microscopy. Examples of doubly myelinated axons were located in both species. In addition, other unusual axon/myelin sheath configurations, similar to those previously observed in rat sympathetic nerves, were located. As suggested for the rat, these configurations may represent stages in a sequence leading to the eventual formation of doubly myelinated axons. Structural evidence suggests that the doubly myelinated axons arise not by a supernumerary Schwann cell forming myelin external to a pre-existing myelinated axon, but by stripping of a Schwann cell and its myelin sheath from the axon by another Schwann cell, with subsequent myelin formation by the latter Schwann cell. Whatever the mechanism of formation of doubly myelinated axons it is interesting that the outer Schwann cell and myelin sheath were structurally intact though lacking close contact with the central axon. 57. Structural variability of optic synapses. By FIuTz-H. GULDNER. Australian National University The synapses of optic nerve afferents were studied in the hypothalamic suprachiasmatic nucleus of the rat. Degeneration and anterograde transport studies have shown them to be recognisable by their striking bright mitochondria showing tubular formations of the inner membrane within an electron-lucent swollen matrix. A morphometrical analysis of this type of synapse revealed a high variability of most of the structural components under apparently uniform conditions. Within the optic presynaptic elements the density of the clear vesicles may vary from 20 to as many as 200/,um2. Dense core vesicles may be present in varying numbers or completely missing. The area of the largest synaptic contacts is about 40 times larger than the area of the smallest contacts. The width of the synaptic cleft varies between 13 and 23 nm and the thickness of the postsynaptic density ranges from virtually 0 up to 60 nm. A minority,
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13-27 % of all optic synapses, belongs to Gray's Type II (narrow cleft and thin or no postsynaptic density), the others belong to Gray's Type I (wide cleft, significant postsynaptic density). A single optic bouton can form several synaptic contacts of different sizes of different types (Gray I and II). According to current knowledge Gray Type I synapses have excitatory effects and Gray Type II synapses have inhibitory effects. The optic boutons and even a single bouton might therefore induce excitatory and inhibitory postsynaptic potentials of very different strengths in the postsynaptic dendrites. 58. Motor neuron multiplicity mainly modulated by muscle mass - indirect evidence from facial motor neuron counts in marsupials. By C. R. R. WATSON and JAN PROVIS. University of New South Wales Counts of facial motor neurons from serial sections of the brains of members of 20 marsupial genera were made in order to determine the relationship between facial motor neuron numbers and brain and body size. The number of cells in the facial nucleus ranges from 2800 in Sminthopsis (body wt 10.5 g) to 12160 in Vombatus (body wt 15-9 kg). On a log-log scale, facial motor neuron number correlates highly with brain weight (r=0-95) and body weight (r= 0-93). There is no evidence of localised enlargement of facial subnuclei that might correlate with specialised facial behaviours in particular animals (e.g. vibrissae whisking in Trichosurus and complex ear movements in Didelphis). These findings suggest that facial motor neuron number is determined by simple facial muscle bulk (which we assume to correlate with body size); the complexity of facial muscle activity does not seem to significantly influence facial motor neuron number. This phenomenon is in sharp contrast to the well-known subnuclear enlargement of sensory nuclei related to behavioural specialisation in mammals. These findings, in conjunction with observations by other workers on avian and amphibian embryos, suggest that many embryonic facial motor neurons die at the time of neuromuscular synaptogenesis; only those nmotor neurons making appropriate neuromuscular synapses survive. In this way the size of the adult population of facial motor neurons would be determined by the amount of facial muscle available for innervation.
59. Experimental low back pain, referred pain and muscle spasm. By N. BOGDUK and R. R. MUNRO. University of Sydney The distribution of local pain, referred pain and muscle spasm following the stimulation of the back was studied in six experiments on the author (N.B.) and three on two other volunteers. The back was stimulated with injections of 6 % saline into the L 5-S I interspinous ligament or the multifidus muscle at the L 5 level. Pain was described subjectively and muscle activity was recorded electromyographically. Local pain was produced in all experiments. It lasted 5 minutes, peaking in severity at 2 minutes. Referred pain occurred in two experiments. It was dull and pressure-like in nature. It occurred in the gluteal region, extending along the iliac crest from the posterior superior spine to the front of the hip joint. It occurred about 30 seconds after stimulation and lasted 5 minutes. Involuntary muscle activity occurred in five experiments. Its distribution varied between experiments, but each of the following muscles demonstrated activity on at least one occasion multifidus, gluteus medius and tensor fasciae latae. The activity started 1 minute after stimulation and lasted 2-5 minutes. Muscle activity occurred in experiments different from those in which referred pain occurred. These results demonstrate that noxious stimulation of structures supplied by dorsal rami can produce independent sensory and motor effects in areas supplied by both dorsal and ventral rami. It is suggested that these 'reflexes' may be the mechanism of referred lower limb pain and so called 'rheumatic nodules' in certain low back pain syndromes.
Proceedings of the Anatomical Society of
60. Localisation of acetylcholinesterase enzyme in the limbic system of the rabbit brain. By S. WANG and M. GIRGIs. Sydney University The aim of this study is to present a detailed and thorough description of acetylcholinesterase enzyme (AChE) content of the limbic structures in the rabbit which is now commonly used in our laboratory for investigating electrographic-behavioural correlations of the cholinergic limbic system. We use 'chemitrodes' for intracerebral microinjections of cholinomimetic drugs; a detailed histochemical study (using Koelle's method) of the distribution of AChE enzyme in the limbic system of the rabbit was deemed necessary. An integral part of such a description must be a comparison with normal cytological material and in this case it was necessary of include a detailed account of the nuclear disposition of certain areas. Such areas as the septum and the amygdala are particularly poorly described in the rabbit and an attempt was made to clarify in some way the confusion that still exists in the literature of the comparative anatomy and exact localisation of these structures. (A completely new stereotaxic atlas of the rabbit brain was actually constructed; Girgis and Wang, poster presentation, this Congress.) On the whole it could be inferred from this study that AChE-bearing neurons in general are better developed in phylogenetically older parts of the brain. Practically all the relays in Papez's 'cortical circuits of emotion' have a fairly high AChE activity. As noted by us in other macrosmatic mammals, the intensity of AChE staining in the rabbit brain is much greater overall than in, say, the microsmatic primates.
61. The role of the M. cricopharyngeus in the suckling lamb. ByJ. C. NEWHOOK and P. T. SMITH. Massey University The influence of sucking attitude on the function of M. cricopharyngeus in the lamb was studied. Concurrent electromyographic and cineradiographic studies were undertaken on a lamb from 18-25 days of age, while it was at rest, swallowing saliva, or sucking a barium sulphate and milk mixture from a teat held either above head level or near floor level. EMG records were made from M. thyropharyngeus, M. cricopharyngeus, and rostral and caudal cervical oesophagus. At rest, M. cricopharyngeus continuously exhibited a very low, irregular, level of basal electrical activity with no tonic characteristics. A salivary swallow was preceded by a slowly increasing level of activity, abruptly terminated by an inhibitory period of 200 msec. This was followed by strong activity which commenced before that of the rostral oesophagus and declined over about 1 second. During sucking, in both head up and head down attitudes, inhibition of M. cricopharyngeus commenced when the bolus reached the pharyngeal isthmus, and bolus transit time through the laryngopharynx was constant. With the head elevated, the duration of inhibition was about 150 msec, being significantly shorter than that preceding a salivary swallow (P < 0-001). The activity which followed was either no more than basal or was weak ( 0 05) sex difference in body weight favouring males appeared by the 18th day postnatally in the young of exercised animals, but not until the 46th day in the young of the control animals. By the 22nd day significant (P > 0-001) differences were also found between the relative adrenal weights of male and female offspring from exercised animals.
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The data indicated that exercise during pregnancy did not affect the myocardial capillary to fibre ratio in offspring up to 56 days postnatally but caused sex differences in body weight and relative adrenal weight at an earlier age in the offspring from exercised mothers. 73. The effect of pineal denervation on crypt cell proliferation in the rat small intestine. By B. D. CALLAGHAN. University of Adelaide Previous investigators have shown that operative removal of the pineal gland in the rat was associated with an increased mitotic rate in a number of tissues, including the small intestine. It has been established that the rat pineal gland depends exclusively for its nerve supply on the integrity of the superior cervical sympathetic ganglion. The purpose of the present study was to determine if the same effect on crypt cell proliferation could be obtained by denervation rather than removal of the pineal gland. Bilateral superior cervical sympathetic ganglionectomy was performed in male albino SpragueDawley rats and, using a stathmokinetic technique, mitotic rates were estimated in the crypts of the jejunum (5 cm distal to the duodenojejunal flexure) and ileum (5 cm proximal to the ileocaecal junction), on the 14th post-operative day. When compared with mitotic rates in a control group, no statistically significant difference was found. These observations suggest that the mitotic rate in the crypts of the jejunum and ileum is not significantly altered by denervation of the pineal gland. A possible explanation for this result is that denervation of the pineal gland does not result in the loss of all of its functions, particularly the glandular. Another possibility is that during removal of the pineal, other neighbouring structures may have been damaged or altered, thus causing the observed effects upon crypt cell proliferation.